Download ZOONOSES –PROTECTION OF PUBLIC AND ANIMAL HEALTH

FACULTY OF PUBLIC HEALTH, SLOVAK MEDICAL UNIVERSITY, BRATISLAVA
SLOVAK EPIDEMIOLOGICAL AND VACCINOLOGICAL SOCIETY
NATIONAL FOCAL POINT OF SLOVAK REPUBLIC FOR SCIENTIFIC AND TECHNICAL MATTERS
FOR EFSA– MINISTRY OF AGRICULTURE AND RURAL DEVELOPMENT OF THE SR
STATE VETERINARY AND FOOD ADMINISTRATION OF THE SLOVAK REPUBLIC
PUBLIC HEALTH AUTHORITY OF THE SLOVAK REPUBLIC
CZECHOSLOVAK SOCIETY FOR MICROBIOLOGY
UNIVERSITY OF VETERINARY MEDICINE AND PHARMACY, KOŠICE
INSTITUTE OF PARASITOLOGY, SLOVAK ACADEMY OF SCIENCES
ZOONOSES –PROTECTION OF PUBLIC
AND ANIMAL HEALTH
REVIEWED ABSTRACTS
FROM
5th SCIENTIFIC CONGRESS
HELD IN
BRATISLAVA,
18 th – 20 th O CTOBER 2016
Zoonoses - Protection of Public and Animal Health
5th Scientific Congress
18th-20th October 2016
COMPILER :
DOC .
MUD R . Z UZANA K RIŠTÚFKOVÁ , P H D., MPH
REVIEWERS :
Č IŽNÁR , D R S C .
DOC . MUD R . A NNA E GNEROVÁ , CS C ., MIM . PROF .
PROF . I NG . I VAN
1. I SSUE
B RATISLAVA , S LOVAK MEDICAL UNIVER SITY IN
B RATISLAVA , 2016
ISBN 978-80-89702-30-5
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Zoonoses - Protection of Public and Animal Health
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18th-20th October 2016
CONTENT
PART I. – ABSTRACTS OF ORAL PRESENTATIONS
OCCURENCE AND DIVERSITY OF BABESIA IN SWEDEN ........................................ 7
ALIMENTARY OUTBREAKS OF TICKBORNE ENCEPHALITIS IN
SLOVAKREPUBLIC .......................................................................................................... 10
THE LONG TERM TREND IN THE OCCURRENCE OF ZOONOSES IN THE
SLOVAK REPUBLIC ......................................................................................................... 15
TEN YEARS OF MONITORING OF THERMOTOLERANT CAMPYLOBACTER IN
THE HUMAN FOOD CHAIN IN THE CZECH REPUBLIC............................................ 22
INCREASE OF CAMPYLOBACTERIOSIS OUTBREAKS DUE TO RAW MILK
CONSUMPTION IN GERMANY ...................................................................................... 26
WASTEWATER – SOURCE OF ANTIBIOTIC RESISTANT BACTERIA .................... 28
OCCURRENCE OF AICHI VIRUS IN MYTILUS GALLOPROVINCIALIS ..................... 32
A SEROSURVEY OF FLAVIVIRUS INFECTION IN HORSES AND BIRDS .............. 36
MOLECULAR ECO-EPIDEMIOLOGY OF BORRELIOSIS IN SLOVAKIA ................ 41
NATIONAL DATABASE OF LISTERIA MONOCYTOGENES..................................... 45
PFGE PROFILES SOURCED FROM FOOD, FEED, ANIMALS, AND THE RELATED
ENVIRONMENT ................................................................................................................ 45
SALMONELLOSIS OUTBREAKS IN 2014 AND 2015 IN THE CZECH REPUBLIC .. 49
MONITORING OF OCCURRENCE TICK-BORNE ENCEPHALITIS IN RAW MILK IN
SLOVAKIA WITHIN 2014-2016 ....................................................................................... 53
MOLECULAR EPIDEMIOLOGY OF LISTERIOSIS - A TOOL FOR OUTBREAK
INVESTIGATION............................................................................................................... 56
PRESENCE OF TICK-BORNE ENCEPHALITIS VIRUS IN TICKS FROM SELECTED
LOCATIONS IN SLOVAKIA ............................................................................................ 60
VECTORS (INSECTA) OF SELECTED ZOONOTIC INFECTIOUS AGENTS –
THE
CURRENT SITUATION IN SLOVAKIA .......................................................................... 65
THE INCIDENCE OF SALMONELLOSIS IN SLOVAKIA - TRENDS AND CURRENT
STATUS .............................................................................................................................. 69
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DETECTION OF HAEMOTROPIC MYCOPLASMAS IN DOMESTIC AND WILD
LIVING ANIMALS IN SLOVAKIA (PRELIMINARY DATA) ....................................... 72
WE SEARCHED FOR ONE, WE DISCOVERED MORE AND WHAT´S NEXT? ........ 76
INFLUENZA – THE PERMANENT THREAT ................................................................. 80
DETERMINATION OF MICROBIAL INDICATORS IN DAIRY PRODUCTS BASED
ON THE DETECTION OF OXYGEN CONSUMPTION.................................................. 87
GROWTH DYNAMICS OF E. COLI ISOLATED FROM DAIRY PRODUCTS ............ 89
PRE-CUT (READY-TO-EAT) VEGETABLES: EVALUATION OF THE
CORRELATION BETWEEN ARCOBACTER SPP. AND ENTEROBACTERIACEAE .... 94
COULD TICK-BORNE ENCEPHALITIS LEAVE BEHIND PERMANENT
CONSEQUENCES? ............................................................................................................ 98
RISK FACTORS OF SPORADIC CAMPYLOBACTERIOSIS IN THE BRATISLAVA
REGION, CASE-CONTROL STUDY.............................................................................. 101
RISK COMMUNICATION WHEN CONTAMINATION TAKES PLACE: FOOD
BUSINESS OPERATOR VIEW ....................................................................................... 104
CAMPYLOBACTER DETECTION DEPENDING ON THE MATRIX, METHOD AND
MEDIUM ........................................................................................................................... 108
THE ANIMAL HEALTH LAW -REGULATION (EU) 2016/429 – AND THE FUTURE
OF FOOD SAFETY AND FREE COMMERCE IN EUROPE ........................................ 111
EARLY LOCALIZED AND DISSEMINATED LYME BORRELIOSIS IN WESTERN
SLOVAKIA ....................................................................................................................... 115
FAMILIAL OCCURENCE OF TICK-BORN ENCEPHALITIS IN ENDEMIC REGION CASE STUDY. .................................................................................................................. 118
ALIMENTARY BOTULISM – OUR EXPERIENCES WITH THE LABORATORY
DIAGNOSTICS. ................................................................................................................ 123
ZIKA VIRUS – SHOULD WE BE AFRAID OF IT? ....................................................... 128
GENOTYPING OF GIARDIA DUODENALIS ISOLATES FROM CHILDREN AND
DOGS IN EASTERN SLOVAKIA ................................................................................... 131
HEPATITIS A AND HEPATITIS E IN READY-TO-EAT SALADS ............................. 134
CANINE BABESIOSIS IN SLOVAKIA .......................................................................... 138
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PART II. –ABSTRACTS OF POSTERS
THE USE OF BACTERIOPHAGES IN THE THERAPY OF CRONOBACTER SPP. ... 142
IDENTIFICATION AND CHARACTERIZATION OF TRICLOSAN RESISTANT
COLIFORMS FROM SEWAGE SLUDGE ...................................................................... 146
DETECTION OF CHLAMYDIA SPP. IN DIFFERENT SPECIES OF BIRDS FROM
SLOVAKIA ....................................................................................................................... 150
COMPARISON OF METHODS USED IN THE DETECTION OF YERSINIA
ENTEROCOLITICA IN MILK AND MEAT COMMODITIES: CULTIVATION
METHOD VERSUS PCR ................................................................................................. 154
FIRST EVIDENCEOF ZOONOTIC SPECIES CRYPTOSPORIDIUM MUSKRAT
GENOTYPES I AND II F IN RODENTS IN SLOVAKIA .............................................. 158
HUMAN GENOTYPES OF ZOONOTIC PATHOGENS CRYPTOSPORIDIUM SPP.
IDENTIFIED IN RODENTS IN SLOVAKIA .................................................................. 161
HARD TICKS AND TICK-BORNE PATHOGENS DANGEROUS TO HUMANS IN
URBAN PARKS OF THE CAPITAL OF UKRAINE ...................................................... 164
TRICHINELLA (NEMATODA, TRICHINELLIDAE) AMONGST WILD AND
DOMESTIC MAMMALS IN UKRAINE ......................................................................... 167
CHARACTERIZATION OF CRONOBACTER STRAINS ISOLATED FROM
DIFFERENT SOURCES ................................................................................................... 169
SIMULTANEOUS DETECTION AND DIFFERENTIATION OF FLAVIVIRUSES BY
PCR AND REVERSE LINE BLOT (RLB) ...................................................................... 173
ALIMENTARY INTOXICATIONS CAUSED BY FOODSTUFF CONTAINING -CN
CHEMICAL GROUP ........................................................................................................ 178
PREVALENCE OF CHLAMYDIA FELIS INFECTION ACROSS VARIOUS
POPULATIONS OF CATS AND ITS IMPACT FOR PUBLIC HEALTH ..................... 182
MOLECULAR CHARACTERISTICS OF GENOTYPES AND SUBTYPES OF
CRYPTOSPORIDIUM HOMINIS IN SLOVAKIA ......................................................... 186
APPLICATION OF MULTILOCUS SEQUENCE TYPING IN THE ANALYSIS
OF BORRELIA BURGDORFERI SENSU LATO ............................................................. 190
LIVER TREMATODES IN WILD RUMINANTS .......................................................... 194
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THE INCIDENCE OF HEPATITIS E VIRUS IN DIFFERENT AGE CATEGORIES OF
DOMESTIC PIGS ............................................................................................................. 197
TAIL FIBER ASSOCIATED DEPOLYMERASES AS A POTENTIAL BIOFILM
DEGRADATION AGENT ................................................................................................ 201
SEROPREVALENCE OF ANAPLASMOSIS IN HUMANS
FROM EASTERN SLOVAKIA ........................................................................................ 204
ANTIBACTERIAL EFFECTS OF HERBAL ESSENTIAL OILS AGAINST SELECTED
FOOD-BORNE PATHOGENS ......................................................................................... 207
LABORATORY DIAGNOSTICS OF LEGIONELLOSIS AND TULAREMIA –
SEROLOGICAL TESTING AND PCR ........................................................................... 212
CHARACTERIZATION OF ESCHERICHIA COLI O157:H7 STRAINS ISOLATED
FROM CATTLE HIDES AT SLAUGHTER .................................................................... 218
SAPROLEGNIA PARASITICA – DANGEROUS FISH PARASITE ............................. 221
THE CURRENT EPIZOOTIOLOGICAL AND EPIDEMIOLOGICAL SITUATION IN
OCCURENCE OF RABIES IN SLOVAKIA ................................................................... 225
MOLECULAR CHARACTERIZATION AND TRACING OF PERSISTENT LISTERIA
MONOCYTOGENES STRAINS IN THE PRODUCTION CHAIN OF TRADITIONAL
MEAT PROCESSING FACILITY IN SLOVAKIA ......................................................... 229
OCCURRENCE OF ESCHERICHIA COLI PRODUCING ESBL-, AMPC- IN
POULTRY, PORK AND BEEF SAMPLES TAKEN FROM THE RETAIL. ................. 233
ENVIRONMENT IN LOW HYGIENIC STANDARDS SETTLEMENT IN PREŠOV
REGION – A POTENTIAL SOURCE OF ZOONOSES .................................................. 236
LEPTOSPIROSES IN SLOVAKIA IN 2006 – 2015 ........................................................ 238
DETECTION OF GENE ENCODING ENTEROTOXINS IN COAGULASE-NEGATIVE
STAPHYLOCOCCI ISOLATED FROM FOODS OF ANIMAL ORIGIN ..................... 242
DETECTION OF TICK-BORNE PATHOGENS IN DOG – FEEDING TICKS IN
BRATISLAVA (SLOVAK REPUBLIC) .......................................................................... 246
MALASSEZIOSIS IN HUMANS AND ANIMALS ........................................................ 250
PREVALENCE OF SALMONELLA IN POULTRY GALLUS GALLUS IN THE SLOVAK
REPUBLIC IN THE YEARS 2011 - 2015. ....................................................................... 254
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IXODES RICINUS TICK, ITS PATHOGENS AND HUMAN ........................................ 259
ANAPLASMA PHAGOCYTOPHILUM – A PATHOGEN CIRCULATING IN WILD
ANIMALS IN NATURAL FOCI OF WESTERN SLOVAKIA ...................................... 263
SHEEP MILK AS A SOURCE OF HUMAN TOXOPLASMOSIS ................................. 266
OCCURRENCE OF STRONGYLOIDES STERCORALIS IN CHILDREN AND DOGS IN
EASTERN SLOVAKIA .................................................................................................... 270
CIGUATERA FISH CONTAMINATION AND CONSEQUENCES OF CIGUATOXIN
CONTAMINATED FISH CONSUMPTION IN HUMANS ............................................ 273
TRANSMISSION AND INTERACTIONS BETWEEN BORRELIA AFZELII AND
RICKETTSIA SPP. IN IXODES RICINUS TICKS AND LABORATORY MICE MODEL
........................................................................................................................................... 277
DETECTION ZOONOTIC SPECIES ENTEROCYTOZOON BIENEUSI IN CALVES .. 281
USE OF ATP BIOLUMINESCENCE METHODS IN THE PREVENTION OF
FOODBORNE DISEASES ............................................................................................... 285
MOLECULAR CHARACTERIZATION AND TRACING OF PERSISTENT LISTERIA
MONOCYTOGENES STRAINS IN THE PRODUCTION CHAIN OF TRADITIONAL
BRYNDZA CHEESE PROCESSING FACILITY IN SLOVAKIA ................................. 289
SANITATION AS AN IMPORTANT TOOL FOR MONITORING ENVIRONMENTAL
HYGIENE IN FOOD PROCESSING PLANTS ............................................................... 293
TULAREMIA IN SLOVAKIA - EPIDEMIOLOGIC AND EPIZOOTIC ISSUES, 2005 2014 ................................................................................................................................... 297
PROS AND CONS OF LYME DISEASE TESTS: AN AGREEMENT ANALYSIS ..... 302
IDENTIFICATION AND CHARACTERIZATION OF LISTERIA MONOCYTOGENES
ISOLATED FROM FISH IN POLAND ........................................................................... 308
MASTITIS PATHOGENS ISOLATED FROM SAMPLES OF MILK IN DAIRY COWS
AND THEIR RESISTANCE ............................................................................................. 312
CHANGES IN THE COMPOSITION OF BACTERIAL MICROFLORA OF PIGEONS
DURING THE RACE SEASON ....................................................................................... 316
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PART I. – ABSTRACTS OF ORAL PRESENTATIONS
OCCURENCE AND DIVERSITY OF BABESIA IN SWEDEN
Andersson O. Martin
Center for Ecology and Evolution in Microbial Model Systems (EEMiS), Linnaeus
University, SE-391 82 Kalmar, Sweden
[email protected]
Introduction: The tick-transmitted intraerythrocytic parasite Babesia is maintained in
zoonotic cycles between vertebrate hosts and tick vectors [1] and most zoonotic species are
maintained in wildlife reservoirs. Various Babesia species have been detected in a wide
range of different mammal species [1]. However, the occurrence in natural mammal hosts
is still incompletely known for several zoonotic species [1]. The most prevalent zoonotic
species, Babesia microti, is mainly reported from USA, and is maintained in various rodent
reservoir hosts. In Europe, most human cases are attributed to the species B. divergens that
is mainly associated with cattle. Moreover, also B. venatorum is known to infect humans in
Europe [2, 3]. This species mainly utilizes roe deer as reservoir hosts [4]. Primarily
Babesia spp. are of veterinary importance and cause severe economic losses in cattle and
other domestic animals worldwide [5–8]. However since several species are also known to
infect humans, babesiosis is considered as an emerging zoonosis in parts of the world [1,
9–11].
In Sweden, the occurrence and diversity of Babesia species is to a large extent unknown,
since no detection have been performed with molecular methods. We therefore
investigated the occurrence and diversity of Babesia species with PCR and sequencing in
ticks and a prospective reservoir host, the roe deer (Capreolus capreolus).
Methods: In order to estimate the exposure to Babesia from infected ticks, we collected
questing Ixodes ricinus ticks from several sites across southern Sweden during two
consecutive field seasons and investigated the occurrence of Babesia species with a PCR
assay targeting the 18s rRNA gene [12]. Furthermore, we investigated the prevalence of
Babesia and diversity of species in roe deer in two sites in south-central Sweden using the
same methods as above. Blood samples were taken from trapped roe deer at two different
study sites, 150 km apart in southern Sweden; Bogesund (59°24’N, 18°12’E) is located at
the inner reaches of the Stockholm Archipelago, surrounded by water and covered by
highly productive mixed coniferous and deciduous forest and farmlands with high deer
densities. Grimsö Wildlife Research Area (59°60’N, 15°16’E) has a roe deer population
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with much lower density, and colder and longer winters due to its inland location. The area
consists primarily of coniferous forest interspersed with bogs, mires and fens.
Results and Discussion: We report for the first time the occurrence of the zoonotic species
Babesia venatorum in Swedish ticks, with a prevalence of 1%. We also detected B. microti
(prevalence 3.2%) and B. divergens (prevalence 0.2%). The incidence of Babesia in
questing ticks is substantially lower than that of several other tick-borne diseases in
Sweden such as Borrelia spp. and Candidatus Neoehrlichia mikurensis. We show with
molecular methods that two Babesia spp. occur in wild roe deer in Sweden, B. capreoli and
B. venatorum. This is to the best of our knowledge the first molecular detection of Babesia
spp. in any wildlife species in Sweden. In total 52% of the individuals (40 out of 77) were
infected with Babesia spp. The prevalence of B. capreoli in the individuals were 44% (34
/77) and the prevalence of B. venatorum was 7.8% (6/77). B. capreoli is seemingly not able
to infect cattle [13], and no reports of infections in humans have been published. This
species is therefore not likely to be a threat to other species than the natural hosts [13].
Infection have been reported from several different deer species [13 and references herein].
Contrastingly, B. venatorum apparently has a broader host range and is also capable of
infecting humans, it is also known to infect chamois (Rupicapra rupicapra) and ibex
(Capra ibex) in the Alpine region [14], and has also been found in a captive reindeer
(Rangifer sp.) in the Netherlands [15]. Several human cases of B. venatorum have been
reported from Europe and more recently from China [16, 17, 2, 3] and the zoonotic
potential of this species requires further investigation to correctly estimate risks for humans
and perhaps domestic animals.
Conclusions: We conclude that babesiosis should not be neglected as a possible diagnosis
following tick bites in humans and animals in Sweden as well as in other part of Europe.
Literature
1.
Yabsley MJ, Shock BC: Natural history of Zoonotic Babesia: Role of wildlife reservoirs. Int J Parasitol
Parasites Wildl 2013, 2:18–31.
2.
Herwaldt BL, Cacciò S, Gherlinzoni F, Aspöck H, Slemenda SB, Piccaluga PP, Martinelli G, Edelhofer
R, Hollenstein U, Poletti G, Pampiglione S, Löschenberger K, Tura S, Pieniazek NJ: Molecular
characterization of a non-Babesia divergens organism causing zoonotic babesiosis in Europe. Emerg
Infect Dis 2003, 9:942–948.
3.
Häselbarth K, Tenter AM, Brade V, Krieger G, Hunfeld KP: First case of human babesiosis in Germany
- Clinical presentation and molecular characterisation of the pathogen. Int J Med Microbiol 2007,
297:197–204.
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Duh D, Petrovec M, Bidovec A, Avsic-Zupanc T: Cervids as Babesiae hosts, Slovenia. Emerg Infect
Dis2005, 11:1121–1123.
5.
Zintl A, Mulcahy G, Skerrett HE, Taylor SM, Gray JS: Babesia divergens, a blood parasite of veterinary
and zoonotic importance. Clin Microbiol Rev 2003, 16:622–636.
6.
Bock R, Jackson L, de Vos a, Jorgensen W: Babesiosis of cattle.Parasitology 2004, 129 Suppl:S247-S269.
7.
Homer MJ, Aguilar-Delfin I, Telford SR, Krause PJ, Persing DH: Babesiosis.Clin Microbiol Rev 2000,
13:451–469.
8.
Purnell RE: Tick-borne diseases of British livestock. Vet Med Rev 1981, 1:58–69.
9.
Gray J, Zintl A, Hildebrandt A, Hunfeld KP, Weiss L: Zoonotic babesiosis: Overview of the disease and
novel aspects of pathogen identity. Ticks Tick Borne Dis 2010, 1:3–10.
10. Gorenflot A, Moubri K, Precigout B, A CE: Human babesiosis.Ann Trop Med Parasitol 1998, 92:489–
501.
11. Kjemtrup AM, Conrad PA: Human babesiosis: An emerging tick-borne disease. Int J Parasitol 2000,
30:1323–1337.
12. Casati S, Sager H, Gern L, Piffaretti J: Presence of potentially pathogenic Babesia sp. for human in
Ixodes ricinus in Switzerland. Ann Agric Environ Med 2006, 13:65–70.
13. Malandrin L, Jouglin M, Sun Y, Brisseau N, Chauvin A: Redescription of Babesia capreoli (Enigk and
Friedhoff, 1962) from roe deer (Capreolus capreolus): Isolation, cultivation, host specificity, molecular
characterisation and differentiation from Babesia divergens. Int J Parasitol 2010, 40:277–284.
14. Michel AO, Mathis A, Ryser-Degiorgis MP: Babesia spp. in European wild ruminant species: Parasite
diversity and risk factors for infection. Vet Res 2014, 45:1–11.
15. Kik M, Nijhof AM, Balk JA, Jongejan F: Babesia sp. EU1 Infection in a Forest Reindeer, the
Netherlands. Emerg Infect Dis 2011, 17:936–937.
16. Sun Y, Li S-G, Jiang J-F, Wang X, Zhang Y, Wang H, Cao W-C: Babesia venatorum Infection in Child,
China. Emerg Infect Dis 2014, 20:896–897.
17. Jiang J-F, Zheng Y-C, Jiang R-R, Li H, Huo Q-B, Jiang B-G, Sun Y, Jia N, Wang Y-W, Ma L, Liu H-B,
Chu Y-L, Ni X-B, Liu K, Song Y-D, Yao N-N, Wang H, Sun T, Cao W-C: Epidemiological, clinical,
and laboratory characteristics of 48 cases of “Babesia venatorum” infection in China: a descriptive study.
Lancet Infect Dis 2015, 15:196–203.
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ALIMENTARY OUTBREAKS OF TICKBORNE ENCEPHALITIS IN
SLOVAKREPUBLIC
Avdičová M.,1 Kerlik J.1, Seligová J.2
1
Regional Public Health Authority in Banská Bystrica, Slovak Republic
2
Regional Public Health Authority in Košice, Slovak Republic
[email protected]
Introduction: Tickborne encephalitis (TBE) is occurring worldwide. The causative agent
the virus (TBEV) is a cause of severe debilitating neurological disorders in humans. In
many countries besides the mode of transmission via ticks as vector, the alimentary mode
of transmission has been described as well. This mode of transmission in endemic areas is
associated with ingesting of unpasteurized dairy products contaminated by TBEV from
domestic infected animals. In Slovakia several cases of TBE are diagnosed every year. The
patient’s anamnesis shows the history of the tick bites or consumption of dairy products
made from unpasteurized goat or sheep milk. TBE transmitted by alimentary route is
occurring mostly in epidemies in both small family types and larger community types. The
sale of the dairy products directly from the farms together with raised preference of
consumers to buy such products is supporting the occurrence of these epidemics.
The Aim of the Study: The goal of the study was to analyse occurrence of TBE in
Slovakia in recent 20 years. We have focused to topology of the TBE as well as to
proportion of alimentary epidemies in the last 5 years. The effectivity of epidemiologic
interventions and measures applied for the control of the epidemies were analyzed.
Similarly the effectivity of the cooperation with veterinary experts during the confirmation
of the suspected sources of the infection was evaluated as well.
Methodology: We have analysed TBEcases occurring in Slovakia during the period from
January 1st 2012 to September 30th 2016. We have focused to characterization of the
occurrence, trends, age related morbidity rate, seasonality, mortality rate and lethality. The
analysis of the epidemies included also the time trend, number of patients, the number of
exposed persons, identification of the transmission factor based on epidemiological
anamnesis data and the laboratory examinations.
Results and Discussion: TBE occurrence in Slovakia follows for the long time mildly
increasing trend. The highest morbidity rate in the recent years was observed in the 2013
(Fig.1). It is evident that endemic occurrence is shifting northward to the regions with
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higher altitude above sea level. We have registered two fatal cases. These will be presented
in a separate lecture. During the recent 5 years we have registered 592 TBE cases and
occurrence reveals a characteristic seasonal profile having two peaks, in May and
September (Fig.2). The disease occurs in all age categories except the 0-age children with
very rare occurrence. The highest age specific morbidity rate was observed in the age
group 20-24 years old persons (Fig.3). Totally 13 alimentary epidemies of TBE were
registered in different parts of Slovakia in recent 5 years with number of patients from 2 to
44 (Fig.4). Totally 112 patients have contracted the TBEV via alimentary route. The
contaminated dairy products were responsible for 19 % of the total number of TBE
patients. The dairy products made of goat, sheep and cow milk are the dominating
anamnestic transmission factor. During the investigation of the source of infection,
specifically the animals and their dairy products, only rarely the contaminated factor of
transmission was confirmed. Similarly in the period of investigation only rarely the
suspected product could be identified. In the suspected animals due to the time shift from
disease to the investigation, the virus cannot be detected and thus only specific antibodies
are estimated. From the 13 analysed epidemies the milk was tested for the TBEV by PCR
method 4 times, all with the negative results. However, in one of the cases the serology for
specific antibodies was positive. The serological testing of the animals showed 6 positive
results, 3 times the serology was not carried on and 2 times the results were not reported to
the Public Health Authority.
Fig.1: TBE occurrence in Slovakia
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Fig.2: TBE seasonal incidence in Slovakia
Fig.3: TBE age-specific incidence in Slovakia
The major cause for the failure to identify the factors of TBE transmission appears to be
methodical. The State Veterinary Service in Slovakia has been using for direct TBEV
detection in animal products, particularly in milk, the PCR method. Several studies have
shown that after infected tick bites the animal the viremia develops in 24 to 48 hours. In
this period the virus is detected in the animal milk. The virus is present in the milk of farm
animal for 6-7 days. If man consumes the contaminated milk, the incubation time for
development of the clinical form of the disease lasts approximately. 14 days (10-28). Then
the patient with clinical symptoms of the infection visits her/his physician or health care
service where biological samples are taken and dispatched to laboratories for examination.
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The results of laboratory examinations are available after three days. Thus totally
minimally 21 to 28 days pass. After this period the examination of the milk for the
presence of TBEV is useless and negative laboratory results are meaningless. Research
studies show that viremia does not last in infected animal more than for one week. Thus
only the detection of specific antibodies in the blood of the animal is a relevant method for
early diagnosis of TBE.
Place of epidemic outbreak
Fig.4: TBE outbreak by district and place of epidemics, SR, 2012-2016.
The present studies have showed that TBEV survive well in milk. Provided such milk is
used for production of cheese, it is a product of a high risk for consumers. One of the
studies has shown that the number of TBEV particles at thermal treatment of the product
reveals a significant drop and virus particles are finally completely inactivated. The
optimal temperature for TBEV survival is between 4-8 ⁰C in a refrigerator. The virus
particles keep the full virulence potential at this temperature. At room temperature the
number of virus particles is decreasing, and the raise of the temperature to 30 ⁰C and
subsequent drop of the temperature to the level standardly used at production of the cheese
decreases the number of the viral particles from 106 to 103. However, this number of the
viral particles represents a sufficient infectious dose to cause a disease. The optimal
production temperature 72 ⁰C used during the process of pasteurization for 15 seconds and
subsequent cooling meet the criteria for production of dairy products without the TBEV.
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Conclusion : Slovakia represents in 2/3 of the territory the large endemic area of the TBE
in animal reservoirs and tick vectors. It seems that the optimal tools for prevention of the
alimentary TBE is either pasteurization of the goat and sheep dairy products and
adjustment of the production technology to higher temperature or vaccination of all
gourmet consumers. Applying this approach appears to be only effective way to prevention
of alimentary TBE epidemies and thus to protect health of the population.
Literature
1.
Analyse of Epidemiological Situation in Slovakia. 2012-2015. Available at: www.epis.sk/informačná
časť/Publikácie/výročné správy 2011,2012, 2013, 2014, 2015.
2.
Cisak, E., Wojcik-Fatla, A., Zajac, V., Sroka, J., Buczek, A., and Dutkiewicz, J. (2010). Prevalence of
tick-borne encephalitis virus (TBEV) in samples of raw milk taken randomly from cows, goats, and
sheep in eastern Poland. Ann. Agric. Environ. Med. 17, 283–286.
3.
European Centre for Disease Prevention and Control. Annual Epidemiological Report 2016 – Tick borne
encephalitis,(Internet), Stockholm; ECDC 2016.
4.
Gresikova, M. (1958a). Excretion of the tick-borne encephalitis virus in the milk of subcutaneously
infected cows. Acta Virol. 2, 188–192.
5.
Gresikova, M. (1958b). Recovery of the tick-borne encephalitis virus from the blood and milk of
subcutaneously infected sheep. Acta Virol. 2, 113–119.
6.
Gresikova, M., Sekeyova, M., Stanislava, S., and Necas, S. (1975). Sheep milk-borne epidemic of tickborne encephalitis in Slovakia. Intervirology 5, 57–61.
7.
Offerdal, D.,Niall,G.,C.,Bloom,M.: Stability of a Tick-Borne Flavivirus in Milk.
Front. Bioeng. Biotechnol., 11 May 2016 | http://dx.doi.org/10.3389/fbioe.2016.
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18th-20th October 2016
THE LONG TERM TREND IN THE OCCURRENCE OF ZOONOSES
IN THE SLOVAK REPUBLIC
Avdičová M.1, Krištúfková Z.2, Námešná J.1,Musilová M.1
1
2
Regional Public Health Authority in Banská Bystrica, Slovak Republic
Faculty of Public Health, Slovak Medical University in Bratislava, Slovak Republic
[email protected]
Introduction: Zoonoses – the diseases transmissible from animals to man cover a large
group of diseases that in the number are having a significant impact on the quality of life.
The number of the diseases has been steadily raising and there are at least 200 known
zoonoses at present. This number represents one third of all infectious diseases (1). The
zoonoses of domestic animals and synanthropic rodents, out of the all zoonoses, are the
most important for man. All mechanisms known for the mode of transmission (ingestion,
aspiration, contact, inoculation and transmission via live vectors) play a role in spreading
of the zoonoses. The importance of zoonoses is supported by a fact that some of them have
shown increasing trend with a occurrence in epidemic form. And some of them with
a severe clinical picture and fatal outcome. For successful prevention and control of the
zoonoses it is important to follow up the changes in spreading mode and to apply effective
preventive measures accordingly.
The aim of the study:The goal of the presentation is to show the long term trends in
occurrence of zoonoses in Slovakia in recent 20 years (2, 3). Further, to compare it with
the occurrence in other European states and point out to changes in epidemiology of
selected zoonoses in recent years (4, 5, 6).
Methodology: We have analysed cases of the disease reported to the information system
EPIS including its previous modifications. We have focused to the incidence, age specific
morbidity, seasonality, type of the occurrence, factors of diseases transmission and other
epidemiological characteristics in zoonoses transmitted by an alimentary road
(salmonellosis, campylobacteriosis, VETEC infections, toxoplasmosis, trichinellosis and
listeriosis). The other zoonoses such as anthrax, rabies, brucellosis and Q fever were
included as well. We have not analysed occurrence trends of infections characterised by
a natural focus of infection. These are analysed in extra presentation due to the equal
importance in Slovakia. In the study we have applied descriptive and comparative methods
as well as analysis of trends.
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Results and Discussion: We have observed following substantial changes over the period
of the study:
The salmonellosis have shown a significant decrease and change in a character of the
epidemies since 1998. The number of epidemies in food services such as cafeterias, dining
halls, canteens have dropped, while the small epidemies of family type were on a raise.
The common factor responsible for the raise of the epidemies is in the underestimated
thermal conditions at preparation of the food from eggs, poultry or other meat. The mild
increase in the epidemies has been observed since 2013. Further studies will be needed to
identify the responsible causal factors (Fig.1.). We have been observing the raise in the
occurrence of rare salmonellosis caused by serovars isolated from turtles, geckos etc. The
incidence of the salmonellosis recorded in 2014 in Slovak Republic (80.9) was 3.5 higher
over an average in EU and was one of the highest in Europe. Totally 52 Salmonella
serovars caused the diseases. The dominating serovars were S. Enteritidis (81.3 %) and S.
Typhimurium (5 %). The most critical health impact factor, the age-specific morbidity,
reveals the highest long term trend in the age group of children 0-1. (Fig.2)
Fig.1: Long-term trend in occurrence of salmonellosis and campylobacteriosis in
Slovakia
The campylobacter infections keep for a long period the occurrence in the form of sporadic
cases. The larger epidemies occur rarely however the steady raise over the period of recent
12 years has been observed (Fig.2). The dominating factor of transmission was identified
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poultry meat and products from no pasteurized milk, particularly from the sheep milk. The
incidence in Slovakia reached 126.8 in 2015, the value exceeding the EU average by 81 %.
The highest age-specific morbidity was recorded in children 0-1 years group (Fig.3).
Fig.2: Age-specific incidence on salmonellosis in 2015.
Fig.3: Age-specific incidence on campylobacteriosis in 2015.
The occurrence of yersiniosis in Slovakia was for a long period low and similar to the
average in EU. The incidence rate of yersiniosis in 2014 reached 3.1/100 000, representing
the occurrence by 64 % higher as the average in EU. The incidence rate then raised in 2015
to 4.1/100 000. As the factor of transmission, reported by patients it was the pork meat,
however the laboratory testing has not proved this association.
The occurrence of infections caused by Vero-toxin producing E. coli was very low (10
times lower than in other EU countries). This can be associated with the low level of
laboratory surveillance of the infections. For comparison in the 2014 the occurrence in
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Slovakia was at the level 26 cases (morbidity rate 0.5/100 000) representing 3 times less
than in EU countries.
The occurrence of toxoplasmosis in Slovakia shows for a long period a high morbidity rate
(4.0/100 000) with an age profile in children and the dominating symptoms on lymph
nodes and eyes. The next were the cases in 25-34 age categories of adult women. This
probably was caused by the examination of the pregnant women. It was interesting to see
the low occurrence of the congenital toxoplasmosis (less than 1 case per 50 000 labours, 0
or 1 case pre year. This probably is associated with the prenatal screening of pregnant
women. The occurrence of congenital toxoplasmosis was surprisingly very low (1 per
50 000 births – 0 or 1 case per year). This result appears to be the consequence of the
antenatal screening of the pregnant women. No endemic occurrence was observed and
more cross sectional cover was observed.
Fig.4: Occurrence of toxoplasmosis by districts in Slovakia 2011-2015.
Tularaemia occurs in a sporadic form in Slovakia. The number of cases reported in 2015 in
Slovakia was 28 (Incidence rate 0.5/100 000), 7 cases were reported in 2014 (0.13/100
000). The EU average in these years was 480 cases with morbidity rate 0.1/100 000. The
occurrence of tularaemia reveals a clearly endemic form in the South-West Slovakia.
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The Q fever was not recorded in Slovakia in 2015. In the recent 20 years only 4 cases of
the Q fever were recorded, with the last case in 2014. In EU countries totally 777 cases
were recorded in this year (0.18/100 000).
Fig.5: Occurrence of tularemia by districts in Slovakia, 2011-2015.
Brucellosis shows in a long term trend only a rare occurrence. Two cases were reported in
1999 both imported from Spain by sheep shearers. In spite of the low occurrence of the
brucellosis for a long period, both cases were confirmed by laboratory examination. The
last case occurred in 2015 however its epidemiology was not clarified neither type of the
brucella was specified.
The occurrence of listeriosis shows characteristics similar to other EU countries. The
disease is presented by a severe clinical picture, some of them with fatal outcome. The
number of the diseases shows a mild decrease and yearly 1-2 cases of congenital form of
listeriosis are reported. In 2014 totally 29 cases (morbidity rate 0.55/100 000) were
reported with two cases of the congenital form. The occurrence of the listeriosis in
Slovakia shows the profile in EU (0.52/100 000).
The last case of rabies in man occurred in 1990 in the County Rožňava after the previous
one case in the County Brezno in 1984. There is a high standard of the prophylactic
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measures applied to the persons wounded by animals suspected from rabies. The control of
situation in occurrence of rabies in animals is also at a high level.
In a group of parasitic diseases transmitted from animals to man the dominating ones are
toxocariasis, trichinellosis and echinococcosis.
Trichinellosis shows for a long time a sporadic and rarer occurrence in epidemic form. The
largest such outbreak was described in Slovakia in 1998 (Trichinella britovi, factor of
transmission were identified sausages with a portion of dog meat, 360 cases). Two cases
were reported in 2014 (morbidity rate 0.04/100 000). In the EU 319 cases occurred at that
time (morbidity rate 0.7/100 000).
Echinococcosis occurrence is recorded rarely in Slovakia. Only 5 cases were reported in
2015 and 8 cases (morbidity rate 0.09/100 000) in 2014. The average in EU was 0.18/100
000. (2, 3, 4, 5, 6)
The zoonoses with a natural focus of infection represent a very important group of
diseases. Their occurrence is a high risk factor for man due to the spreading to endemic
areas, the spreading via alimentary road and epidemic occurrence. The example is the tick
encephalitis. Due to the severity and heavy burden they will be presented in a special
section.
Analysis of zoonoses by the profession showed that the professionally acquired zoonoses
occur only rarely. In close future it will be necessary to incorporate into the category of
zoonotic disease the recently described zoonoses such as hepatitis E. Recently in Slovakia
10-15 cases are recorded every year (2, 3).
The challenging issue for public health experts specialized in zoonoses is a potential to
identify and implement measures in cases of the old diseases and new ones that may spread
as a consequence of demographic factors such as population density raise, migration, food
shortage and war conflicts. Increased contacts with rodents and free animals and their
parasites as well as risky behaviour activities such as camping in wild conditions and
consumption of a raw foodstuff of animal origin without thermal processing represent
other area deserving a special attention. Therefore an education of inhabitants, the
upgrading of their awareness regarding the potential risks and preventable measures has
been an important activity for prevention of the zoonoses (1).
Conclusion: The presented analysis has shown that from the point of view of impact on
the population health the occurrence of salmonellosis, campylobacteriosis, and
toxoplasmosis represent a serious problem. It is not only due to the quantity of the cases,
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but primarily due to the high proportion of hospitalized patients, particularly children. The
occurrence of zoonoses in Slovakia compared with zoonoses in other EU countries is
different. While in salmonellosis and campylobacteriosis the morbidity is the second one
highest, after the Czech Republic, the other zoonoses by morbidity are at the level of
average in EU. This group of zoonoses with lower morbidity can be divided into two
subgroups. One encompasses the zoonoses with low or zero morbidity due to the high
quality of surveillance by veterinary services covering brucelosis, bovine tuberculosis and
partially also rabies. The second group of zoonoses is presented with a lower occurrence
where the diagnostics in human does not reach the top quality (infections caused by
VETEC). Therefore it is important to keep surveillance both in veterinary and human field
at the high standard, to keep and to focus epidemiological surveillance to zoonoses and to
be prepared for new and new emerging infections.
Literature
1.
Bauerfeind R.:Zoonoses: Infectious Diseases Transmissible from Animals to Humans. 2016.532 pp.
ISBN 9781555819255.
2.
Analysis of Epidemioligical Situation in Slovakia, 2014. Available at: www.epis.sk/informačná
časť/Publikácie/výročné správy 2015
3.
Analysis of Epidemiological Situation in Slovakia, 2015. Available at: www.epis.sk/Informačná
časť/Publikácie/Výročné správy/2014
4.
European
Centre
for
Disease
Prevention
and
Control.
Annual
Epidemiological
Report
2016.(Internet),Stockholm;ECDC 2016. Available at: www.ecec.europa.eu/Annual epidemiologigal
report 2016.
5.
EU summary report on zoonoses, zoonotic agents and food-borne epidemies 2014.EFSA Journal
2015;13(12):4329. Available at: www.efsa.europa.eu/
6.
Report on Zoonoses, alimentary infections and water infections in Slovakia 2014. Ministry of
Agriculture and Rural Development, Slovakia. ISBN 978-80-89738-05-2.
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Zoonoses - Protection of Public and Animal Health
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18th-20th October 2016
TEN YEARS OF MONITORING OF THERMOTOLERANT CAMPYLOBACTER
IN THE HUMAN FOOD CHAIN IN THE CZECH REPUBLIC
J. Bardoň
State Veterinary Institute Olomouc,Jakoubka ze Stříbra 1, Olomouc, Czech Republic
[email protected]
Introduction: Bacteria of the genus Campylobacter considerably contribute to human
foodborne illnesses in both the Czech Republic and Europe. These bacteria are widespread
in nature, with the main reservoir being the intestinal tract of birds and mammals, both
domestic and wild. In the vast majority of cases, human infections are foodborne. The
main sources are meat and offal of poultry, pigs or cattle. An important cause of less
common family outbreaks is cross contamination of foods and kitchen utensils in
consumers’ households. Cases have even been reported of diseases caused by consuming
raw milk or dairy products made from unpasteurized milk (fresh cheese). The vast majority
of them are individual cases; in Europe, Campylobacter spp. do not significantly contribute
to the development of epidemics. When epidemic infections do occur, the most frequent
source was poultry meat [1]. Another potential sources of infections, mainly in children,
are infected pets, in particular dogs and cats. Interhuman transmission of the infection is
uncommon [2]. Yet another source of the diseases is water reservoirs, with the infection
being linked to water sports. Survival of Campylobacter in water is facilitated by their
entry into the protozoa Tetrahymena pyriformis and Acanthamoeba castellanii, where the
bacteria are better protected from the external environment [3]. A risk factor for humans is
the long-term use of proton pump inhibitors which impairs the gastric mucosal barrier by
increasing the pH of gastric contents [4]. The age group most affected by foodborne
infections in Europe is young children [1,2]. The State Veterinary Administration of the
Czech Republic has monitored the prevalence of thermotolerant Campylobacter spp. in
selected sample groups (poultry, pigs and milk) in the country since 2006.
The aim of the study: Monitoring the prevalence of thermotolerant Campylobacter spp. in
poultry, pigs and raw cow’s milk. Testing the resistance of isolates to selected antibiotics.
Methodology: As part of surveillance for zoonoses, the State Veterinary Administration of
the Czech Republic has monitored the prevalence of thermotolerant Campylobacter spp. in
selected foods (poultry, pigs and cow’s milk) since 2006. The groups and numbers of
samples examined in 2006 to 2015 are shown in Table 1.
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Zoonoses - Protection of Public and Animal Health
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Table 1 – Trends in the prevalence of thermotolerant Campylobacter spp. in the Czech
Republic between 2006 and 2015 in the monitored sample groups.
Year
Samples examined
Site of sampling
No. of
Prevalence
samples
(%)
2006
Broiler cloacal swabs
slaughterhouses
189
49%
2007
Broiler cloacal swabs
slaughterhouses
246
45%
2008
Broiler ceca
slaughterhouses
422
61%
Broiler neck skin
slaughterhouses
422
70%
Neck skin – fresh poultry
supermarkets
120
75%
Neck skin – frozen poultry
supermarkets
120
37%
Broiler ceca
slaughterhouses
134
72%
Milk filters (cow’s milk)
dairy farms
252
3%
2011
Broiler ceca
slaughterhouses
145
63%
2012
Broiler ceca
slaughterhouses
125
60%
2013
Broiler ceca
slaughterhouses
119
55%
2014
Broiler ceca
slaughterhouses
281
56%
2015
Broiler ceca
slaughterhouses
146
65%
Pig ceca
slaughterhouses
195
72%
2009
2010
The detection of Campylobacter spp. was performed in accordance with ISO 10272-1 (a
qualitative method) [5]. Selective media for Campylobacter spp. were used; a modified
selective medium (CCDA, Trios) was microaerophilically incubated (GENbox Microaer,
bioMérieux). Species identification of isolates was performed using PCR and, later,
matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (Biotyper
Microflex, Bruker). The isolates were tested for resistance to selected antibiotics
(erythromycin, ciprofloxacin, tetracycline, streptomycin, gentamicin and nalidixic acid)
using a microdilution method (Campy, Trios; Sensititre, Eucamp2, TREK). The inoculated
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plates were incubated in a microaerophilic atmosphere (GENbox Microaer, bioMérieux)
for 48 hours at 37°C.
Results and Discussion: The prevalence of thermotolerant Campylobacter spp. in
monitored samples is seen in Table 1. The table shows that a high proportion of poultry at
retail sale is superficially contaminated with Campylobacter spp. (75% of fresh poultry).
High rates of positive findings were also observed in cecal contents from broilers and pigs.
Among the monitored foods, the lowest rates (3% of positive samples) were detected when
testing milk filters. The results showed high resistance to quinolone antibiotics and
ampicillin in C. jejuni and C. coli. Moreover, the latter also showed high resistance to
tetracycline and streptomycin. Each year, data on the prevalence of Campylobacter
infections in humans and prevalence of Campylobacter spp. in food animals and foods are
published by the European Food Safety Authority (EFSA) in its summary report which
also compares data from the EU member states (1). In some parameters, the data from
individual member states vary considerably. With respect to monitoring of the prevalence
of Campylobacter spp. in fresh poultry meat at retail sale (e.g. Campylobacter in fresh
broiler meat, 2013), the definition of fresh meat as stated in Regulation (EC) No. 853/2004
of the European Parliament and of the Council must be considered [6]. According to this
definition, fresh meat, including meat that is vacuum-wrapped or wrapped in a controlled
atmosphere, has not undergone any preserving process other than chilling, freezing or
quick-freezing. It means that monitoring of Campylobacter spp. in fresh poultry meat in
accordance with the EFSA might include samples collected from both chilled and frozen
broilers. Apparently, there are significant differences in the rates of positive results
between the two categories. Therefore, data on the percentages of positive findings cannot
be reliably compared among the member states without more detailed descriptions of the
samples. Consistently with our results, high resistance of Campylobacter spp. to quinolone
antibiotics is seen throughout Europe [7].
Conclusion: The results of ten-year monitoring of thermotolerant Campylobacter spp. in
the Czech Republic show high rates of these bacteria in poultry and pig ceca as well as on
the skin of fresh broilers at retail sale. The prevalence in raw milk is very low. The isolates
are highly resistant to quinolone antibiotics, with C. coli being also resistant to tetracycline
and streptomycin.
Supported by the State Veterinary Administration of the Czech Republic.
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Literature
1.
EFSA and ECDC (European Food Safety Authority and European Centre for Disease Prevention and
Control), 2015. The European Union Summary Report on Trends and Sources of Zoonoses, Zoonotic
Agents
and
Food-borne
Outbreaks
in
2013.
EFSA
Journal
2015;13(1):3991,
165
pp.
doi:10.2903/j.efsa.2015.3991
2.
Táborská J. Kampylobakterové infekce. In: Beneš J. a kol: Infekční lékařství. Praha; Galén, 2009:242244.
3.
Patrick, M. E., Gilbert, M. J., Blaser, M. J. et al. Human infections with new subspecies of
Campylobacter fetus. Emerg Infect Dis 2013;19:1678-1680.
4.
Feodoroff B, de Haan C. P, Ellström P, et al. Clonal distribution and virulence of Campylobacter jejuni
isolates in blood. Emerg Infect Dis 2013;19:1653-1655.
5.
ISO 10272-1:2006 Microbiology of food and animal feeding stuffs-Horizontal method for detection and
enumeration of Campylobacter spp.
6.
Evropský parlament a Rada Evropské unie. Nařízení Evropského parlamentu a rady (ES) č. 853/2004 ze
dne 29. dubna 2004 stanovující zvláštní hygienické předpisy pro potraviny živočišného původu. Úřední
věstník Evropské unie. 2004: 12.
7. EFSA (European Food Safety Authority) and ECDC (European Centre for Disease Prevention and
Control), 2016. The European Union summary report on antimicrobial resistance in zoonotic and
indicator bacteria from humans, animals and food in 2014. EFSA Journal 2016;14(2):4380, 207 pp.
doi:10.2903/j.efsa.2016.4380
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INCREASE OF CAMPYLOBACTERIOSIS OUTBREAKS DUE TO RAW MILK
CONSUMPTION IN GERMANY
Becker N, Heckenbach K, Schewe T, Raschke D, Luber P
Federal Office of Consumer Protection and Food Safety, Mauerstraße 39 – 42, 10117
Berlin, Germany
[email protected]
Introduction: The watching and warning system for crisis prevention at the German
Federal Office of Consumer Protection and Food Safety (BVL) observes since 2015 a
trend towards raw milk consumption in Germany. Identified drivers were at one hand the
abolition of the milk quota in Germany in April 2015, which accelerated the decline of the
price of milk. At the other hand, some studies concerning health-promoting effects of raw
milk were published and used for raw milk commercial. An early trend analysis indicated
an increased risk for campylobacteriosis disease in districts with milk vending machines
compared to rural districts without milk vending machines.Based on this observation and
the foodborne outbreak data a risk communication for consumers concerning the protective
effect of decocting raw milk and a FAQ for raw milk consumption were released by the
German Federal Institute for Risk Assessment (BfR).
Methods: The number of campylobacteriosis cases in the time period week 9 to 29 of the
year 2015 was compared with the number of campylobacteriosis cases in the same period
of time in the year 2014. The trend analysis was based on the assumption that most of the
raw milk vending machines have been constructed in springtime 2015. The location of raw
milk vending machines in Germany was investigated online by the search for the terms
“Milchtankstelle”, “Rohmilch”, “Hof” (“milk vending machine”, “raw milk”, “farm”) in
Google, DuckDuckGo and PolyMeta. The assignment of campylobacteriosis cases to
districts with and without raw milk vending machines was made using post codes. The
accessibility of the raw milk vending machines within one hour from rural district with
campylobacteriosis cases was calculated using a route planer (Falk).
The ODDS Ratio for districts with campylobacteriosis cases without increase of more than
5 cases and districts with an increase of at least 5 cases with and without raw milk vending
machines was calculated.The trend analysis did not include the exposure of consumers to
raw milk by farm- or organic markets or raw milk vending machines which were not listed
online. Some vending machines existed alreadybefore 2015.
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Results and Discussion: Campylobacteriosis cases have been reported in 338 of 446
German districts in the investigated time period. In 39 districts an increase of at least 5
cases was observed in the year 2015 in comparison to the year 2014. 88 raw milk vending
machines were identified. They were annotated to 70 districts. The ODDS Ratio analysis
revealed that the probability of an increase of more than 5 campylobacteriosis cases is 70
% higher in districts with raw milk vending machines compared to districts without
vending machines (ODDS Ratio (1.7 95 % CI 0.8 – 3.7), Table 1).
Table 1 Analysis of Campylobacteriosis cases in German districts with and without raw
milk vending machine.
With
Without
∑
9
30
39
61
346
407
70
376
446
Increase ≥ 5 cases
district
bacteriosis in
Campylo-
District and raw milk vending machine
Decrease or Increase
of < 5 cases
∑
In nine districts, the number of cases increased by more than 5 and there was a raw milk
vending machine located. In further nine districts a vending machine could be reached in
less than one hour. Including these districts into the calculation increased ODDS ratio to
4.8 (95 %, CI 2.8 – 8.3).
The analysis of foodborne outbreak data of the year 2015 revealed an increase of
campylobacteriosis outbreaks caused by the consumption of raw milk. Based on these
observations as a risk management measure the BfR released a risk communication for
consumers concerning the protective effect of decocting raw milk and an FAQ for raw
milk consumption.
Conclusion: In districts with raw milk vending machines there were early indications of an
increase of human campylobacteriosis cases in 2015. The German collection of foodborne
outbreak data confirmed the indicated increase of campylobacteriosis cases due to raw
milk consumption. In order to mitigate the risk a specific risk communication was released
to increase the awareness of German consumers about the risks associated with raw milk
consumption.
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WASTEWATER – SOURCE OF ANTIBIOTIC RESISTANT BACTERIA
Birošová, L., Mackuľak, T., Medveďová, A., Nagyová, K.
Department of Nutrition and Food Quality Assessment, Faculty of Chemical and Food
Technology, Slovak University of Technology, Radlinského 9, 81237 Bratislava, Slovakia
[email protected]
Introduction: Urban wastewater contain wide spectrum of different anthropogenic
pollutants such toxic metals, persistent organic pollutants, pharmaceuticals, pathogenic
microorganisms as well as antibiotic resistant bacteria (ARB) and antibiotic resistant genes
(ARG) (1). Chemical pollutants represent selective pressure for bacteria ending up in
development of mutations leading to antibiotic resistance (2). Beside this phenomenon,
antibiotic resistance can be spread also by horizontal gene transfer. Hospital effluent is
a complex mixture of chemical and biological substances which are continually
discharged.
Effluents
from
hospitals,
industry,
municipal
organizations,
and
urban/agricultural runoff in many developing countries represent a significant source of
emerging contaminants (metals, ARGs, ARB) in the receiving environment as the effluents
are discharged into sewer systems, rivers, lakes, and seas without prior treatment which
may then accumulate in sediments (1). Some bacteria are able to attach to surface and form
biofilm. High bacterial density and diversity are found in biofilms from wastewater
systems, especially from activated sludge of sewage treatment plants. Biofilms are also
generated in surface water and drinking water distribution systems (3). It is also well
known that microorganisms attached to a surface in the form of a biofilm exhibit
remarkable resistance to all types of antimicrobial challenge, compared with the same
microorganisms grown as freely suspended cells (4).
The aim of the study: The aim of the research presented in this paper is to assess
prevalence of antibiotic resistant coliform bacteria, enterococci and Staphylococcus aureus
in wastewater from hospital effluents, wastewater treatment plant (WWTP) influent and
effluent as well as in sewer biofilm in capital city of Slovakia. Second aim is identification
and characterization of coliform isolates from the perspective of antibiotic susceptibility
and ability to form biofilm.
Material and Methods: The paper analysed the wastewater from WWTP Petržalka
housing estate and from one hospital situated in Bratislava flowing into WWTP Petržalka.
Characterization of WWTP Petržalka was reffered in article of Mackuľak et al. (5).
Sampling was performed in December in 2014. Samples of wastewater were collected
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according to Mackuľak et al. (6). Occurrence of antibiotic resistant bacteria in wastewater
and biofilm samples was also detected according to Mackuľak et al. (6). Isolated coliform
bacteria were identified by MALDI-TOF mass spectrometry analysis. Spectra were
analysed by using MALDI BioTyper software (v 2.0) (BioTyper Library v 3.0; Bruker
Daltonics). Resistant strains isolated from diagnostic media with antibiotic were then tested
for cross-resistance to
other antibiotics (ampicillin, tetracycline,
ciprofloxacin,
chloramphenicol, gentamicin) using a plate dilution drop method (104 CFU/drop) using the
agar dilution method (7). Changes in biofilm formation were determined according
Beenken et al. (8) in 96-well polystyrene microtiter plates.
Results and Discussion
Tab 1. Prevalence of total and resistant bacteria in wastewater and sewerage biofilm
Hospital
effluent
Log
CFU/mL
CFB
ENT
SA
Total
A
G
C
CH
T
Total
A
G
C
V
Total
A
G
C
CH
T
M
E
CEF
P
V
2,8
2,78
2,72
1,38
0,125
1,56
0
0
0
0
0
2,53
0
0
0
0
0
2
0
0
1,5
2,2
WWTP Petržalka
Influent
Effluent
water
water
Log
Log
CFU/mL
CFU/mL
4,55
1,56
4,42
1,1
3,56
0
3,28
0
1,9
0
0
0
3,07
0,23
1,48
0
1,78
0
1,3
0
1,6
0
2,8
2,9
0
2,5
0
1,2
0
0
0
0
0
2,5
1,4
2,1
0,6
1,6
0
1,5
1,4
1,9
1,1
1,4
CFB – coliform bacteria ENT – enterococci
Biofilm
Influent
Log CFU/g
Effluent
Log CFU/g
7,42
6,62
5,2
5,44
4,7
5,2
4,28
0
0
0
3,48
3,6
1,7
0
0
0
0,7
2,4
0,7
0,8
1,3
1,8
8,66
7,64
6,78
6,48
6,13
6,83
5,74
5,48
5,73
5,61
5,41
4,3
3
0,6
0,8
0,2
2,7
3,3
2,7
1,6
2,4
2,6
SA – Staphylococcus aureus
A – ampicillin, G – gentamicin, C – ciprofloxacin, CH – chloramphenicol, T – tetracycline,
V – vancomycin, M – methicillin, E – erythromycin, CEF – cefoxitin, P - penicillin
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Hospital effluent contains high number of bacteria and pharmaceuticals. In this study, we
have examined wastewater from hospital situated near WWTP Petržalka. This hospital has
its own disinfection of wastewater. This reflects number of bacteria observed in studied
samples (Tab 1). Number of total CFB was relatively low (2.8 CFU/mL). Majority of CFB
were resistant to ampicillin and gentamicin. No enterococci were detected. In case of
S.aureus was detected only presence of methicillin, vancomycin and penicillin resistant
strains. WWTP influent contained much higher number of bacteria. In case of CFB
resistance to tetracycline was not detected. Enterococci were resistant to all studied
antibiotics including vancomycin. Lower number of total bacteria was observed in effluent
samples. Only exception was determined in case of S.aureus. This could be caused by
release of bacteria from sewerage biofilm, which possess very high number of antibiotic
resistant bacteria.
From WWTP influent wastewater were isolated 27 strains of coliform bacteria. Majority of
isolates was identified as Morganella morganii (12 isolates) or Citrobacter sp. (11
C.freundii; 2 C.braakii; 1 C.youngae). One strain was identified as Escherichia coli.
Twenty isolates were multidrug resistant, including E.coli. Two strains were very strong
producers of biofilm, 20 strains belong to strong producers and 5 strains were moderate
biofilm producers.
Conclusion: Anthropogenic activity contributes to dissemination of antibiotic resistance to
the environment. Municipal wastewater is hotspot of different multi drug resistant bacteria,
which are able to pass WWTP and disseminate resistance genes on the sensitive bacteria
and thus close the circular flow of resistance.
Acknowledgement: This study was financially supported by Research and Development
Agency of Slovak Republic, contract No. APVV-0122-12 and by STU grant for young
scientists, contract No. 1618/16.
References
1.
Laffite, A., Kilunga, P.I., Kayembe, J.M., Devarajan, N., Mulaji, C.K., Giuliani, G., Slaveykova, V.I.,
Poté, J.: Hospital effluents are one of several sources of metal, antibiotic resistance genes, and bacterial
markers disseminated in Sub-Saharan urban rivers. Front. Microbiol. 7, 1 – 14, 2016.
2.
Birosova, L., Mikulasova, M.: The effect of environmental pollutants and food processing on the
development of antibiotic resistance. Biomed. Pap. 158, 315-320, 2014.
3.
Schwartz, T., Kohnen, W., Jansen, B., Obst U.: Detection of antibiotic-resistant bacteria and their
resistance genes in wastewater, surface water, and drinking water biofilms. FEMS Microbiol. Ecol., 43,
325-335, 2003.
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4.
18th-20th October 2016
Mara, D., Horan, N.J.: Handbook of water and wastewater microbiology, Academic Press, London,
ISBN 0080478190, 832, 2003.
5.
Mackuľak, T., Birošová, L., Gál, M., Bodík, I., Grabic, R., Ryba, J., Škubák, J.: Wastewater analysis: the
mean of the monitoring of frequently prescribed pharmaceuticals in Slovakia, Environ. Monit Assess.
188, 1-12, 2016.
6.
Mackuľak, T., Vojs, M., Grabic, R., Golovko, O., Vojs Staňová, A., Birošová, L., Medveďová, A.,
Híveš, J., Gál, M., Kromka, A., Hanusová, A.: Occurrence of pharmaceuticals, illicit drugs, and resistant
types of bacteria in hospital effluent and their effective degradation by boron-doped diamond electrodes,
Monatsh. Chem.-Chemical Monthly 147, 97-103, 2016.
7.
Olejníková, P., Kurucová, M., Švorc, L. U., Marchalín, Š.: Induction of resistance in Mycobacterium
smegmatis. Can. J. Microbiol., 59, 126-129, 2012.
8.
Beenken, K.E., Blevins, J.S., Smeltzer, M.S.: Mutation of sarA in Staphylococcus aureus limits biofilm
formation. Infect Immun.,71, 4206-4211, 2003.
31
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OCCURRENCE OF AICHI VIRUS IN MYTILUS GALLOPROVINCIALIS
Bottaro, M., Di Pinto, A., Mottola, A., Marchetti, P., Di Martino, B., Terio, V.
Department of Veterinary Medicine, University of Bari, Strada provinciale per
Casamassima km 3, 70010 Valenzano (Ba) – Italy
[email protected]
Introduction: Aichi virus 1 (AiV-1), a human enteric virus belonging to the genus
Kobuvirus, is a member of the family Picornaviridae. AiV-1 is a small, non-enveloped
virus of approximately 27–30 nm in diameter with a single-stranded, positive polarity
RNA genome [13]. It was first recognized in 1989 as the cause of oyster-associated nonbacterial gastroenteritis in humans in Aichi Prefecture, Japan [18].
AiV-1 has been proposed as a causative agent of human gastroenteritis; clinical signs and
symptoms of Aichi virus infection include diarrhoea, abdominal pain, nausea, vomiting
and fever [18].
AiV-1 is globally distributed and it has been detected in various types of environmental
samples, such as sewage, surface water, groundwater and shellfish [7]. AiV-1 transmission
is thought to occur through direct contact, by faecal-oral routes, or through consumption of
contaminated food or water. In most cases, documented outbreaks associated with
consumption of foods have involved oysters or other seafood [4, 8, 14].
Filter-feeding shellfish are an important source for transmission of enteric viral diseases,
since they are able to accumulate and concentrate waterborne pathogens, especially when
they are grown in coastal areas contaminated by sewage [9]. In the European Countries, the
microbiological quality of commercially harvested shellfish intended for human
consumption must comply with the EU Food Hygiene Regulations (EC 2073/2005), which
rely exclusively on bacterial indicators (Escherichia coli and Salmonella). Although the
faecal indicator system has been in place for many years, it has been very well documented
that this system does not adequately index for the presence of the “traditional” viral agents
transmitted by shellfish (Norovirus and Hepatitis A virus) [3] or of the “emerging” foodborne viruses such as Aichi virus.
The aim of the study: To date, in Italy, there have been no epidemiological data regarding
the circulation of AiV-1 genotype A in shellfish and its potential association with diarrheal
cases or outbreaks.
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In this preliminary study, we assessed the presence of AiV-1 in Italian retail shellfish by
screening a collection of bivalve molluscs by using bio-molecular method (Reverse
Transcriptase-PCR).
Methods: Shellfish sampling
A total of 70 shellfish samples, including only Mytilus galloprovincialis, were examined.
All the samples were collected from open-air markets, hypermarket and fish chops in the
North and Centre of Apulia (SE Italy) from May 2015 to April 2016. All the mussel
samples were taken at local retail stores during ten independent sampling times separated
by at least 2 week.
Viral nucleic acid extraction
The viral and nucleic acid extraction process were performed according to ISO/TS
15216:2013 [5].
Detection of AiV by RT-PCR
AiV-1 was detected by RT-PCR using the primer set Ai6261 and Ai6779, targeting the
RNA polymerase gene to amplify a 519-bp fragment at the 3CD junction. A nested PCR
was performed with the primer pair C94b-246k to amplify a 223-bp segment within the
3C-3D junction region [12]. The amplified products were analysed by gel electrophoresis
on Green Gel Safe 10000X-stained 1.5% agarose. Several procedures were adopted during
RNA extraction and PCR amplifications to avoid cross-contaminations.
Sequencing and phylogenetic analysis
For phylogenetic analysis, sequence alignment and clustering were performed by using
Geneious software package (Geneious version 6.1.8 created by Biomatters). The sequences
were preliminary analyzed using free access sequence databases by BLAST
(http://www.ncbi.nlm.nih.gov) and FASTA (http://www.ebi.ac.uk) and therefore were
compared to a selection of sequences representative of recent epidemic strains circulating
worldwide.
Results and Discussion: Viral contamination in shellfish is a serious problem and recent
papers have demonstrated contamination of different bivalve molluscs worldwide [2, 16,
17]. Although Norovirus and Hepatitis A virus, causing gastroenteritis and hepatitis, are
regarded as the most common causes of food-borne infections, in recent years other viruses
with zoonotic potential emerged.
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In this study, 70 shellfish samples collected in Italy between the years 2015-2016 were
screened for AiV-1. All the samples tested negative in the first-round RT-PCR with primer
pair Ai6261- Ai6779. However, in the nested PCR with primers C94b-246k, AiV-1 RNA
was detected only in one (0.7%) sample. The sample was collected in June 2015 from a
fish shop located in the North of Apulia (Foggia).
Phylogenetic analysis of a partial genomic region (223-bp segment within the 3C-3D
junction region) of the AiV-1 allowed its characterization as genotype A. The
predominance of genotype A strains in the human population has been described in
previous studies in Europe and in Asia [1, 11, 19].
To our knowledge, this study confirmed, for the first time in Italy, the presence of RNA
AiV-1 in Mytilus galloprovincialis. Although the low incidence (0.7%; 1/70) of AiV-1
genotype A that we observed in our survey, our result suggests that this virus is present in
the water environment. The current study is consistent with the seroepidemiologic data
collected worldwide [6, 10] and highlights the role of AiV-1 as an emerging virus
implicated in food related gastroenteritis caused by contaminated bivalves.
Conclusion: Considering the increasing interest in emerging pathogen infections
worldwide [15] and the increase in food-borne disease related to the consumption of raw or
lightly cooked mussels, an intensive and continuous monitoring of zoonotic viral agent
such as Aichi virus is strongly recommended in order to assess the human health risk.
Our findings suggest that AiV-1 could potentially be an appropriate indicator of viral
contamination in the environment. A better knowledge of shellfish contamination will help
improve shellfish safety, thus protecting consumers.
Structured surveillance studies on the occurrence of AiV-1 in environments, in particular,
in shellfish, possibly in combination with clinical studies in human patients, are needed for
a better understanding of AiV-1 epidemiology, with regards to its temporal and
geographical distribution, environmental stability and potential health risks to humans.
Literature
1.
Ambert-Balay K, Lorrot M, Bon F, Giraudon H, Kaplon J, Wolfer M, Lebon P, Gendrel D, Pothier P
(2008). Prevalence and genetic diversity of Aichi virus strains in stool samples from community and
hospitalized patients. J Clin Microbiol 46:1252–58
2.
Benabbes L, Ollivier J, Schaeffer J, Parnaudeau S, Rhaissi H, Nourlil J, Le Guyader FS (2013).
Norovirus and other human enteric viruses in Moroccan shellfish. Food Environ Virol, 5:35-40
3.
Formiga-Cruz M., Allard AK, Conden-Hansson AC, Henshilwood K, Hernroth BE, Jofre J, Lees DN,
Lucena F, Papapetropoulou M, Rangdale RE, Tsibouxi A, Vantarakis A, Girones R (2003). Evaluation
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of potential indicators of viral contamination in shellfish and their applicability to diverse geographical
areas. Appl. Environ. Microbiol. 69:1556-63
4.
Hansman GS, Oka T, Li TC, Nishio O, Noda M, Takeda N (2008). Detection of human enteric viruses in
Japanese clams. J. Food Prot.71:1689–95
5.
ISO/TS 15216-2 (2013). Microbiology of food and animal feed-horizontal method for determination of
hepatitis A virus and norovirus in food using real-time RT-PCR. Part 2: Method for qualitative detection
International Organization for Standardization, Geneva, Switzerland
6.
Khamrin P, Maneekarn N, Okitsu S, Ushijima H (2014). Epidemiology of human and animal
kobuviruses. VirusDis. 25:195–200
7.
Kitajima M, Gerba CP (2014). Aichi Virus 1: Environmental occurrence and behaviour. Pathogens 2015,
4, 256-268
8.
Le Guyader FS, Le Saux JC, Ambert-Balay K, Krol J, Serais O, Parnaudeau S, Giraudon H, Delmas G,
Pommepuy M, Pothier P, Atmar RL (2008). Aichi virus, norovirus, astrovirus, enterovirus, and rotavirus
involved in clinical cases from a French oyster related gastroenteritis outbreak. J Clin Microbiol
46:4011–17
9.
Le Guyader FS, Haugarreau L, Miossec L, Dubois E, Pommepuy M (2000). Three year study to assess
human enteric viruses in shellfish. Appl. Environ. Microbiol. 66:3241–48
10. Lodder WJ, Rutjes SA, Takumi K, Husman de Roda AM (2013). Aichi Virus in sewage and surface
water, the Netherlands. Emerging Infect. Dis, 19
11. Oh DY, Silva PA, Hauroeder B, Diedrich S, Cardoso DD, Schreier E (2006). Molecular characterization
of the first Aichi viruses isolated in Europe and in South America. Arch Virol 151:1199–1206
12. Pham NT, Khamrin P, Nguyen TA, Kanti DS, Phan TG, Okitsu S, Ushijima H (2007). Isolation and
molecular characterization of Aichi Viruses from fecal specimens collected in Japan, Bangladesh,
Thailand, and Vietnam. J Clin Microbiol, 2287–88
13. Reuter G, Boros A, Pankovics P (2011). Kobuviruses - A comprehensive review. Rev. Med. Virol. 21,
32–41
14. Sdiri-Loulizi K, Hassine M, Aouni Z, Gharbi-Khelifi H, Sakly N, Chouchane S, Guediche MN, Pothier
P, Aouni M, Ambert-Balay K (2010). First molecular detection of Aichi virus in sewage and shellfish
samples in the Monastir region of Tunisia. Arch Virol 155:1509–13
15. Skovgaard N (2007). New trends in emerging pathogens. Int. J. Food Microbiol. 120:217-24
16. Terio V, Martella V, Moschidou P, Di Pinto P, Tantillo G, Buonavoglia C (2010). Norovirus in retail
shellfish. Food Microbiol 27:29–32
17. Woods JW, Calci KR, Marchant-Tambone JG, Burkhardt III W (2016). Detection and molecular
characterization of norovirus from oysters implicated in outbreaks in the US. Food Microbiol 59:76-84
18. Yamashita T, Kobayashi S, Sakae K, Nakata S, Chiba S, Ishihara Y, Isomura S (1991). Isolation of
cytopathic small round viruses with BS-C-1 cells from patients with gastroenteritis. J Infect Dis
164:954–57
19. Yamashita T, Ito M, Tsuzuki H, Sakae K (2001). Identification of Aichi virus infection by measurement
of immunoglobulin responses in an enzyme-linked immunosorbent assay. J Clin Microbiol 39:4178–80
35
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A SEROSURVEY OF FLAVIVIRUS INFECTION IN HORSES AND BIRDS
Csank, T.1, Bakonyi, T.2, Korytár, Ľ. 3, Major, P.4, Gyuranecz, M.5, Pistl, J.1
1
University of Veterinary Medicine and Pharmacy in Košice, Department of Microbiology
and Immunology, Komenského 73, 041 81 Košice, Slovakia, [email protected]
2
University of Veterinary Medicine, Department of Microbiology and Infectious Diseases,
Hungária krt. 23 – 25, 1143 Budapest, Hungary
3
UVMP in Košice, Department of the environment, veterinary legislation and economy
4
5
UVMP in Košice, Clinic for birds and exotic animals
Hungarian Academy of Sciences, Institute for Veterinary Medical Research, MTA Centre
for Agricultural Research, Hungária krt. 21, 1143 Budapest, Hungary
Introduction: Flaviviruses are arboviruses.Based on transmission,flaviviruses are
classified as mosquito-, tick-borne and flaviviruses without vector transmission. They have
a wide range of host spectrum. West Nile virus (WNV) and Usutu virus (USUV) are
antigenically close mosquito-borne viruses mostly associated with birds but occasionally
transmitted to horses and humans. The course of infection is often subclinical; however, in
some cases severe neurological disease may occur [1]. TBEV is a tick-borne flavivirus.
Small mammals act as reservoirs. Ruminants and humans act as accidental hosts. TBEV
may cause CNS infection in humans after biting by infected tick, or through consumption
of raw goat or sheep milk and milk products [2]. Serological diagnosis in areas where
several flaviviruses co-circulate is difficult because of cross reaction. The aim of our work
was to assess the prevalence of WNV, USUV and TBEV infections in horses and birds
from Slovakia.
Material and methods: Serum samples. Horse sera (n=145) were collected during 2013 in
counties Banská Štiavnica (n=11), Bratislava (n=30), Košice (n=4), Levice (n=16), Myjava
(n=4), Pezinok (n=27), Prešov (n=2), Prievidza (n=1), Rožňava (n=14), Spišská Nová Ves
(n=10) and Žiar nad Hronom (n=16). Twenty-two horses have not been abroad Slovakia
and have not been vaccinated against WNV. None of the tested horses had clinical signs of
West Nile fever.
Seventy-five bird serum samples have been originated from counties Banská Bystrica
(n=2), Košice (n=16), Košice okolie (n=31), Levice (n=8), Nitra (n=2), Poltár (n=1),
Prešov (n=2), Rimavská Sobota (n=1), Spišská Nová Ves (n=1), Trebišov (n=8), Vranov
nad Topľou (n=1), Zvolen (n=2). Birds were either caught by mist netting or were patients
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in the Clinic for birds and exotic animals. Information on mist netting localities are
available in [3] and information on patients are available on request.
ELISA. Horse and bird sera were tested for WNV by blocking ELISA kit INGEZIM WNV
COMPAC (Ingenasa, Spain) following the manufacturer´s protocol. Positive and dubious
horse sera were further tested for the presence of WNV IgMs by capture ELISA kit
INGEZIM WNV IgM (Ingenasa). TBEV antibodies in horses were detected by competitive
ELISA kit EIA TBEV Ig (TestLine, Czech Republic) without modifications.
Micro-virus neutralization test. Horse and bird sera were inactivated at 56 °C for 30
minutes and prediluted to 1:10 in EMEM. Serial two-fold dilutions of prediluted sera were
prepared in duplicates in a volume of 25 μl. An equal volume of 100 TCID50 of virus
strains WNV 578/10 and USUV 939/01 (kindly provided by professor Norbert Nowotny,
Veterinärmedizinische Universität, Wien) were added into each well. After 1-hour
incubation at 37 °C a 50 μl of 10 % EMEM supplemented by antibiotics containing 1×104
Vero E6 cells were added into each well. Virus inoculum was back-titrated during each
sample batch. Plates were incubated for 5 days at 37 °C at 5 % CO2 atmosphere. Positive
result for WNV or USUV in cross reactive samples was assigned if the neutralizing
antibody titer of the certain virus was at least 4-fold higher.
Results: Seventeen out of 145 (11.7 %) horse serum samples reacted positively in ELISA
and 12 (8.3 %) were confirmed by VNT (Table 1). Seropositive horses were from counties
Banská Štiavnica (n=2), Bratislava (n=5), Levice (n=2), Pezinok (n=2) and Žiar nad
Hronom (n=1). Five VNT negative sera had lower PI then 50 % and were negative in
VNT. Three sera cross reacted in TBEV ELISA with N/S [%] 204 – 442. Three out of 26
WNV dubious sera had N/S [%] of 436, 652 and 721 in TBEV ELISA. None of the WNV
positive and dubious horse serum samples had WNV IgMs. Six horses (samples 211., 258.,
259., 352., 353., 413.) which have not been abroad Slovakia were positive (4.1 %) for
WNV neutralizing antibodies.
There was no horse serum sample positive for neutralizing antibodies against USUV.
Nine (6.2 %) horse serum samples were positive in TBEV ELISA (Table 2) from counties
Bratislava (n=3), Levice (n=4), Košice (n=1) and Pezinok (n=1). Among these samples,
two cross reacted with WNV ELISA positive and one WNV ELISA and VNT WNV
positive horses. Three out of 10 (6.9 %) TBEV dubious horse serum reacted positively in
WNV ELISA with PI 93.8 – 96.7 % and VNT WNV titer 1:160 – 1:640 from counties
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(Table 1). There were no USUV antibodies and no cross reaction of TBEV positive sera
with USUV.
Table 1 WNV antibody prevalence in horses
Sample
WNV Ig (PI)
VNT WNV
VNT USUV
TBEV Ig (N/S [%])
135.
+ (46)
-
-
+ (442)
151.
+ (41)
-
-
- (143)
180.
+ (48)
-
-
- (115)
211.
+ (95)
1:320-640
-
- (131)
258.
+ (94)
1:320
-
D (160)
259.
+ (95)
1:640
1:10
- (147)
330.
+ (94)
1:160
-
D (157)
339.
+ (96)
1:320-640
-
- (115)
348.
+ (45)
-
-
- (114)
351.
+ (43)
-
-
+ (204)
352.
+ (90)
1:20
-
+ (264)
353.
+ (87)
1:10-1:20
-
- (119)
395.
+ (97)
1:640
-
D (162)
399.
+ (96)
1:160-320
-
- (128)
410.
+ (96)
1:320
-
- (136)
413.
+ (86)
1:20-1:40
-
- (129)
423.
+ (68)
1:40
-
- (117)
Legend: WNV Ig – positive (+) if percentage of inhibition (PI) ≥ 40 %, negative (-) if PI ≤ 30 % and dubious
(D) if PI 30 – 40 %. TBEV Ig –positive if % N/S > 200, negative if % N/S < 150 and dubious if % N/S is 150
– 199.
Table 2 TBEV antibody prevalence in horses
Sample
TBEV Ig
WNV Ig
VNT WNV
VNT USUV
135.
+ (442)
+ (46)
-
-
155.
+ (353)
- (23)
-
-
287.
+ (652)
D (32)
-
-
344.
+ (436)
D (35)
-
-
350.
+ (721)
D (37)
-
-
351.
+ (204)
+ (43)
-
-
352.
+ (264)
+ (90)
1:20
-
376.
+ (355)
- (8)
-
-
383.
+ (443)
- (12)
-
-
Thirteen (17.3 %), mostly raptor bird serum reacted positive in WNV ELISA of which 9 (12 %) was
confirmed by VNT (Table 3). Four of these samples were collected during 2013 and 5 during 2014. Sample
214 could not be evaluated as WNV or USUV positive, as neutralizing antibody titers did not reach 4-fold
difference (Table 3).
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Table 3 WNV antibody prevalence in birds
Sample
Bird species
WNV Ig
VNT WNV
VNT USUV
103.
Western marsh-harrier
79
1:10
-
210.
White stork
74
1:160
-
214.
Northern goshawk
84
1:80
1:20
274.
Northern goshawk
92
1:40-1:80
-
275.
Northern goshawk
41
-
-
448.
Northern goshawk
92
1:160
-
484.
Common kestrel
94
1:10-1:20
-
485.
Common kestrel
87
-
-
523.
Osprey
96
1:80
-
524.
Peregrine falcon
91
-
-
525.
Eurasian eagle-owl
94
1:160
-
528.
Golden eagle
93
1:40
-
563.
House sparrow
95
1:80
-
Legend: WNV Ig – positive (+) if percentage of inhibition (PI) ≥ 40 %, negative (-) if PI ≤ 30 %
and dubious (D) if PI 30 – 40 %. TBEV Ig –positive if % N/S > 200, negative if % N/S < 150 and
dubious if % N/S is 150 – 199.
WNV ELISA and VNT WNV positive birds were from counties Košice (n=3), Košice
okolie (n=2), Prešov (n=1), Spišská Nová Ves (n=1) and Trebišov (n=2). A house sparrow
(sample 563.) was caught in county Trebišov, the rest were patient samples. The golden
eagle has been live in captivity and showed signs of aspergillosis. The rest of the birds
were found in the nature and showing apathy, or had trauma. Three samples had dubious
PI and were negative in both neutralization assays. A pooled serum sample of 4 Eurasian
great tits had neutralizing USUV antibody titer 1:40. These birds were caught at county of
Levice during 2013.
Discussion: Overall prevalence of WNV infection in horses during 2013 was 11.7 % out
of which 8.3 % was confirmed by VNT. A former study showed 15.1 % WNV neutralizing
antibodyprevalence in horses from Slovakia, out of which 4.8 % were autochthonous [4].
Here, similar proportion of horses showed autochthonous WNV infection. USUV infection
was described in horses in Morocco and Croatia [5 – 6]; however, we did not detect any
USUV positive horses sampled during the study period. TBEV antibodies were detected in
2.1 % of horses. In the Czech Republic during 2011 and 2013 a 0.2 % prevalence TBEV
antibodies, respectively was observed [7].
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Recently, we described WNV circulation in the bird population in Slovakia but no USUV
[3]. Most of the WNV seropositive birds in this study were raptors and one pooled serum
sample of Eurasian great tits contained USUV antibodies.
Results of this study confirmed WNV infection in horses and birds. Further study is needed
to clarify the circulation of WNV USUV and TBEV in livestock animals and birds in
Slovakia.
Acknowledgement: This work was supported by projects VEGA 1/0729/16, ITMS
26220220185 (MediPark) and Domus Hungarica Scientiarum et Artium senior scholarship
of the Hungarian Academy of Sciences.
References
1. Hubalek, Z. (2008). "Mosquito-borne viruses in Europe." Parasitol Res 103 Suppl 1: S29-43.
2. Dobler, G. (2010). "Zoonotic tick-borne flaviviruses." Veterinary Microbiology 140(3-4): 221-228.
3. Csank, T. et al. (2016). "Detection of West Nile virus and tick-borne encephalitis virus in birds in
Slovakia, using a universal primer set." Archives of Virology 161(6): 1679-1683.
4. Hubalek, Z. et al. (2013). "West Nile Virus Equine Serosurvey in the Czech and Slovak Republics."
Vector Borne Zoonotic Dis 13: 733-738.
5. Barbic, L. et al. (2013). "Demonstration of Usutu Virus Antibodies in Horses, Croatia." Vector-Borne and
Zoonotic Diseases 13(10): 772-774.
6. Durand, B. et al. (2016). "Seroprevalence of West Nile and Usutu viruses in military working horses and
dogs, Morocco, 2012: dog as an alternative WNV sentinel species?" Epidemiology and Infection 144(9):
1857-1864.
7. Sedlak, K. et al. "Surveillance of West Nile fever in horses in the Czech Republic from 2011 to 2013."
Epidemiol Mikrobiol Imunol 63(4): 307-11.
40
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MOLECULAR ECO-EPIDEMIOLOGY OF BORRELIOSIS IN SLOVAKIA
Derdáková, M.a, Chvostáč, M.a, Vaculová, T.a, Václav, R.a, Ďurovská, J.b,c, Pancák J.c,
Rusňáková Tarageľová, Va.
a
Institute of Zoology Slovak Academy of Sciences, Dúbravská cesta 9, 84506 Bratislava,
Slovakia,bAmbulance for Lyme Borreliosis, Teslova 33, Bratislava, Slovakia, c1st.
Department of Neurology, Medical faculty of Commenius University and Universtiy
hospital of Bratislava, Slovakia
[email protected]
Introduction: Borrelia spirochetes belong to the most common and epidemiologically the
most important bacteria that are transmitted by ticks. Genus Borrelia is formed by two
groups that are morphologically indistinguishable but differ in the vectors, clinical
symptomatic in patients and ecology. Members of the Borrelia burgdorferi sensu lato (s.l.)
group are the causative agents of Lyme borreliosis, which is the most common tick-borne
disease in Europe (1). Second group is represented by more than 20 spirochetes that cause
relapsing fever and are transmitted mostly by soft ticks (2). B. burgdorferi s.l. complex
currently encompasses 20 genospecies out of which eight were confirmed in Europe. The
prevalence of B. burgdorferi s.l. in questing ticks in Europe varies between 0-50%. The
mean prevalence rate is around 13-20% (3). Human disease is caused by Borrelia
burgdorferi sensu stricto (s.s.), Borrelia spielmanii, B. afzelii, B. garinii, Borrelia
bavariensis and Borrelia bissettii. All the genospecies of B. burgdorferi s.l. are maintained
via zoonotic transmission cycles involving vertebrate reservoir hosts and ixodid ticks.
Humans can act as accidental hosts.
Borrelia miyamotoi belongs to the relapsing fever spirochetes. It is the only known species
from this group that is transmitted by the hard ticks from the genus Ixodes. Since its
discovery in 1995 in Ixodes persulcatus ticks from Japan it was detected in Ixodes ticks in
the USA, Asia and Europe so it shares the same vector with the Lyme borreliosis
spirochetes (4). Its pathogenicity was confirmed in Russia in 2011 (5). Human cases were
recorded in Europe, the USA and Asia. Possible co-infection of B. burgdorferi s.l. and B.
miyamotoi was described in patients from Japan (6).The prevalence in ticks across the
Europe is between 0-3% (7). The most important reservoir hosts are probably rodents (8).
Presence of this pathogen in Slovakia was confirmed in 2012 by Subramanian et al. (9),
however its distribution, genetic variability and ecology is unknown.
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The aim of the study: The aim of the study was to identify the prevalence and genetic
variability of Borrelia burgdorferi s.l. and occurence of recently recognized pathogen B.
miyamotoi in questing I. ricinus ticks in various habitats of Slovakia including sylvatic and
urban sites. Moreover, since 2013 we have started to test the Borrelia prevalence and its
heterogeneity in ticks that were detached from patients presented at the Ambulance for
Lyme borreliosis in Bratislava.
Methodology: Questing ticks were collected by flagging the vegetation on various types of
habitats and directly preserved in 70% ethanol. Human feeding ticks were transported alive
or in ethanol to our laboratory. In questing ticks, the DNA was isolated using alkaline
hydrolysis method and/or by DNAeasy tissue kit (Qiagen) in blood fed ticks from patients.
B. burgdorferi s.l. was detected using nested or single PCR targeting the 5S–23S intergenic
spacer (IGS) (10,11) or by Real-time PCR (12). Spirochetes were assigned to a species by
RFLP (10) or reverse line blot assay (11). B. miyamotoi was detected by Real-time PCR of
16S rRNA gene (5). Representative samples of B. burgdorferi s.l. genospecies were
sequenced. Phylogenetic analyses were conducted using Maximum likehood method in
Mega 6 (13). Statistical analyses of B.burgdorferi s.l. genospecies association with the
types of habitat were analysed using constrained correspondence analysis.
Results and Discussion: We have been studying prevalence and genetic variability of B.
burgdorferi s.l. in questing and host-feeding I. ricinus for over than 15 years using various
molecular methods (10, 11, 14, and 15). Furthermore in recent years we have tested the
prevalence of B. miyamotoi in questing ticks. More than 7739 questing I. ricinus ticks were
sampled from 10 diverse habitats including mountain spruce forest, lowland deciduous
forest, xerothermic steppe, suburban forest, urban park, game reserve and woodlandfarmland ecotone. 2969 ticks collected during 2011-2013 were screened for the presence of
B. myiamotoi. 226 feeding I. ricinus ticks were obtained from patients in 2013-2016. We
have confirmed spread of I. ricinus ticks to higher altitudes over the 1000 m. asl (15). In
total 1355 out of 7739 (18%) questing I. ricinus ticks were infected with B. burgdorferi s.l.
which corresponds to the mean European prevalence (3). The overall prevalence of B.
burgdorferi s.l. between different sites varied from 4.4 % at suburban Park in Bratislava in
2016 to 46% at Martinské hole in 2010. B. afzelii, B. garinii, and B. valaisiana were
detected at each studied site as the most prevalent with the few exceptions. In submountain area of central Slovakia, B. lusitaniae predominated. It was an unexpected result
that sites located in the mountains represented natural foci of B. lusitaniae, a genospecies
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that has usually been associated with lizards and xerothermic habitats (16). B. burgdorferi
s.s.was not detected at every site, but it was found in urban parks from both western and
eastern Slovakia. At these sites, B. bavariensis and B. spielmanii were detected. Borreliapositive ticks in urban habitats harbored mostly B. garinii and B. valaisiana assigning
blackbirds population an essential role for circulation of borrelia in towns. The lowest
prevalence of Borrelia was found at urban area with very low density of rodents where
deer supplemented the blood meal for the I. ricinus larvae. B. myiamotoi was detected in
23 out of 2969 (0.8%) tested questing ticks from various sites in Slovakia. 18.1% (41/226)
ticks removed from patients were positive for B. burgdorferi s.l. in contrast B. myiamotoi
was found only in 2 ticks. Higher prevalence of B. burgdorferi s.l. was found in adult
females (32.7%) as in nymphs (16.8%). B. afzelii predominated, followed by B. garinii and
B. valaisiana. B. lusitaniae and B. spielmanii were detected in single tick.
Conclusion: Profound understanding of the eco-epidemiology of Lyme borreliosis is
crucial part of accurate diagnostics, treatment and estimation of risk of infection.Different
clinical manifestation and ecological cycles has been assigned to different genospecies
therefore it is crucial to exactly identify borrelia. We have confirmed spread of Borrelia
infected ticks in towns as well as in mountains and high genetic variability affected by
local habitats. Recent evidence of B. miyamotoi in questing and human feeding ticks points
out on possible risk of the infection in Slovakia.
Acknowledgements: This study was financially supported by the projects VEGA
no.2/0108/13 and APVV 14-0274. We thank K. Hanincová, J. Koči, R. Ivanová and L.
Mahríková for collaboration.
Literature
1.
Hubálek, Z., 2009. Epidemiology of Lyme borreliosis. Curr Probl Dermatol.37, 31-50.
2.
Barbour, A.G., Hayes, S.F.1986.Biology of Borrelia species.Microbiol Rev. 50, 381-400.
3.
Rauter, C, Hartung, T. 2005.Prevalence of B. burgdorferi sensu lato genospecies in Ixodes ricinus ticks
in Europe: a metaanalysis. Appl Environ Microbiol. 71, 7203–7216.
4.
Fukunaga, M, Koreki, Y. 1995.The flagellin gene of B. miyamotoi sp. nov and its phylogenetic
relationship among Borrelia species. FEMS Microbiol Lett. 134, 255–8.
5.
Platonov, AE, Karan, LS, Kolyasnikova, NM. et al. 2011. Humans Infected with Relapsing Fever
Spirochete Borrelia miyamotoi. Emerg Infect Dis. 17, 1816–1823.
6.
Sato, K, Takano, A, Konnai, S. et al. 2014. Human infections with Borrelia miyamotoi, Japan.Emerg
Infect Dis. 20, 1391–3.
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7.
18th-20th October 2016
Crowder, CD, Carolan, HE, Rounds, MA. et al. 2014. Prevalence of B. miyamotoi in Ixodes ticks in
Europe and the United States.Emerg. Infect. Dis. 20, 1678–82.
8.
Burri, C, Schumann, O, Schumann, C. et al. 2014. Are Apodemus spp. mice and Myodes glareolus
reservoirs for Borrelia miyamotoi, Candidatus Neoehrlichia mikurensis, Rickettsia helvetica, R.
monacensis and Anaplasma phagocytophilum? Ticks Tick Borne Dis. 5, 245–51.
9.
Subramanian, G, Sekeyova, Z, Raoult, D. et al. 2012. Multiple tick-associated bacteria in Ixodes ricinus
from Slovakia. Ticks Tick Borne Dis. 3, 406-10.
10. Derdáková, M, Beati, L, Pet’ko, B. et al. 2003. Genetic variability within B. burgdorferi sensu lato
genospecies established by PCR–SSCP analysis of the rrfA-rrlB intergenic spacer in I. ricinus ticks from
the Czech Republic. Appl Environ Microbiol.69,509–516.
11. Hanincová, K, Schäfer, SM., Etti, S. et al. 2003. Association of Borrelia afzelii with rodents in Europe
Parasitology 126, 11–20.
12. Courtney JW, Kostelnik LM, Zeidner NS. Et al. 2004. Multiplex real-time PCR for detection of A.
phagocytophilum and B. burgdorferi.Clin Microbiol. 42, 3164-8.
13. Tamura K, Stecher G, Peterson D, et al. 2013. MEGA6: Molecular Evolutionary Genetic Analysis
Version 6.0. Molecular Biology and Evolution 30,2725-2729.
14. Tarageľová V, Koči J, Hanincová K. et al. 2008. Blackbirds and song thrushes constitute a key reservoir
of Borrelia garinii, the causative agent of borreliosis in central Europe. Appl. Environ. Microbiol. 74,
1289–1293.
15. Rusňáková Tarageľová, V., Mahríková, L., Selyemová, D. 2016. Natural foci of Borrelia lusitaniae in a
mountain regionof Central Europe Ticks Tick-borne Dis. 7, 350–356.
16. De Michelis S, Sewell HS, Collares-Pereira M. et al. 2000. Genetic diversity of Borrelia burgdorferi
sensu lato in ticks from mainland Portugal. J. Clin. Microbiol. 38, 2128–2133.
44
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NATIONAL DATABASE OF LISTERIA MONOCYTOGENES
PFGE PROFILES SOURCED FROM FOOD, FEED, ANIMALS, AND THE
RELATED ENVIRONMENT
Filipova M., Kubicová Z., Cabanová L.
Veterinary and Food Institute Dolny Kubin, Janoskova 1611/58, 026 01 Dolny Kubin
[email protected]
Introduction: Opening of free-trade area in European Union has caused increase in
listeriosis incidence over the last decade (1). Investigations of food-related listeriosis cases
are necessary because they can lead to control and limit the spread of outbreak by rapidly
identifying Listeria monocytogenes source. Although many typing methods have become
part of routine strain characterisation, active international surveillance system with
database of typing and epidemiological data was missing for a long time. The crucial role
of surveillance system is evident mainly for predicting the clone spread as well as early
recognition of wide-dispersed strains. Indeed, one of the most successful Pulse Field Gel
Electrophoresis based surveillance network (PulseNet USA) demonstrated that sharing
typing data is the way to link different epidemiological cases. Due to evident success this
network expanded very soon to other countries including Europe. However, PulseNet
Europe ended up after setting up due to lack of funding (2). The continued need to
exchange typing data led to creation TESSy (The European Surveillance system) by the
European Center for Diseases Prevention and Control (ECDC) in November 2012. This
database collects molecular typing data for Salmonella, Listeria, verotoxigenic Escherichia
coli and multi-drug resistant Mycobacterium tuberculosis from human sector. In parallel of
TESSy, distinctive database hosted by Reference Laboratory of European Union for L.
monocytogenes (EURL Lm DB) was set up for centralising and sharing typing data from
food and food production sector. Moreover, European Commission asked in 2013 the
European Food Safety Agency (EFSA) and ECDC for technical support regarding
collection of data on molecular typing by developing and managing joint database.
Cooperating EFSA-ECDC database is in pilot phase now and it will start to work fully in
2017 (3). Creation of new complex database is essential for proper outbreak investigations
because of direct linking of epidemiological typing data from food and health sector.
The aim of the study: Slovak National Reference Laboratory (NRL) for L.
monocytogenes has been hosted by Veterinary and Food Institute in Dolny Kubin since
2007. To NRL responsibilities belongs inter alia collecting and typing L. monocytogenes
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isolates from food sector on national level. Our aim was to meet the requirements of EURL
and EFSA for involving Slovak NRL in EURL Lm DB and EFSA-ECDC database.
Becoming Slovak national database part of European databases is important step for our
contribution in early detection of multinational outbreaks and epidemic potential prediction
of wide dispersed clones.
Methodology: PFGE method together with profile interpretation was consolidated
according to standard operating procedures (SOP) developed by EURL (4). The
competence for PFGE typing of L. monocytogenes was recognised by participation in
proficiency testing trials focused not only on PFGE method but also on profile analysis and
interpretation. As the principle of exchange data in EURL Lm DB is based on internet
communication via dedicated software, BioNumerics v6.6 software (Applied Maths,
Belgium) was purchased for proper sharing of typing data in this database. L.
monocytogenes isolates acquired in 2014-2015 period were examined by harmonised
PFGE and molecular typing data with epidemiological information were sent to EURL for
curation process. Accepted data were signed as confirmed and involved in EURL Lm DB.
Results and discussion: Harmonisation of PFGE method and criterion for profile
interpretation with EURL led to consolidation of molecular typing results and reducing of
operator-dependent subjectivity. The ability of Slovak NRL to do molecular typing by
PFGE was proved by participation in proficiency testing trials. In one of them, organised in
2012, we received satisfactory results and we were included among 16 successful
laboratories out of 28 participants (5).
Tab. 1 Content of national database of L. monocytogenes managed by NRL
No. of isolates with PFGE
No. isolates accepted in
profile
EURL Lm database
89
84
52
2015
86
78
41
2016
37*
Year
No. of isolates
2014
*to 31 August, 2016
As we fulfilled all EURL requirements, our national database became the part of EURL
Lm database by signing of Memorandum of Understanding about EURL Lm DB in 2014.
Since then we have typed 162 L. monocytogenes isolates by PFGE (Tab. 1) and after
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elimination of duplicates, 96 profiles were successfully submitted to EURL Lm DB. In
conclusion, 3 pulsotypes were assigned as unsatisfactory during curation process and their
submission to EURL Lm DB was rejected. The source of submitted isolates in 2014,
respectively in 2015, was as follows: food (n = 44; 28), animals (n = 7; 9), feed (n = 0; 1)
and related environment (n= 1, 2). Milk and milk products followed by meat and meat
products were the most frequent food source of L. monocytogenes isolates accepted in
EURL Lm database (Tab. 2).
Tab. 2 Number of isolates distributed among different categories of food/food
products
Milk and milk
Meat and meat
Fish and fishery
Elaborated food
Processing
products
products
products
products
environment
2014
21
11
1
3
8
2015
15
8
0
0
6
Year
Involvement of national database hosted by Slovak NRL in active surveillance system has
allowed us to participate in investigations of several outbreaks in Europe. As not all PFGE
profiles are sharing in EURL Lm database, we were asked for searching national database
to find related PFGE profiles in case of investigation of food origin for sporadic human
listeriosis cases regularly observed since 2013 in Germany and severe outbreak in January
2015 in Italy. Strains with indistinguishable or closely related matching profiles with
available epidemiological data were sent to concern NRL for deeper study. Moreover, we
were approached to match some PFGE profile selected for the project of EURL focused on
using “Next Generation Sequencing” method in epidemiological study and subtyping of L.
monocytogenes. In sum, 20 closely related isolates were already sent to EURL for “Next
Generation Sequencing” analysis.
Conclusion: Our skills with EURL Lm database as well as collaboration with other NRLs
in epidemiological investigations predispose our NRL to be officially nominated by our
Competent Authority to participate in building of EFSA database. We became
representatives of Slovakia for submitting molecular typing data on L. monocytogenes,
verotoxigenic E. coli and Salmonella sourced in animals, feed, food and related
environment. As EFSA database will be the part of joint EFSA-ECDC database, it opens
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new opportunities for not only for our NRL but also for Slovakia itself in case of foodborne pathogens.
Literature
1.
EFSA-ECDC (2013) The European Union summary report on trends and sources of zoonoses, zoonotic
agents and food-borne outbreaks in 2013. EFSA Journal 2015; 13(1): 3991, 1-165
2.
Swaminathan B., Gerner-Smidt P., Ng L.K., Lukinmaa S., Kam K.M., Rolando S., Gutierrez E.P.,
Binszetein N. (2006) Building PulseNet International: an interconnected system of laboratory networks
to facilitate timely public health recognition and response to foodborne disease outbreaks and emerging
foodborne diseases. Foodborne Pathog. Dis. 3, 36-50
3.
EFSA (2014) Technical specifications for the pilot on the collection of data on molecular testing of foodborne pathogens from food, feed and animal samples. EFSA supporting publication 2014, 1-58
4.
Félix B., Dao T.T., Grout J., Lombard B., Assere A., Brisabois A., Roussel S. (2012) Pulsed-field gel
electrophoresis in Listeria monocytogenes sub-typing: harmonisation at the European Union level. In:
Magdeldin, Sameh (Ed.), Gel Electrophoresis: Principles and Basics. Vol.1 INTECH, Japan, 241-254
(Niigata University)
5.
Félix B., Niskanen T., Vingadassalon N., Dao T.T., Assere A., Lombard B., Brisabois A., Roussel S.
(2013) Pulsed-field gel electrophoresis proficiency testing trials: toward European harmonisation of
typing of the typing of food and clinical strains of Listeria monocytogenes. Foodborne Pathog. Dis 10,
873-881
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SALMONELLOSIS OUTBREAKS IN 2014 AND 2015 IN THE CZECH REPUBLIC
Gelbíčová T., Karpíšková R.
Veterinary Research Institute, Hudcova 70, 621 00 Brno, Czech Republic,
[email protected]
Introduction: In the Czech Republic, human non-typhoidal salmonellosis is the second
most frequently reported food-borne illnes, with about 10,000 cases and several outbreaks
reported annually. In 2014, the highest notification rates of salmonelllosis in EU were
reported by the Czech Republic (126.1 cases per 100,000inhabitants) and was
accompaniedby an increase in the number of Salmonella outbreaks [1].The aim of this
study was to perform the characterization of Salmonella isolates from selected
epidemiologically linked cases in the Czech Republic during the years 2014 and 2015.
Matherials and methods: Isolates: In total,243Salmonella isolates (232 originated from
human and 11 from food) reported as a part of an outbreak were testedin 2014 (Table 1)
and 2015 (Table 2).
Serotyping: The antigenic structure was determined by slide agglutination method with
commercial antisera using the Kauffmann-White-LeMinor scheme [2].
Phage typing: Phage typing of S. Enteritidis and S. 4,5,12,i:- was carried out according to
the recommendations of the Health Protection Agency, Colindale, United Kingdom
[3].Antimicrobial susceptibility testing:Salmonella strains were tested using disk
diffusion method in accordance with the Clinical and Laboratory Standard Institute
guidelines [4], resistance to 18 antimicrobial agents was tested.
Results and Discussion: The majority of outbreaks were caused by S. Enteritidis PT8
(10/16, 63%). S. Enteritidis PT8 is for a long time the the most prevalent phage type in the
Czech Republic [5].The other (6) outbreaks were caused by S. Enteritidis PT13, PT13a and
PT14b, S. 4,[5],12:i:- and one rare serotype, S. Itami (Table 1 and 2). Majority of the
Salmonella isolates in our study were susceptible to all antimicrobial agentstested.
In the context with the outbreak caused by S. Enteritidis PT13 in the South Moravia region
in June 2014 (Table1), 40 people were affected. Initially unrelated salmonellosis cases
were discovered in one hospital and restaurant from different cities. Dumplings originating
from the one local producer were considered as a vehiculum of infection. Contaminated
egg melange was determined as a suspect source of the infection [6]. S. Enteritidis PT13 is
relatively rare in the Czech Republic and also in the EU. However, another
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epidemiologically linked isolates were discovered in the same region in November 2014. It
is possible that the source of these cases could be identical. All the strains of S. Enteritidis
PT13 were resistant to ampicillin. The plasmid mediated resistance to ampicillin is typical
for this phage type in the Czech Republic [7]. Dumplings containing raw eggs were
confirmed as the suspect vehiculum of this infection caused by S. Enteritidis PT8 in the
Central Bohemia region in February 2014 [8].
During the year 2014 (July and September), strains of S. Enteritidis PT14b were detected
in human cases (Table 1). According to the alert of ECDC (2014) and on the basis of
MLVA typing [9], it was revealed that the strains were part of the international outbreak
caused by eggs originating from breedings of layer hens in Germany. Outbreak caused by
S. Enteritidis PT14b in the Central Bohemia region in September 2014, affected 113
people, suspect vehiculum of the infection was goulash with pasta served at the primary
school, which might be contamined either by eggs or by an infected personnel [10].
The only outbreak with laboratory confirmed vehiculum (spaghetti carbonara) was noted in
Plzeň region in summer 2015. Eggs contaminated by S. Enteritidis PT13a were the source
of infection. Spaghetti and egg shells sampled in the restaurant kitchen were positive for S.
Enteritidis PT13a , however, the presence of S. Enteritidis PT13a was not confirmed in the
poultry farm from where the eggs came from. Hens were positive for the presence of S.
Enteritidis but different phage type PT8. The source of this outbreak was not clarified.
Conclusion: Czech Republic belongs to the countries with the large number of
salmonellosis cases. However, according to the Manual for reporting on food-borne
outbreaks in accordance with Directive 2003/99/EC for information deriving from the year
2015[11], our country fails in the determination of strong evidence Salmonella outbreaks
with the laboratory confirmed vehiculum. For the efficient outbreak investigation it is
important to establish a team of closely cooperating specialists at local level. Important is
also the real time molecular epidemiology approach.
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Table 1: Characteristics of strains from Salmonella outbreaks in the Czech Republic in
2014
Month
Region
No. of tested
human/food
strains
Salmonella spp.
Phenotypic
AMR pattern
February
Liberec
35
S. Enteritidis PT8
susceptible
February
Plzeň
21
S. Itami
susceptible
February
Vysočina
2/3
S. Enteritidis PT8
susceptible
February
Central Bohemia
13
S. Enteritidis PT8
April
Zlín
6
S. Enteritidis PT8
susceptible
susceptible
April
Zlín
4
S. Enteritidis PT8
susceptible
June
South Moravia
28
S. Enteritidis PT13
resistant - A
susceptible
susceptible
susceptible
susceptible
resistant - A
July
South Bohemia
4
August
Hradec Králové
5/1
September
Central Bohemia
6
September
Zlín
17
November
South Moravia
11
AMR pattern – antimicrobial pattern, A – ampicillin
S. Enteritidis PT14b
S. Enteritidis PT8
S. Enteritidis PT14b
S. Enteritidis PT8
S. Enteritidis PT13
Table 2: Characteristics of strains from Salmonella outbreaks in the Czech Republic in 2015
No. of tested
human/food
strains
Salmonella spp.
Phenotypic AMR
pattern
Central Bohemia
10
S. Enteritidis PT8
susceptible
June
South Moravia
7
S. Enteritidis PT8
susceptible
July
Plzeň
36/7
S. Enteritidis PT13a
susceptible
July
South Bohemia
6
S. Enteritidis PT8
August
Liberec
21
S. 4,[5],12:i:- DT193
susceptible
resistant -ASSuT
Month
Region
May
AMR pattern – antimicrobial pattern, A – ampicillin, S- streptomycin, Su - sulphonamides, T tetracycline
References
1.
EFSA (European Food Safety Authority) and ECDC (European Centre for Disease Prevention and
Control) 2015. The European Union summary report on trends and sources of zoonoses, zoonotic agents
and
food-borne
outbreaks
in
2014.
EFSA
Journal
2015;13(12):4329,
191pp.
doi:10.2903/j.efsa.2015.4329
2.
Grimont, P., Weill, F.X. 2007. Antigenic formulae of the Salmonella serovars. 9th edition. WHO
Collaborating Centre for Reference and Research on Salmonella, Paris, France, p.166.
3.
Ward, L.R., De Sa, J.D.H., Rowe, B. A phage-typing scheme for Salmonella Eneritidis. Epidemiology
and Infection, 1987, vol. 99, p. 291-294.
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4.
18th-20th October 2016
CLSI. Performance standards for antimicrobial susceptibility testing: twenty two informational
supplement. CLSI Document M100-S22. Clinical and Laboratory Standard Institute, USA,
Pennsylvania, 2012.
5.
Myšková, P., Karpíšková, R. Diverzita humánních izolátů salmonel v Jihomoravském kraji v letech
2009-2012. Epidemiologie, mikrobiologie, imunologie, 2014, vol. 63, no. 2, p. 116-120.
6.
Ciupek, R. Závěrečná zpráva ze dvou souvisejících epidemických výskytů salmonelózy v „Nemocnici“ a
„Restauraci“ v Jihomoravském kraji. Zprávy CEM, 2014, vol. 23, no. 10, p. 351-357.
7.
Hradecká, H., Koláčková, I., Karpíšková, R., Rychlík, I. An outbreak of human salmonellosis caused by
ampicilin-resistant Salmonella enterica serovar Enteritidis PT13 in the Czech Republic. Epidemiology
and Infection, 2006, vol. 134, no. 4, p. 737-740.
8.
Juřičková, J., Tkadlecová, H. Závěrečná zpráva z epidemie salmonelózy u konzumentů knedlíků
z restaurace v Kunovicích. Zprávy CEM, 2014, vol. 23, no. 9, p. 313-315.
9.
Myšková, P. Molekulárně epidemiologická charakteristika bakterií rodu Salmonella na území České
republiky, Disertační práce, Masarykova univerzita, Brno, 2015, 150 pp.
10. Kavalírová, E., Rumlová, L. Závěrečná zpráva o epidemickém výskytu salmonelózy (dg. A02)
v základní škole a mateřské škole v Mladé Boleslavi. Zprávy CEM, 2015, vol. 24, no. 2, p. 46-50.
11. EFSA (European Food Safety Authority), 2016. Manual for reporting on food-borne outbreaks in
accordance with Directive 2003/99/EC for information deriving from the year 2015. EFSA supporting
publication 2016:EN-989. 43 pp.
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MONITORING OF OCCURRENCE TICK-BORNE ENCEPHALITIS IN RAW
MILK IN SLOVAKIA WITHIN 2014-2016
Jurovčíková J.
State veterinary and food institute, Veterinary and food institute in Dolny Kubin,
department of molecular-biological analysis, Jánoškova 1611/58, 026 01 Dolný Kubín,
Slovakia
[email protected]
Introduction: Tick-borne encephalitis virus (TBEV) causes one of the most important
flaviviral infections of the human central nervous system in Europe and Asia. Three
subtypes of TBE virus are known: the European, the Siberian and the Far Eastern subtype
named according to their main area of circulation. The European suptype is found in all
European countries except the Benelux and Great Britain (1). The TBEV subtypes are
associated with different severity of disease. The mortality rate is 1-2% in Europe and from
6% to 40% in Asia (2). The European subtype (the main vector is tick Ixodes ricinus)
typically causes biphasic illness. The first phase is characterized by influenza-like
symptoms, followed by an asymptomatic interval and a second phase when the central
nervous system is affected. (3). The major route of virus transmission is tick bites, but
TBEV also can be transmitted during consumption of unpasteurized milk and milk
products (cheese, butter, yoghurt) from infected animals, primarly sheep and goats
(4).About 5% of the TBE annual human cases (between <20 and 100) in Slovakia are
acquired through the consumption of unpasteurized raw sheep and goat milk products
(5,6). Thelarge domestic animals have a closer contact to TBEV during grazing and can
excrete TBEV into milk during the viremic phase for 3-7 days, beginning as early as the
second or third day postinfection (7). The infection remains sub-clinical in the animals
followed by full recovery (4,5). The virus originating from one or a few animals can infect
numerous people. Raw milk consumption can pose a considerable health risk, particularly
in areas where TBE cases frequently occur (8).
Since the clinical
features of TBE are non-specific, the actual diagnosis must be
established in the laboratory. In the first viremic phase of disease before seroconversion
TBE virus can be detected in serum and milk by PCR (polymerase chain reaction),
although virus load decreases with the appearance of IgM antibodies. Therefore, PCRbased TBEV diagnostics is an important alternative to serological methods, especially in
the early stages of disease (9).
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The aim of the study: The aim of this study was to investigate the viral prevalence of
TBEV in raw sheep and goat milk in Slovakia within 2014-2016.
Methodology: Mostly pooled samples of unpasteurized sheep and goat milk collected
within 2014-2016 were examined for the presence of RNA of TBEV by using highly
specific real-time RT-PCR in Veterinary and Food Institute in Dolny Kubin (VFI DK). The
samples came from all part of Slovakia, however mostly from Žilina, Banská Bystrica and
Prešov region.
Results and Discussion: A total of 409 samples of raw milk (85% sheep, 15% goat) were
examined for the presence of RNA of TBEVin 2014. RNA of TBEV was confirmed in 4
samples of sheep milk: districts – Detva, Brezno, Banská Bystrica. In all cases it was the
large producers of unpasteurized milk.
These positive samples were linked with the cases of tick-borne encephalitis in humans.
In 2015 a total of 184 samples of raw milk (86% sheep, 14% goat) were examined. RNA
of TBEV was not confirmed in any samples of milk.
The same way as two last years a total of 441 samples of raw milk (86% sheep, 14% goat)
were examined in 2016. RNA of TBEV was confirmed in 1 sample of sheep milk from
district Ilava and 1 sample of goat milk from district Bytča. The positive sample of sheep
milk was originated from the large producer and was taken for the purpose of self-control
without linking with the cases of tick-borne encephalitis in humans. The positive sample of
goat milk originated from small farm was linked with unspecified health problems
residents of the farm.
This results indicate that the consumption of raw milk from sheep and goats may be
associated with risk of infection with TBEV.
Conclusion: In recent years the incidence ofTBE has permanently been increasing
throughout Europe and the virus has emerged in areas formerly known as no-risk, possibly
due to climate changes (10). According to some erlier publications, the role of the
alimentary route in spreding tick-borne encephalitis is insignificant from the
epidemiological aspect, because these infections are less frequent than the ones originating
from tick bite (8). However, milk-borne cases accur every year in Slovakia and these are
not only isolated cases but also family oubreaks and outbreaks involving lot of people.
Human milk-borne infections can be avoided both by vaccination of people and
pasteurizing milk So far, diagnostic evidence and epidemiological studies rely usually on
serological testing. However, correct interpretation of serological tests is often complicated
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by a number of factors suchas cross reactions with antibodies directed against other virus
species, past infection with TBEV or recent vaccination. Furthermore, and most
importantly, antibody assays may be negative in the early phase of the disease when
clinical symptoms are manifest (11,12).Therefore, PCR-based TBEV diagnostics is an
important alternative to serological methods, especially in the early stages of disease (9).
Literature
1.
SŰSS J.: Tick-borne encephalitis 2010: epidemiology, risk areas, and virus strains in europe and Asia-an
overview, Tick and Tick-Borne Diseases, 1,2, 2-15 (2011).
2.
GRIDSUN T.S., GOULD E.A.: Infectious transcripts of tick-borne encephalitis virus generated in days
by T-PCR, Virology, 214, 611-618 (2003).
3.
GRIDSUN T.S., FROLOVA T.M., ZHANKOV A.I., ARMESTO M., TURNER S.L., FROLOVA M.P.,
POGODINA M.M., LASHKEKEVICH V.A., GOULD E.A.: Characterization of a siberian virus
isolated from a patient with progressive chronic tick-borne encephalitis. J.Virol, 774, 25-36 (2003).
4.
SŰSS J.: Epidemiology and ecology of TBE relevant to the production of effective vaccines. Vaccine,
21:S19-35. DOI: 10.1016/S0264-410X(02)00812-5 (2003).
5.
LABUDA M., ELEČKOVÁ E., LIČKOVÁ M., SABÓ A.: Tick-borne encephalitis virus foci in
Slovakia. Int.J.Med.Microbiol. 291, Suppl.33,43-47 (2002).
6.
FREY S., ESSBAUER S., ZÖLLER G., KLEMPA B., DOBLER G., PFEFFER M.: Full genome
sequences and preliminary molecular characterization of three tick-borne encephalitis virus strains
isolated from ticks and a bank vole in Slovak.Virus Genes, 48:184. Doi:10.1007/s11262-013-0985-0
(2014).
7.
GRESIKOVA M.: Recovery of the tick-borne encephalitis virus from the blood and milk of
subcutaneously infected sheep. Acta Virol., 2: 113-9 (1958).
8.
BALOGH Z., FERENCZI E., SZELES K., STEFANOFF P., WLODZIMIERZ G., SZOMOR K.N.,
TAKACS M., BERENCSI G.: Tick-borne encephalitis outbreak in Hungary due to consumption of raw
goat milk. J. of Vir.Met.,163, 481-485 (2010).
9.
HOLZMANN H.: diagnostic of tick-borne encephalitis. Vaccine 21, S1/36-S1/40 (2003).
10. DONOSO MANTKE O., SCHADLER R., NIEDRIG M.: A survey on cases of tick-borne encephalitis in
European countries. Eurosurveillance 13 (17), pii: 18848 (2008).
11. DUMPIS U., CROOK D., OKSI J.: Tick-borne encephalitis. Clin.Infect.Dis., 28, 882-890 (1999).
12. SCWAIGER M., CASSINOTTI P.: Development of a quantitative real-time RT-PCR assay with internal
control for the laboratory detection of tick borne encephalitis virus (TBEV) RNA. Journal of Clinical
Virology, 27, 136-145 (2003).
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MOLECULAR EPIDEMIOLOGY OF LISTERIOSIS - A TOOL FOR OUTBREAK
INVESTIGATION
Karpíšková, R., Gelbíčová, T., Zobaníková, M., Tomaštíková, Z.
Department of Bacteriology, Veterinary Research Institute, Brno, Czech Republic
[email protected]
Introduction: Listeria monocytogenes is an emerging foodborne pathogen, causative agent
of listeriosis. This disease is mostly transmitted via oral route, affecting vulnerable groups
of the population such as the elderly, individuals with weak immune system, pregnant
women and newborns. The high case fatality rate of 20-30% makes this pathogen a leading
cause of food-borne related mortality in humans [1].
Listeriosis is usually spread by consumption of foods that contain L. monocytogenes, more
frequently such as smoked fish, cold meats or soft cheeses. Ready-to-eat foods are often
the source of listeria infections especially those with long shelf life that support the
bacterial growth. Such foods are usually stored in refrigerator and consumed without any
additional heating or cooking.In 2014, EU member states reported 2,161 confirmed human
cases of listeriosis. The EU notification rate was 0.57 cases per 100,000 population, the
rate in the Czech Republic was a bit lower 0.36 cases, but the number of cases is rising
both in EU and CR [2].
Listeriosis usually occur as sporadic, epidemic cases are rare, small outbreaks may remain
unrecognized. Molecular biology techniques have become increasingly integrated into the
practice of infectious disease epidemiology and molecular epidemiology approach is an
efficien tool in outbreak investigation, namely in listeriosis outbreaks.
Aim of the study: The aim of the study was to characterize the human isolates of Listeria
monocytogenes and with appropriate methods identify related cases and detect cases with
epidemic contexts.
Material and methods: Bacterial isolates
Altogether 26 human and 3 food isolates of L. monocytogenes of pulsotype 810 were
collected in years 2012 – 2016.
Serotyping
Isolates were serotyped by slide agglutination method (Denka Seiken, Japan) in
combination with multiplex PCR [3].
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Antimicrobial sensitivity test
Isolates were tested for their sensitivity to seven antimicrobial agents by disk diffusion
method. The following antibiotics and concentration per disk were used: penicillin (1U),
erythromycin (15 µg), gentamicin (10 µg), meropenem (10 µg), tetracycline (30 µg),
trimethoprim-sulphametoxazole (25 µg) and ampicillin (2 µg). Reactions were evaluated in
accordance with EUCAST and Vela et al. [4,5].
Macrorestriction analysis
Macrorestriction analysis was performed in accordance with PulseNet Europe protocol [6]
using AscI and ApaI enzyme (New England BioLabs, USA). Results were analyzed by
Bionumerics 5.1 software (AppliedMaths, Belgium).
Clonogrouping
Isolates were divided by PCR clonogrouping into specific clonal complexes [7].
cgMLST
Core genome multilocus sequence typing (cgMLST) and data analysis using the Ridom
SeqSphere+ software (Ridom GmbH, Germany) were performed according to the study
Ruppitsch et al.[8].
Results and discussion: In the Czech Republic serotype 1/2a belongs to the most frequent
ones in both human population and isolates obtained from food. Since 2012 an increased
number of listeriosis has been reported from the Moravian-Silesian Region (Figures 1 and
2). Molecular methods have confirmed the occurrence of identical clones in patients. It was
a serotype 1/2a, pulsotype 810 (AscI) /12 (ApaI), clonal complex CC8, sequence type ST8.
This clone of L. monocytogenes occurred mainly in the described area.
9
8
reported cases
7
6
5
4
3
2
1
0
2012
2013
2014
2015
year
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Figure 1: The number of reported cases of listeriosis in the Moravian-Silesian region
by the year of onset
Figure 2: Distribution and number of cases caused by the epidemic clone 810 in the
period from 2012 till 2016 in the Czech Republic
Currently, the epidemiological investigation and investigation in food premises in Frýdek
Místek locality has been carried out in order to identify the possible sources of infection.
Acknowledgements: The study was supported by projects
IGA VFU Brno
206/2016/FVHE and MZ AZV 16-31488A.
References
1.
Nyarko, E.B., Donnelly, C.W. Listeria monocytogenes: strain heterogeneity, methods, and challenges of
subtyping.Journal of Food Science, 2015, vol. 80, no. 12, p. M2868-78.
2.
EFSA (European Food Safety Authority) and ECDC (European Centre for Disease Prevention and
Control) 2015. The European Union summary report on trends and sources of zoonoses, zoonotic agents
and food-borne outbreaks in 2014. EFSA Journal 2015;13(12):4329, 191pp.
doi:10.2903/j.efsa.2015.4329
3.
Doumith, M., Buchrieser, C., Glaser, P., Jacquet, C., Martin, P. Differentiation of the major Listeria
monocytogenes serovars by multiplex PCR. Journal of Clinical Microbiology. 2004, vol. 42, no. 8, p.
3819 – 3822.
4.
The European Committee on Antimicrobial Susceptibility Testing. Breakpoint tables for interpretation of
MICs and zone diameters. Version 6.0, 2016. http://www.eucast.org.
5.
Vela, A.I., Fernández-Garayzábal, J.F., Latre, M.V., Rodríguez, A.A., Domínguez, L., Moreno, M.A.
Antimicrobial susceptibility of Listeria monocytogenes isolated from meningoencephalitis in sheep.
International Journal of Antimicrobial Agents. 2001, vol. 17, no. 3, p. 215-220.
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6.
18th-20th October 2016
PulseNet Europe protocol (2013), http://www.pulsenetinternational.org/assets/PulseNet/uploads/pfge/PN
L04_ListeriaPFGEProtocol.pdf.
7.
Chenal – Francisque, V., Maury, M.M., Lavina, M., Touchon, M., Leclercq, A., Lecuit, M., Brisse, S.
Clonogrouping, a rapid multiplex PCR method for identification of major clones of Listeria
monocytogenes. Journal of Clinical Microbiology. 2015, vol. 53, no. 10, p. 3355 – 3358.
8.
Ruppitsch, W., Pietzka, A., Prior, K., Bletz, S., Fernandez, H.L., Allerberg, F., Harmsen, D., Mellmann,
A. Defining and evaluating a core genome multilocus sequence typing scheme for whole-genome
sequence-based typing of Listeria monocytogenes. Journal of Clinical Microbiology, 2015, vol. 53, no.
9, p. 2869-2876.
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PRESENCE OF TICK-BORNE ENCEPHALITIS VIRUS IN TICKS FROM
SELECTED LOCATIONS IN SLOVAKIA
Kerlik J.1, Avdičová M.1, Musilová M.1, Dobler G.2, Molčányi T.3, Csank T.4,
Drzewnioková P.4, Majláthová V.5, Majláth I. 6
1
Department of Epidemiology, Regional Authority of Public Health, Banská Bystrica,
Slovakia
2
Department of Virology and Rickettsiology, Bundeswehr Institute of Microbiology,
Munich, Germany
3
4
Office of the General Surgeon, Armed Forces of Slovak Republic, Ružomberok, Slovakia
Department of Microbiology and Immunology, University of Veterinary Medicine, Košice,
Slovakia
5
6
Institute of Parasitology, Slovak Academy of Sciences, Košice, Slovakia
Institute of Biology and Ecology, Pavol Jozef Šafárik University, Košice, Slovakia
[email protected]
Introduction: The Czechoslovak Republic was one of the first countries in Europe where
the tick-borne encephalitis virus (TBEV) was confirmed in Ixodes ricinus in 1947 (1). In
1951 it was the first time - again in Czechoslovakia – during the TBE alimentary outbreak
in Rožňava when TBEV transmission was demonstrated to humans from the milk of
infected animals (2). Since then the arbovirus research in Czechoslovakia began to have a
longterm tradition. The Department of Virus Ecology from the Virology Institute of the
Slovak Academy of Sciences (SAS) studied a group of arboviruses including TBEV for
more than 50 years. Especially on the basis of data of workers from the Institute of
Virology about the isolation of TBEV from ticks and reservoir animals during years 19641997 as well as indirectly, on the basis of the data about the location of TBE infection in
infected individuals reported for the last 30 years, a list of approximately 50 natural TBEV
foci in Slovakia was set up in 2002. During the last years, however, the TBEV study in
Slovakia declined. As epidemiologists, we see a shift of human TBE cases from the south
to the northern areas of the country especially in the basin of the river Váh, but also in the
eastern Slovakia during last years. Parasitologists from the SAS confirmed that at present
the limit of distribution of ticks moved up to 1000-1400 m a. s. l., what presents an
increase of 400 to 600 vertical meters for the last 30 to 50 years (3). It is believed that the
most important factor behind the tick shift as well as the changes of TBE incidence in
Slovakia is the climate change (4).
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Aim: The aim of our study was to update data on the prevalence of TBEV as well as
rickettsias in selected locations in Slovakia.
Methods: Two studieson detection TBEV in collected ticks in Slovakia consecutive year
have been done. The pilot study was carried out for one week in the second half of May
(18. - 21.5.) 2015. Ticks were collected from 6 locations in Banská Bystrica county. The
follow up study was realised at the turn of June and July (28.6-1.7) 2016 at 10 locations in
five counties of Slovakia. Sampling locations in both studies were selected on basis of
number of outbreak occurrence and according to TBE incidence in humans during last
years, where ticks as TBEV vectors have not been confirmed yet. Ticks were collected by
flagging method. Examination of ticks for TBEV was performed by RT-qPCR (5). In the
pilot study other pathogens in ticks were also examinated. Rickettsias were tested by PCR
and specified by nucleid acid sequencing at the same place. In the follow up study
rickettsias from the ticks have not been tested yet.
Results: In the follow up study 1 931 ticks were tested.No positive TBEV was found in all
the selected locations in Slovakia. In the previous pilot study we collected 828 ticks in the
selected areas from Banská Bystrica county. We found one positive TBEV sample in the
area of Lešť. Phylogenetic analysis of the virus strain reveals a close relationship with a
virus strain from southeastern Germany. In the pilot study rickettsias were confirmed from
all selected areas. From 54 positive rickettsias there was Rickettsia helvetica confirmed in
51 cases and Rickettsia monacensis in 3 cases.
Discussion: The prevalence of TBEV infected ticks in populations of Ixodes ricinus is
often low. In European areas where TBEV is endemic, the prevalence of the virus in
Ixodes ricinus populations varies from 0,1 to 5,0% (6). Negative findings in the follow up
study from the selected areas in Slovakia, where TBE human cases and alimentary
outbreaks have occurred during last years, confirm that testing of ticks for TBEV is
necessary to examine earlier at the time of ticks´ highest activity, i.e. at the time of the
most probable detection TBEV in the ticks. Although the area of tick collection carried out
nearly at the end of May in the previous pilot study was smaller and also tick sample set
was smaller, there was positive TBEV found in one sample from the area of Lešť. To
detect the TBE natural foci more detailed data on the infestation with ticks in humans or
exact places of grazing of goat and sheep flocks at the time of potential infection are
necessary. As natural foci of TBE seem to be small and restricted (Dobler G. unpublished
results) having the detailed information is a pre-requisite for tick sampling to be succesful.
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Summary: In the follow up study we examinated a larger tick sample set than in the pilot
study. However, since the term of tick collection was set later at the turn of June and July
comparing tick collection nearly at the end of May in the previous pilot study, the results
of the follow up study are not surprising. It points out that in order to test TBEV in ticks
from suspicious foci, the tick collection should be done at the time of ticks´ highest activity
(the end of April or latest at the beginning of the May). In the case of alimentary outbreaks
TBEV in ticks from the incrimated areas should be tested as soon as possible, i.e. as soon
as the first TBE cases in outbreak appear. In the next study we plan to collect the ticks
from the same selected areas in Slovakia as in the following study. However, the collection
will be carried out earlier at the end of April or latest at the beginning of May, i.e. at the
time of the ticks´ highest activity and when the most TBE infection cases and outbreaks
occur.
There is substantial temporal variation in the prevalence of TBEV and newly established
foci will not necessarily persist through time (7). Continual update of TBEV foci in
Slovakia is therefore necessary to establish in order to know if the risk of infection remains
or it has disappeared. Virologists, microbiologists, veterinarians together with
epidemiologists should cooperate more for the continuous clarification of the risk
locations, where the preventive measures (awareness of population, immunization against
TBE, regular control of product pasteurization in challets, etc.) can be established in order
to prevent TBE infection.
References
1.
Rampas J., Gallia F. [Isolation of tick-borne encephalitis virus from ticks Ixodes ricinus]. Čas. Lék. čes.,
1949, 88: 1179-80. [Article in Czech]
2.
Blaškovič, D. 1954 [Outbreak of tick-borne encephalitis in Rožňava natural focus].
3.
Bratislava: Vydavateľstvo SAV, 1954, 314 p. [Book in Slovak]
4.
Peťko B., Majláth I., Majláthová V. [Impact of the climate change on expansion of ticks in the
mountainous regions of Slovakia]. Zborník príspevkov z vedeckého kongresu „Zoológia 2014“, 19.
Feriancove dni. Prešovská univerzita v Prešove, 2014, p. 178, ISBN 978-80-555-1140-5 [Abstract in
Slovak]
5.
Danielová, V., Kliegrová, S., Daniel, M., and Beneš, Č. Influence of climate warming on tick-borne
encephalitis expansion to higher altitudes over the last decade (1997- 2006) in the Highland Region
(Czech Republic). Cent. Eur. J. Public Health, 2008, 16: 4-11.
6.
Schwaiger M, Cassinotti P., Development of a quantitative real-time RT-PCR assay with internal control
for the laboratory detection of tick borne encephalitis virus (TBEV) RNA. J Clin Virol. 2003
Jul;27(2):136-45.
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7.
18th-20th October 2016
Süss J. Tick-borne encephalitis 2010: epidemiology, risk areas, and virus strains in Europe and Asia-an
overview. Ticks Tick Borne Dis. 2011, 2: 2-15. 10.1016/j.ttbdis.2010.10.007
8.
Randolph SE: The shifting landscape of tick-borne zoonoses: tick-borne encephalitis and Lyme
borreliosis in Europe. Philos Trans R Soc Lond B Biol Sci. 2001, 356: 1045-1056.
10.1098/rstb.2001.0893.
77 ticks:
0 TBEV
4 Rickettsia helvetica
190 ticks:
1. Čierny
Balog
6. Slovenská
Ľupča
0 TBEV
7 Rickettsia helvetica
2. Lom nad
Rimavicou
151 ticks:
0 TBEV
16 Rickettsia helvetica
1 Rickettsia monacensis
4. Zaježová
77 ticks:
0 TBEV
6 Rickettsia helvetica
5. Ábelová
282 ticks:
0 TBEV
14 Rickettsia helvetica
53 ticks:
1 Rickettsia monacensis
3. Lešť
1 TBEV
4 Rickettsia helvetica
Fig. 1 Analysis of collected ticks for TBEV and rickettsia from the selected locations
of Banská Bystrica county.
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Fig. 2 Selected locations of 5 counties in Slovakia for testing TBEV in collected ticks.
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VECTORS (INSECTA) OF SELECTED ZOONOTIC INFECTIOUS AGENTS –
THE CURRENT SITUATION IN SLOVAKIA
Kočišová A., Bocková E., Weissová T.*
Institute of Parasitology, University of Veterinary Medicine and Pharmacy in Košice,
* Department of Internal Diseases of Small Animals, UVMP in Košice,
Komenského 73, 041 81 Košice, Slovak Republic
[email protected]
Introduction: Although infectious diseases in Slovakia do not represent the primary health
problem, thanks to the media, at least once a year we succumb to the panic of global
epidemic. It is particularly the case, if diseases are not yet known (“emerging diseases“),
and their causative agents are not accurately defined, we do not know their route of
transmission or available treatment. That is why the current trends in the research of
veterinary entomology are focused mainly on the spread of causative agents of diseases
transmitted by insects.
Objective: The objective of this paper is to provide a review of the occurrence of potential
insect vectors of pathogens in Slovakia, focused on the possible ways of parasite
transmission.
Materials and methods: Since 2002, entomological research of insect vectors is actively
performed, mainly in Eastern Slovakia, in animal breeding premises as well as in the
communal sphere. The trapping was carried out while respecting the methodical principles
of separation, conservation, and storage of insect, depending on individual species [1].
Species were identified according to morphological signs, while using available
identification keys [2, 3].
Results and discussion: Over the last few years, we observe a rising trend of the
occurrence of lice, especially among preschool children, but also in marginalised areas of
Slovakia. In this century, however, the transmission of bacterial pathogens by lice to
humans was not observed. A similar situation is with the occurrence of bed-bugs (Cimex
lectularius), the population of which is more and more frequently diagnosed in apartments,
houses, but also in lodging houses and hospitals, as well as in social care homes. A bedbug is an unpleasant parasite, sucking blood primarily from humans, invoking thus
dermatitis of various degrees, depending on a host’s susceptibility. Fortunately,
speculations on bed-bugs being vectors of hepatitis B and HIV have not been confirmed so
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far. As for fleas, we have examined 2,604 fleas from animals and humans (Table 1) and
only in one case (0.04%) we observed a cysticercoid, a developmental stage of Dipylidium
caninum and Hymenolepis spp. tapeworms, in the body cavity of a dog flea
(Ctenocephalides canis); in two cases, the presence of Bartonella spp. (0.08%) was
observed.
Table 1 Lice, bed-bugs, and fleas as vectors of selected pathogens in Slovakia.
? The route of transmission of a pathogen by a vector to humans in Slovakia – not
confirmed.
Despite the information, broadcasted by various commercial media, that in Slovakia there
are several dozens of cases every year, transmission from a flea to a human or occurrence
of a disease were not scientifically proved, similarly to the transmission of bacteria of
plague and typhoid fever. Great attention is also paid to the research of diptera which are
the second most important vectors of pathogens, following the ticks. As for mosquitoes,
over the last ten years, we were mostly trapping calamity species, such as Aedes vexans
and Ochlerotatus sticticus, and species with the outbreak tendency during repeated floods,
such as Culex pipiens. In Slovakia, there are 6 mosquito species of Anopheles genus, able
to transmit the causative agents of malaria under many other conditions (Table 2). Our
research confirmed that Aedes vexans is the primary vector of filariae of Dirofilaria genus
in Slovakia. In addition to mosquitoes, we also monitored the occurrence of biting midges,
most important of them being the representatives of Culicoides genus, participating in the
transmission of haemosporidian blood parasites, filariae, and many viruses. Black flies
(Simuliidae) are significant in Europe as the species bothering animals and humans.
Mosquitoes, as vectors, participate in the transmission of a wide range of causative agents
of viral and parasitary diseases. Recently, Europe (Austria, Hungary, and Italy) witnesses a
higher occurrence of cases of the West Nile fever.
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Table 2 Diptera as vectors of selected pathogens in Slovakia.
* WHO declared the Slovak Republic as a malaria-free area in 1963;
? The route of transmission of a pathogen by a vector to humans in Slovakia – not
confirmed.
As a result of climatic warming, health hazard is also caused by other viral diseases, e.g.
the Dengue fever, spreading from Africa to warm European regions. Due to long-lasting
global thermal changes of the environment, there is increasing probability of spreading and
adaptation of unoriginal species of mosquito-vectors to new territories and introduction of
originally exotic diseases. A typical example of introduced mosquito’s species, not only to
Europe but also to Slovakia [4], is Aedes albopictus. Successful spread and adaptation to a
new environment are also significantly facilitated by global and local climatic changes.
Another one of the introduced mosquitoes in Europe is Aedes koreicus[5], a potential
vector of arboviruses and filariae. In hatching sites similar to those of Ae. albopictus,
created by human activities, also another species of exotic mosquito is developing in
Europe, Ochlerotatus japonicus [6]. It is only a question of time that another transmissible
parasitary disease, leishmaniasis, emerges in Slovakia. The latest cases of presence of
vectors of Leishmania (Psychodidae: Phlebotominae) were confirmed in Belgium,
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Germany, Austria, and Hungary, and, moreover, autochthonous cases of canine
leishmaniasis were observed in Germany and in Hungary [7, 8, 9].
Conclusion: The climate affects the survival and the reproduction rate of vectors,
influences suitability of the environment, distribution, count, and time activity of a vector,
intensity of attacks throughout a year, and the rate of development, survival, and
reproduction of a pathogen inside a vector. Our research confirmed the occurrence of
potential insect vectors, particularly from among the Diptera and Siphonaptera insect
orders.
Acknowledgements: The paper is supported by the grant task VEGA No. 1/0080/15, the
project titled “The Spread of Harmful Organisms in Terms of Human and Animal Health“
of EFSA`s FP in SR, and the elementary research of the National Reference Laboratory for
pesticides of the University of Veterinary Medicine and Pharmacy in Košice.
References
1.
SCHAUFF M.E.: Collecting and preserving insects and mites: Techniques & Tools. USDA, 1986, 69 pp.
2.
BECKER N., PETRIC D., ZGOMBA M., BOASE C., MADON M., DAHL CH., KAISER A.:
Mosquitoes and their control. Second edition, Springer–Verlag Berlin, Heidelberg, 2010, 577 pp.
3.
CHVÁLA M., HŮRKA K., CHALUPSKÝ J., KNOZ J., MINÁŘ J., ORSZÁGH I.: Krev sající mouchy
a střečci – Diptera. Fauna ČSSR svazek 22. ACADEMIA Praha, 1980, 538 s.
4.
BOCKOVÁ E., KOČIŠOVÁ A., LETKOVÁ V.: First record of Aedes albopictus in Slovakia. Acta
Parasitologica, 2013, 58: 603-606. DOI: 10.2478/s11686-013-0158-2.
5.
CAPELLI G., DRAGO A., MARTINI S., MONTARSI F., SOPPELSA M., DELAI N., RAVAGNAN
S., MAZZON L., SCHAFFNER F., MATHIS A., DILUCA M., ROMI R., RUSSO F.: First report in
Italy of the exotic mosquito species Aedes (Finlaya) koreicus, a potential vector of arboviruses and
filariae. Parasites & vectors, 2011, 4: 188;
6.
HUBER K., JOST A., PLUSKOTA B., BECKER N.: Surveillance and control of the invasive and
established species Ochlerotatus japonicus in south Germany. The 6. EMCA workshop, Budapest, Book
of abstract, 2011: 51.
7.
FARKAS R., TÁNCZOS B., BONGIORNO G., MAROLI M., DEREURE J., READY P.D.: First
surveys to investigate the presence of canine leishmaniasis and its phlebotomine vectors in Hungary.
Vector Borne Zoonotic Dis. Larchmt. N, 2011, 11: 823–834
8.
POEPPL W., OBWALLER A.G., WEILER M., BURGMANN H., MOOSEDER G., LORENTZ S.,
RAUCHENWALD F., ASPÖCK H., WALOCHNIK J., NAUCKE T.J.: Emergence of sandflies
(Phlebotominae) in Austria, a Central European country. Parasitol. Res., 2013, 112: 4231–4237.
9.
TÁNCZOS B., BALOGH N., KIRÁLY L., BIKSI I., SZEREDI L., GYURKOVSKY M., SCALONE
A., FIORENTINO E., GRAMICCIA M., FARKAS R.: First record of autochthonous canine
leishmaniasis in Hungary. Vector Borne Zoonotic Dis. Larchmt. N, 2012, 12: 588–594.
68
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THE INCIDENCE OF SALMONELLOSIS IN SLOVAKIA - TRENDS AND
CURRENT STATUS
A. Kološová
Regional Public Health Authority, Mederčská 39,945 01 Komárno
[email protected]
Introduction: Salmonelloses have been presented by the highest mortality rate and are the
most frequent cause of epidemics in Slovakia(1). The health care providers and
microbiological diagnostic laboratories are obliged to report the human cases of
salmonellosis to official authorities (2). The epidemiological surveillance started its
history in the
60s of the past century. The Epidemiological Information System in
Slovakia (EPIS) has been in operation since January 1st 2007. Reporting of the
transmissible diseases to EPIS is based on the link of all appropriate authorities and users
to the Central Registry via internet. In the Central Registry all infectious diseases are
recorded and stored according to the legislative of Slovak Republic. The automatic
reporting (on-line) and registration of the diagnostic results from the biological samples
analysis is also available. The system enables to obtain an immediate view of the
transmissible diseases occurence in the country and contributes to effective application of
preventive tools for control of the spreading of infectious diseases (3).
Goal of the study:The goal of the study was to analyse the data in the EPIS system related
to the epidemiological situation and to the trends in the development of salmonellosis
morbidity rates in Slovak Republic. We focused our attention also to the important factors
having an impact on the process of the disease spreading and thus contributing to the
effectiveness of intervention activities.
Methods: The study is based on the all-state data collected by the EPIS system. The data
in the EPIS system represent cases reported by health care providers and diagnostic
laboratories, as well as the information from the subsequent epidemiological examination
carried on by public health experts. The detailed epidemiological examination is carried on
not only during the epidemic occurence of the disease, but in the sporadic cases as well.
The study was carried on during the period of 10 years (2006-2015). The data were treated
by the decriptive approach.
Results and Discussion: Totally 59481 salmonellosis cases were diagnosed in Slovak
Republic during the study period. The number range was spread from 4153 cases in the
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year 2013 (morbidity rate 76.7/100 000 inhabitants) to 9542 cases in the year 2007
(morbidity rate 176.9/100 000 inhabitants). While a significant drop of the morbidity was
observed during the years 2007 – 2009, the increase in the morbidity started in the year
2013 with 8% in the year 2014 and 16% in the year 2015.
The highest number of cases during the study period was observed in the age category 1 –
4 years old children (22.4%), followed by the age category 5-9 years old (13.5%). In
almost 95% of cases the salmonellosis was presented with gastrointestinal symptoms. The
asymptomatic sheding of Salmonella was observed in 2.2% of cases, and in 0.6% cases
non-intestinal infections were diagnosed. Complicated septicemia was diagnosed in 0.2%
cases. In 2% cases no clinical form of the salmonelosis was estimated. From the total
number of cases 84.2% were confirmed by laboratory data. Most frequent isolates of
Salmonella from the study samples were as follows: S. Enteritidis (83.5%), S.
Typhimurium (7.7%), S. Infantis (2.0%), S. Enterica (0,8%), S. Bovisorbificans (0.4%), S.
Derby (0.3%), S. Virchow (0.2%), S. Montevideo (0.2%) other isolates (2.6%) and rest of
the non-identified isolates (1%). With the raising trend of Salmonellosis, the increase in
occurence of S. Enteritidis and drop in S. Typhimurium has been observed since the year
2013. In the cases without positive laboratory data the diagnosis was based on the clinical
symptoms and the epidemiological associations. The most important factors of
transmission were identified eggs and egg products (25.4%).
No association between eggs from local farms and supermarket stores was observed. The
second important factor of Salmonella tramsmision was identified meat and meat products
(10.9%), particularly the chicken meat. Next followed with 7.5% mixed food, milk and
milk products 2.7%, sweets and confectionary products 1.4%, contaminated items,
environment and hands 0.8%. Other factors of transmission were recorded in 2.4%. Totally
in 47.6% cases the factor of transmission was not identified. There were 510 (0.8%)
imported cases during the study period. From Czech Republic 87 cases, from Chroatia 64
cases, from Bulgaria 56, from Turkey 51 and from Hungary 47 cases. During the study
period of 10 years, 349 epidmics of salmonelloses have occured, all with more than 5
patients. Transregional spreading was identified in 10 epidemics.
Results of our study do not let us to make a conclusion regarding the identification of
causes of the trend development in the human salmonelosis in Slovakia. However the study
points out to several important events. No significant difference was observed in
participation of the individual factors of transmission in the recent three years when
compared with the previous years. There was no increase in the the number of epidemics in
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the study period, that could be responsible for the increase of the morbidity. Therefore we
hypothesize that for the increased trend in Salmonellosis occurence in Slovakia the
contaminated food chain reaching the consumers is responsible.
Conclusion: The salmonelloses occuring in the Slovak Republic have been increasing
since 2013 with the dominating Salmonella Enteritidis. In order to elucidate the causative
association of the presented trend in salmonelloses, the project on prospective case-control
study with the more detailed anamnestic data related to origin of food chain products
would be desired.
Literature
1.
Annual Report 2015. Activities of Public Health Authorities of Slovak Republic. Slovak Public Health
Authority, 2016, p. 35, 354.
2.
Law No 355/2007 on protection, support and development of public health. Department of Justice,
Slovak
Republic,
2007,
Add.
No
5,
p
147.
/Internet
link
https://www.slov-
lex.sk/static/pdf/2007/355/ZZ_2007_355_20160701.pdf
3.
Mikas, J., Sláčiková, M. Avdičová, M. et all., Surveillance of Infectious Diseases. Public Health
Authority of Slovak Republic. Project: Creation and implementation of educational programms for
Regional Public Health Authorities in Slovak Republic. Funded by EU, and state budget of SR. 2010,
s 6-7, ISBN 978-80-7159-200-6
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DETECTION OF HAEMOTROPIC MYCOPLASMAS IN DOMESTIC AND WILD
LIVING ANIMALS IN SLOVAKIA (PRELIMINARY DATA)
Komjáti-Nagyová M., Víchová B.
Department of Vector-Borne Diseases, Institute of Parasitology, Slovak Academy of
Sciences, Hlinkova 3, 040 01 Košice, Slovak Republic
[email protected]
Introduction: Haemotropic mycoplasmas, so-called haemoplasmas, previously known
as Haemobartonella orEperythrozoon, are placed in genus Mycoplasma based on lack of a
cell wall, 16S rRNA gene sequences and use of the UGA codon to encode tryptophan.
Mycoplasmas are epierythrocytic parasites, observed as small blue cocci, rings or rods in
blood smears. They attach to erythrocytes, but some species penetrate into the host cell or
appear free in the plasma (1, 2).
The haemoplasmas infect a wide variety of vertebrates worldwide and several Mycoplasma
species have a zoonotic potential, for example, M. pneumoniaeand/or M. genitalium.
These organisms cause a wide variety of clinical symptoms and vary in their ability to
cause clinically significant hemolytic anemia.More severe acute anemias are associated
with splenectomy, immunodeficiency or concurrent diseases. The typical clinical signs
include lethargy, anorexia, fever etc.Infected animals remain permanent carriers of
parasites.
Parasitaemia
may
re-emerge
when
the
animal
is
stressed
or
immunocompromised. Subsequently,the chronic carriers can represent a source of
infection for other animals (3, 4). Several veterinary important species cancausesignificant
economic losses in livestock farming (cattle, goats and sheep, pigs).
The real distribution of haemoplasmas is unknown since only a few studies havebeen
published so far. Haemoplasmas may be transmitted by the transfer of infected blood, via
arthropod vectors, vertical transmission - from mother to offspring andalso direct
transmission associated with fighting games between infected animals (5, 6).
The aim of the study: The aimof this study was to determine the diversity and prevalence
of Mycoplasmaspecies in domestic (cattle, goats) and wild (red foxes) animals, because, no
data from Slovakia have been published yet.
Material and Methodology: During the spring months of 2016, altogether 79 EDTAtreated blood samples of cattle, 157 blood samples of goats and 300 tissue samples (liver,
spleen or blood) from wild carnivores were obtained. Genomic DNA was extracted from
200 μl of blood sample or tissue sample, using a commercial DNA extraction kits
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(NucleoSpin Blood/Tissue kit, Machery Nagel, Germany). Briefly, for the molecular
detection of Mycoplasma spp., PCR was performed using the primers 322s and 938as
targeting a 600bp region of the 16S rRNA gene (7). Following the amplification, thePCR
products were visualized by electrophoresis on 1.5% agarose gels stained with GoodView
Nucleic Acid Stain (Beijing SBS Genetech, Beijing, China).
Positive PCR products were purified using a purification kit (Qiagen, Hilden, Germany)
and sequenced. Nucleotide sequences were manually edited in MEGA 6(Tamura et al.,
2013) and further compared with GenBank entries by BLAST (8). For the alignment of the
homologous nucleotide sequences, the ClustalW program was used. Obtained sequences
were submitted to GenBank database.
Results and Discussion: In total, 536 samples of domestic and wild living animalswere
examined. The presence of Mycoplasma spp. was confirmed in 64 tested samples, which
represents the prevalence of nearly 12%.
In cattle, the prevalence of Mycoplasma infection was relatively high, almost 57%. Two
distinct
species
have
been
identified,
namely
Mycoplasma
wenyonii
and
CandidatusMycoplasma haemobos, which are often responsible for hindlimb edema,
inflammation of mammary gland and reduction in milk production (9).
Overall, 6 out of 157 blood samples of goats tested positive, with Mycoplasma ovis being
the most common species. Infection in goats and sheep is often asymptomatic, however,
chronic infection may result in poor weight gain and decreased wool production (10).
In wild living canine carnivores, the prevalence of Mycoplasma infection was over 4%.The
spectrum of Mycoplasma species was wider. In red foxes (Vulpes vulpes) C. M. turicensis,
M. haemobos, M. haemocanis and M. haemofeliswere identified.
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Fig.1: Map of Slovakia, red stars represent the localities with proven Mycoplasma infection
in tested animals
Conclusions: The main objective of this study was to obtain the picture of the occurrence
and, diversity of Mycoplasma species in Slovakia.This study brings the preliminary results,
and represent the first data because there are no similar information about circulation of
haemoplasmasin Slovakia published. Of course, further research is needed to identify the
possible risk of infection for wild living and domestic animals and pets.
Literature
1.
Neimark, H., Johansson, K.E., Rikihisa, Y., Tully, J.G., 2001. Proposal to transfer some members of the
genera Haemobartonella and Eperythrozoon to the genus Mycoplasma with descriptions of ‘Candidatus
Mycoplasma haemofelis’ ‘Candidatus Mycoplasma haemomuris’ ‘Candidatus Mycoplasma haemosuis’
and ‘Candidatus Mycoplasma wenyonii’. Int. J. Syst. Evol. Microbiol. 50, 891–899.
2.
Chalker VJ. Canine mycoplasmas. Research in Veterinary Science 2005;79(1):1–8.
3.
Sykes JE, Ball LM, Bailiff NL, Fry MM. ‘Candidatus Mycoplasma haematoparvum’, a novel small
hemotropic mycoplasma from a dog. International Journal of Systematic and Evolutionary Microbiology
2005;55(Pt 1):27–30.
4.
Wengi N, Willi B, Boretti FS, Cattori V, Riond B, Meli ML, et al. Realtime PCR-based prevalence
study, infection follow-up and molecular characterization of canine hemotropic mycoplasmas.
Veterinary Microbiology 2008;126(1–3):132–41.
5.
Smith, J.A., Thrall, M.A., Smith, J.L., Salman, M.D., Ching, S.V., Collons, J.K., 1990. Eperythrozoon
wenyonii infection in dairy cattle. J. Am. Vet. Med. Assoc. 196, 1244–1250.
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6.
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Hornok, S., Meli, M.L., Erdos, A., Hajtós, I., Lutz, H., Hofmann-Lehmann, R., 2009. Molecular
characterization of two different strains of haemotropicmycoplasmas from a sheep flock with fatal
haemolytic anaemia andconcomitant Anaplasma ovis infection. Vet. Microbiol. 136, 372–377.
7.
Compton, S.M., Maggi, R.G., Breitschwerdt, E.B., 2012. Candidatus Mycoplasma haematoparvum and
Mycoplasma haemocanis infections in dogs from the United States. Comparative Immunology,
Microbiology and Infectious Diseases 35, 557–562.
8.
Tamura, K., Stecher, G., Peterson, D., Filipski, A., Kumar, S., 2013. MEGA6: molecular evolutionary
genetics analysis version 6.0. Mol. Biol. Evol. 30, 2725–2729.
9.
http://www.merckvetmanual.com/mvm/circulatory_system/blood_parasites/hemotropic_mycoplasmas.ht
ml#v3257737
10. Stuen, S., 2016. Haemoparasites in small ruminants in European countries: Challenges and clinical
relevance. Volume 142, Pages 22–27.DOI: http://dx.doi.org/10.1016/j.smallrumres.2016.03.005.
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WE SEARCHED FOR ONE, WE DISCOVERED MORE AND WHAT´S NEXT?
Košťanová Z.,
Regional Public Health Authority in Žiar nad Hronom, Slovak Republic
[email protected]
Introduction: The best knowledge of the characteristics of tick borne diseases in our own
region is the most effective tool of prevention. Before the beginning of our study in 3
districts of Regional Public Health of Authority in Žiar nad Hronom (RPHAZH), we had
only the knowledge of results from studies of the ticks, the vectors borne disease and the
reservoirs in Slovakia. The results pointed out the circulation of the Tick-borne
encephalitis virus (TBE) in natural foci in our neighbor districts.
According to the
morbidity it seemed that we should be concerned only about Lyme borreliosis (LB) in our
region. In 2002 our RPHAZH established co-operation with Institute of Virology of the
Slovak Academy of Sciences, Bratislava (IV SAS BA) with primary aim to find an answer
to the question if we also have endemic foci of TBE virus in our districts. The years of our
co-operation showed the fact that we are quite interesting region. There are not only
natural foci of LB and TBE virus but also the natural foci of Anaplasma phagocytophilum
(AP), Coxiella burnetii and Rickettsia SFG in our region.
Aims: The main aim was to sum up the results of study of natural foci of the vectors borne
diseases in years 2002 – 2011 in regions of RPHAZH and to give answers to the question
what have we done with the obtained data in our daily practice. We wanted also to point
out the importance of mutual co-operation of various organizations for the purpose of
solving such complex problems such as tick borne diseases.
Methodology: The employees of epidemiology department collected 112 ticks (Ixodes
ricinus) by flagging of the vegetation in the selected some areas with increased morbidity
of LB in 2002. The collection was realized in 4 localities of the district Žarnovica (ZC), in
4 localities of the district Banská Štiavnica (BS) and in 3 localities of the district Žiar nad
Hronom (ZH). The samples were delivered to IVSAS BA for the investigation. In the
following years the tick collection (approximately 600) and the capture of 67 wild rodents
(Apodemus flavicolis, Clethrionomys glareolus a Microtus arvalis) were realized by the
personnel of IVSAS in 22 selected localities.
At the same time the personnel of RPHAZH was provided with 205 human blood sera in
localities where the circulation of monitored pathogens was confirmed. The blood sera
were investigated by IVSAS BA for TBE virus, Borrelia burgdorferi s.l. (Bbs.l.), Rickettsia
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SFG, Cocxiella burnetii and AP using the following methods: PCR and DNA sequencing
methods, Westernblot IgG and IgM kits, cultivation, HGE IgG IFA, ELISA, complement
fixation, hemagglutination inhibition. Twenty blood serums were examined for antibodies
against AP in Slovenia thanks to the personnel of IVSAS BA in 2002. In the co-operation
with the personnel of Regional Veterinary and Food Institute, Žiar nad Hronom (RVFI ZH)
secured the collection of 80 spleen samples from wild animals (the boars, red deer, road
deer, moufflons). These samples were examined for the TBEvirus (in 2002), Bbs.1., AP,
Rickettsia SFG and Coxiella burneti by personnel of IV SAS BA. We used individual,
group and mass forms in order to fulfill the aim of our study.
Results: The investigation of human blood serums for the presence antibodies against TBE
virus was in 2,08% positive and from samples of ticks in 4,93%. Based on these results
there were more endemic foci of TBEvirus added to our attraction zone of RPHAZH.
Besides the locality of Bartošova Lehôtka, which was known for being the endemic foci
based on morbidity, there were added also more endemic foci such as Trnavá Hora,
Bzenica, Štiavnické vrchy and the mountain Vtáčnik.
The percentage of infected ticks by Borrelia burgdorferi sensu lato was 1,78% in 2002
and 4,35 % in 2004. In 2 localities (Trnavá Hora, Bzenica) Borrelia afzelii was detected.
In 3 localities (Malá Lehota, Hodruša - Hámre and Horná Ves) Bbs.l was detected. A
patient with the diagnosis of LB from Žiar nad Hronom was diagnosed with Borrelia
garinii from a removed tick.
From the tick samples from 22 localities Anaplasma phagocytophila was detected in 5,77
% (the highest number of AP was in the village of Lutila - 35,71 % and the lowest number
in Bzenica - 2,17 %). The examined sample of wild rodents were in 6,06% positive for AP
in Lutila and in 5% in Hodruša Hámre. 11,25 % of deer spleen samples were positive. IgG
antibodies against AP were detected in 30,38 % of the examined human serums. The
investigation of ticks for the presence of antibodies against Rickettsia SFG from 22
localities showed the prevalence from 1,5% to 7,38%. 1,75% samples of wild rodents were
positive for Rickettsial infectedness. None of the deer samples were positive. From the
examined samples of human serums there were antibodies IgG against Rickettsia SFG
detected in 9,43% of inhabitants, all of them from Kremnica and its surroundings.
The examination of ticks for Coxiella burnetii confirmed the circulation in 2,1% in
Bzenica, of wild rodents in 1,49% in Hodruša-Hámre and of deer in 1,25% in the village
Kľak.
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We should take into account also the risk of dual infections in the natural foci with the
circulation of various pathogens. There were 10 mixed natural foci detected in the
attraction zone of RPHAZH. The circulation of 4 pathogens was confirmed in 1 location
(Bzenica), of 3 pathogens in 3 locations (Hodruša-Hámre, Malá Lehota, Trnavá Hora) and
of 2 pathogens in 6 locations.
We informed continuously about the results of our research activities not only the
professional public at the conferences, seminars, specialized magazines or in the
ambulances of general practitioners, but primarily the non-professional public. The
decrease of our research activities was the beginning of more intense health-educational
activities, which were the main reason of the realization of the research part. For the
intensive prevention in this area we use individual, group and the mass forms of
intervention.
Discussion: The prevention of tick-borne diseases is very complex process and because of
the existence of vectors and reservoirs sometimes almost impossible to be influenced. The
personal protection of the individuals is the only way how to minimalize the risk of tickborne disease. Its basis is the constant and objective public awareness. Before the
beginning of our co-operation we hadn´t had the answer to the question if in our attraction
zone we had besides TBEvirus also some other natural foci of tick-borne diseases.
The prevalence of ticks in endemic foci with the TBE virus is stated to be 1-2% (1). In our
small sample of 142 ticks there were 4,98% of ticks infected with TBEvirus. The study
carried out in southeastern and northeastern Slovakia showed the prevalence of ticks of
10,15% with Bb.s.l. From our sample it was around 1,78% in 2002 and 4,35% in 2004 that
also pointed out the variable percentage of infected tiks in the particular years. The
prevalence of ticks with AP in Slovakia is from 1,1 to 7,8%, in our region 5,77% (2,3,4).
The prevalence of wild rodents with AP (6,06% in Lutila and 5% in Hodruša Hámre) was
lower than reported from the study from eastern Slovakia (5). From the samples of human
serums there were 30,38% positive for antibodies IgG against AP. These results that
IVSASBA and RPHAZH published in 2008 (9) had not been published in Slovakia before
then. The first case of Human granulocytic anaplasmosis from Slovakia was published in
2010 (6). The prevalence of ticks with Rickettsia SFG in our region depending on the
region in particular was from 1,5% to 7,38% which was lower percent than the results of
the study in which the prevalence of examined ticks in Slovakia was 10% (7). The
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antibodies of IgG were detected in 9,43% of patients that corresponded with the results of
the study in Slovakia (8).
Conclusion: The results of investigation of human serums, ticks, wild rodents and spleens
of deer in IVSASBA definitely showed the fact that the districts of BS, ZC and ZH are the
regional areas where the intensive prevention against the tick-borne diseases has its
foundation.
Acknowledgement: We couldn´t have done this study without the team of IVSAS in
Bratislava, primarily without RNDr. Elena Kocianová, DrSc., RNDr. Katarína Schwarzová
CSc., Mgr. PhD. Katarína Štefanidesová, without the general practitioners from the
districts of ZH, ZC and BS, without the employees of the Department of Clinical
Microbiology in ZH, without RPHAZH in ZH.
Literature
1.
http://www.solen.sk/pdf/f10fa3829ae4d404d487e5c7eaeb3afa.pdf Eva Máderová, Tick-borne encephalitis
in Slovakia, Public Health Authority in Slovakia , Bratislava, Via pract., 2005, roč 2 (1): 51-54,
2.
Derdáková M, Štefančíková A at all: Emergence and genetic variability of Anaplasma species in small
ruminants and ticks from Central Europe. Vet Microbiol. 2011, 153: 293-298. 10.1016/j.vetmic.2011.05.044.
3.
https://parasitesandvectors.biomedcentral.com/articles/10.1186/1756-3305-6-238,
Lucia Pangrácová,
Markéta Derdáková at all : Ixodes ricinus abundance and its infection with the tick-borne pathogens in urban
and suburban areas of Eastern Slovakia.
4.
https://parasitesandvectors.biomedcentral.com/articles/10.1186/s13071-015-0880-8
Zuzana Svitálková,
Danka Haruštiaková at all.: Anaplasma phagocytophilum prevalence in ticks and rodents in an urban and
natural habitat in southwestern Slovakia.
5.
http://link.springer.com/article/10.1007%2Fs12223-008-0077-z, A.Štefančíková, M. Derdáková at all.:
Serological and molekular detection of Borrelia burgdorferi sensu lato and Anaplasmetaceae in rodents, Folia
microbiologica, november 2008, Volume 53, Issue 6, pp 493-499
6.
Nováková M, Víchová B. a kol.: First case of Human granulocytic anaplasmosis from Slovakia, Ann Agric
Environ Med 2010, 17,173-175
7.
http://www.ncbi.nlm.nih.gov/pubmed/23043605 : Sekeyová Z, Mediannikov O at all.: Isolation of rickettsia
helvetica from ticks in Slovakia
8.
Z.Sekeyová at all.: Update on Rickettsioses in Slovakia, Acta virologica 57:180-199,2013
9.
E. Kocianová, Z. Košťanová, K. Štefanidesová at all., Serologic evidence of Anaplasma phagocytophilum
infections in patients with a history of tick bite in central Slovakia, Wien Klin Wochenschr (2008), 120/1314:427-431)
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INFLUENZA – THE PERMANENT THREAT
Krištúfková Z.
Faculty of Public Health, Slovak Medical University in Bratislava,
Slovak Republic
Introduction: Influenza is a serious infectious disease with a high morbidity and mortality
rates worldwide. It is an underestimated disease both in layman and educated population.
The causative agent, the influenza virus, particularly type A is highly variable, causing
epidemics every year with serious social and economic consequences. The death toll in
European Union depending on the size of the epidemics has been estimated between
40 000 to 220 000 persons(1).
The influenza is causing besides theevery year epidemics also pandemics. The WHO
defines influenza pandemic as an epidemic that meets two criteria: it is caused by a new
influenza subtype of type A virus and it affects the population worldwide.
Aim: The goal of the study was to evaluateimpact of the pandemic in 2009-2011 on the
population of Slovakia and to document the present risks of the animal viruses
transmission to man.
Pandemy in the years 2009 – 2011: The recent pandemy that had spread during the years
2009 to 2010 was caused by virus A(H1N1)pdm09. The virus wasreassortant of two
different strains of swine, one human and ane bird strain. Such strain was a new one in the
world, never identified previously (2, 3, 8). Its mode of spreading met the criteria of
pandemic virus. Most of the cases were presented as a mild form of the influenza though
some heavy cases of the influenza with a fatal outcome were recorded as well.
The pandemy was declared on June 11th, 2009 by the Director General of WHO, and
declared terminated by August 8th, 2010 together with the recommended measures as
defined by the WHO International Health Recommendations. This pandemic strain was
declared to be the seasonal type and already has been circulating in population and
causing a disease including the death of patients.
The new influenza strain was isolated and identified from patients on March 28th 2009 in
Mexico.The virus has disseminated quickly worldwide. The first case was identified in
Slovakia on May29th, 2009. Subsequently all patients having the symptoms of influenza
were under investigation, reported and followed. The first pandemic wave hit Slovakia in
November.Due torapid spreading of the pandemy and number of new cases it was not
possible to report and investigate individually all cases. Therefore the reporting of acute
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respiratory diseases, influenza and influenza-like diseases switched to weekly cumulative
reporting since November 11th, 2009. The reporting of acute serious respiratory diseases
under acronym SARI was introduced on November 13th, 2009. The SARI was defined as
diseases with a sudden raise of body temperature, sore throat, shorten or difficult breath
andthe patient was send to hospital due to respiratory complications. The laboratory
examination was carried on only in hospitalized patients with SARI, dead patients and
from sentinel physicians(9, 10, 11).
Totally 436 severe acute respiratory disease cases were reported in Slovakia between
November 3rd, 2009 and August 8th, 2010. The number of dead patients was 108
(24.8%). The laboratory examination confirmed pandemic influenza in 165 from the total
number 435 patients. From the confirmed influenza patients 54 (32.7%) had died (Tab.1.).
We assume with a high probability that all cases of SARI were caused by the pandemic
virus.
Tab. 1:Morbidity and mortality rates from SARI. Slovakia,
November 3rd, 2009 – August 10th, 2010
SARI
Diseases with confirmedA(H1N1)pdm09
SARI
abs.
%
abs.
%
Diseases
436
100,0
165
100,0
Deaths
108
24,8
54
32,7
Reference:Mikas, 2012
The total morbidity rate from SARI reached 8.0/100 000. The highest was in age
category 0-4 years old children (11.5/100 000), the lowest in 5-9 years old children
(3.5/100 000). In higher age groups the morbidity was raising, reaching the highest value
in 55-64 years old category (11.0/100 000) (Chart 1.) The total mortality rate for SARI was
2.0/100 000, the highest in the age group 45 – 54 reaching 3,6/100 000 inhabitants.
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morbidity, mortality rates 100 000
14,0
12,0 11,5
chorobnosť
11,0
10,7
úmrtnosť
10,0
10,0
4,9
3,9
3,5
4,0
3,6
3,0
2,5
1,7
2,0
10 – 14 15 – 19 20 – 24 25 – 34 35 – 44 45 – 54 55 – 64
65 + Celkom
1,4
1,0
0–4
5–9
1,4
1,2
0,7
0,0
0,0
6,1
5,7
6,0
2,0
8,0
7,8
8,0
Age groups
Chart 1: The morbidity and mortality rates from SARI by age groups, Slovakia,
November 3rd, 2009 – August 10th, 2010
Reference: Krištúfková, 2014
The lethality from SARI was high reaching 24.8%. In the laboratory confirmed
cases the lethality rate was even higherreaching 32.7%. The highest lethality rate was
recorded in the age group 45 – 54 years (34.1%) and in the age group 35 – 44 years
(32.3%). No death was recorded in the 5 – 9 age group of SARI patients.
40,0
35,0
32,3
34,1
SARI
29,9
Letality rate %
30,0
25,0
25,0
25,0
24,8
23,2
18,2
20,0
15,8
15,0
10,0
9,1
5,0
0,0
0,0
0–4
5 – 9 10 – 14 15 – 19 20 – 24 25 – 34 35 – 44 45 – 54 55 – 64
65 + Celkom
Age groups
Chart 2: The lethality rate from SARI by the age groups, Slovakia, November 11th,
2009 – August 10th, 2010.
Reference:Krištúfková, 2014
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During the pandemy 13 pregnant women had acquired the pandemic virus and developed
the disease. Six of these patients had died (Chart 2.) (6, 7). Some of the gene segments
from the pandemic strains were identical with the segments from the virus that caused the
pandemy in 1918. The pandemic virus from 1918 circulated in population until 1950.
Therefore the older population having a contact with circulating virus developed an
immunitythat partially played a protective role against the pandemic strain (4).
Tab. 2: The diseases and deaths from SARI in pregnant women, Slovakia, November
3rd 2009 – August 10th, 2010.
SARI pregnant women
abs.
%
Diseases
13
100
Deaths
6
46,2
Reference: Mikas, 2012
Our analysis carried on in Slovakia, similarly as other studies abroad,showed
statisticallymost significant impact of pregnancy on the outcome of pandemic influenza.
Other health disorders such as cardiovascular diseases, chronic obstructive pulmonary
disease, malignant diseases and age in both sexes reveal an impact on the severity and
squealof the pandemic influenza as well. (5).
The vaccine against the pandemic influenza was imported to Slovakia on December 15th
2009. The composition of the vaccine was the same as vaccine Vaxigrip that have been
applied in vaccination program against the seasonal influenza every year. However, instead
of the seasonal strain the vaccine contained the pandemic strain. The efficacy of the
vaccine was very high and the vaccine was safe. After vaccination, no unexpected side
reactions were reported. The first doses were applied to health care workers, then after
January 13th, 2010 pregnant women were immunised and adults persons with serious
chronic diseases as well. Further, children from 6 months, adolescents with serious chronic
diseases, persons responsible for safety of economic and public life and from February 5th
vaccine was available for every citizen (Chart 3.).After this date 25 citizens died of SARI.
In four cases the pandemic strain was confirmed. None of these 25 citizens was vaccinated
against influenza.
By the reported cases from the outpatient clinics and vaccinating physicians there were
23 044 inhabitants vaccinated against pandemic influenza, corresponding to the 0.4 % of
Slovak population (7). The worldwide carried on antivaccination campaign had a negative
impact on the attitude of the inhabitants to vaccination and hadcast doubts on the pandemy.
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WHO has confirmed in May 2010 the worldwide death toll of 12 000 patients from
pandemy. Simultaneously it confirmed that the high efficacy and safety of the vaccine
helped to stop spreading of the pandemy.
Chart 3:Death from SARI and death from SARI with laboratory confirmed pandemic virus in Slovakia according to the
weeks and availability of the vaccine.
References:PHA SR
Transmission of the animal viruses to man: The transmission of the animal viruses to
man can occur very rarely. For instance the highly contagious bird influenza virus
A(H5N1) infected 18 persons in Hong Kong in 1997. Six of the infected patients died.
Then the virus had spread worldwide. The migratory birds were the key transmission
factor. The worldwide occurrence of the disease was recorded by WHO with registered
851 laboratory confirmed infections in man in 16 states between 2003 and middle of 2016.
The heavy viral pneumonia with fatal outcome was registered in 450 patients (lethality
52,9%). The occurrence of the virus was highest in Egypt in 2015. Virus A(H5N1) killed
millions of poultry and many farms had to be closed and poultry farming stopped in order
to break the spreading of the disease. Epizootic spreading of the virus in poultry industry
was accompanied by the severe negative impact on the livelihood, economy and
international trade in the affected countries. The bird virus A(H5N6) has been confirmed in
poultry in China besides the strain A(H5N1). Due to the close contacts of inhabitants with
poultry the diseases has occurred. China reported 14 confirmed diseases and 6 fatal cases
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since 2014 when the bird virus A(H5N6) was first time identified in a child. Other virus
transmissible from animals to man was identified as the bird virus A(H7N9). After the
virus was identified first time in 2013 as a causative agent of influenza in three persons in
Shanghaj the WHO recorded 781 disease cases in man with 313 fatal cases (40,1%).The
infection of the man can be contracted by a direct contact with infected poultry, or
contaminated environment most often at the live poultry markets. This is a case in China
(5). The virus of bird flu has not been transmitted from man to man. Such transfer can
occur only when the genome of the virus contains besides animal also part of the human
influenza virus genome.
Conclusion: The selection pressure generally has an impact of the evolution of the viruses.
Influenza viruses with a potential to exchange the genetic information mutually, represent
an etiological agents hence causing repeatedly epidemics and pandemics. The influenza is
a disease with no chance to be eradicated and thus it always will represent a risk for man.
The effective vaccination appears to be only effective tool to prevent its spreading. The
personal attitude is an important phenomenon for our health.
Literature
1.
Beran J, Havlík J. The influenza, clinical picture, prevention and therapy. Praha, Maxdorf 2005, 175 s.
2.
COHEN J., 2009. Swine flu outbreak out of Mexico, Scientists ponder swine flu origin. In Science.
ISSN 0036-8075, 2009, vol. 324, no. 5928, p. 700-702
3.
ECDC 2009a; ECDC. 2009a. Risk Assessment – 2009 influenza A(H1N1) pandemic, Version 7.
[online].
Stockholm:
ECDC,
2009.
[cit.
2010.10.06].
Dostupné
na:
http://www.ecdc.europa.eu/en/healthtopics/Documents/0908_Influenza_AH1N1_Risk_Assessment.pdf.
4.
GARTEM, R.J., Davis C.T., Russel C.A. et al. 2009. Antigenic and genetic characteristic of swine –
origin 2009 A (H1N1) influenza virus circulating in humans. In Science. ISSN 0036-807, 2009, vol. 325,
no 5937, p. 197-201.
5.
HLAVINKOVÁ L., MIKAS J., KRIŠTÚFKOVÁ Z.: Risk factors for severe outcome of cases with
pandemic influenza A(H1N1)pdm09 in Slovakia, BratislLekListy 2015; 116 (6), p. 389 – 393, DOI:
10.4149/BLL_2015_074
6.
KRIŠTÚFKOVÁ Z., OLEÁR V.: Influenza Surveillance PRO BanskáBystrica, 2014, ss.104, ISBN
978-80-89057-57-3
7.
MIKAS J.: Influenza pandemieofA(H1N1)pdm09 in Slovak Republic. Theses 2012, Univerzita Pavla
Jozefa Šafárika v Košiciach, Lekárska fakulta
8.
TRIFONOV V., KHIABANIAN H., RABADAN R. 2009, Influenza A (H1N1) virus. In The New
England Journal of Medicine. ISSN 0028-4793, 2009, vol. 361, p. 115-119
9.
ÚVZ SR. 2009a. Annual Report of Public Health Authority in Slovakia 2009. [online]. Bratislava: ÚVZ
SR, 2009.
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10. ÚVZ SR. 2009b. The Regulation on modification of measures associated with spreading of the influenza
virus A(H1N1) 2009. ÚVZ SR, 2009.
11. ÚVZ SR. 2010. Annual Report of Public Health Authority in Slovakia 2010. Bratislava, ÚVZ SR, 2010.
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DETERMINATION OF MICROBIAL INDICATORS IN DAIRY PRODUCTS
BASED ON THE DETECTION OF OXYGEN CONSUMPTION
V. Lehotová, M. Petruláková, Ľ. Valík
Department of Nutrition and Food Quality Assessment, Faculty of Chemical and Food
Technology STU, Radlinského 9, 812 37Bratislava, Slovakia
[email protected]
Introduction: The primary responsibility for the setting out the necessary hygiene
conditions for production foods, safe and suitable for consumption, were always directed
by the legislation, and accepted by manufactures. The compromise approaches or
deficiencies in the application of the principles of good manufacturing/hygiene practices
have a direct impact on the incidence of microorganisms in the eatables. Therefore, there is
a need to obtain the information about microbiological quality in time, as quickly as
possible.
The aim of the study: In this work, we had the opportunity to introduce the results and
evaluate the experience with the GreenLight™ equipment (Mocon Inc., Minneapolis, MN,
USA) aimed on determination total viable counts as microbiological food quality indicator
based on the detection of metabolic activity of reproduction-competent cells present in a
test sample.
Methodology: Method for the determination is based on the fluorescence detection of
oxygen consumed by the population of microorganisms present in a food sample during
incubation at 30 °C.
Results and Discussion: New analytical techniques, the GreenLight™ system for rapid
enumeration of total viable counts (TVC) were used to estimate the numbers of bacteria
inoculated in different levels in broth nutrient media. The new detection methodology was
compared with agar plating EN ISO 4833:2003 method showing excellent correlation. The
following coefficients of determination R2 = 0.985 and 0.999 were calculated for aerobic
Pseudomonas aeruginosa and facultative anaerobic E. coli, respectively. In the method
yeast and moulds, coefficient of determination reached the value 0.91 in all cases sour milk
products contaminated with different species of yeasts. Lower coefficients of
determination for the linear dependence between the time to reach a threshold and the
number of microorganisms were ascertained in the set of samples with a limited number
and content of microorganisms contained in a narrow range of values.
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Conclusion: After calibration, the system based on the principle of quenching of
luminescence intensity and lifetime of an oxygen-sensitive dye by sample O2 consumed
during microbial growth enables to determine the number of microorganisms within less
than 24 hours. The higher microbial load the shorter time for determination of viable count
is needed. In case of simple food matrix for example, the results can be reached even
within one shift of production.
Acknowledgement: The authors would like to thank to Slovak Research and Development
Agency (SRDA) project APVV-15-0006 and company MOCON Inc. (Minneapolis, MN,
USA) for providing of equipment GreenLight™ Model 930 and APCheckTM vials.
Literature
1.
Lehotová, V., Petruláková, M., Valík, Ľ. (2016).Application of a new method to control microbial
quality of foods based on the detection of oxygen consumption. Acta Chimica Slovaca, 9 (1), 19-22.
2.
EN ISO 4833-1 (2014). Microbiology of the food chain: Horizontal method for the enumeration of
microorganisms. Part 1: Colony count at 30 °C by the pour plate technique.
3.
STN ISO 21527-1 (2008). Microbiology of food and animal feeding stuffs -- Horizontal method for the
enumeration of yeasts and moulds. Part 1: Colony count technique in products with water activity greater
than 0.95.
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GROWTH DYNAMICS OF E. COLI ISOLATED FROM DAIRY PRODUCTS
A. Medveďová, V. Bartalošová, T. Mančušková, Ľ. Valík
Department of Nutrition and Food Quality Assessment, Faculty of Chemical and Food
Technology, Slovak University of Technology in Bratislava, Slovak Republic
[email protected]
Introduction: Outbreaks of Escherichia coli-related food-borne diseases are often
connected to the consumption of dairy products. The densities of E. coli of about 107
CFU/ml may be reached during manufacture of dairy products, especially of raw milk
cheese. E. coli is a relevant indicator of potential faecal contamination, due to its
commensal relationship with the intestines of human and other mammals. Moreover, poor
hygienic practices together with possible faecal contamination during milking may result
in contamination of raw milk. So it is a very common part of raw milk and also of
processed milk and dairy products. As reported Donelly et al. [1], E. coli can contaminate
raw milk on the farm with contamination levels of 4.2-10 %, however in 23-65 % of milk
samples was E. coli detected in the range of 104-106CFU/ml [2,3]. In Slovak raw milk
cheese the presence of E. coli was reported in 20-33 % of all samples [4]. The possibility
of E. coli transmission through the consumption of raw milk as well as raw milk dairy
products has been repeatedly documented [5]. Raw milk, insufficiently pasteurized or
secondary contaminated pasteurized milk were identified as the most frequent source of E.
coli contamination [6].
The aim of the study was to characterise the presence of E. coli in dairy products and to
describe the growth potential of isolates in dependence on temperature and origin of
isolates.
Methodology:Isolates of E. coli were collected from ewes’ lump cheese from raw milk
(9), cows’ lump cheese from raw milk (2), raw cows’ milk (8) and „Bryndza“ cheese (9).
Their identity was confirmed by Gram staining, microscopic examination, growth on
Chromocult Coliform and EMB agars, citrate, oxidative-fermentative, Voges-Proskauer,
indole and catalase-presence tests. Resistance of isolates against ampicillin, gentamicin,
ciprofloxacin, chloramphenicol and tetracycline was determined according to EUCAST
resistance breakpoints by the use of cultivation method. Growth dynamics of isolates was
analysed by the use of their cultivation in milk at 15 °C. Obtained growth curves were
modelled with the mechanistic DM-fit model and secondary Gibson model was used for
fitting growth parameters of E. coli BR isolate as a function of temperature. Also the
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growth dynamics of E. coli BR isolate in the presence of Fresco starter culture (in addition
ranging from 3 to 6 log CFU/ml) was studied during their cultivation in milk at 15 °C.
Obtained growth curves were modelled as described previously.
Results and Discussion: 28 isolates of E. coli were collected from dairy products from
September 2014 to December 2016. The average density of E. coli was 3.4 102 CFU/ml in
raw cows’ milk, 5.3 102 CFU/g in ewes’ lump cheese from raw milk, 6.4 102 CFU/g in
cows’ lump cheese from raw milkand 3.0 102 CFU/g in „Bryndza“ cheese. It is in
accordance with Hill et al. [7] who reported that in majority of milk samplesthe density of
E. coli is less than 102 CFU/ml.Identification of isolates was confirmed by microbiological
and biochemical methods. All isolates were Gram negative rods, with typical growth on
Chromocult Coliform agar and on EMB agar. They were also all catalase positive, able to
produce indole from tryptophane, unable to produce acetoine and diacetyl from glucose
and to assimilate citrate as an only source of carbon. All of them utilised glucose, lactose
and sucrose with the production of acids and gases. Based on the antibiotic resistance
examination, it can be concluded that the most effective antibiotic was ciprofloxacin (11 %
resistant isolates) and the less effective antibiotic was gentamicin (94 % resistant isolates).
Only 3 isolates were sensitive to all studied antibiotics, on the other hand, there was no
isolate resistant to all 5 antibiotics.
As it is seen in Fig. 1, the average growth rate (Gr) of all isolates in milk at 15 °C was
0.120 ±0.010 log CFU/ml.h (n = 84; CV = 10.1 %). The lag phase ranged from 1.9 h to
11.1 h and the maximal counts in stationary phase ranged about 8.43 ±0.33 log CFU/ml.
Based on obtained results it can be concluded that the growth dynamic of E. coli in milk
can be determined with a high degree of reproducibility. Moreover, the origin of the isolate
did not affect the growth ability to a great extant what is a prerequisite for risk assessment
associated with E. coli in milk and dairy products by predictive microbiology.
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40
100%
90%
35
80%
30
70%
frequency
25
60%
20
50%
40%
15
30%
10
20%
5
10%
0
0%
Gr (log CFU.ml-1.h-1)
Fig. 1: Histogram of growth rate values of 28 E. coli isolates during growth in milk at 15
°C
Within an empirical approach to model the effect of the incubation temperature on the
growth rate of E. coli BR, secondary Gibsonmodel was used to include all growth rates in
temperature range from 8-46 °C. The values of the specific growth rate of E. coli BR were
plotted against calculated Tw-values and fitted with a regression model that was represented
by the equation ln 𝜇 = −0.296𝑇𝑤2 + 1.655𝑇𝑤 − 1.606 (𝑅 2 = 0.967; 𝑇𝑤 = √𝑇𝑚𝑎𝑥 − 𝑇),
whose graphical representation is depicted in Fig. 2. By using this model, optimal
temperature Topt = 38.8 °C for E. coli BR growth in milk was calculated.
1
0,5
0
-0,5
ln m
-1
-1,5
-2
-2,5
-3
-3,5
5
10
15
20
25
30
35
40
45
50
T (°C)
Fig. 2: Plots of ln μ of specific growth rates versus 𝑇 for E. coli BR. Symbols indicate the
natural logarithm of specific growth rate calculated from growth curves at each incubation
temperature for E. coli BR. The continuous line indicates the fitted ln μ vs. T function.
91
9
9
8
8
7
7
6
6
5
4
3
EC + 3FR; 15 °C
log CFU/ml; pH
log CFU/ml; pH
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2
5
4
3
EC + 6FR; 15 °C
2
EC_Fr
FR_Ec
pH
EC_Fr
E. coli
1
a
18th-20th October 2016
FR_Ec
pH
E. coli
1
0
6 12 18 24 30 36 42 48 54 60 66 72 78
time (h)
b
0
6 12 18 24 30 36 42 48 54 60 66 72 78
time (h)
Fig. 3a, 3b: Growth curves of E. coli in monoculture (◊) and in co-culture (♦) with Fresco
starter culture (■) in initial addition of 3 log CFU/ml (3a) and 6 log CFU/ml (3b) in milk at
15 °C with pH value changes (▲)
Growth responses of E. coli as affected by the presence of Fresco culture was studied at 15
°C in culture addition ranged from 103 CFU/ml to 106 CFU/ml. Based on the obtained
results (Fig. 3a and 3b), the adequate addition and active metabolism of lactic acid bacteria
resulting in rapid pH value decreasing to the value that may play a significant role in a
partial inhibition of E. coli in milk. The initial concentration of Fresco culture higher than
106 CFU/ml were able to inhibit the E. coli growth, as presented the up growth of E. coli
about 102 CFU/ml, compared to up growth of 106 CFU/ml in monoculture at the same
temperature.
Conclusion: It can be concluded that the growth dynamics of E. coliisolates is not affected
by their origin, even not by the culture media, and consequently the growth dynamics can
be estimated with 4–11 % error in the prediction.Moreover, the adequate addition of
Fresco culture curing cheese manufacture and the early pH decrease on the levels lower
than 5.0 may inhibit the E. coli growth and minimise risk of E. coli-related food-borne
outbreaks.
This work was supported by contract No. APVV-15-0006.
Literature
1.
Donelly CW: Growth and survival of microbial pathogens in cheese. In: Fox P, McSweeney P, Cogan T,
Guince T (eds.) Cheese: Chemistry, Physics and Microbiology, 3rd ed. Academic Press, Hardbound,
2004.
2.
Chye FY, Abdullah A, Ayob MK: Bacteriological quality and safety of raw milk in Malaysia. Food
Microbiology 21:535-541, 2004.
3.
Lues JFR, Venter P, van der Westhuizen H: Enumeration of potential microbiological hazards in milk
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from a marginal urban settlement in central South Africa. Food Microbiology 20:321-326, 2003.
4.
Valík
Ľ:
Risk
assessment
of
pathogen
bacteria
presence
in
raw
milk.
http://www.mpsr.sk/sk/index.php?navID=525&navID2=525&sID=111&id=8, 44p, 2013.
5.
European Food Safety Authority: European Centre for Disease Prevention and Control; The European
Union Summary Report on Trends and Sources of Zoonoses, Zoonotic Agents and Food-borne
Outbreaks in 2011; EFSA Journal 11(4):3129, 2013.
6.
Kousta M, Mataragas M, Skandamis P, Drosinos EH: Prevalence and sources of cheese contamination
with pathogens at farm. Food Control 21:805-815, 2010.
7. Hill B, Smythe B, Lindsay D, Shepard J: Microbiology of raw milk in New Zealand. International
Journal of Food Microbiology 157:305-308, 2012.
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PRE-CUT (READY-TO-EAT) VEGETABLES: EVALUATION OF THE
CORRELATION BETWEEN ARCOBACTER SPP. AND
ENTEROBACTERIACEAE.
Mottola A., Bonerba E., Bozzo G., Lippolis V., Marchetti P., Tantillo G., Di Pinto A.
Department of Veterinary Medicine, University of Bari Aldo Moro, Prov. le Casamassima,
km 3, 70010 Valenzano, Bari, Italy
[email protected]
Introduction: The number of foodborne illness outbreaks linked to vegetables has
increased in the last years, and in almost every case bacteria belonging to the family of
Enterobacteriaceae and Campylobacteraceae have been involved (3, 4).
Several factors, such as (i) the ability of some pathogens to adhere to plant surfaces, or
become internalized through cut edges, (ii) the complex surface and porosity of vegetables,
(iii) the use of contaminated irrigation water or organic fertilizers, (iv) contact with
surfaces, (v) the washing of vegetables in water, and (vi) human handling, may lead to the
contamination of food by pathogens from the intestinal tract of humans or animals during
production, harvest and processing steps (10).
Currently, the microbiological quality of pre-cut ready-to-eat (RTE) vegetables is based on
the evaluation of Salmonella spp., Listeria monocytogenes and E. coli only, as laid down
by Commission Regulation (EC) No. 1441/2007. However, other members belonging to
the Enterobacteriaceae family such as E. coli O157, Shigella, and Yersinia have also been
involved in the outbreaks (3, 4, 9). In addition, Campylobacteraceae members, such as
Campylobacter and Arcobacter spp., have been gaining in importance for public health. In
particular, Arcobacter spp., classified as emerging foodborne pathogens and serious
hazards to human health (7), encompass some pathogenic species (A. butzleri, A.
cryaerophilus and A. skirrowii) associated with human (e.g., diarrhoea; enterocolitis;
bacteraemia) and animal illnesses (e.g., gastrointestinal disorders, mastitis, reproductive
problems), and have been commonly isolated in human and animal stools (1,11,12).
The aim of the study: Given the importance of Enterobacteriaceae and Arcobacter spp.
for public health, the aim of the present study was to evaluate the relationship of the
presence of Arcobacter spp. with Enterobacteriaceae in RTE vegetables.
Methodology: Sampling- A total of 80 pre-cut RTE lettuces were obtained from
supermarkets in the Apulia region (SE Italy) between May and August 2016. The samples
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were taken to the laboratory in cooled containers (+4 °C) and processed within 24h of
purchase.
Cultural analysis
Salmonella spp. – The microbiological determination of Salmonella spp. was performed
using standard culture methods (UNI EN ISO 6579:2008).
Listeria monocytogenes – Analysis ofL. monocytogenes was performed following the ISO
11290-1:2004 reference method.
E. coli –The enumeration of E. coli was performed following ISO 16649-2:2001.
Enumeration of Enterobacteriaceae –The enumeration of Enterobacteriaceae was
performed following the ISO 21528-2:2004 reference method.
Isolation of Arcobacter spp. – The microbiological determination of Arcobacter spp. was
performed using the method described by Mottola et al., 2016 (10).
Biomolecular analysis
m-PCR – The presence and the identity of the Arcobacter isolates was determined using a
Multiplex-PCR assay as described by Houf et al. (2000) (6).
Statistical analysis
In order to analyze the correlation between the number of Enterobacteriaceae and the
presence of Arcobacter strains and between the number of E. coli and Arcobacter spp., a
Logit Regression Model was performed. The significance of results was indicated by the
Likelihood ratio. A P value of 0.05 was considered as statistically significant.
Results and Discussion: The results of this study showed improper management along the
RTE vegetable food chain. Although L. monocytogenes and Salmonella spp. were not
detected in any of the samples, the study reveals that E. coli was not compliant with Reg.
EC 1441/07, showing values higher than 1000cfu/g in 5/80 (25%) samples.
Enterobacteriaceae were found to vary from 1.2 x 102 to 3.8 x 10⁸ cfu/g, and the presence
of Arcobacter butzleri was observed in 26/80 (32.5%) of samples analyzed (Table 1).
Also, the statistical analysis, confirmed the association between Arcobacter spp. and
Enterobacteriaceae (P-value <0.05), and between Arcobacter spp. and E. coli (P-value
<0.05), in accordance with Collado et al. (2008). In addition, it revealed that samples
positive for Arcobacter spp. showed a significantly higher Enterobacteriaceae and E. coli
concentration than negative ones.
Considering that minimally-processed vegetables that have previously been washed,
selected, cut and packed in modified atmospheres, and commonly eaten without further
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processing, have been gaining in popularity among consumers (5,8), the presence of A.
butzleri in RTE vegetables is likely to be correlated with improper hygienic conditions
during production on the farm (e.g. manure, faeces, soil, microorganisms or postharvest
contamination sources) (8), and represents a potential risk for consumer’s health. Thus,
appropriate implementation of food safety management systems such as Good Agricultural
Practices (GAPs), Good Manufacturing Practices (GMPs) and Good Hygienic Practices
(GHPs) and Hazard Analysis and Critical Control Point (HACCP) are required.
Table 1. Microbiological and biomolecular analysis results.
Enterobacteriaceae E. coli
Salmonella
spp.-positive
samples
L. monocytogenes
-positive samples
Arcobacter
spp.-positive
samples*
N° of samples
with incidence
ranging
from <10 - 102
cfu/g
6
75
0
0
1
N° of samples
with incidence
ranging from
103 - 104 cfu/g
9
3
0
0
1
N° of samples
with incidence
ranging from
105 - 106 cfu/g
38
2
0
0
6
N° of samples
with incidence
ranging from
107 - 108 cfu/g
27
0
0
0
18
0
0
26
Total
*All samples were identified as A. butzleri
Conclusion: In conclusion, the results obtained confirmed that the evaluation of E. coli is
used
as
an
indicator
of
faecal
contamination.
However,
the
evaluation
of
Enterobacteriaceae contamination should be used as an index of the potential presence of
pathogens and that further studies are required in order to assess the association between
Arcobacter spp. and Enterobacteriaceae for a complete safety approach for RTE
vegetables.
Literature
1.
Collado, L., Figueras, M. J., 2011. Taxonomy, epidemiology, and clinical relevance of the genus
Arcobacter. Clin. Microbiol. Rev. 24, 174-192.
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2.
18th-20th October 2016
Collado, L., Inza, I., Guarro, J., Figueras, M.J., 2008. Presence of Arcobacter spp. in environmental
waters correlates with high levels of fecal pollution. Environ. Microbiol. 10, 1635-1640.
3.
EFSA (European Food Safety Authority), (2013). Scientific Opinion on the risk posed by pathogens in
food of non-animal origin. Part 1 (outbreak data analysis and risk ranking of food/pathogen
combinations). EFSA Journal 2013;11(1):3025.
4.
European Commission Scientific Committee on Food. (2002). Risk profile on the microbial
contamination
of
fruits
and
vegetables
eaten
raw.
Retrieved
1
September
2016,
from
www.europa.eu.int/comm/food/fs/sc/scf/out125_en.pdf.
5.
Falomir, M.P., Gozalbo D., Rico, H., 2010. Coliform bacteria in fresh vegetables: from cultivated lands
to consumers. Current Research, Technology and Education Topics in Applied Microbiology and
Microbial Biotechnology. A. Méndez-Vilas (Ed.) Formatex 2010.
6.
Houf, K., Tutenel, A., De Zutter, L., Van Hoof, J., Vandamme, P., 2000. Development of a multiplex
PCR assay for the simultaneous detection and identification of Arcobacter butzleri, Arcobacter
cryaerophilus and Arcobacter skirrowii. FEMS Microbiol. Lett. 193, 89-94.
7.
ICMSF, 2002. Microorganisms in food, 7. Microbiological testing in food safety management. Kluwer
Academic-Plenum Publishers, New York.
8.
Jeddi, M.Z., Yunesian, M., Gorji, M.E, Noori, N., Pourmand, M.R., Khaniki, G.R.J, 2014. Microbial
Evaluation of Fresh, Minimally-processed Vegetables and Bagged Sprouts from Chain Supermarkets. J.
Health Popul. Nutr. 32, 391-399.
9.
MacDonald, E., Heier, B.T., Nygård, K., Stalheim, T., Cudjoe, K.S., Skjerdal, T., Wester, A.L., Lindstedt,
B.A., Stavnes, T.L., Vold, L., 2012. Yersinia enterocolitica outbreak associated with ready-to-eat salad
mix, Norway, 2011. Emerg Infect Dis. 18, 1496-1499.
10. Mottola, A., Bonerba, E., Bozzo, G., Marchetti, P., Celano, G.V., Colao, V., Terio, V., Tantillo, G.,
Figueras, M.J., Di Pinto, A., 2016. Occurrence of emerging food-borne pathogenic Arcobacter spp.
isolated from pre-cut (ready-to-eat) vegetables. Int. J. Food. Microbiol. 236, 33-37.
11. Van Driessche, E., Houf, K., Van Hoof, K., De Zutter, L., Vandamme, P., 2003. Isolation of Arcobacter
species from animal faeces. FEMS Microbiol. Lett. 229, 243-248.
12. Webb, A.L., Boras, V.F., Kruczkiewicz, P., Selinger, L.B., Taboada, E.N., Inglis, G.D., 2016.
Comparative detection and quantification of Arcobacter butzleri in stools from diarrheic and nondiarrheic human beings in South western Alberta, Canada. J. Clin. Microbiol. 54,1082-1088.
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COULD TICK-BORNE ENCEPHALITIS LEAVE BEHIND PERMANENT
CONSEQUENCES?
J. Pertinačová
Chief Public Health Officer of the Ministry of Interior of the Slovak Republic
[email protected]
Introduction: Tick-born meningoencephalitis (TBME) is a severe acute viral disease of
central nervous system, manifested by neurological disorders and in some cases by death
of a patient. The disease is endemic in many countries of Europe. It is the most serious
disease transmitted by arthropods on the continent.
Goal of the study: The primary goal of the study was to inform on occurence ofTBME in
Slovakia as well as to point out to severity of the disease and to the risk of permanent
damage of the patient`s health.
Methods: We have analysed clinical and epidemiological data of 92 patients hospitalized
in 12 Hospital Infectious Departments in Slovakia in 2011. The diagnosis was based on
clinical symptoms and laboratory data on inflamation of CNS and positive IgM and IgG
specific antibodies in serum. None of the patients was vaccinated against TBME.
Results: From the total number of 92 TBME cases, 68 were male (73.9%) and 24 female
(26,1%). The average age in male group was 44.1 years, almost 6 years higher than in
female group (38.4). The biphasis course of the disease was observed in 66 cases (71.7%)
more often in
patients with meningitis (34 cases, 51.5%) than in patients with
meningoencephalitis (28.8%). The clinical picture of meningitis was observed more often
(50.0%) than the clinical picture of the meningoencephalitis (30 patients, 32.6%). The
paretic complications developed in 12 patients (13.0%). One of the patients had died.
Postenecephalitic syndrom was diagnosed in 25 patients (27.2%), the age of four patients
was below 35 years (16, 18, 21, and 26), the age of the rest of the patients was in a range
40 – 76 years (average 47 years).
The most often complaints of the patients were as
follow: headache (52.0%), tremor HK (32%), fatigue (24.0%) and lack of concentration
(20.0%). The tick biting was reported by 68 patients (73.9%).
Discussion: Even the noncomplicated course of the TBME deserves reconvalescence
period for several weeks. In the case of complications and subsequent consequences, the
disease outcome is having a serious impact on the patient`s life quality that may persist for
a long time or even for all the life. The disease occurs two times more in male, than in
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female (1). The typical biphasic course of the disease is diagnosed by 65 – 70% more
often in meningeal form (1, 2). In our study we have observed 2.8 times higher occurence
of this form in male than in female. The clasical biphasic course was observed in 71.7% of
patients and have reach a higher frequency in patients with meningitis (51.5%). Meningeal
form is typical mainly for younger age groups and by the reports from other studies the
occurence is reported between 47 to 55% (3, 4). In our study we have observed the disease
in a form of meningitis by 50%, and particularly in the younger age group (average age
37.7 years). The complications in patiens with meningitis were observed only in one case
(paresis n. facialis in 28 years old female). The head nerves paresis described in a low
number of patients with meningitis was reported in some studies (1, 5). In our study group
of 30 patients with meningoencephalitis, the paresis symptoms occured in 5 patients
(20.0%), out of them 3 cases of paresis n. facialis, one case of n. oculomotorius and one
case of a light hemiparesis. Our study is presenting a higher number of such cases in
a comparison with other studies abroad (5-12% of patients) (1, 5). Quadriparesis was
diagnosed in all six patients with meningoencephalitis similarly as it was reported only in
older
patients
(43-74
years,
an
average
59.2
years)
in
other
studies.
Themeningoencephalitis accompanied by a quadriparesis occurs in children and adolescent
very rarely and only a few case are described in literature (1, 6). We have not identified a
case in our study.
After acute phase of the disease, the 35 – 58% of patients suffer for a long period from
postencephalitic syndrom characterised by neurological and mental signs (7, 2). Studies
published by other authors confirmed the presence of postencephalitic syndrom in 25 –
39% patients. The most frequent disorders described in these studies were as follow:
concentration and memory problems, headache, tremor, vertigo and ataxie (4, 8). In our
study we have diagnosed postencephalitic syndrom in 27.2% patients with most often
reported headache, tremor HK, fatique and lack of concentration. The letality rates
described in literature are in the range 1 – 3% . One patient in our study group had died
(1.1%).
Conclusion: Slovakia is a country with endemic occurence of the TBME. The disease
occurs in all age categories. Our results show the higher frequency of the disease in adult
persons and seniors also with a more serious forms of the disease. One third of the patients
after the active phase of the disease showed a various neural and mental disorders. These
for a long time have impaired the quality of the life with negative impact on the cost99
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benefit outcome of the health care. There is no specific therapy for the disease, however
the active vaccination is the most powerfull tool for prevention.
References
1.
Kaiser R.: The clinical and epidemiological profile of tick-borne encephalitis in southernGermany 1994–
1999. A prospective study of 656 patients. Brain, 1999, 122, s. 2067-2078
2.
Kaiser R.: Tick-borne encephalitis: Clinical findings and prognosis in adults. Wiener medizinische
Wochenschr. 2012 Jun, 162(11-12), s.239-243
3.
Kaiser R.:Tick-borne encephalitis (TBE) in Germany and clinical course of the disease.Int J Med
Microbiol. 2002, Jun, 291 Suppl 33, s.58-61.
4.
Günther G., Haglund M., Lindquist L. et al.: Toick-borne encephalitis in Swedwn in relation to aseptic
meningo-encephalitis of other etiology: a prospective study of clinicaland outcome. J Neurol 1997, april,
244(4), s.230-238
5.
Haglund M., Forsgren M., Lindh G., Lindquist L.: A 10-year follow-up study of tick-borneencephalitis
in the Stockholm area and a review of the literature: need for a vaccinationstrategy. Scand J Infect Dis.
1996, 28(3), s.217-224
6.
Štruncová V., Sedláček D.: Klíšťová encefalitida u dětí. Pediatrie pro praxi, 2009, 10(2),s.70-71
7.
Roháčová H.: Klíšťová meningoencefalitida, aktualizace 2012. Practicus, roč.11, č. 4/2012,s.26-28,
ISSN 1213-8711.
8.
Chmelík V.: Klíšťová meningoencefalitida. Med. Pro Praxi 2008, 5(3), s.105-108
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RISK FACTORS OF SPORADIC CAMPYLOBACTERIOSIS IN THE
BRATISLAVA REGION, CASE-CONTROL STUDY
J. Pertinačová
Chief Public Health Officer of the Ministry of Interior of the Slovak Republic
Chief Hygienist, Ministry of Interior, Slovak Republic
[email protected]
Introduction: The thermotolerant Campylobacter sp. particularly Campylobacter jejuni is
the most often
cause of acute bacterial enteritis both in developing and developed
industrial countries. The occurence of campylobasteriosis reveals an increasing trend
worldwide. In the countries with a well established surveillance system the number of
campylobacterioses is higher than the occurence of salmonella infections.
The goal of the study: The goal of the study was to identify risk and protective factors in
sporadic cases of Campylobacter infections in Bratislava district. The method of casecontrol study was applied for this purpose.
Methods: We have performed a case-control study in 8 counties in the Bratislava region in
2013-2014. We have included into the study 119 cases confirmed by the cultivation
method and associated 238 controls (ratio 1:2). Selected controls were adapted to each
case regarding age, gender and residence. Expected exposures were grouped in 5
categories (food consumption, kitchen hygiene and contact with an animal, leisure
activities, history and medicines).
Results: We identified several independent risk factors in each category using linear
logistic regressive analysis for occurrence of the Campylobacter infection. A statistically
significant association with the disease was found in food exposure after the consumption
of chicken meat, kebab, chicken livers, insufficiently thermally cooked chicken,
unpasteurizedfresh milk or milk bought in machines, fresh strawberries and cucumber.
Other exposures that represented increased risk included the purchase of cooled or
homemade chicken, insufficient hygiene when manipulating with raw chicken meat in the
household, farm visits, swimming in natural water and antacid usage. Independent factors
connected with risk decrease (protective factors) include the consumption of ham, fish,
yoghurt, unpasteurized milk, unpeeled apple and pear, contact with an animal outside of
the household and the play of a child in a park or garden.
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Discussion: The consumption of kebab, chicken livers, chicken meat and poorly cooked
chicken meat (red-brown along the bones) was identified to be the statistically significant
independent risk factor in our study. Several case-control studies have described
the
chicken meat as the high risk factor for the disease (1,2,3,4). The results of these studies
point out to important role of poultry meat in an epidemiology of human campylobacter
infection. Also the consumption of fresh strawberies and cucumbers was identified as an
important independent risk factor for contraction of the disease. These results are
consonant with several case-control studies confirming the consumption of raw vegetable
or soft fruits as a high risk factor (5, 6). The C. jejuni survives on the contaminated fresh
vegetable and fruits for a long period and can be the risk factor for consumers (7). The
consumption of the raw milk and dairy products from unpasteurized milk has been well
known risk factor for the contraction of the disease (8). In the presented study the
consumption of the home made cheese and domestic raw milk was also identified as an
important independent risk factor. Handling of the raw chicken meat in a household
represents in our study an important risk factor. The association of the raw meat handling
in a household was described as an important risk factor for contraction of the disease in
other studies as well (9, 10).
Campylobacter survive in a raw meat, milk, water, and
during improper handling in kitchen can secondary contaminate food that already was
thermally processed (1, 8, 11). The cross contamination during the processing of the
chicken meat is the dominant factor of disease transmission. (1, 8). Our findings
confirming that the contact with poultry outside of the household and visit of the farm or
ZOO Garden are the independent risk factors for a contraction of the disease. These results
are in agreement with results of the other case-control studies (9, 12). Our results indicate
that contact with poultry outside of the household represents a higher risk than a visit of the
farm.
The study of the risk factors for human infections has been carried on in many
epidemiological projects, however due to the wide spreading of campylobacters in animals
the epidemiology of campylobacterioses remains with many open questions. The well
known risk factors help to explain not more than half of the cases (1, 5).
Conclusion: Our results refer to insufficient hygiene management in all stages of the food
chain. Educating consumers about the found risk factors presents an important action to
decrease the incidence of Campylobacter infections.
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References
1.
Friedman C.R., Hoekstra R.M., Samuel M. et al. (2004). Risk factors for sporadic
Campylobacter
infection in the United States: A case-control study in FoodNet sites. Clin Infect Dis.38 Suppl 3, s.285296.
2.
Stafford R.J., Schluter P.J., Kirk M.D., Wilson A., Unicomb L., Ashbolt R., Gregory J. (2007). A multicentre prospective case-control study of campylobacter infection in persons aged 5 years and older in
Australia. Epidemiology and Infection, August 2007, 135 (06), s. 978-988
3.
Danis K., Di Renzi M., O’Neill W., Smyth B., McKeown P., Foley B., Tohani V., Devine M. (2009).
Risk Factors for sporadic Campylobacter infection: An all-Ireland case control study. Eurosurveillance,
Vol 14, (7) s. 1-8
4.
Mughini G. L., Smid J.H. et al. (2012). Risk factors for campylobacteriosis of chicken, ruminant, and
environmental origin: a combined case-control and source attribution analysis. PLoS One. 2012, 7(8),
s.75-9
5.
Evans M.E., Ribeiro C.D., Roland L., Salmon R.L. (2003). Hazards of Healthy Living: Bottled Water
and Salad Vegetables as Risk Factors for Campylobacter InfectionEmerg Infect Dis. 9(10), s. 1219–
1225.
6.
Fullerton E., Ingram L., Jones T.F., Anderson B.J., McCarthy P. et al. (2007).Sporadic Campylobacter
Infection in Infants: A Population-Based Surveillance Case-Control Study. Periatric Infectious Disease
Journal, 2007, 26(1), s. 19-24
7.
Kärenlampi R., Hänninen M.L. (2004). Survival of Campylobacter jejuni on various fresh produce. Int J
Food Microbiol. 2004 Dec 15;97(2), s.187-195.
8.
Wilson D.J., Gabriel E., Leatherbarrow A.H., Cheesbrough J., Gee S., Bolton E., Fox A., Fearnhead P.,
Hart A.C., Diggle P.J. (2008).Tracing the Source of Campylobacteriosis. PLoS Genet. 2008, 4(9), s. 1023. ISSN 1553-7404
9.
Kapperud G., Espeland G., Wahl E., Walde A., Herikstad H., Gustavsen S., Tveit I., Natas O., Bevanger
L.,
Diranges
A.
(2003).Factors
Associated
with
Increased
and
Decreased
Risk
of
CampylobacterInfection: A Prospective Case-Control Study in Norway. Am. J. Epidemiol. 158 (3), s.
234-242.
10. Fajo –Pascual M., Godoy P., Ferrero-Cancer M., Wymore K. (2010).Case–control study of risk factors
for sporadic Campylobacter infections in northeastern Spain. Eur J Public Health (2010) 20 (4), s. 443448.
11. Nelson A., Cox, L. Richardson J., Musgrove T.M.( 2010).Campylobacter jejuni and Other
Campylobacter’s. Pathogens and Toxins in Foods Challenges and Interventions Edited by V. K. Juneja
and J. N. Sofos, 20 10 ASM Press, Washington, DC ,Chapter 2
s.20-30, [online]
http://ddr.nal.usda.gov/bitstream/10113/40399/1/IND44350891.pdf [cit. 2. 2. 2015].
12. Neimann J., Engberg J., Mølbak K., Wegener H.C. (2003).A case-control study of risk factors for
sporadic campylobacter infections in Denmark. Epidemiol Infect. Jun 2003, 130(3), s.353-66.
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RISK COMMUNICATION WHEN CONTAMINATION TAKES PLACE: FOOD
BUSINESS OPERATOR VIEW
Peter Raspor,
Guest professor of Food Safety at The University of Natural Resources and Life Sciences,
Vienna, 2006Guest professor of Modern Bio-Technology in Food Production at University Vienna,
2008Guest professor of Food Quality and Safety at Faculty of Biosistemic Sci. at University of
Maribor, 2009Guest professor of Food Safety at Faculty of Health Sciences at University of Ljubljana,
2011Retired professor of Microbiology and Food safety from University of Primorska, 20142016
Retired professor of Industrial Microbiology and Biotechnology from University of
Ljubljana, 1986-2013
Retired professor of Food Biotechnology from Budapest Corvinus University, 1994-2006
Neubergerjeva 13, Ljubljana, Slovenia. [email protected]
Introduction: A foodborne illness event creates a unique communication environment, as
foodborne illness is both a risk to individuals not infected and a crisis to those experiencing
symptoms what shapes perceptions of risk. Consumer perceptions of risk are essential.
Food and health professionals, government officials, industry representatives, and others
who communicate with consumers about risks may struggle to understand why people
make the decisions they do(1). The communicator can become frustrated when the
consumer does not read or listen carefully to and understand potential risks, or does not
seek out additional information in order to make informed decisions. In daily life
consumers do not see and think about risk in the same way that experts think about it (2).
Consumer filter risk information through a variety of ways mostly based on their
experience that affect what they hear, how they process and come to understand the
information, what they conclude, how they react upon being informed and finally how they
actually act. For the average consumer, risk is highly subjective and also the wording is not
always well understand (3, 4). Risk communication rises to the challenge of bridging this
divide between expert analysis of the risk equation on one side and public reaction and
action on the other.
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The aim of the study: This paper has the aim to present state of the art in the area of
unique communication environment when foodborne illness is starts due to contamination
with food and request very specific behaviour and reaction of managers of food supply
chains towards consumers.
Methodology: The methods of research are based on various arching machines on the
selection of key words and its combinations. We applied searching platfiormes like science
direct: (http://www.sciencedirect.com/) pub med http://www.ncbi.nlm.nih.gov/pubmed
and http://scholar.google.com/ in preselected sets of key terms relevat for this study. Then
the articales were analised for the purpose fof this paper.
Results and Discussion: Hazard – something that can go wrong; Probability – likelihood
of it happening; Consequences – implications of hazard; Value – subjective evaluation of
the relative importance of what might be lost (5). We have to deal with two faces of the
same issue. Risk communication historically has been develop in 4 phases. Phase 1 (19751985): emphasis on comparative risk estimates, “arrogance of technical expertise”. Phase 2
(1985-1995): emphasis on successful communications, practices from modern marketing,
limited success due to lack of trust.Phase 3 (1995-2005): emphasis on social context, trust
through commitment. Phase 4 (2005-now): emphasis on comprehensive approach
involving new communication means on global scale . Crisis communication is marked by
three distinct phases: pre-crisis, acute crisis, and post-crisis. The pre-crisis stage is marked
with messages intended to mitigate harm and encourage preparation for the crisis.
Communication in the acute phase of a crisis involves disseminating instructing and
adjusting information to help individuals cope with the crisis event. Finally, post-crisis
communication provides an opportunity for communication after activities have returned to
normal, specifically providing an opportunity to explain organizational learning and
renewal (e.g., what led to the contamination, what is being done to ensure that another
contamination will not occur, and information about their turned safety of the
contaminated product). Risk Perception Model and Good Practices for Risk
Communication seeks for solutions which should not be restricted to negative messages
and warnings but should include positive ‘educational messages’(6,7).
Results showed that consumers demonstrated judgements of `optimistic-bias' and the
`illusion of control', as well as notions of perceived invulnerability to food poisoning from
self-prepared foods. Statistical associations between perceptions of personal risk, control
and responsibility and risk and control attributed to `other people' have been identified.
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Findings may have negative implications for the effectiveness of consumer food safety
education initiatives. It is suggested that consideration of such judgements and associations
during the development of future consumer food safety risk communication strategies may
increase their effectiveness also at home(8, 9, 10).
Conclusion: Consequently is necessary to respect all good practices in food supply
chains/ nets (11) and at least major Rules of Risk Communication i.e.: Accept and involve
public as a legitimate partner, Listen to the public’s specific concerns, coordinate and
collaborate with other credible sources
Acknowledgment: Support of European Union’s Seventh Framework Programme under
grant agreement no 613912 with project TRAFoon is kindly acknowlged to induce this
thinking.
Literature
1.
M. Jevšnik, V. Hlebec, P. Raspor, Consumers’ awareness of food safety from shopping to eating, Food
Control, Volume 19, Issue 8, August 2008, Pages 737–745
2.
Witte, K. (1992). Putting the fear back in to fear appeals: The extended parallel process model.
Communication Monographs, 59, 329-349.
3.
Ambrožič, M., Jevšnik, M., Raspor, P. Inconsistent terminology in food safety field : a permanent risk
factor?. Journal of food and nutrition research, ISSN 1336-8672, 2010, vol. 49, no. 4, str. 186-194
4.
3 A. Wilcock, M. Pun, J. Khanona, M. Aung, Consumer attitudes, knowledge and behaviour: a review of
food safety issues, Trends in Food Science & Technology, Volume 15, Issue 2, February 2004, Pages
56–66
5.
Jevšnik, M., Hlebec, V., Raspor, P.. Food safety knowledge and practices among food handlers in
Slovenia. Food control, ISSN 0956-7135. [Print ed.], 2008, vol. 19, no. 12, str. 1107-1118
6.
Nedović, V. (editor), Raspor, P. (editor), Lević, J. (editor), Tumbas Šaponjac, V. (editor), BARBOSACÁNOVAS, Gustavo V. (editor). Emerging and traditional technologies for safe, healthy and quality
food, (Food engineering series, 1571-0297). Cham [etc.]: Elsevier, 2016. XVIII, 478 str., ilustr. ISBN
978-3-319-24038-1. ISBN 978-3-319-24040-4.
7.
Jevšnik, M., Hlebec, V., Raspor, P.. Consumer interpretation of the term food safety. Acta alimentaria,
ISSN 0139-3006, 2008, vol. 37, no. 4, str. 437-448.
8.
Raspor, P.. Total food chain safety : how good practices can contribute?. Trends in food science &
technology, ISSN 0924-2244. [Print ed.], 2008, vol. 19, str. 405-412.
9.
Redmond, E. C., C. J. Griffith, Consumer perceptions of food safety risk, control and responsibility,
Appetite, Volume 43, Issue 3, December 2004, Pages 309–313
10. Jevšnik, M., Ovca, A., Likar, K., Raspor, P. Food safety at home : the last step of food supply chain. V:
WALSCH, Marion B. (ur.). Food supplies and food safety : production, conservation and population
impact, (Food and beverage consumption and health). New York: Nova Science, cop. 2011, str. 73-95,
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11. Raspor, P., Ambrožič, M., Jevšnik, M-. Food chain safety management systems : the impact of good
practices. V: YANNIOTIS, Stavros (ur.). Advances in food process engineering research and
applications, (Food engineering series, 1571-0297). New York [etc.]: Springer, 2013, str. 607-625.
12. RASPOR, P. Food chain safety management systems : the impact of good practices. V: Obuchitelen
seminar "Savremenni analitichni podhodi za osiguryavane na kachestvoto i bezopasnostta na hrani,
napitki i furazhi", Plovdiv, UHT, 21-24.06.216. Plovdiv: UHT - [Univerzitet po hranitelni tehnologii],
2016, str. [18-41].
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CAMPYLOBACTER DETECTION DEPENDING ON THE MATRIX, METHOD
AND MEDIUM
Repérant E., Laisney M. J., Nagard B., Quesne S., Rouxel S., Le Gall F., Chemaly M.,
Denis M.
Anses, French Agency for Food, Environmental and Occupational Health Safety,
French NRL for Campylobacter, BP 53, 22440 Ploufragan, France
[email protected]
Introduction: Campylobacteriosis represents the most frequent foodborne zoonosis in the
European Union (EU) since 2005 (EFSA 2015). The predominant Campylobacter species
causing this illness are known to be Campylobacter jejuni (C. jejuni)and Campylobacter
coli (C. coli), (Park SF, 2002) and most Campylobacter strains infecting humans originate
from
the
poultry
reservoir
(EFSA
2011).
Therefore
quantitative
data
from
naturallycontaminated chicken samples are of direct interest to public health. Moreover,
Campylobacter culture being fastidious, its optimal detection and isolation requires
adequate methods and media.
Thus, we investigated the performance of three enrichment durations in Bolton broth (0, 24
and 48 hours) and compared four isolation media (mCCDA, Butzler no.2, Karmali and
Campyfood agar (CFA)) for the detection of Campylobacterspp.in naturally contaminated
chicken samples.
Methodology: Ninety chicken samples from retail and slaughterhouse were analysed
according to the ISO 10272 method (2006), part 1 for detection and part 2 for numeration
(30 thigh skins, 30 carcass neck skins and 30 caeca, pools of ten caeca each). Numeration
(direct isolation) and streaking of the broths after 24 and 48 hours enrichment (E24H and
E48H) were performed on four isolation media (mCCDA, Butzler no.2, Karmali and
CFA).
Additional 1213 chicken samples from two surveys (the first with 426 neck skins and 426
caeca,pools of ten caeca each and the second with 121 thigh skins, 120 carcass neck skins
and 120 chicken breasts) were also analysed for Campylobacter detection and
identification after direct isolation on mCCDA and E48H on mCCDA and Butzler no.2.
Identification of Campylobacterspecies was done by multiplex PCR using primers and
PCR conditions as described by Denis et al.(1999). Statistical analyses were carried out
using R software (R Core Team, 2013). The distribution of numeration between samples
and media was compared using Krukall-Wallis test whereas the distribution of
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Campylobacterpositive/negative samples and C. jejuni /C. coli were compared using
Pearson’s test.
Results and discussion: The overall analysis of our results from the four selective agar
media and after E24H and E48H revealed that 97.8% of our samples were contaminated
with Campylobacter spp.. As already shown by Scherer et al. (2006), Sampers et al. (2008)
and Guyard-Nicodème et al. (2015), the level of contamination after numeration was
significantly higher in caeca (median = 8.28 log10 cfu/g) and decreased from neck skins
(median = 3.59 log10 cfu/g) to thigh skins (median = 1.65 log10 cfu/g) with a p value <
2.2.e-16.
The numeration remained identical for each matrix for the four selective media tested.
However, the detection of Campylobacter spp. varied following the method (direct
isolation, E24H, E48H) and the media used (mCCDA, Butzler no.2, Karmali and CFA).
When considering all media and samples, the highest number of positive samples was
found before enrichment (p value = 1.21.e-15) with 89.4% after direct isolation and 77.2%
and 80% after E24H and E48H respectively. This decrease after enrichment could be
explained by the growth of other bacteria in Bolton broth and thus prevent Campylobacter
growth particularly if Campylobacter was present in small amount initially.
When considering all methods and samples, Butzler no.2 was the most efficient medium
for detecting Campylobacter (p value = 6.82.e-9). Butzler no.2 allowed the detection of
90% of positive samples followed by CFA (76.6%), mCCDA (71.5%) and Karmali
(68.5%).
Moreover, Campylobacter detection varied between the type of matrix (caeca, neck skin,
thigh skin). For caeca and neck skins, positive samples were higher after direct isolation
(112 and 120 respectively) and decreased after enrichment (102 and 82 at E24H, and 64
and 74 at E48H, respectively). For thigh skins, there was no difference between methods
(92 positive samples after direct isolation vs.94 at E24H and 90 at E48H).
Out of the 3679 Campylobacter isolates collected using various matrices, methods and
media, 44.1% were identified as C. jejuni and 55.9% as C. coli. The distribution of these
two Campylobacter species varied with the method and the media used. C. jejuni was
mostly represented after direct isolation reaching 65.8% of the positive samples whereas it
dropped to 39.1% after E24H and to 16.6% after E48H (p value < 0.05). Enrichment in
Bolton broth clearly disadvantaged C. jejuni. The reverse effect was observed for C. coli
which seemed to be promoted by the enrichment period. The same results were also
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obtained with samples from the EU and national surveys. Finally, when considering all
methods and matrices, Butzler no.2 appeared to be the most efficient medium for detecting
C. jejuni (49% vs 50.9% C. coli) compared to the 3 other media (p value < 0.05).
Conclusion: These results highlight the influence of both the detection method and the
isolation media not only on Campylobacter detection but also on Campylobacter species
identification. Our results confirm the need to adapt the method used to the samples to be
analysed as it will be described in the future version of the ISO 10272 method.
Literature
1.
EFSA 2015. The European Union Summary Report on Trends and Sources of Zoonoses, Zoonotic
Agents and Food-borne Outbreaks in 2013. EFSA Journal 2015;13 (1): 3991, 162 pp.
2.
Park S.F., 2002. The physiology of Campylobacter species and its relevance to their role as foodborne
pathogens. Int. J. Food Microbiol. 74, 177-188.
3.
EFSA 2011. Scientific Opinion on Campylobacter in broiler meat production: control options and
performance objectives and/or targets at different stages of the food chain. EFSA Journal 2011;
9(4):2105.
4.
ISO 10272 (2006). Microbiology of food and animal feeding stuffs-horizontal method for detection and
enumeration of Campylobacter spp. Part 1: detection method. Part 2:Colony-count technique.
5.
Denis, M., Soumet, C., Rivoal, K., Ermel, G., Blivet, D., Salvat, G., Colin, P. 1999. Development of a
m-PCR assay for simultaneous identification of Campylobacter jejuni and C. coli. Lett. Appl. Microbiol.
29, 406-410.
6.
R Core Team (2013). R: A language and environment for statistical computing. R Foundation for
Statistical Computing, Vienna, Austria. URL http://www.R-project.org/.
7.
Scherer, K., Bartelt, E., Sommerfeld, C., Hildebrandt, G. 2006. Comparison of different sampling
techniques and enumeration methods for the isolation and quantification of Campylobacter spp. in raw
retail chicken legs. Int. J. Food Microbiol. 108, 115-119.
8.
Sampers, I., Habib, I., Berkvens, D., Dumoulin, A., Zutter, L.D., Uyttendaele, M., 2008. Processing
practices contributing to Campylobacter contamination in Belgian chicken meat preparations. Int. J.
Food Microbiol.128, 297-303.
9.
Guyard-Nicodème,M., Rivoal, K., Houard, E., Rose, V., Quesne, S., Mourand, G., Rouxel, S., Kempf, I.,
Guillier,L., Gauchard, F., Chemaly, M., 2015. Prevalence and characterization of Campylobacter jejuni
from chicken meat sold in French retail outlets. Int. J. Food Microbiol. 203, 8–14
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THE ANIMAL HEALTH LAW -REGULATION (EU) 2016/429 – AND THE
FUTURE OF FOOD SAFETY AND FREE COMMERCE IN EUROPE
Ruffo G., Fossati P., Locatelli V.
Department of Health, Animal Science and Food Safety, Università degli Studi di Milano,
via Celoria 12, 20133, Milano, Italia
[email protected]
Introduction:The Authors analyze the Regulation (Eu) 2016/429 on transmissible animal
diseases and amending and repealing certain acts in the area of animal health (‘Animal
Health Law’) and compare it with the European Food Legislation and Safety Food.The
study regards the subject matter and aim, scope, definition, listing of transmissible
diseases, and disease notification and measures to surveillance and to eradicate.
The aim of the study:The aim of this study is to analyze contents of Reg. (EU) No.
429/2016, to ensure an efficient application of the disease prevention rules and control
rules laid down in this Regulation into Member State, the impact on public health and food
safety Legislation (Regulations EC No. 178/2002, No. 882/2004, No. 853/2004) and the
impact on effective functioning of the internal market.
The study regards the TFEU procedure to delegate to the Commission the power to adopt
non-legislative acts of general application that supplement or amend certain non-essential
elements of a legislative act.The Authors analyze the official controls by competent
authorities to ensure the human health, the animal health and to protect the environment.
Methodology: The Authors analyze the following legislation:
-Regulation (Eu) 2016/429, ‘Animal Health Law’,
-Treaty of Lisbon -TFEU- (2009),
-Regulation (EC) No 178/2002 laying down the general principles and requirements of
food law,
-Regulation (EC) No 882/2004 official controls on feed and food law, animal health and
animal welfare rules,
-Regulation (EC) No 853/2004 laying down specific hygiene rules for food of animal
origin.
The Regulation (EU) No. 429/2016 considerates the links between animal health and
public health, the environment, food and feed safety, animal welfare, food security,
together with economic, social and cultural aspects. The animal health measures provided
in this Regulation are taken on the basis of the risk assessment and of the available
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scientific evidence; it’s very important the work of the European Food Safety Authority
(EFSA).With the goal to ensure the correct and harmonised application of the Regulation
(EU) No. 429/2016 and to ensure the same animal health status in all State Members, the
European legislator has established a list of transmissible animal diseases which pose a risk
to animal or public health in the Union (Article 5 and Annex II) and has delegated to the
Commission the power to adopt acts amending the Annex II (Articles 6, 7).
The Listing of Article 5 and Annex II contain the following diseases:
Foot and mouth
disease;
Rinderpest
(cattle plague)
Sheep pox or goat
pox
Classical swine fever
African swine
fever
Swine vesicular
disease
Lumpy skin
disease
Highly pathogenic
avian influenza
Bluetongue
African horse
sickness
Teschen disease
Vesicular
stomatitis
Contagious bovine
pleuropneumonia
Newcastle
disease
Bovine
tuberculosis
Venezuelan equine
viral
encephalomyelitis
Bovine brucellosis
(B. abortus)
Haemorrhagic
disease of deer
Ovine and caprine
brucellosis
(B. melitensis)
Anthrax
Rabies
Echinococcosis
Campylobacterios
is
Listeriosis
Trichinellosis
Viral
haemorrhagic
septicæmia (VHS)
Infectious
haematopoietic
necrosis (IHN)
Infection with
Perkinsus marinus
Infection with
Microcytos mackini
Salmonellosis
(zoonotic
salmonella)
Epizootic
haematopoietic
necrosis in fish
(EHN)
Taura syndrome
in crustaceans
Infectious salmon
anaemia (ISA)
Infection with
Marteilia refringens
Infection with
Bonamia ostreae
Sheep and goat
plague
Rift Valley fever
Epizootic
ulcerative
syndrome in fish
(EUS)
Yellowhead
disease in
crustaceans
White spot
disease in
crustaceans
Transmissible
spongiform
encephalopathies
(TSE)
Verotoxigenic E. coli
Infection with
Bonamia exitiosa
Koi herpes virus
disease (KHV)
A disease is considered to be ‘emerging disease’when it is not yet included in listing
disease for Article 5.
The Article 18 regulate disease notification. Disease notification is compulsory for
following cases. When there are any reasons to suspect the presence in animals of a listed
disease (Article 5), or where the presence of such a disease is detected in animals, the
operators immediately notify the competent authority. Those diseases do not normally
occur in the Union and for their immediate eradication, specific measuresmust be taken.
[point (a) of Article 9(1)]
When there are any reasons to suspect the presence in animals of a listed disease for which
there is a need for surveillance within the Union, or where the presence of such a disease is
detected in animals, the operatorsnotify disease the competent authorityas soon as
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practicable [point (e) of Article 9(1)].
Notification within Member States
When there are the presence of abnormal mortalities and other signs of serious disease or
significant decreased production rates with an undetermined cause, the operators notify
them a veterinarian; he shall carry out further investigation, including sampling for
laboratory examination. The State Member may decide that the notifications provided for
in late point may be directed to the competent authority.
The Commission shall adopt rulesto prevent and to control disease, with regard torisk
assessment and seriousness of disease.
TheReg. (EU) n. 429/2016 lays down the Commission shall adopt delegated acts or
implementing acts (Articles 291 and 290 of the TFEU), respecting the requirements
provided for same Regulation.
The Article 270 of Reg. (EU) n. 429/2016 repeals a lot of Decisions, Directives and few
Regulation (No. 1760/2000), with the goal to ensure the correct application of legislation
on veterinary and zootechnical matters.
Results and Discussion: The EU has experienced in 1994 the health emergency with BSE.
This experience has led the European legislature to use the instrument of the Regulation in
order to harmonize within the EU legal behavior in order to ensure a different level of
human health and ensure the functioning of the internal market through trade in live
animals and foods from animals. The Reg. (EC) No. 178/2002, was born in a time of
emergency, and rappresented the foundation of Food Law and declared the principles of
food safety in all countries of the European Union and also in third countries (import /
export). With Reg. (EC) No. 178/200, the Union governed directly Food Safety, and it
gave power to Member State. However the legislation on food safety was missing an
essential and primary aspect: the harmonization of animal health, the uniformity of
measures in cases of infectious diseases that had repercussions on human health.
Animal Health was regulated so messy, confusing and not harmonized, divided between
directives and decisions, and animal health rules of the individual member states, which,
depending on the internal health problems, decided independently what were the infectious
diseases which are notifiable and what were the restriction health measures of animal
movements.
Conclusion: As the Reg (EC). No. 178/2002 on Food Safety, the new Reg.(EU) No.
429/2016 represents a starting basis for future legislation or acts on the protection of
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animal health
The list of notificable diseases for Reg. (EU) No. 429/2016 modifies indirectly but
immediately all lists of infectious diseases contained in the directives and decisions of the
EEC and the single laws of the Member States, to proceedthe harmonization of the health
status of the animals in European Union, toensure the internal market through trade in live
animals, withuout movement restrictions imposed by the national veterinary laws of single
Member States.
Reg. (EU) No. 429/2016, which shall apply from 21 April 2021, is very important in
veterinary legislation, becauseit will be integrated with the regulations on food safety with
regard to the management of biosecurity in animal breeding and derived products.
According this regulation also animal health will no longer be governed by individual
Member States but be directly the European Union will directly order measures to fight
against trasmissible diseases of animals.
Literature
1.
Regulation (EC) No 178/2002 of the European Parliament and of the Council of 28 January 2002 laying
down the general principles and requirements of food law, establishing the European Food Safety
Authority and laying down procedures in matters of food safety
2.
Treaty of Lisbon on the Functioning of the European Union (TFEU) (2009)
3.
Regulation (EU) No 182/2011 of the European Parliament and of the Council of 16 February 2011
laying down the rules and general principles concerning mechanisms for control by Member States of
the Commission's exercise of implementing powers
4. Regulation (Eu) 2016/429 of the European Parliament and of the Council of 9 March 2016 on
transmissible animal diseases and amending and repealing certain acts in the area of animal health
(‘Animal Health Law’)
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EARLY LOCALIZED AND DISSEMINATED LYME BORRELIOSIS IN
WESTERN SLOVAKIA
K. Schwarzova, I. Waczulikova
Institute of Microbiology, Faculty of Medicine and University Hospital, Sasinkova 4, 811
08 Bratislava
[email protected]
Department of Nuclear Physics and Biophysics, Faculty of Mathematics, Physics, and
Informatics, Mlynska Dolina F1, 842 48 Bratislava, Slovakia
[email protected]
Introduction: Despite improvements in prevention, diagnosis and treatment, Lyme
borreliosis (LB) is still the most common arthropod-borne disease in northern hemisphere,
with risk of infection associated with occupation (forestry work) and outdoor recreational
activities. In Europe, LB is caused by infection with one or more pathogenic European
genospecies of Borrelia burgdorferi sensu lato, mainly transmitted by the tick Ixodes
ricinus. Clinically, the progression of LB is divided into early localized, early disseminated
and late stages.
The aim of the study: The primary aim was the direct detection of Borrelia pathogenic
species in biopsy material of patients with dermatological problems and suspected LB. The
risk of obtaining false-negative/positive results in serological assays was investigated and
the results were analysed by StatsDirect software.
Methodology:The study population consisted of 239 patients who presented to the
dermatologist of the University Hospital, Bratislava
between 2013-2015 with early
localized or early disseminated disease; dermatitis, urticaria, erythema nodosum, localized
sclerodermia – sclerodermia circumscripta, fever and other diseases (e.g. nonspecific
dermatitis). ELISA and Western blot (Anti-Borrelia IgG, IgM ELISA and WB,
Euroimmun) were compared on these patient samples mainly focusing on the IgG and IgM
antibodies presence. Immunodominant proteins were evaluated by EUROLineScan
software. Skin samples from patients with non-specific dermatitis directly from the lesions
and blood were collected for potential spirochete cultivation in BSK-H medium and
analyzed by PCR and multilocus sequence typing (MLST).
Results and Discussion: Results revealed overall conformity of 50,21% between ELISA
and WB for the anti borrelia IgM antibody presence; 42,26% of tested samples suggested
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false positive and 7,53% false negative results. For the anti borrelia IgG antibodies
presence the overall conformity between these two tests was 52,27%; with 15,91% false
positive and 31,82% false negative results. Immunodominant proteins p17, p19, p21,
OspC, p30, OspA, BmpA, p83 and VlsE were mainly documented, depending on diagnosis
(Fig.1).
Borrelia burgdorferi s.l., B. valaisiana, B. spielmanii and B. garinii (also in coinfection)
were identified in skin lesions and serum samples of patient with superficial or nonspecific
dermatitis, however, the attempts to cultivate live spirochetes from patient samples failed.
False-negative/positive results for IgM and IgG serologic tests can occur as well. Falsepositive serologic results may have been caused by antibody cross-reactivity, falsenegative results can occur due to early acute disease or immunocompromised states.
Conclusion: To date, no single sensitive and highly specific laboratory diagnostic test for
LB is available. Regional differences among borrelia isolates contributes as well to this
problem. Sensitivity of serodiagnostic tests (ELISA, WB) varied with stage of the disease.
Specific diagnosis can help to improve prognosis of the disease particularly in
controversial cases or in the case of the negative results obtained by the standard
serological tests.
Literature
1.
Schwarzova K. et al. Detection of Borrelia burgdorferi sensu stricto and Borrelia garinii DNAs in
patient with Hyperkeratosis lenticularis perstans (Flegel disease). Folia Microbiol, 61:359–363, 2016
2.
Middelveen MJ et al. Exploring the association between Morgellons disease and lyme disease
identification of Borrelia burgdorferi in Morgellons disease patients. BMC Dermatol.; 15(1): 1, 2015
3.
Matlahová K., Schwarzová K., Waczulíková I. Diagnostická výkonnosť ELISA testov štandardne
používaných v mikrobiologickej diagnostike Lymskej boreliózy. Interaktívna konferencia mladých
vedcov 2015 [7.] - Bratislava, 5.5.-6.6.2015.
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Documentation
Fig. 1 Nonspecific superficial dermatitis:
ELISA: IgM negative, IgG 37 RU/ml positive (cut-off : 20 RU/ml)
WB: IgM negative, IgG positive (17, 19 kDa, OspC, BmpA, VlsE)
Borrelia burgdorferi s.l. was detected in biopsy material and serum
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FAMILIAL OCCURENCE OF TICK-BORN ENCEPHALITIS IN ENDEMIC
REGION - CASE STUDY.
Štefkovičová M.1,2, Kopilec Garabášová, M.1, Šimurka P.2,3, Oleár V.2
1
Regional Public Health Authority Trenčín, Nemocničná 4, 911 01 Trenčín, Slovakia
[email protected]
2
School of Health Care, Alexander Dubcek University of Trencin, Trencin
3
Teaching Hospital in Trenčín
Introduction: Tick-born encephalitis is one of the most frequentflavoviral infection of the
central nervous system in Europe (1). In Slovak Republic around 10% of the country (700
– 1200m altitude) is colonized by ticks. The incidence rate of tick-born encephalitis in
Slovakia is 2.09/100 000 inhabitants (range 1.69 – 3.05). The highest morbidity rate has
been reported for the age-group 55-64. In the recent years the occurence of tick-born
encephalitis is concentrated in one locus in Central Považie Valley (Trenčín and Žilina
Regions). Totally 419 cases were registered (61.9%) out of the 677 notifiable cases in
Slovakia over the 2010 – 2015. The highest concentration of cases was observed in three
counties around the cities Púchov, Považská Bystrica and Bytča. The concentration was 10
times higher than an average incidence in Slovakia during the period 2010 – 2015 (2). The
presented study describes the familial occurence of the TBE from this endemic area.
The aim of the study: The goal of the study was to show the fatal outcome of the TBE in
a patient after the kidney transplantation and subsequent immunosuppresive therapy. The
disease with a characteristic double phase picture with a fast recovery was at that time
observed also in the patient`s son.
Methodology: We are describing 2 cases of TBE in a family of 5 members that occured in
July 2015, under the mountain range Strážovské hory in the valley stredné Považie. The
disease was diagnosed in father and son. Both were living in the endemic area of the
stredné Považie. The most probable factor of transmission was a raw goat milk from
a domestic animal bought by a family in an effort to improve, by the nutritional value of
the milk, the health status of the sick father.
Results and Discussion: The first case was 14 years old son with a double phase course of
the disease. The body temperature since June 6th was 39 ⁰C, accompanied by headache,
photo/phonophobia, repeatedly vomitting, and hospitalized with suspected neuroinfection.
After 10 days of hospitalization the patient was released in a good conditions from
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hospital. The diagnosis was based on data from biochemical analysis of spinal fluid
indicating to aseptic menigitis (spinal fluid elements 474, CB 0,84) and positive ELISA
test for IgM and IgG serum antibodies. The test for spinal fluid antibodies was negative.
Five days (on June 6th) before the hospitalization the tick was removed from the boy`s left
upper back. The mother informed that the boy was from time to time drinking a raw goat
milk from the domestic animal.
The second case, the 48 years old patient, the father of the family, after kidney
transplantation (2013) and immunosupressive therapy was hospitalized with a suspected
neuroinfection. His health status deteriorated, in spite of the limited immunosupesive
therapy, patient was somnolent, with intermittent coma, MRI-hypersignal zones in a brain
grey matter, stepwise epi-paroxysms, quadriparesis ascendant left, coma vigil, after 19
weeks patient died. Due to the severe conditions at the admission to the hospital it was not
possible to determine whether the patient has had the viremic first phase of the disease at
home, or he was admitted with a severe monophasic disease course. The symptoms in this
case could be mitigated by the already applied immunosupressive therapy. The wife of the
patient had informed that the patient was drinking the raw goat milk in association with his
basic diagnosis. The TBE diagnosis was estimated based on the biochemical testing of the
spinal fluid confirming the aseptic meningitis (spinal fluid elements 2480, CB 0,65), the
positive ELISA test for serum IgM and IgG antibodies. The IgM and IgG antibodies in
a spinal fluid at the admission to the hospital were negative. Most probably the patient was
tick bitten around June 24th.
There are two specific factors of alimentary transmission. One is an accumulation of the
cases in one place over the short period and the second one is a short incubation time (3 – 4
days) similarly as at the transmission by the tick. The highest risk is represented by the
consumption of the nonpasteurized goat milk or its products. The alimentary transmission
covers around 1% of all TBE cases. The epidemies caused by consumption of
nonpasteurized milk have been more often reported from East Europe and Baltic region
than from the Central Europe (3). In Czech Republic 64 cases were reported in 1997 –
2008. Half of them (56.3%) were caused by the consumption of nonpasteurized goat milk
(4). The alimentary transmission of the TBE, after consumption of a raw goat milk, was
described in Slovenia in 2012 (5). In Austria 6 out of the 7 exposed persons were
diagnosed with TBE in 2008 (6). The epidemy of TBE was reported in Hungary in 2007.
Out of 154 exposed persons 25 were diagnosed with the diseases, all of them consumed
raw goat milk (7). In Estonia 27 cases of TBE were reported from two counties, all
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associated with consumption of the nonpasteurized goat milk as a part of healthy diet
campaigne. All cases were confirmed by the laboratory data (8). The first information on
the occurence of TBE caused by consumption of the cows milk in EU appeared in 2011 in
Hungary. There were notified 11 patients out of 103 exposed persons. The nonpasteurized
cows milk was identified as a transmission factor (9). In Chroatia were notified 7 cases of
TBE in April 2015, all in association with the consumption of raw milk and/or cheese from
a small goat farm (1). Slovakia has notified most probably the highest number of
alimentary cases of TBE. In an epidemy in the Rožňava city in 1951 the infection occured
in 660 persons. A raw goat milk mixed with a cow´s milk was identified as the factor of
transmission (4, 10). Thirteen years later an epidemy was described in Závada in 1974 and
then in 1994 a familial epidemy associated with a consumption of raw goat milk occured in
region of Považská Bystrica (13).
The predictive risk factors for severe TBE are a high age, genetic factors and severe
clinical disease during the acute phase of of TBE (3). The serious clinical diseases are
represented for instance by immunodefciency. In the literature we can find a number of
cases described with a fatal outcome as described further. The twelve years old boy was
hospitalized with suspected hemophagocytic lymphohistiocytosis without any sign of TBE.
The immunosupressive therapy was initiated and after 8 days a relaps of high temperature
accompanied with development of meningeal signs, progreding quadruplegia and coma
followed. In spite of the intensive care after 31 weeks the child died (12). Other case was
reported from the Netherland. Fourty eight years old female originaly was diagnosed with
cerebral vasculitis at systemic lupus erythematosus and immunotherapy was initiated. The
serology showed positive IgM and IgG antiviral TBE antibodies and therefore the
diagnosis was changed to polyradiculoencephalitis. The immunotherapy was modified
accordingly, however, the patient died after 90 days (13). Hudopisk (5) described a case of
31 years old man with a transplanted kidney and on immunosupresive therapy. The disease
was presented by a double phase course with a picture of the mild to severe meningitis and
the patient recovered. It is interesting that in the intial meningo-encephalitic phase of the
disease the specific IgM and IgG serum antibodies are always present, while the antibodies
in a spinal fluid are present only in about 50% cases (1, 4). The younger patient in our
study also showed the presence of specific IgM and IgG antibodies in the initial meningoencephalitic phase of the disease, however his father showed only IgM antibodies in the
serum. Specific antibodies in the spinal fluid were not detected in both cases.
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Our study has documented the tragic impact of the raw milk consumption for the farmer´s
family. The benefits of the nonpasteurized milk and dairy products consumption should be
presented together with the health risks associated with such dietary habits particularly in
the endemic areas of the TBE occurence. Education of the population in endemic areas on
non-specific preventive measures associated with the outdoor activities (such as sport,
social, touristic) and about the vaccination as well should be incorporated into the public
health programs for control of the vector-born diseases.
Conclusion: Every year more than 100 cases of TBE are notified in Slovakia. Besides the
transmission via tick biting, also alimentary route of transmission due to the consumption
of the raw milk and nonpasteurized dairy products particularly in the natural foci of TBE
are registered. To get under the control the incidence of the TBE a common effort of
veterinary and public health authorities, experts and scientists aiming to protect the health
of the population is desired.
Literature
1.
MARKOVINOVIĆ L. et al. An outbreak of tick-borne encephalitis associated with raw goat milk and
cheese consumption, Croatia, 2015. In Infection. 2016 Jun 30
2.
AVDIČOVÁ, M. a kol. 2016. Analýza epidemiologickej situácie a činnosti odborov epidemiológie v
Slovenskej republike za rok 2015. RÚVZ so sídlom v Banskej Bystrici, 2014.
3.
BOGOVIC, P. STRLE, F. Tick-borne encephalitis: A review of epidemiology, clinical characteristics,
and management. In World J Clin Cases. 2015 May 16; 3(5): 430–441.
4.
KRÍZ, B. et al. Alimentary transmission of tick-borne encephalitis in the Czech Republic 1997-2008. In
Epidemiol Mikrobiol Imunol. 2009 Apr;58(2):98-103.
5.
HUDOPISK, N., et al. Tick-borne Encephalitis Associated with Consumption of Raw Goat Milk,
Slovenia, 2012. In Emerg Infect Dis. 2013 May; 19(5): 806–808.
6.
HOLZMANN, H. et al. Tick-borne Encephalitis from Eating Goat Cheese in a Mountain Region of
Austria. In Emerg Infect Dis. 2009 Oct; 15(10): 1671–1673.
7.
BALOGH, Z. et al. Tick-borne encephalitis outbreak in Hungary due to consumption of raw goat milk.
In Journal of Virological Methods. 2010; 163(2):481-485
8.
KERBO, N. et al. Tickborne encephalitis outbreak in Estonia linked to raw goat milk, May-June 2005. In
Euro Surveill. 2005 Jun 23;10(6):E050623.2.
9.
CAINI, S. et al. Tick-borne encephalitis transmitted by unpasteurised cow milk in Western Hungary –
September to October 2011. In Euro Surveill. 2012 Mar 22;17(12). pii: 20128.
10. Epidémia encefalitídy v rožňavskom prírodnom ohnisku nákaz. Sborník príspevkov o epidémii
encefalitídy prenesenej mliekom a o komplexnom výskume prírodného ohniska nákaz.. SAV 1954 s. 314
11. KOHL, O. et al. Family outbreak of alimentary tick-borne encephalitis in Slovakia associated with a
natural focus of infection. In Eur J Epidemiol. 1996 Aug;12(4):373-5.
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12. CHMELÍK, V. et al. Fatal tick-borne encephalitis in an immunosuppressed 12-year-old patient. In J Clin
Virol. 2016 Jan;74:73-4.
13. DE BRUIJN, M. et al. Tick-borne' encephalitis in an immunocompromised patient. In Ned Tijdschr
Geneeskd. 2015;159:A9067. (Article in Dutch).
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ALIMENTARY BOTULISM – OUR EXPERIENCES WITH THE LABORATORY
DIAGNOSTICS.
Strhársky, J.1, Maďarová, L. 1, Dorner, M. 2,3, Dorner, B. 2,3, Fatkulinová, M. 1, Avdičová,
M. 1, Sedliačiková, I. 1, Klement, C. 1,4, Donáth, V.5
1
Regional Authority of Public Health Banská Bystrica, Department of Medical
Microbiology, Banská Bystrica, Slovakia
2
Robert Koch Institute, Biological Toxins / Consultant Laboratory for Clostridium
botulinum, Berlin, Germany
3
Robert Koch Institute, Centre for Biological Threats and Special Pathogens, Berlin,
Germany
4
5
Slovak Medical University, Faculty of Public Health, Bratislava, Slovakia
F. D. Roosevelt Teaching Hospital, Department of Neurology, Slovak Medical University,
Banská Bystrica, Slovakia
Introduction: On 12th August 2015 we reported an outbreak of food-borne botulism
occurred in 42 years old man lives in Banská Bystrica (Slovakia). Clostridium botulinum
was isolated from the leftover packaging of commercial hummus. The product was
withdrawn from the market and a warning through the RASFF portal was issued
immediately. Further investigation in cooperation with Robert Koch Institute in Berlin
revealed the unusual subtype A3.
Aim of the study: The aim of the study was to draw attention on diagnosis of the rare
illness such as food-borne botulism is. The next objective was to highlight the need for
improving diagnostic procedures and interpretation of laboratory results at national level in
a suspected case of botulism.
Methodology: The combination of culture and molecular-biology based methods was used
for the laboratory confirmation of Clostridium botulinum. The culture was performed using
modified STN EN ISO 7937:2005. Multiplex PCR assay for the detection of genes
encoding botulinum neurotoxin types A, B, E and F (3) using agarose gel electrophoresis
was performed according to the STN P CEN ISO/TS 17919:2013. Further molecular
analyses as well as the sequencing were carried out in cooperation with Robert Koch
Institute in Berlin, Germany (2).
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Results:
Clinical investigation:
OnAugust 2015, a 42 years old man was admitted to the Department of Neurology of F. D.
Roosevelt Teaching Hospital in Banská Bystrica complaining nausea, vomiting, double
vision, unsteadiness with walking, dizziness, generalized weakness, and difficulties to
swallow after the consumption of three bags of commercial hummus spread. Due to the
nature and presentation of clinical symptoms the food-borne botulism was suspected. The
very next day his condition deteriorated rapidly and the patient wastransferred to the
Department of Anesthesiology and Resuscitation. Treatment consisted mainly of intensive
symptomatic care, including respiratory support and intravenous administration of horse
trivalent anti-A, B, E antitoxin. The gastric lavage for the laboratory investigation for the
presence of botulinum toxin using the mouse lethality assay was performed 48 hours after
the onset of clinical symptoms. This sample was stored at 4°C for one week before
negative laboratory analysis at the Public Health Institute Ostrava, Czech Republic.
Unfortunately serum and stool samples, which have been proposed for most reliable
diagnosis (1), have not been taken.
Epidemiological investigation
On 13th August 2015, epidemiologists conducted epidemiological investigation and
sampling in the patient's household. According to the patient and his mother, there were
signs of gas production in the packaging of hummus spreads. Epidemiologists notified the
Regional Veterinary and Food Administration of the Slovak Republic.
Laboratory investigation
The Regional Authority of Public Health in Banská Bystrica (RAPH) received various
food items from the household of the patient including opened and almost empty
packaging of hummus (spread made from chickpeas, tofu, rapeseed oil, onion, salt, maize
starch, and spices). RAPH was able to isolate three strains with typical C. botulinum
morphology from each packaging (Fig. 1). Analysis with a multiplex PCR revealed two of
them positive for the bont/A gene. The positive result of multiplex PCR reaction is
illustrated on Fig. 2. All isolates had been transferred to the Robert Koch Institute (RKI)
for further molecular biological investigation. At RKI, results were confirmed by
quantitative multiplex PCR for bont/A, B, E, and F(4) and the non-toxic nonhaemagglutinin (ntnh) gene as a surrogate marker for BoNT-producing clostridia (5).
Sequencing of the 16S rRNA gene (6) and comparison with GenBank entries revealed that
the 16S sequence of all three strains were >99 % identical to 16S sequences of C.
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botulinum group I/C. sporogenes which are virtually undistinguishable on the 16S level
(1). For subtype identification the bont/A genes of the two toxinogenic isolates were
sequenced (6) and compared to GenBank entries. Both bont/A sequences (GenBank
KU376389) were found to be identical and showed highest identity to bont/A sequences of
the unusual subtype A3. The expression of the toxin was confirmed by an in-house
multiplex ELISA (8).
Fig. 1: A. Culture on TSC agar plate, typical
morphology of Clostridium spp. B. Spores after
the culture on TSC agar plate, typical
Discussion
subterminal spores, Gram stain.
Fig. 2: Result of PCR,1) Ladder; 2) Sample; 3) - 6)
Positive controls: 3) C. botulinumserotype A; 4) C.
botulinum serotype B; 5) C. botulinum serotype E;
6) C. botulinum serotype F; 7) Negative control; 8)
Ladder
Botulism is a paralytic life threating illness caused in humans by botulinum neurotoxins
(BoNTs) producing by Clostridium botulinum Groups I–IV, C. baratii, and C. butyricum
strains. Seven different serotypes (BoNT/A–G) are recognized which all lead to flaccid
paralysis due to a blockade of neurotransmitter release at cholinergic neurons. BoNT types
A, B, E and F are mainly responsible for botulism in humans.
Initially diagnosis depends on the clinical picture but laboratory confirmation and/or an
epidemiological link to other cases is necessary to confirm the suspected clinical diagnosis.
There is a high degree of variation in severity, presumably related to exposure to different
amounts of toxin; however, those patients most severely affected require prolonged acute
care including assisted respiration, sometimes for several months (7). In the presented case,
thanks to the treatment, respiratory support and intravenous administration of horse
trivalent anti-A, B, E antitoxin the patient survived, though with the permanent health
consequences.
Although C. botulinum spores are ubiquitous in the environment, the growth and
production of botulinum toxin in foods only occurs under particular conditions (anaerobic,
low-salt, low-acid). In the presented case initially, suspected botulism could not be
confirmed by toxin testing from gastric lavage. Moreover serum and stool samples have
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not been taken. However, subsequent analysis of food items revealed the presence of C.
botulinum in two of three consumed hummus packs, thereby confirming food-borne
botulism. Hummus has been involved in other food-borne botulism outbreaks, e.g. in the
UK, possibly due to time and temperature abuse (11). Further laboratory investigations
confirmed a case of food-borne botulism based on the unusual subtype BoNT/A3. To our
knowledge, this is the first case due to this subtype in almost 100 years: in 1922 eight
people died due to contaminated wild duck paste in Loch Maree, Scotland (9).
Conclusions: We live in a world of rapidly evolving and pursuing of food industry.
Therefore, continuous mandatory notification of botulism and immediate recalls of
suspected food items will help to ensure the safety of customers across Europe.Related to
our experiences with the laboratory diagnostics of alimentary botulism, we have to
highlight the need for harmonizing and improving diagnostic procedures and interpretation
of laboratory results at national level in a suspected case of botulism. Better cooperation
between resorts is necessary as well.
Acknowledgments: We are grateful to our colleges from the RKI (Berlin, Germany) for
their professional and helpful approach with the further molecular-biology based analysis;
and tothe Public Health Institute Ostrava, Czech Republic, for the laboratory investigation
of the presence of botulinum toxin using the mouse lethality assay.
References
1.
Peck MW. Biology and genomic analysis of Clostridium botulinum. Adv Microb Physiol. 2009;55:183265, 320.
2.
Dorner MB, Schulz KM, Kull S, Dorner BG. Complexity of botulinum neurotoxins: challenges for
detection technology. Curr Top Microbiol Immunol. 2013;364:219-55.
3.
De Medici D, Anniballi F, Wyatt GM, Lindström M, Messelhäußer U, Aldus CF, et al. Multiplex PCR to
detect botulinum neurotoxin-producing clostridia in clinical, food and environmental samples. Appl
Environ Microbiol. 2009;75(20):6457-61.
4.
Kirchner S, Krämer KM, Schulze M, Pauly D, Jacob D, Gessler F, et al. Pentaplexed quantitative realtime PCR assay for the simultaneous detection and quantification of botulinum neurotoxin-producing
clostridia in food and clinical samples. Appl Environ Microbiol. 2010;76(13):4387-95.
5.
Raphael BH, Andreadis JD. Real-time PCR detection of the nontoxic nonhemagglutinin gene as a rapid
screening method for bacterial isolates harboring the botulinum neurotoxin (A-G) gene complex. J
Microbiol Methods. 2007;71(3):343-6.
6.
Hill KK, Smith TJ, Helma CH, Ticknor LO, Foley BT, Svensson RT, et al. Genetic diversity among
botulinum neurotoxin-producing clostridial strains. J Bacteriol. 2007;189(3):818-32.
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McLauchlin J, Grant KA, Little CL. food-borne botulism in the United Kongdom. journal of Public
health, 2006, 28(4): 337-342.
8.
Pauly D, Kirchner S, Stoermann B, Schreiber T, Kaulfuss S, Schade R, et al. Simultaneous
quantification of five bacterial and plant toxins from complex matrices using a multiplexed fluorescent
magnetic suspension assay. Analyst. 2009;134(10):2028-39.
9.
Scottish Board of Health. Botulism at Loch Maree. Analyst. 1923;48(564):118-20.
10. Dowell VR, Jr., McCroskey LM, Hatheway CL, Lombard GL, Hughes JM, Merson MH.
Coproexamination for botulinal toxin and clostridium botulinum. A new procedure for laboratory
diagnosis of botulism. J Am Med Assoc. 1977;238(17):1829-32.
11. Peck MW, Goodburn KE, Betts RP, Stringer SC. Assessment of the potential for growth and neurotoxin
formation by non-proteolytic Clostridium botulinum in short shelf-life commercial foods designed to be
stored chilled. Trends Food Sci Technol. 2008;19(4):207-216.
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ZIKA VIRUS – SHOULD WE BE AFRAID OF IT?
Striežová E¹, Krištúfková Z²
Regional Public Health Authority in Žiar nad Hronom, Department of Epidemiology,
Slovak Republic, [email protected]
Faculty of Public Health, Slovak Medical University in Bratislava, Slovak Republic²
Introduction: The Zika virus (ZIKV) was first identified in 1947 in a monkey in the Zika
forest of Uganda and was first isolated in humans in 1952 in Uganda and the United
Republic of Tanzania (1). It belongs to the Flaviviridae family and the Flavivirus genus,
and is thus related to the dengue, yellow fever, Japanese encephalitis, and West Nile
viruses. The main route for ZIKV transmission to a human is through a mosquito bite with
Aedes aegypti and Aedes albopictus as the most common vectors. Although ZIKV was
described 70 years ago, it only became a public health problem at the end of 2015, when an
outbreak in Brazil was associated with a significant increase of microcephaly cases in fetus
and newborns (2). From 2007 (identified in Asia) to 2016, the virus spread eastward, across the
Pacific Ocean to the Americas.
Figure 1. Countries or territories with reported confirmed autochthonous cases of
Zika virus infection in the past three months, as of 19 August 2016
In February 2016, The World Health Organization declared ZIKV infection as a Global Public
Health Emergency.
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Goal: Zika virus is common in Africa, South America, Asia and Pacific Region. Mosquito
Aedes aegypti is the key vector for the ZIKV, however the spreading of this mosquito is
not observed in Europe and thus the infections by ZIKV are imported only by travelers
from the regions. The goal of our study was to point out to importance of awareness and
knowledge of health care providers about epidemiological situation related to the disease
around the world, its spreading, symptoms and signs during the management of the
patients suspected with the ZIKV disease. For travelers to countries with the occurrence of
the infection it is important to be informed about preventive measures and tools before the
travel as well as after returning from the country.
Methods: The key document with important information for health care providers and
travelers regarding the ZIKV virus infection is the Regulation issued in 2016 by the Chief
Public Health Authority of Slovak Republic. It contains information about ZIKV, clinical,
laboratory and epidemiological criteria and the classification of the disease case. Further
the information includes the world wide spreading of the diseases, situation in SR,
laboratory diagnosis, basic rules for collecting samples of biological material and
management of patients with suspected diseases. The recommended preventive measures
and tools for travelers as well as advices for persons returning from countries of ZIKV
occurrence are included also (3,4).
Results: The epidemiologists have organized seminars on this topic in hospitals for
practitioners and physicians of the I. line. At the regional level the seminars were
organized also for the emergency rescue staff. The Regulation issued by the Chief Public
Health Authority was distributed to the general practitioners, specialized physicians in
gynecology and obstetrics, neurologists and physicians in hospitals. There were three cases
of imported ZIKV infection in Slovakia up to the August 18th 2016. Al three cases were
confirmed by the laboratories abroad (4).
Discussion: A study showed that both Aedes aegypti and Aedes albopictus were
susceptible to ZIKV infection but they also depended on the mosquito population in each
region or country (5). Ae. aegypti was previously founded sporadically in Europe in the
first half of the 20th century. Thus it appears that there are not climatic conditions that
would prevent spreading and survival of the mosquito Ae. aegypti provided it would reach
the Southern Europe . It is clear that in such situation it would have a significant impact on
public health in Southern Europe (6).
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Conclusion: The spreading of the Zika virus depends on the mosquitoes, that are the key
vector transmitting the virus to humans. The mosquitoes breed in warm climatic conditions
in stagnant waters, marshes and any places and items that hold the stagnant water. The key
activity thus remains on the reduction of the mosquitoes breeding by elimination of the
places with stagnant waters and by application of repellents. The surveillance of the
infection is of a paramount importance for effective prevention regarding the interhuman
spreading of the virus in countries where no confirmed cases of autochtonous ZIKA
infection were recorded.
Literature
1.
Interim Report 27 May 2016 WHO/ZIKV/SRF/16.2
2.
Journal of Medical Virology 88:1291(2016)
3.
The Regulation issued by the Chief Public Health Authority of Slovak Republic regarding the ZIKA
virus infection, February 26th, 2016.
4.
Amendment of The Regulation issued by the Chief Public Health Authority of Slovak Republic
regarding the ZIKA virus infection, August 23 rd, 2016.
5.
Chouin-Carneiro et al.,2016. Differential susceptibilities of Aedes aegypti and Aedes albopictus from the
Americas to Zika virus. PLoS Negl Trop Dis 10:e0004543
6.
http/ecdc.europa.eu/en/healthtopics/vectorsúmosquitoes/pages/aede-aegypti.aspx
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GENOTYPING OF GIARDIA DUODENALIS ISOLATES FROM CHILDREN AND
DOGS IN EASTERN SLOVAKIA
G. Štrkolcová1, M. Goldová1, M. Maďar2
1
Department of Parasitology, 2Department of Microbiology and Immunology, University of
Veterinary Medicine and Pharmacy in Košice, Komenského 73, 041 81 Košice, Slovakia
[email protected]
Introduction: Giardia is a ubiquitous intestinal protozoan parasite and one of the major
intestinal pathogens in humans which is present in tropical as well as temperate zones. It
occurs in all age groups, but the most endangered group consists of children younger than
2 years [1]. Giardiasis has often an asymptomatic course. Increase in the giardiasis
occurrence in those institutions is why giardiasis is referred to as a ʺre–emerging diseaseʺ
in developed world [2]. Out of eight known assemblages A–H [3], the A and B possess the
most important potential to infect humans and the remaining assemblages were found in
various mammals and only exceptionally (C, E, F) in humans [4, 5, 6]. However, as much
as 99.2% of human giardiasis cases are caused by assemblages A (G. duodenalis) and B
(G. enterica) they are able to infect also a large number of various animals including cats,
dogs, livestock and wildlife [7].
The aim of the study: The aim of this study was to determine the assemblages of G.
duodenalis in children aged 1–14 years old attending different preschools and schools and
domestic pets in two towns (Medzev, Košice) in Eastern Slovakia. In the present work,
genotyping was carried out at the triosephosphate isomerase-gene loci (positive samples)
for Giardia.
Methodology: 13 microscopically positive faecal samples (5 from the segregated
settlement, 3 from the orphanage and 1 from an autistic child, 2 from dogs and cats from
standard conditions) were used for genotyping the Giardia assemblages. DNA was
extracted using the ZR Fecal DNA MiniPrep™ kit (Zymo Research, USA), in compliance
with the manufacturer`s instructions, and then subjected to a nested PCR. The nested PCR
protocol was used to amplify a partial sequence of the triosephosphate isomerase (tpi)
gene. Obtained tpi sequence of our tested samples, were submitted to the GenBank and
accepted for publishing in GenBank databases under the accession numbers.
Results and Discussion: The tpi sequences of nine Giardia positive samples (from
children) were submitted to the GenBank under the accession numbers KR10539 ̶
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KR105401, KP866783 ̶ KP866786. A comparison of our sequences with the existing
GenBank data showed that six samples had 99–100% similarity to Giardia enterica
(assemblage B) and three to G. duodenalis (assemblage A). In Poland, Giardia enterica
(syn. G. intestinalis) assemblage B has been identified in four isolates of human origin in
Poland [8]. In Hungary assemblage A was found [9].The tpi sequences of four Giardia
positive samples (from pets) were submitted to the GenBank under the accession numbers
KP866787 ̶ KP866790. A comparison of our sequences with the existing GenBank data
showed that three samples had 99–100% similarity to Giardia canis (assemblage C) and
one to G. cati (assemblage F). In Italy molecular characterization showed occurrence 49 %
assemblage D in dogs, 36.1 % assemblage C and 4.2 % assemblage A [10]. In Hungary
[11]confirmed the presence of G. duodenalis in 7.5% of dogs assemblages C and D.
Conclusion: Our results represent the first report of the occurrence of assemblages A and
B of Giardia in children aged 1–14 years old attending different preschools and schools in
two towns (Medzev, Košice) in Eastern Slovakia. Sympatric occurrence of both
assemblages A, B was found in children from the orphanage in Medzev. In various
regions, the Giardia assemblages A and B occurred in humans and their virulence varies –
the A assemblage is usually associated with intermittent diarrhoea, while the B assemblage
is the cause of persistent diarrhoea. We are currently lacking comprehensive information
on the giardiasis epidemiology in the wider territory of Slovakia and processes of a
possible transfer of this infection among the population groups living in various social and
economic conditions.
Acknowledgments: This study was funded by the projects of the Scientific Grant Agency
of the Ministry of Education of the SR and the Slovak Academy of Sciences, VEGA
1/0455/15.
Literature
1.
Farthing MJ (1996) Giardiasis. Gastroenterol Clin N 25:493–515 doi: 10.1016/S0889-8553(05)70260-0
2.
Thompson RCA, Monis PT (2004) Variation in Giardia: Implications for taxonomy and epidemiology.
Adv Parasitol 58:69–137.doi: 10.1016/S0065-308X(04)58002-8
3.
Lasek-Nesselquist E1, Welch DM, Sogin ML (2010) The identification of a new Giardia duodenalis
assemblage in marine vertebrates and a preliminary analysis of G. duodenalis population biology in
marine systems. Int J Parasitol. 2010 Aug 1; 40(9):1063-74. doi: 10.1016/j.ijpara.2010.02.015. Epub
2010 Mar 30.
4.
Gelanew T, Lalle M, Hailu A, Pozio E, Caccio SM (2007) Molecular characterization of human isolates
of Giardia duodenalis from Ethiopia. Acta Trop 102:92–99. doi: 10.1016/j.actatropica.2007.04.003
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Foronda P, Bargues MD, Abreu-Acosta N, Periago MV, Valero MA, Valladares B, Mas-Coma S (2008)
Identification of genotypes of Giardia intestinalis of human isolates in Egypt. Parasitol Res 103:1177–
1181. doi: 10.1007/s00 436-008-1113-2
6.
Štrkolcová G, Maďar M, Hinney B, Goldová M, Mojžišová J, Halánová M(2015) Dog´s genotype of
Giardia duodenalis in human: first evidence in Europe. Acta Parasitol 60:796–799. doi: 10.1515/ap2015-0113
7.
Xiao L, Fayer R (2008) Molecular characterization of species and genotypes of Cryptosporidium and
Giardia
and
assessment
of
zoonotic
transmission.
Int
J
Parasitol
38:1239–1255.
doi:
10.1016/j.ijpara.2008.03.006
8.
Solarczyk P, Majewska AC (2007) Molecular identification of Giardia spp. and Cryptosporidium spp. in
humans and animals. Ann Parasitol 53:110
9.
Plutzer J, Törökné A, Szénási Z, Kucsera I, Farkas K, Karanis P (2014) Detection and genotype analysis
of Giardia duodenalis from asymptomatic Hungarian inhabitants and comparative findings in three
distinct locations. Acta Microbiol Immunol Hung. 61:19–26. doi: 10.1556/AMicr.61.2014.1.3
10. Pipia AP, Varcasia A, Tamponi C, Sanna G, Soda M, Paoletti B, Traversa D, Scala A (2014) Canine
giardiosis in Sardinia Island, Italy: prevalence, molecular characterization, and risk factors. J Infect Dev
Ctries 8:655–660
11. Szénási Z, Marton S, Kucsera I, Tánczos B, Horváth K, Orosz E, Lukács Z, Szeidemann Z (2007)
Preliminary investigation of the prevalence and genotype distribution of Giardia intestinalis in dogs in
Hungary. Parasitol Res 101:145–152
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HEPATITIS A AND HEPATITIS E IN READY-TO-EAT SALADS
Terio V., Bottaro M., Di Pinto A., Mottola A., Marchetti P., Bonerba E., Tantillo G.
Department of Veterinary Medicine, University of Bari,
Strada provinciale per Casamassima km3, 70010 Valenzano (BA)- Italy
[email protected]
Introduction: Over the last few years, public health promotion and healthier lifestyles
have led to increased demand of ready-to-eat (RTE) vegetables because they provide
vitamins, minerals and phyto-nutrients. They can be obtained from fresh products through
selection, washing, peeling, cutting sanitization, rinsing, drying and packaging in order to
extend shelf life and preserve their nutrition properties, because they are eaten raw (11).
The consumption of fresh products like RTE salads, is often linked to the numerous
foodborne virus outbreaks. Enteric viruses such as norovirus (NoV), hepatitis A virus
(HAV), hepatitis E virus (HEV), rotavirus (RV) and astrovirus (AstV) may contaminate
vegetables, during cultivation before harvest or post-harvest.
During pre-harvest cultivation, contamination can occur by application of organic wastes
as fertilizer, contamination of water used for irrigation with faecal material, contact with
inadequately-treated sewage or sewage-polluted water (5).
Water is the main critical vehicle of contamination in the farm-to-fork continuum.
Spraying, washing or immersion of fruits and vegetables in water are common practices
during post-harvest processing (5). Moreover, minimal processing may induce crosscontamination of clean products during cutting, washing and packaging (12).
Recently, HAV has been found in six EU countries (Belgium, Czech Republic, Germany,
Italy, the Netherlands and Spain) in semi-dried tomatoes, dates, frozen strawberries,
strawberry, frozen mixed berry and frozen red currants (4; 13).
A recent multi-laboratory investigation conducted in three European countries (Greece,
Serbia and Poland) demonstrated that leafy green vegetables destined to the market were
contaminated with HEV (9). Limited data exist in literature on the prevalence of foodborne
viruses in RTE vegetables.
The aim of the study: The purpose of this study was to investigate the presence of HAV
and HEV in RTE vegetables available for sale in Italy, bacterial pathogens (Salmonella
spp. and Listeria monocytogenes) and process hygiene criteria (E. coli count) according to
European (EU) Regulation 1441/2007.
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Methodology:
RTE sampling
A total of 40 RTE mix salads samples belonging to different brands and purchased from
supermarkets in Apulia, were examined during February-May 2016. All the samples were
labelled as “Pre-washed and ready-to-eat”. All the samples were obtained in their original
packaging and analysed before the expiration date (up to 8 days).
Isolation of Salmonella spp., Listeria monocytogenesand Escherichia coli enumeration
Salmonella spp. and Listeria monocytogenes isolation was performed following EN/ISO
6579 and EN/ISO 11290-2, respectively. Escherichia coli count was performed following
theISO 16649-2.
Virus concentration and nucleic acid extraction
HAV and HEV RNA was extracted following the ISO/TS 15216-2:2013.
Hepatitis A and Hepatitis E virus qPCR
Reverse transcription of viral RNA and real time one-step PCR were performed using
Invitrogen RNA Ultrasense one step qRT-PCR system (Life technology, Italy), according
to the manufacturer’s instructions.
The RT-qPCR reactions were performed in duplicate. All tests included negative controls
for virus and for an internal amplification control (IAC).
Hepatitis A qPCR
This assay was performed using the primers and conditions described in the ISO/TS
15216-2 method with the inclusion of an internal amplification control (IAC).
Hepatitis E qPCR
HEV assay used the primers and conditions described by Jothikumar et al. (2006) with the
inclusion of an internal amplification control (IAC).
Result
and
discussion:
Isolation
of
Salmonella
spp.,
Listeria
monocytogenesandEscherichia coli enumeration
The pathogenic foodborne microorganisms Salmonella spp. and L. monocytogenes,
according to EU food safety criteria (2), were not detected in any RTE salads. All samples
were complied with Hygiene criteria (E.coli) of EU Reg., 1441/2007.
Detection of foodborne viruses by RT-qPCR
The mean viral extraction efficiency of the process was > 1% for each sample.
HAV was the most prevalent pathogenic enteric virus, detected in 7.5% of samples (3/40),
and followed by HEV in 2,5% of samples (1/40).
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In this study, Salmonella spp. and L. monocytogenes were not detected in RTE salads, in
compliance with RTE salad safety criteria as set out in the European Regulation.
On the opposite, the virological investigations carried out in this study detect the presence
of HAV and HEV in 7,5% and 2,5% of the analysed samples, respectively. To our
knowledge, this is the first time that HAV and HEV were identified in RTE salads, in Italy.
The HAV and HEV positive samples were mixed salads that are characterized by a
production process that requires intense manipulation during collection. Viral
contamination may occur in several times and points of the production chain. As viruses
are not able to replicate extracellulary, their presence can only be accounted for by preharvesting contaminations of vegetables or by post-harvesting recontaminations by food
handlers (1). The presence of viral acid nucleic revealed by the molecular assays,
represents a clear index of contamination and potential risk for consumers. Moreover, this
contamination represents an unacceptable risk that the food handlers have to evaluate in
the risk assessment plan (3).
Conclusion: This study indicates that introducing additional microbiological criteria is
necessary to guarantee safety of RTE vegetables. Indeed, screening for enteric viruses,
which are not correlated to the presence of E. coli and Salmonella spp. (10), is crucial
when assessing the health risks related with RTE salads.
Appropriate implementation of food-safety management systems including Good
Agricultural Practices (GAP), Good Hygiene Practices (GHP) and Good Manufacturing
Practices (GMP) should be the primary objective of operators producing RTE salads.
Literature
1.
Berger, C.N., Sodha, S.V., Shaw, R.K., Griffin, P.M., Pink, D., Hand, P., Frankel, G., 2010. Fresh fruit
and vegetables as vehicles for the transmission of human pathogens. Environ. Microbiol. 12, 2385–2397.
2.
European Commission (EC) regulation N° 1441/2007 of 5 December 2007 amending Regulation (EC)
No 2073/2005 on microbiological criteria for foodstuffs.
3.
European Commission (EC) regulation N° 178/2002 of 28 January 2002 laying down the general
principles and requirements of food law, establishing the European Food Safety Authority and laying
down procedures in matters of food safety.
4.
European Food Safety Authority, 2014a. Tracing of food items in connection to the multinational
hepatitis A virus outbreak in Europe. EFSA Journal 12, 3821, 186.
5.
Gandhi, K. M., Mandrell, R.E., Tian, P., 2010. Binding of virus-like particles of Norwalk virus to
romaine lettuce veins. Appl. Environ. Microbiol. 76, 7997–8003.
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ISO 16649-2, 2001. Microbiology of food and animal feeding stuffs — horizontal method for the
enumeration of beta-glucuronidase-positive Escherichia coli. Part 2: Colony-count Technique at 44 °C
Using 5-bromo-4-chloro-3-indolyl beta-D-glucuronide.
7.
ISO/TS 15216-2, 2013. Microbiology of food and animal feed—horizontal method for determination of
hepatitis A virus and norovirus in food using real-time RT-PCR. Part 2: Method for qualitative detection
International Organization for Standardization, Geneva, Switzerland.
8.
Jothikumar, N., Cromeans, T. L., Robertson, B. H., Meng, X. J., Hill, V. R., 2006. A broadly reactive
one-step real-time RT-PCR assay for rapid and sensitive detection of hepatitis E virus. J. Virol.
Methods131, 65–71.
9.
Kokkinos, P., Kozyra, I., Lazic, S., Bouwknegt, M., Rutjes, S., Willems, K., Moloney, R., de Roda
Husman, A.M., Kaupke, A., Legaki, E., D'Agostino, M., Cook, N., Rzeżutka, A., Petrovic, T.,
Vantarakis, A., 2012. Harmonised investigation of the occurrence of human enteric viruses in the leafy
green vegetable supply chain in three European countries. Food Environ. Virol.4, 179–191.
10. Krog, J.S., Larsen, L.E., Schultz, A.C. 2014. Enteric porcine viruses in farmed shellfish in Denmark. Int.
J. Food Microbiol. 186, 105–109.
11. Little, C. L., and Gillespie, I. A., 2008. Prepared salads and public health. J. Appl. Microbiol.105, 17291743.
12. Lopez-Velasco, G., Davis, M., Boyer, R.R., Williams, R.C., Ponder M. A., 2010. Alterations of the
phylloepiphytic bacterial community associated with interactions of Escherichia coli O157:H7 during
storage of packaged spinach at refrigeration temperatures. Food Microbiol. 27, 476-486.
13. Terio, V., Bottaro, M., Di Pinto, A., Catella, C., Chironna, M., Bozzo, G., Kingsley, D. H., Bonerba, E.,
Morea, A., Martella, V., 2015. Outbreak of Hepatitis A in Italy associated with frozen red currants
imported from Poland: A Case Study. Food Environ. Virol. 7, 305–308.
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CANINE BABESIOSIS IN SLOVAKIA
Víchová, B., Blaňarová, L., Komjáti-Nagyová, M.
Institute of Parasitology, Slovak Academy of Sciences, Hlinkova 3, 04001 Košice, Slovakia
[email protected]
Introduction: Babesiosis is an emerging, tick-transmitted, zoonotic disease caused by
haematotropic parasites of the genus Babesia. Babesia and Theileria (Apicomplexa) are
some of the most ubiquitous and widespread blood parasites of considerable worldwide
economic, medical, and veterinary importance. These intraerythrocytic protozoans cause a
hemolytic, malaria-like diseases. There are over 100 species of Babesia identified so far
and many species infect only non-human mammalian hosts, most commonly dogs, cattle,
horses, and sheep.
In Slovakia, first autochthonous report of canine babesiosis, caused by Babesia canis
canis, was described in 2001, in the south-eastern part of the country (1). Since then, the
number of cases is growing exponentially every year, and currently, Slovakia is considered
an area with the endemic occurrence of canine babesiosis.
Agents of canine babesiosis cause severe epizootics in areas with the endemic occurrence
of D. reticulatus, infection of which may locally reach up to 40% (2). In the past, D.
reticulatus occurred primarily in western Slovakia and in some areas of eastern Slovakia.
Bullová et al. (2009) (3) confirmed the spread of the ornate dog tick (D. reticulatus), the
epidemiologically most important vector of large B. canis, to the north and by more than
300 meters in higher altitudes.
Based on the knowledge of an extension and above-mentioned changes in the distribution
of arthropod vectors, we have previously assumed that all cases of canine babesiosis in
Slovakia are caused by B. canis which is endemic in local dogs.
Due to global climate change, new locations suitable for the tick survival are established in
areas which were not favorable in the past. These changes support the emergence and
expansion of various vector-borne pathogens.
The aim of the study: The main goal of the study was to assess the prevalence rate of
Babesia in dogs suspected for canine babesiosis with a history of tick bites, and/or nonspecific febrile conditions.
Material and Methodology: From April 2013 until the present, altogether 397 EDTA
treated blood samples originated from dogs suspected for babesiosis were screened by
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molecular methods for the presence of babesia. The samples of blood were obtained from
vet practitioners from multiple locations in Slovakia.
Genomic DNA was extracted from 200 μl of the blood sample, using a commercial DNA
extraction kit (NucleoSpin Blood kit, Machery Nagel, Germany). For the molecular
detection of Babesia spp., PCR amplification of approximately 450 bp long fragment of
18S rRNA gene, spanned by a reverse BJ1 and forward BN2 primer was performed
according to Casati et al. (2006) (4).In each PCR reaction, sequenced DNA from the
Babesia-positive dog was used as positive control and nucleases free water was added as
the template in the negative control. The PCR products were visualized by electrophoresis
on 1. 5% agarose gels stained with GoodView Nucleic Acid Stain (Beijing SBS Genetech,
Beijing, China).
Randomly chosen positive PCR products were purified using a purification kit (Qiagen,
Hilden, Germany) and sequenced. Nucleotide sequences were manually edited in MEGA 6
(5). and further compared with GenBank entries by BLAST (6). For the alignment of the
homologous nucleotide sequences, the Clustal W program was used.
In order to rapidly discriminate B. gibsoni and B. canis, without further sequencing,
a restriction fragment length polymorphism (RFLP) analysis was optimized. 18S rRNA
gene PCR products, with a length of approximately 450 bp, have been analyzed. Samples
were digested at 37°C/3.5 hours with HinfI (Promega, Madison, USA) restriction enzyme
as described previously (7). Products of digestion were visualized by electrophoresis
(175V/25 min) on 2% agarose gel stained with the GelRed Nucleic Acid Gel Stain
(Biotium, CA, USA) in TBE (TrisBorateEDTA) buffer.
Results and Discuss: In total, the presence of blood parasites was confirmed in 44 tested
samples, representing the prevalence of 11.1%. In 5 samples, the presence of A.
phagocytophilum was recorded. No simultaneous infection has been noticed in the tested
file.
In June 2013, a 3 years old mixed pit bull male was presented to the veterinary clinic in
Bratislava, Slovakia, with a history of massive haemoglobinuria and blood transfusion
repeated twice during the previous treatment at another clinic. A few months later, the
owner visited the veterinary clinic again,with a 2 years old mixed pit bull terrier with a 3
days history of lethargy, weakness, and repeated syncopes.
Genomic DNA was isolated and subjected to PCR analysis, which confirmed the presence
of Babesia spp. Without further sequencing, based on experiences and routine from the
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previously diagnosed cases of B. canis – induced babesiosis, with anemia and similar
clinical signs, the PCR results were regarded as a B. canis – infection. Treatment with the
most widely used anti-piroplasm drug imidocarb dipropionate (Imizol), at a dose of 6
mg/kg intramuscularly, was initiated. This treatment, however, did not produce expected
therapeutic response. An ultrasonography (USG) revealed the splenomegaly, thus the
splenectomy was carried out and blood transfusion was performed on the second dayafter
admission to the clinic. Subsequently, the dog was discharged with antibiotics and Imizol.
Five days later, fever and significant haemoglobinuria returned. At this point, a blood
smear was prepared from the whole EDTA treated venous blood sample. The veterinarian
observed small basophilic inclusions suspected to be B.gibsoni trophozoites inside
erythrocytes and a blood sample was taken for the PCR. Higher doses of Imizol in
combination with antibiotics, analgesics and intravenous fluids were administered.
Nevertheless, the health status of the dog did not improve.
The BLAST analysis of sequences obtained after repeated PCR analysis confirmed 100%
similarity of the sequences with B. gibsoni isolated from dogs worldwide. After RFLP,
distinct restriction patterns were observed for B. canis and B. gibsoni, respectively.
In Europe, B. canis is considered to be the most common species responsible for the canine
babesiosis, a severe and potentially life threatening infection.
Here we present data indicating rising incidence of canine babesiosis caused by B. canis as
well as the first molecular detection of B. gibsoni in two mixed pit bull terriers with a
clinical and hematological signs of babesiosis. Polymerase chain reaction (PCR),
Restriction Fragment Length Polymorphism (RFLP) analysis and further sequencing of the
18S rRNA gene fragments isolated from blood samples of both dogs confirmed 100%
similarity to each other, and 100% identity with B.gibsoni isolatesthat infect dogs
worldwide.
Consequently, this study confirmed that B. canis is not the only species responsible for
canine babesiosis in Slovakia. Although infections reported in western Slovakia cannot be
clearly considered as autochthonous, we present the first molecular characterization of
clinical infections caused by small babesia, suggesting that B. gibsoni may be
underdiagnosed here.
Conclusions: As reported above, we observe progressively rising incidence of canine
babesiosis caused by B. canis in Slovakia. The results of the study should also alert
veterinarians of a new infection that previously did not occur in Slovakia. As this infection
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requires different treatment than the one caused by B. canis, differential diagnosis and
accurate identification of causative agents is crucial for the selection of an appropriate
therapy and successful treatment, especially in dogs with hemolytic anemia and febrile
conditions.
Literature
1.
Chandoga, P., Goldova, M., Baranova, D., Kozak, M., 2002. First cases of Canine babesiosis in the
Slovak Republic. Vet. Rec. 150, 82–84.
2.
Majlathova, V., Majlath, I., Vichova, B., Gul’ova, I., Derdakova, M., Sesztakova, E.,Pet’ko, B., 2011.
Polymerase chain reaction confirmation of Babesia canis canisand Anaplasma phagocytophilum in dogs
suspected of babesiosis in Slovakia.Vector Borne Zoonotic Dis. 11, 1447–1451.
3.
Bullova, E., Lukan, M., Stanko, M., Petko, B., 2009. Spatial distribution of Dermacentor reticulatus tick
in Slovakia in the beginning of the 21st century.Vet. Parasitol. 165, 357–360.
4.
Casati, S., Sager, H., Gern, L., Piffaretti, J.C., 2006. Presence of potentially pathogenic Babesia sp. for
human in Ixodes ricinus in Switzerland. Ann. Agric. Environ.Med. 13, 65–70.
5.
Tamura, K., Stecher, G., Peterson, D., Filipski, A., Kumar, S., 2013. MEGA6: molecular evolutionary
genetics analysis version 6.0. Mol. Biol. Evol. 30, 2725–2729.
6.
Altschul, S.F., Madden, T.L., Schaffer, A.A., Zhang, J., Zhang, Z., Miller, W., Lipman,D.J., 1997.
Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids
Res. 25, 3389–3402.
7.
Solano-Gallego, L., Trotta, M., Carli, E., Carcy, B., Caldin, M., Furlanello, T., 2008.Babesia canis canis
and Babesia canis vogeli clinicopathological findings and DNA detection by means of PCR-RFLP in
blood from Italian dogs suspected of tick-borne disease. Vet. Parasitol. 157, 211–221.
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PART II. –ABSTRACTS OF POSTERS
THE USE OF BACTERIOPHAGES IN THE THERAPY OF CRONOBACTER SPP.
Belanová V. (1), Kajsík M. (2), Oravcová L. (1), Šoltýs K. (2),
Drahovská H. (1)
(1) Faculty of Natural Sciences, Comenius University in Bratislava, Mlynská dolina,
Ilkovičova 6, 842 15 Bratislava 4, Slovakia,[email protected]
(2) Comenius University Science Park, Ilkovičova 8, 842 16 Bratislava, Slovakia
Introduction: Cronobacter spp. are considered opportunistic pathogenic bacteria
belonging to Enterobacteriaceae family (1) associated with rare but fatal neonatal
infections such as meningitis and necrotising enterocolitis (2). Infections also occur in
older children and adults (3) causing septicemia, pneumonia, osteomyelitis, wound
infections, and splenic abscesses (4). Cronobacter spp. are ubiquitous in the environment
and have been isolated from plant materials, fruits and vegetables, among other sources.
However, the proposed route of infection in infants is through the consumption of
contaminated powdered infant formula (PIF) which has been the most common vehicle
implicated in Cronobacter infections (5). One possible way how to solve the problems
involving pathogen contamination in food is the application of bacteriophages (6). Phages
have been used to control pathogens due to their high specificity and efficiency. However,
before application, it is necessary to obtain sufficient information about phages used as a
controlling agent to guarantee their safety and reliability (7, 8).
The aim of the study: In this study, we characterized properties of Pet-CM3-4 phage
which infects broad range of Cronobacter strains and its spontaneous mutant Pet-CM3-1
possesing narrowed host specificity. We identified host cell receptors recognized by PetCM3-4 and Pet-CM3-1 phages. These data are fundamental for its application in food
control.
Methodology: Bacteriophage Pet-CM3-4 was isolated from wastewater (9) on indicator C.
malonaticus 161007/29 and Pet-CM3-1 phage was isolated as spontaneous mutant during
phage cultivation. The purification of phage preparates was made by ultracentrifugation in
CsCl gradient (10). The host specificity of phages was determined by phage titer analysis
on 50 Cronobacter strains. Phage DNA was isolated using Phage DNA Isolation Kit
(Norgen, Biotek Corp., Canada) according to manufacturers protocol and sequenced on
MiSeq (Illumina, USA). Identification of
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a transposon mutant library of C. malonaticus 161007/29 prepared by EZ-Tn5™<DHFR1>Tnp Transposome™ Kit (Epicentre, Illumina) according to manufacturers protocol.
Transposon mutants were incubated in the presence of phages Pet-CM3-1 or Pet-CM3-4
and single colonies were isolated on the LB plate. The site of transposon insertion was
identificated by single primer PCR or by whole genome sequencing.
Results and Discussion: In this study we characterised the new bacteriophage Pet-CM3-4
infecting Cronobacter spp. This phage has been classified as T4-like phage belonging to
Myoviridae family. The phage genome is 172 kb (GC= 39,8%) long with 279 proteincoding genes and 18 tRNA genes. Sequence analysis has revealed that Pet-CM3-4 shares
95% similarity with PG7 phage and 92% similarity with CC31 phage of Myoviridae family
also infecting Enterobacteriaceae.During incubation of Pet-CM3-4 phage on indicator
strain we identified its mutant Pet-CM3-1 with narrowed host specificity. By testing host
specificity of both phages we detected that Pet-CM3-4 was able to infect 94% of tested
Cronobacter strains. On the other hand Pet-CM3-1 phage infected only 12% Cronobacter
strains. To explain the altered host specificity of Pet-CM3-1 phage we performed the
comparison of both genomes. We identified six missense mutations in the whole genome,
one of them was substitution K163Q in gp257gene encoding the receptor-recognising
protein localized of long tail fiber, which was responsible for narrowed host specificity of
Pet-CM3-1 phage. To identify the bacterial cell
receptors recognised by phages we
prepared the transposon mutant library of the C. malonaticus 161007/29 and selected 80
mutants growing in the presence of Pet-CM3-1 (40 CM1 mutants) or Pet-CM3-4 (40 CM4
mutants) phages. In most CM1 clones we obtained the transposon insertion in rfb cluster
encoding for O-antigen of Cma O:3 (Csa O:5) serotype and in waaL gene encoding for Oantigen ligase which mediates the ligation of the O-antigen to lipid A-core. The majority of
CM4 transposon mutants had the insertion in waaL (Table 1). Growth inhibition and
adsorption methods have shown, that mutants with transposon inserted into genes of rfb
cluster were able to grow in the presence of Pet-CM3-1 phage and adsorption revealed the
lower levels. On the other hand the growth of these mutants was inhibited by Pet-CM3-4
phage as this phage was able to adsorb on them. Mutants with transposome inserted into
waaL gene were able to grow in the presence of both phages and both phages adsorbed
with lower efficiency on these mutnants. According to these results we suppose that the
bacterial receptor recognised by Pet-CM3-1 phage is O-antigen LPS falling into the
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serotype Cma O:3 and Pet-CM3-4 phage use the inner part (core) of LPS as bacterial
receptor.
Table 1: Identification of the transposition site in transposon mutants selected to resistance
to phages Pet-CM3-1 and Pet-CM3-4.
Selection
Gene
Function
Number of mutants
O-antigen ligase
14
rfb
O-antigen
10
ECA
enterobacterial common antigen
2
gltK
glutamate/aspartate permease
2
hsl
QS transcriptional regulator
1
Pet-CM3-1 waaL
11
Non-identified
Pet-CM3-4 waaL
O-antigen ligase
13
rfb
O-antigen
1
carB
carbamoyl phosphate synthase
1
ccpA
capA protein
1
msrA
methionine sulfoxid reductase
2
gpmA
glycerol phosphate mutase
1
ompE
outher membrane protein ompE
1
puuD
utilisation of putrescine
2
uds
undecaprenyl diphosphate synthase
1
mfp
permease
1
ycjW
utilisation of maltodextrine
1
15
Non-identified
Conclusion: Pet-CM3-4 phage and its spontaneous mutant with altered host specificity
Pet-CM3-1 phage were isolated from wastewater. According to the sequence analysis these
phages were classified as T4-like phages falling into Myoviridae family. Sequence analysis
has revealed that altered host specificity of Pet-CM3-1 phage was caused by substitution
K163Q in gp257gene encoding the receptor-recognising protein. For identification of the
bacterial cell surface receptors recognised by phages we made the transposome mutants
library and we suppose that the probable bacterial receptor recognised by Pet-CM3-1
phage is O-antigen LPS falling into the serotype Cma O:3 (Csa O:5) and Pet-CM3-4 phage
use the inner part (core) of LPS as bacterial receptor.
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Literature
1.
Forsythe S. J., Dickins B., Jolley K.A. (2014) Cronobacter, the emergent bacterial pathogen
Enterobacter sakazakii comes of age; MLST and whole genome sequence analysis. BMC Genomics.
16;15:1121.
2.
Ogrodzki P., Forsythe S. (2015) Capsular profiling of the Cronobacter genus and the association of
specificCronobacter sakazakii and C. malonaticus capsule types with neonatal meningitis and
necrotizing enterocolitis. BMC Genomics. 16; 758-773.
3.
Holý O., Forsythe S. (2014) Cronobacter spp. as emerging causes of healthcare-associated infection.
Journal of Hospital Infection. 86; 169–177.
4.
Patrick M.E., Mahon B.E., Greene S.A., Rounds J., Cronquist A, Wymore K., Boothe E., Lathrop S.,
Palmer A., Bowen A. (2014) Incidence of Cronobacter spp. Infections, United States, 2003–2009.
Emerging Infectious Diseases. DOI 10.3201/eid2009.140545.
5.
Endersen L., Guinane M.C., Johnston CH., Neve H., Coffey A., Ross R.P., McAuliffe O., O’Mahony J.
(2015) Genome analysis of Cronobacter phage vB_CsaP_Ss1 reveals an endolysin with potential for
biocontrol of Gram-negative bacterial pathogens. Journal of General Virology. 96; 463-477.
6.
Goodridge L.D., Bisha B. (2011) Phage-based biocontrol strategies to reduce foodborne pathogens in
foods. Bacteriophage. 1; 130-137.
7.
Abbasifar R., Kropinski A.M., Sabour P.M., Ackermann H.-W., Villa A.A., Abbasifar A., Griffiths
M.W. (2013) The Genome of Cronobacter sakazakii Bacteriophage vB_CsaP_GAP227 Suggests a New
Genus within the Autographivirinae. Genome Announcements. DOI:10.1128/genomeA.00122-12.
8.
Sulakvelidze A. (2013) Using lytic bacteriophages to eliminate or sig.nificantly reduce contamination of
food by foodborne bacterial pathogens. J Sci Food Agric 93; 3137–3146.
9.
Chovanová R. (2012) Izolácia a charakterizácia bakteriofágov vhodných na elimináciu kmeňov
Cronobacter spp. Diplomová práca. Univerzita komenského, Bratislava.
10. Kleiner M., Hooper L. V., Duerkop B. A. (2015) Evaluation of methods to purify virus-like particles for
metagenomic sequencing of intestinal viromes. BMC Genomics, 16:7.
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IDENTIFICATION AND CHARACTERIZATION OF TRICLOSAN RESISTANT
COLIFORMS FROM SEWAGE SLUDGE
Nagyová K., Kurtulík M., Klčo S., Olejníková P., Mackuľak T., Birošová L.
Department of Nutrition and Food Quality Assessment, Faculty of Chemical and Food
Technology, Slovak University of Technology, Radlinského 9, 81237 Bratislava, Slovakia
[email protected]
Introduction: Triclosan is antibacterial compound found in a range consumer products
including antibacterial soups, tooth pastes, dress or chopping boards. Resistance to
biocides such triclosan is well documented, which yields concern that cross-resistance to
clinically relevant antibiotics could develop from antimicrobial-bacteria interaction (1).
The high concentrations of triclosan in municipal wastewater and anaerobic digesters pose
a particular concern because of their interaction with a rich and diverse community of
bacteria. Triclosan is the most abundant pharmaceuticals (mean concentration of 39.433
μg/kg and 16.097 μg/kg respectively) found in US wastewater biosolids out of 145
chemicals surveyed (2). In September 2016, the FDA announced that effective September
2017, it would prohibit the sale of non-medical soap containing triclosan or 18 other
ingredients marketed as antimicrobials due to the FDA's findings of the lack of efficacy in
these products (3). In EU the use of triclosan is banned in food contact plastics only in
Germany. However this compound is relatively stable, through years of its use, it is
accumulated in the environment and can effect development of antimicrobial resistance.
The aim of this work was to monitor prevalence of triclosan resistant coliform bacteria
(CFB) in samples of sewage sludge. Isolated resistants were identified with MALDI-TOF.
Consequently we have examined their sensitivity to certain antibiotics of different classes
and their ability to form biofilm.
Material and Methods: Samples of sewage sludge were taken from 3 wastewater
treatment plants (WWTP) in Slovakia. WWTP Petržalka currently serves for 125 000
inhabitants and treats up to 33000 m3/day of municipal wastewater, WWTP Senec treats
wastewater from 68 000 inhabitants, WWTP Bratislava -Vrakuňa is the biggest in Slovakia
and serves to 400 000 inhabitants. Prevalence of triclosan resistant coliforms in sewage
sludge was determined according Birosova et al. (4). Triclosan resistant isolates were
identified by MALDI-TOF mass spectrometry analysis (Bruker Daltonics Inc., USA).
Resistant strains isolated from diagnostic media with antibiotic were then tested for crossresistance to other antibiotics (ampicillin, tetracycline, ciprofloxacin, chloramphenicol,
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gentamicin) using a plate dilution drop method (104 CFU/drop) using the agar dilution
method (5). Biofilm formation was detected according Žemlička et al. (6).
Results and Discussion: The minimal inhibitory concentration of triclosan for most
enterobacteria range between 0.5 to 64 µg/mL. Prevalence of triclosan resistant bacteria
from sewage sludge was evaluated in much higher concentration range 100-300 µg/mL.
The amount of CFB resistant to triclosan was the highest in sample from smallest WWTP
Senec, the lowest amount was found in sample from WWTP Petržalka (Fig. 1). In sewage
sludge from WWTP Petržalka was observed also presence of CFB resistant to highest
applied triclosan concentration.
7
6
log KTJ/g
5
CFB
4
c1
3
c2
c3
2
1
0
Vrakuňa
Petržalka
Senec
Fig. 1Total and triclosan resistant coliform bacteria in samples of sewage sludge.
CFB – coliform bacteria; concentrations of triclosan: c1 = 100 mg/L; c2 = 200 mg/L; c3 =
300 mg/L
Most isolates were identified as Citrobacter freundi (50 %) and Serratia sp. (25 %) (Tab
1). Isolate from Petržalka sewage sludge was identified as Morganella morganii, a
common inhabitant of gastrointestinal tract of mammals and reptiles. One isolate was
identified as Providencia alcalifaciens which belongs to gram-negative enterobacteria.
Isolated triclosan resistant coliforms showed resistance to antibiotics, especially to
ampicillin, gentamicin and chloramphenicol. Almost all isolates have intrinsic ampicillin
resistance. Only four strains isolated from Petržalka sewage possessed acquired resistance.
No multidrug resistance was observed.The ability to form biofilm by isolated resistants
was evaluated according to criteria given by Taniguchi et al. (7).None of the isolates
reached the ability to form the biofilm as P.aeruginosa however the differences were
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notable even among bacteria of the same species. Almost all strains belong to moderate or
strong producers of biofilm.
Tab.1Susceptibility of triclosan resistant isolates to antibiotics and their ability to
form biofilm
triclosan
WWTP
Petržalka
Identified strain
(µg/mL)
AMP
GEN
CIP
CHL
TET
Biofilm
M. morganii
100
R3
R3
S
R1
R2
SP
P. alcalifaciens
300
R3
S
S
R1
R2
SP
C. freundii
100
R1
S
S
S
S
SP
C. freundii
300
R1
S
S
S
S
SP
C. freundii
300
R1
S
S
S
S
SP
C. freundii
200
R3
S
R3
R1
S
SP
C. freundii
300
R3
S
S
S
S
SP
C. freundii
200
S
R3
S
S
S
SP
S. fonticola
200
R3
S
S
S
S
MP
S. liquefaciens
200
R1
S
S
S
S
SP
S. liquefaciens
200
R1
S
S
S
S
SP
Senec
Vrakuňa
AMP – ampicillin; GEN – gentamicicn; CIP – ciprofloxacin; TET – tetracycline; R1 – resistance breakpoints
according to EUCAST; R2 – resistance breakpoints according to CLSI; R3 – high-level resistance (above
CLSI); S – sensitive; SP – strong producer of biofilm; MP – moderate producer of biofilm.
Conclusion: A big part of the sewage sludge is applied to soil, what can lead to
widespread of resistance in the environment. According to our results it should be
reconsidered further use of sludge as well as technological improvement of wastewater
treatment plants.
Acknowledgement: This study was financially supported by Research and Development
Agency of Slovak Republic, contract No. APVV-0122-12 and by STU grant for young
scientists, contract No. 1618/16.
References
1.
Giuliano, C., Rybak, M.J.: Efficacy of triclosan as an antimicrobial hand soap and its potential impact on
antimicrobial resistance: a focused review, Pharmacotherapy 35 (3), 328–336 (2015).
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Carey, D.E., McNamara, P.J.: Altered antibiotic tolerance in anaerobic digesters acclimated to triclosan
or triclocarban, Chemosphere 163, 22-26 (2016).
3.
McGinley, L.: "FDA bans common ingredients in antibacterial soaps and body washes". Washington
Post. Retrieved September 8 (2016).
4.
Birošova, L., Mackuľak, T., Bodík, I., Ryba, J., Škubák, J., Grabic, R.: Pilot study of seasonal
occurrence and distribution of antibiotics and drug resistant bacteria in wastewater treatment plants in
Slovakia, Sci. Total Environ. 490, 440-444 (2014).
5.
Olejníková, P., Kurucová, M., Švorc, L. U., Marchalín, Š.: Induction of resistance in Mycobacterium
smegmatis. Canadian journal of microbiology 59(2), 126-129 (2012).
6.
Žemlička, L., Fodran, P., Lukeš, V., Vagánek, A., Slováková, M., Staško, A., Dubaj, T., Karabín, M.,
Birošová, L., Rapta, P.: Physicochemical and biological properties of luteolin-7-O-glucoside
(cynaroside) isolated from Anthriscus sylvestris (L.) Hoffm, Monatsh Chem 145 (8), 1307-1318 (2014).
7.
Taniguchi, L., de Fátima Faria, B., Rosa, R.T., de Paula, E., Carvalho, A., Gursky, L.C., Elifio-Esposito,
S.L., Parahitiyawa, N., Samaranayake, L.P., Rosa, E.A.: Proposal of a low-cost protocol for colorimetric
semi-quantification of secretory phospholipase by Candida albicans grown in planktonic and biofilm
phases. J Microbiol Methods. 78(2), 171- 4 (2009).
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DETECTION OF CHLAMYDIA SPP. IN DIFFERENT SPECIES OF BIRDS FROM
SLOVAKIA
Čechová L., Halánová M., Halán M., Danišová O., Kalinová Z., Babinská I., Čisláková L.
Pavol Jozef Šafárik University, Faculty of Medicine, Department of Epidemiology,
Šrobárova 2, 041 80 Košice, Slovakia
[email protected]
Introduction: The obligate intracellular bacteria Chlamydia spp. are the etiological agents
of chlamydiosis in wild and domestic birds, mammals and humans (3, 7, 9, 12, 13). The
family Chlamydiaceae currently contains the single genus Chlamydia (C.), which includes
12 recognized species, (16).
Chlamydia psittaci is one of the most important zoonotic species from epidemiological
point of view. There is the causative agent of avian chlamydiosis in birds and
ornithosis/psittacosis in humans. Until recently, C. psittaci was considered to be the only
pathogenic chlamydial species in birds, but recent evidence suggested that avian
chlamydiosis may also involve C. gallinacea and C. avium (15; 17), C. abortus (10), as
well as C. pecorum or C. trachomatis (18). Although most avian chlamydial infections
remain asymptomatic (6), they can lead to respiratory, enteric and ocular disease under
appropriate conditions (19; 8). Transmission can occur either through inhalation, ingestion
or via direct contact with the infected birds (1).
Important sources of ornithosis/psittacosisfor humans are feral pigeons, which have been
ranked as the second major reservoir of C. psittaci.C. psittacican cause also respiratory
disease in poultry, mainly in turkeys and ducks. Reports on C. psittaci outbreaks on
chicken farms or on zoonotic transmissions linked to contact with C. psittaci-infected
chickens are extremely rare. Maybe, chickens seldom become infected and/or strains
infecting chickens are less virulent, presenting a minor risk for humans (4; 20; 21).
Objective: The aim of this study was to investigate the chicken and pigeons for the
presence of Chlamydia spp. from pharyngeal and cloacal swabs, and in the case of positive
result to examine the identity of the obtained sequences.
Methodology: A total of 138 chicken and 14 clinically healthy pigeons were examined for
the presence of Chlamydia spp. Cloacal and pharyngeal swabs, 2 from each pigeon and
chicken, were collected using sterile cotton swabs. We used method PCR on detection of
Chlamydia with primers, which amplified domain I 23S rRNA gene. This segment is a
signature sequence for chlamydial species, genera and families. PCR products were
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analysed by electrophoresis and visualized fragments were compared with the positive
control and the 100 bp DNA ladder. In case of positive result the identity of the obtained
sequences was examined by a BLAST search.
Results and discussion: A total of 276 cloacal and pharyngeal swabs of 138 chicken and
14 cloacal and pharyngeal swabs of 14 pigeons were examined for the presence of
chlamydial infection. Of the total number of 138 examined samples of chicken, 19 (6.9 %)
showed positivity for C. psittaci infection. 12 of whom were from pharyngeal swabs (63
%) and 7 from cloacal swabs (37 %). Any from 14 examined pigeons were positive.
There are 12 closely related and distinct Chlamydia species and most animals are
susceptible to multiple Chlamydia species, resulting in often asymptomatic infections
with low bacterial burdens.Chlamydia is a genus that includes an important zoonotic
pathogen that causes acute diseases in birds and mammals, including humans. They may
lead to a wide range of clinical manifestations including ocular, pulmonary, genital,
articular and intestinal illness, but very often they induce persistent, chronic, or subclinical
infections.
C. psittaci has long been considered the main chlamydial species in poultry. In one study,
C. psittaci was the dominant species only in pigeons, while in chicken was predominant
chlamydial agent C. gallinacea.This study indicates that C. gallinacea is the endemic
chlamydial species in chickens, whereas C. psittaci dominates only in pigeons. The
positivity of Chlamydia spp. in this study is considerably higher than in other reports (5).
But in our study was predominant chlamydial agent in chicken C. psittaci. 276 samples
coming from 138 asymptomatic chicken, as potential source of infection for humans, were
examined for the presence of Chlamydia spp. by PCR method with 6.9 % prevalence. A
higher positivity was observed in samples from pharynx compared to samples from cloaca
(63 % vs. 37 %). No pigeon were positive. Epidemiological situation in occurrence of
ornithosis/psitaccosis in Slovakia is relatively favourable, for the period of last 10 years
was officially reported 22 cases in humans only (2).
Conclusion: C. psittaci infections are apparently emerging in chickens. Accurate
diagnostic monitoring and reporting of infections in poultry and should be promoted.
Additionally, an efficient veterinary vaccine, preventive measures, and information
campaigns could be beneficial to public health. There is a need for higher awareness and
for efficient risk assessment and management.
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Acknowledgements: The study was supported by the Slovak Grant Committee VEGA,
Grant No. 1/0390/12 and partially supported by the Research and Development Support
Agency, contract No. APVV-15-0134.
Literature
1.
Geens T, Dewitte A, Boon N, Vanrompay D. Development of a Chlamydophila psittaci species-specifi
c and genotype-specifi c realtime PCR. Vet Res. 2005;36:787–97
2.
Bakoss P, Macháčová E, Jareková J. et al. Surveillance zoonóz: S R, Leptospirózy 2009-2011: ornitóza
- psitakóza a cicavčie chlamydiózy 2004-2011. Bratislava: ŠVPS SR, 1. ed., 2013; 104 pp. - 29
3.
Dean D., Rothschild J., Ruettger A. et al. Zoonotic Chlamydiaceae species associated with trachoma,
Nepal. Emerg. Infect. Dis. 19, 1948–1955 (2013).
4.
Dickx, V., Geens, T., Deschuyffeleer et al., 2010. Chlamydophila psittaci zoonotic risk assessment in a
chicken and turkey slaughterhouse. J. Clin. Microbiol. 48, 3244–3250.
5.
Guo W. et al., Chlamydia gallinacea, not. C. psittaci, is the endemic chlamydial species in chicken
(Gallus gallus), Scientific Reports 6, 19638, doi:10.1038/srep 19638 (2016)
6.
Kaleta EF., Taday E. M. A. Avian host range of Chlamydophila spp. based on isolation, antigen
detection and serology. Avian Pathol. 32, 435–462 (2003).
7.
Knittler M. R., Sachse K. Chlamydia psittaci: update on an underestimated zoonotic agent. Pathog.
Dis. 73, 1–15 (2015).
8.
Knittler M. R. et al. Chlamydia psittaci: new insights into genomic diversity, clinical pathology, hostpathogen interaction and antibacterial immunity. Int. J. Med. Microbiol. 304, 877–893 (2014).
9.
Laroucau K. et al. Isolation of a new chlamydial agent from infected domestic poultry coincided with
cases of atypical pneumonia among slaughterhouse workers in France. Infect. Genet. Evol. 9, 1240–
1247 (2009).
10. Pantchev A., Sting R., Bauerfeind R., Tyczka J., Sachse K. New real-time PCR tests for species-specific
detection of Chlamydophila psittaci and Chlamydophila abortus from tissue samples. Vet. J.181, 145–
150 (2009).
11. Reinhold P., Hartmann H., Constable P. D. Characterization of acid-base abnormalities in pigs
experimentally infected with Chlamydia suis. Vet. J. 184, 212–218 (2010).
12. Rodolakis, A., Yousef Mohamad, K. (2010). Zoonotic potential of Chlamydophila. Vet Microbiol 140,
382–391.
13. Rohde G., Straube E., Essig A. et al. Chlamydial zoonoses. Dtsch. Ärztebl. Int.107, 174–180 (2010).
14. Gaede, W., Reckling, K.F., Dresenkamp, et al. 2008. Chlamydophila psittaci infections in humans
during an outbreak of psittacosis from poultry in Germany. Zoonoses Public Health 55, 184–188.
15. Sachse K., Laroucau K. Avian chlamydiosis: two more bacterial players discovered. Vet. J. 200, 347–
348 (2014).
16. Sachse K. et al. Emendation of the family Chlamydiaceae: proposal of a single genus, Chlamydia, to
include all currently recognized species. Syst. Appl. Microbiol. 38, 99–103 (2015).
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17. Sachse K. et al. Evidence for the existence of two new members of the family Chlamydiaceae and
proposal of Chlamydia avium sp. nov. and Chlamydia gallinacea sp. nov. Syst. Appl. Microbiol. 37,
79–88 (2014).
18. Sachse K., Kuehlewind S. et al. More than classical Chlamydia psittaci in urban pigeons. Vet.
Microbiol. 157, 476–480 (2012).
19. Vanrompay D., Ducatelle R., Haesebrouck F. Chlamydia psittaci infections: a review with emphasis on
avian chlamydiosis. Vet. Microbiol. 45, 93–119 (1995).
20. Zhang, F., Li, S., Yang, J. et al., 2008. Isolation and characterization of Chlamydophila psittaci isolated
from laying hens with cystic oviducts. Avian Dis. 52, 74–78.
21. Zhou, J., Qiu, C., Lin, G. et al., 2010. Isolation of Chlamydophila psittaci from laying hens in China.
Vet. Res. 3, 43–45.
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COMPARISON OF METHODS USED IN THE DETECTION OF YERSINIA
ENTEROCOLITICA IN MILK AND MEAT COMMODITIES: CULTIVATION
METHOD VERSUS PCR
Čuvalová, Z., Kubicová Z., Cabanová L., Kantíková. M., Mojžišová A.
Reference Laboratory for Yersinia spp. – State veterinary and food institute, Dolný Kubín
Introduction:
Yersinia
enterocolitica, gramnegative,
oxidase-negative,
fakultative
anaerobic bacteria is very heterogenous and can be divided into several serotypes
Y.enterocoliticastrains connected with human yersiniosis belong to biotypes 1B, 2, 3, 4, 5.
All virulent Yersinia strains carry approximately 70 kb plasmid called PYV (plazmid
na Yersinia virulence), which is crucial for bacteria survival and their multiplication in
lymphoid issues. Several studies show that even the strains of biotype 1A can cause
gastrointestinal illnesses with the same symptoms as the pathogenic biotypes even they do
not carry virulent plasmid (pYV).
Food poisoning caused by Y. enterocoliticais calledyersiniosis–acute infection zoonosis.
The most risky food are all types of raw material or products without heat treatment. They
are mainly raw meat (meat products, minced meat, smoked products, poultry), raw milk
and dairy products, contaminated water and vegetables and fruit. Even some outbreaks
after consummation of raw juices have been published.
Aim: Prevalence comparison of pathogenicY. enterocolitica in meat, vegetable and dairy
products, suitability assessment of the cultivation and PCR method.
Methodology:
Cultivation method
Detection of Y. enterocolitica was performed by the cultivation method ISO STN 10273.
Five typical colonies were chosen from each sample for further confirmatory testing. The
suspect colonies were further sub-cultured on blood agar and Engo agar and incubated at
30 °C for 24 hours. The suspect Yersinia colonies were identified with oxidase (ErbaLachema), Kligler iron (Immuna) and urea (Oxoid) tests. Oxidase-negative, glucosepositive, H2S-negative and urease-positive colonies were finally identified with API 20E (
bioMérieux) strips and ENTEROtest 24 (ErbaLachema). Serotyping was performed by
slide agglutination with commercially available O-antisera for the serogroups O: 1-2, O: 3,
O: 5, O: 8 and O: 9 involved in most cases of human yersiniosis in Europe (Y.
enterocolitica Antisera, Denka Seiken, Japan). The colonies that produced typical reaction
patterns were further confirmed as Y. enterocolitica and biotyped according the revised
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scheme of Waut et al. using the following tests: indole production and fermentation of
xylose, salicin, trehalose and aesculin hydrolysis.
Real-Time PCR
Qualitative real-time PCR method according to Lambertz et al., 2008 was used for
detection and identification of pathogenic Y. enterocolitica from enrichment media or from
bacterial culture. Bacterial DNA was extracted by means of heat shock. The principle of
the method is enzymatic amplification of gene fragment using technology of PCR with
primers and TaqMan® probe. Internal experimental method validation in VFI Dolný Kubín
determined LOD of the method less than or equal 5 DNA copies (LOD ≤ 5 DNA copies).
Results and Discussion: During 2013-2015 there were two monitoring programmes
organised by SVFA and MA SR where Y. enterocolitica was analysed in meat, vegetable
and dairy products which represent the risk for the consumer. Samples had been taken by
the official inspectors from district veterinary and food administration. As scientific
literature declares that using the PCR methods there is a higher percentage of positives,
Reference laboratory for Yersinia spp. in Dolný Kubín recommended to examine samples
concurrently by cultivation and PCR methods.
Concerning the examination of all 166 samples there was only one positive case detected
in chilled raw pork meat. Sample had been taken in retail and Poland was the country of
origin. This sample represents 1,49% of meat matrix (1/67). Even though the isolated
strain of Y. enterocolitica was not possible to examine with sera available in our institute,
according the Wauters scheme it was identified as biotype 1A – not pathogenic, but using
the PCR method the same isolate was confirmed as pathogenic.
Even though Y. enterocolitica is ubiquitous microorganism, majority of the isolates
obtained from non- symptomatic carriers, food and environmental samples are nonpathogenic. This is the main reason for identification the isolate pathogenicity.
Biotyping was performed by several fenotypic tests which is time consuming and not
always reliable but using Real-Time PCR the pathogenicicty of Y. enterocolitica isolates
can be revealed quickly and specifically.
Analysing 66 samples of raw cow milk for detection Y. enterocolitica there was no any
positive finding using the cultivation method. One of the reasons can be the seasonality of
bacteria which is considered to be a microbe with affinity to the cold months and the
monitoring program had been running from June to August. Neither in the vegetable
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matrices there was no any isolation of Yersinia spp. although these samples had been
analysed from October to November.
All 166 samples were analysed for detection of Y. enterocolitica by Real-time PCR.
Presence of DNA specific for pathogenic Y. enterocolitica was presented in 16 samples, 13
of them were meat and 2 of vegetable origin and 1 of raw milk. Taking into account the all
samples, 16% was positive (16/166), 19,4% were positive of meat products (13/67), 6,06%
were positive of the vegetable origin (2/33) and 1,51% raw cow milk were positive (1/66).
Since 2009 incidence in Slovakia is over the average in EÚ/EFTA, where is varies about
2,1/100 000 residence. In 2015 there were 218 cases in Slovakia (illness 4,02/100 000)
which increases in 33,7% in comparison with 2014. Therefore, it is important to keep
monitoring the risky food. Examination of pathogenic Y. enterocolitica in animals and food
should be performed together with exact case history of yersiniosis in humans.
Summary: Based on the study for detection the pathogenic Y. enterocolitica by Real-Time
PCR there were 16% positive findings. Comparing to cultivation method where no any
pathogenic and 1% of non-pathogenic bacteria were isolated, the sensitivity is 16-times
higher.
Cultivation and isolation of yersinia is complicated, time-consuming and labour, the
occurrence of high number of psychrotrophic bacteria gets worse the isolation from food,
bacteria from genus Pseudomonas and Proteus complicate the process in meat.
This study confirmed the first scientific opinions that method based on molecularbiological analysis using Real-Time PCR in comparison with cultivation method is much
more sensitive, reliable and faster.
Conclusion: As the cultivation method is considered not to be reliable mainly because of
the sensitivity and specificity and it is very time-consuming and labour, PCR methods are
recommended to be used. Not only the isolation but even the typing and pathogenicity
identification based on the phenotype is long-lasting and often not specific enough. In the
laboratories there is possible to provide the combination of molecular analysis by RealTime PCR together with cultivation method. First method is used for screening and
cultivation method would be used for the samples with positive PCR results. This
combination would increase the capture of pathogenic Y. enterocolitica with cultivation.
As the prevalence of Y. enterocolitica was the highest in meat food samples it would be
reasonable to monitor these risky products.
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Literature
1. Cabanová, L., Čuvalová, Z., 2014: Does Yersinia enterocolitica represent risk in our meat products? In:
Lectures and posters book from International Hygiena alimentorum XXXV, Štrbské Pleso, 21.-23.5.2014,
UVLF Košice, p. 68-72, ISBN 978-80-8077-401-1.
2. Cabanová, L., Čuvalová, Z., 2014. Results of the first Yersinia enterocolitica monitoring
programe in
Slovakia. Folia Veterinaria, ISSN 0015-5748, 2014, vol. 58, supplem. II, s. 13-15
3. Cabanová, L., Čuvalová, Z., 2014. Monitoring Yersinia enterocolitica in risky food– do we have anything
to worry about? Maso, ISSN 1210-4086, 2014, vol.5, s. 46-48
4. Cabanová L., 2013. Monitoring evaluation of presence of Yersinia enterocolitica – Final report of SVFI
in Dolný Kubín. In: Department of food safety and nutrition, Ministry of Agriculture and Rural development
Bratislava, date 30.11.2013, pp. 1-3
5. EFSA Journal 2015; 13(12):4329, str. 101 -104
6. Fredriksson-Ahomaa, M., Korkeala, H., 2003. Clinical Microbiology Reviews 2003, 16:2, pp.220-229
7. Hrušková, V., Kaclíková. E., 2009. Rapid and sensitive detection of pathogenic Yersinia enterocolitica
strains in food using selective enrichment and real-time PCR. In Journal of Food Nutrition Research 2009,
48:2, pp. 100-108
8. Lambertz, S. T., Nilsson, C., Hallanvuo, S., Lindblad, M., 2008. Real-Time PCR Method for Detection of
Pathogenic Yersinia enterocolitica in Food. In Applied and environmental microbiology 2008, 74:19, pp.
6060-6067
9. Mäde, D., Reiting, R., Strauch, E., Ketteritzsch, K., Wicke, A., 2008. A real-time PCR for Detection of
Pathogenic Yersinia enterocolitica in food combined with Universal Internal Amplification Control System.
In Journal für Verbraucherschutz und Lebensmittelsicherheit 2008, 3:2, pp. 141-151
10. STN EN ISO 10273 (2004) Microbiology of food and feed. Horizontal method for detection suspected
pathogenic bacteria Yersinia enterocolitica
11. TENNANT, S. M., T. H. GRANT, AND R. M. ROBINS-BROWNE (2003) Pathogenicity of Yersinia
enterocolitica biotype 1A. In FEMS Immunol. Med. Microbiol. 38:127–137
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FIRST EVIDENCEOF ZOONOTIC SPECIES CRYPTOSPORIDIUM MUSKRAT
GENOTYPES I AND II F IN RODENTS IN SLOVAKIA
O. Danišová1, A. Valenčáková1, M. Stanko2
1
Department of Biology and Genetics, University of Veterinary Medicine and Pharmacy,
Komenského 73, 041 81 Košice, Slovak Republic
2
Institute of Parasitology and Institute of Zoology, Slovak Academy of Science,
Hlinková 3, 040 01 Košice, Slovak Republic
[email protected]
Introduction: Cryptosporidium spp. is an important causative agent of intestinal
parasitoses-induced diarrhoea in humans and animals worldwide.1,2 Rodents (small
mammals) are globally expanded and overpopulated, which increases the risk of transfer of
human and zoonotic pathogens from the genus Cryptosporidium. Worldwide, the
prevalence of cryptosporidium infection is 1 – 62 % in mice and 2.3 – 32.8 % in rats.310
Rodents multiply rapidly and, may serve as a food source and as a reservoir of infection.
That is why their presence anywhere near the farms and holdings, sharing habitat with
livestock, occurrence near reservoirs of drinking water and residential areas, increases the
risk of transmission of infection cryptosporidiosis.
The aim of the study: In this study, Cryptosporidium was detected in wild
immunocompetent asymptomatic small mammals. Altogether 35 faecal samples were
collected from Hyľov-Hlboká valley areas in eastern Slovakia from four different rodent
species (Myodes glareolus, Apodemus agrarius, Apodemus flavicollis, Microtus
subterraneus), three samples originated from insectivore species (Sorex araneus).
Methodology: The samples were examined using a method modified in our laboratory,
based on the isolation of DNA with modification protocol and use combination of specific
primers pair on a small subunit rRNA (18S rRNA) gene for species identification.11-13After
DNA sequencing we were able to compare our sequences with the GenBank sequences.
Results and Discussion: The following species after sequencing were identified:
Cryptosporidium muskrat genotype I (n=3); Cryptosporidium muskrat genotype II (n=3).
Their sequences were identical with isolates from the environment and from animals living
in water or in its proximity. In animals these genotypes were identified for the first time in
muskrats (Ondatra zibethicus14-16), after which they were named. Shortly afterwards, were
identified both genotypes in humans.17Compared with other studies, the positivity in our
study reached 15.78 % in the identification of two species, which indicates the importance
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of this study, although it was conducted in a
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relatively small area of Slovakia, and the
significance of wild rodents acting as a source of cryptosporidium infection.This infection
of Cryptosporidium genotype muskrat I and Cryptosporidiummuskrat genotype II was
recorded in Slovakia for the first time and pointing out that the actual prevalence of
cryptosporidiosis of zoonotic species may be high.
Conclusion: The results of this study indicate low host specificity of the detected
Cryptosporidium species and imply the importance of free living small mammals in urban
and suburban habitats as a potential source of human cryptosporidiosis.
The study was supported by grantsAPVV-15-0134, APVV-14-0274 and VEGA 1/0061/16.
Literature
1.
Goodstein FR, et al., 1989. Bronchial and gastrointestinal cryptosporidiosis in AIDS. J Am Ostiopath
Assoc, 89, 195–197.
2.
McAnulty JM, et al., 1994. A community-wide outbreak of cryptosporidiosis associated with swimming
at a wave pool. J Am Med Associat, 272, 1597–1600.
3.
Klesius PH, et al., 1986. Infectivity of Cryptosporidium sp. isolated from wild mice for calves and mice.
J Am Vet Med Assoc, 189, 192–193.
4.
Sinski E, et al., 1993. Occurence of Cryptosporidium parvum infection in wild small mammals in
District of Mazury Lake (Poland). Acta Parasitol, 38, 59–61.
5.
Chalmers RM, et al., 1997. The prevalence of Cryptosporidium parvum and C. muris in Mus domesticus,
Apodemus sylvaticus and Clethrionomys glareolus in an agricultural system. Parasitol Res, 83, 478–482.
6.
Chilvers BL, et al., 1998. The prevalence of infection of Giardia spp. and Cryptosporidium spp. in wild
animals on farmland, southeastern North Island, New Zealand. Int J Environ Health Res, 8:59–64.
7.
Foo C, et al., 2007. Novel Cryptosporidium Genotype in Wild Australian Mice (Mus domesticus). Appl
Environ Microbiol, 73, 7693–7696.
8.
Kimura A, et al., 2007. Detection and genotyping of Cryptosporidium from brown rats (Rattus
norvegicus) captured in an urban area of Japan. Parasitol Res, 100, 1417–1420.
9.
Feng Y, et al., 2009. Cryptosporidium Genotype and Subtype Distribution in Raw Wastewater in
Shanghai, China: Evidence for Possible Unique Cryptosporidium hominis Transmission. J Clin
Microbiol, 47, 153–157.
10. Lv C, et al., 2009. Cryptosporidium spp. in Wild, Laboratory, and Pet Rodents in China: Prevalence and
Molecular Characterization. Appl Environ Microbiol, 75, 7692–7699.
11. Xiao L, et al., 1999. Genetic diversity within Cryptosporidium parvum andrelated Cryptosporidium
species. Appl Environ Microbiol, 65, 3386–3391.
12. Leetz AS, et al., 2007. An evaluation of primers amplifying DNA targets for the detection of
Cryptosporidium spp. using C. parvum HNJ-1 Japanese isolate in water samples. Parasitol
Res, Sep;101(4), 951–962.
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13. DanišováO, et al., 2016. Detection and identification of 6 Cryptosporidium species in livestock in
Slovakia by amplification of ssu and gp60 genes with the use of PCR analysis. Ann Agric Environ Med,
23(2), 254–258.
14. Ruecker
NJ, et
al., 2012. Molecular
and
phylogenetic
approaches for
assessing sources
of Cryptosporidium contamination in water.Water Res, Oct 15;46(16), 5135–5150.
15. Ruecker NJ, et al., 2007. Tracking host sources of Cryptosporidium spp. in raw water for improved
health risk assessment.Appl Environ Microbiol, Jun 73(12), 3945–3957. Epub 2007 May 4.
16. Wilkes G, et al., 2013. Spatiotemporal analysis of Cryptosporidium species/genotypes and relationships
with other zoonotic pathogens in surface water from mixed-use watersheds.Appl Environ Microbiol,
2013;Jan 79(2), 434–448.
17. Jelison KL, et al., 2009. Source tracking identifies deer and geese as vectors of humaninfectious Cryptosporidium genotypes in an urban/suburban watershed.Environ. Sci Technol, Jun
15,43(12), 4267–4372.
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HUMAN GENOTYPES OF ZOONOTIC PATHOGENS CRYPTOSPORIDIUM SPP.
IDENTIFIED IN RODENTS IN SLOVAKIA
O. Danišová1, A. Valenčáková1, E. Hatalová1, M. Stanko2, L. Luptáková1
1
Department of Biology and Genetics, University of Veterinary Medicine and Pharmacy,
Komenského 73, 041 81 Košice, Slovak Republic
2
Institute of Parasitology and Institute of Zoology, Slovak Academy of Science,
Hlinková 3, 040 01 Košice, Slovak Republic
[email protected]
Introduction: The most common human cryptosporidiosis is caused by zoonotic
species/genotypes C. hominis, C. parvum and C. meleagridis.1 Recently, however,
cryptosporidium infections in humans, caused by species/genotypes of C. felis, C. canis C.
muris, C. suis, C. andersoni a C. ubiquitum,C. baileyi, C. saurophilum, C. nasorum,
C.serpentis, C.wrairi, C. cuniculus, C. horse genotype, C. skunk genotype, C. hominis
monkey genotype, C. guinea pig genotype, C. koala genotype, C. chipmunk genotype and
also C. horse, C. rabbit, C. skunk and C. chipmunk genotype I have been detected
repeatedly.2 Infections by species C. parvum andC. hominis were reported in several places
in Europe: the Czech Republic3,England4, Denmark5, France6, the Netherlands7, Northern
Ireland8, Switzerland9,10, Scotland11, Slovak Republic12
The aim of the study: In this study, we focused on genotyping species of
Cryptosporidium parvum and Cryptosporidium hominis, which we identified in
asymptomatic immunocompetent wild small mammals. Individual genotypes were
compared with the detected genotypes on the territory Sovak Republic and even in
neighboring countries with genotypes of zoonotic potential.
Methodology: The nested PCR was conducted using a protocol modified in our laboratory
to amplify of GP60 gene coding 60-kDa glycoprotein for genotype determination. On the
basis of tandem repeats of serine-encoding trinucleotides: TCA, TCG and TCT at the 5
'end of the gene, and not to repeat variations in the number of trinucleotide repeats, we
divided C. parvum and C. hominis into several subtype families.13
Results and Discussion: The following species were identified: Cryptosporidium parvum
genotypes IIaA18G3R1, IIcA5G3a and IIiA10. From one of the rodent, the species
Cryptosporidium hominis genotype IbA10G2 was identified for the first time.
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Species
C. parvum
C. hominis
Genotypes/Subgenotypes
IIaA18G3R1
Hosts - first evidence
Human
Cattle
IIiA10
IIcA5G3a
Human
Human
IdA10G2
european hedgehog
Human
european hedgehog
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Locality
UK
Netherlands
Australia
New South Wales
Uganda
Japan
Australia
Australia
Germany
Australia
Australia
Australia
Poland
References
14
15
16
17
18
19
20
21
22
21
23
24
25
All genotypes of the species C. parvum and C. hominis identified in small rodentswere
detected in people, rodents and insectivores in many different places (the above citations)
which raises concern about zoonotic transmission in whichrodents and insectivores serve
as a vector of cryptosporidiosis as it was demonstrated in this study.
Conclusion: Exact identification of parasite to the species and genotype level is very
important for human and veterinary parasitology including taxonomy, diagnostics,
treatment,prevention of this parasite, which gives us an opportunity to exactly determine
the source of infection26.
The study was supported by grants APVV-15-0134, APVV-14-0274 and VEGA 1/0061/16.
Literature
1.
Morgan-Ryan UM, et al., 2002. Cryptosporidium hominis n sp. (Apicomplexa: Cryptosporiidae) from
Homo sapiens. J Eukaryot Microbiol, Nov-Dec, 49(6), 433–440.
2.
Xiao L, et al., 2004. Cryptosporidium Taxonomy: Recent Advances and Implications for Public Health.
Clin Microb Rev, 17, 72–97.
3.
Hajdušek O, et al., 2004. Molecular identification of Cryptosporidium spp. in animal and human hosts
from the Czech Republic. Vet Parasitol, 122, 183–192.
4.
Leoni F, et al., 2006. Genetic analysis of Cryptosponidium from 2414 humans with diarrhoea in England
between 1985 and 2000. J Med Microbiol, 55, 703–707.
5.
Enemark HL, et al., 2002. Molecular characterization of Danish Cryptosporidium parvum isolates.
Parasitol,125, 331–341.
6.
Guyot K, et al., 2001. Molecular characterization of Cryptosporidium isolates obtained from humus in
France. J Clin Microbiol,39, 3472–3480.
7.
Homan W, et al., 1999. Characterization of Cryptosporidium parvum in human and animals faeces by
single-tube polymerase chain reaction and restriction analysis. Parasitol Res, Aug, 85(8-9), 707–712.
8.
Lowery CJ, et al., 2001.
Molecular genotyping of human cryptosporidiosis in Northern Ireland:
epidemiological aspect and review. Ir Med Sci, 170(4), 246–250.
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9.
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Fretz R, et al., 2003. Genotyping of Cryptosporidium spp. isolated from human stool samples
in Switzerland. Epidemiol Infect, Aug, 131(1), 663–667.
10. Glaeser C, et al., 2004. Detection and molecular characterization of Cryptosporidium spp. isolated from
diarrheic children in Switzerland. Pediatr Infect Dis, J. Apr, 23(4), 359–361.
11. Mallon M, et al., 2003. Population structures and the role of genetic exchange in the zoonotic pathogen
Cryptosporidium parvum. J Mol Evol, 56, 407–417.
12. Petrincová A, et al., 2015. Molecular characterization and first report of Cryptosporidium genotypes in
human population in Slovak Republic. Electrophoresis, 00, 1–6.
13. Xiao L., 2010. Molecular epidemiology of cryptosporidiosis: an update. Exp Parasitol, 80-89.
14. Jex AR, et al., 2011. Oxford Textbook of Zoonoses: Biology, Clinical Practice, and Public Health
Control, Oxford University Press, Oxford 2011, 536–568.
15. Wielinga, PR, et al., 2008. Molecular epidemiology of Cryptosporidium in humans and cattle in The
Netherlands. Int J Parasitol, Jun, 38(7), 809–817. Epub 2007 Nov 4.
16. Ng J, et al., 2011. Molecular characterization of Cryptosporidium and Giardia in pre-weaned calves in
Western Australia and New South Wales. Vet Parasitol, Mar 10, 176(2-3), 145–150.
17. Ng JS, et al., 2012.. Evidence of Cryptosporidium transmission between cattle and humans in northern
New South Wales. Exp Parasitol, Apr, 130(4), 437–441.
18. Akiyoshi DE, et al., 2006. Subtype analysis of Cryptosporidium isolates from children in Uganda.J
Parasitol, Oct, 92(5), 1097–1100.
19. Abe N, et al., 2006. Subgenotype analysis of Cryptosporidium parvum isolates from humans and animals
in Japan using the 60-kDa glycoprotein gene sequences.Parasitol Res. Aug 99(3), 303–305. Epub 2006
Mar 25.
20. Waldron LS, et al., 2009. Glycoprotein 60 diversity in C. hominis and C. parvum causing human
cryptosporidiosis in NSW, Australia. Exp Parasitol, Jun, 122(2), 124–127.
21. Pangasa A, et al., 2010. Highly sensitive non-isotopic restriction endonuclease fingerprinting of
nucleotide variability in the gp60 gene within Cryptosporidium species, genotypes and subgenotypes
infective to humans, and its implications.Electrophoresis, May, 31(10), 1637–1647.
22. Dyachenko V, et al., 2010. Occurrence and molecular characterization of Cryptosporidium spp.
genotypes in European hedgehogs (Erinaceus europaeus L.) in Germany. Parasitol, Feb, 137(2), 205–
216.
23. Waldron LS, et al., 2011. Molecular epidemiology, spatiotemporal analysis, and ecology of sporadic
human cryptosporidiosis in Australia. Appl Environ Microbiol, Nov, 77(21), 7757–7765.
24. Koehler AV, et al., 2013. Genetic characterization of selected parasites from people with histories of
gastrointestinal disorders using a mutation scanning-coupled approach. Electrophoresis. Jun, 34(12),
1720–1728.
25. Krawczyk AI, et al., 2015. Presence of zoonotic agents in engorged ticks and hedgehog faeces from
Erinaceus europaeus in (sub) urban areas.Parasit Vectors, Apr 9, 8, 210.
26. Peng MM, et al., 2001. A comparison of Cryptosporidium subgenotypes from several geographic
regions. J Eukaryot Microbiol, 28–31.
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HARD TICKS AND TICK-BORNE PATHOGENS DANGEROUS TO HUMANS IN
URBAN PARKS OF THE CAPITAL OF UKRAINE
Didyk Yu. M.1,2, DerdákováM.1
1
Institute of Zoology SAS, Dúbravská cesta 9, Bratislava, 845 06, Slovakia,
[email protected]
2
Schmalhausen Institute of Zoology NAS of Ukraine, B. Khmelnytskogo Str., 15, Kyiv-30,
01030, Ukraine
Introduction: Hard ticks transmit a broad range of viral, bacterial and protozoan
pathogens to humans and animals. Among epidemiologically the most important diseases
are tick-borne encephalitis (TBE), Lyme borreliosis, granulocytic anaplasmosis, spotted
fever group rickettsiosis, tularemia and babesiosis (Rizzoli et al., 2014). In Europe, the
increase of tick-borne diseases are reported. It’s due to changes in human behaviour,
ecology and climate changes. Moreover, new studies show that tick-borne pathogens occur
not only in natural environments but also in European urban areas such as parks and small
green zones inside the city (Pangrácová et al., 2013; Schorn et al., 2011). That means city
central parks and gardens frequently used for leisure activities represent a perspective zone
for a potential contact with infected ticks, and for transmission of tick-borne pathogens to
humans and animals.
12 species of hard ticks occur in the capital of Ukraine Kyiv: I. trianguliceps, I. crenulatus,
I. kaiseri, I. arboricola, I. lividus, I. ricinus, I. apronophorus, I. laguri, D. reticulatus,
H. punctata, H. concinna, R. rossicus (Akimov & Nebogatkin, 2002). The most abundant
and epidemiologically the most important hard tick species are I. ricinus and
D. reticulatus. They are the main vectors of a wide spectrum of viral, bacterial and
protozoan pathogens (Rizzoli et al., 2014; Schorn et al., 2011).
Despite rising interest to tick-borne diseases in Europe, very little is known about the
prevalence of tick-borne pathogens in ticks from Ukraine. One study focused on engorged
I. ricinus and D. reticulatus ticks removed from dogs in the veterinary clinics in Kyiv
(Hamel et al., 2013) and the other one on questing D. reticulatusticks from the Chernobyl
exclusion zone (Karbowiak et al. 2014).
The aim of the studywas to investigate the prevalence of tick-borne pathogens dangerous
to humans and domestic animals in hard ticks from urban parks of Kyiv, Ukraine.
Methodology: During 2015 – 2016, hard ticks were collected by flagging the vegetation in
7 Kyiv parks: city park "Babyn Yar", Fomin Botanical Garden, Kytaivs'ki stavky,
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Holosiivskyi National Park, Gryshko National Botanical Garden, Darnytcia park and park
“Puchovka”. Collected ticks were identified to species and stages according to a taxonomic
key using a stereomicroscope (Emchuk, 1960). Genomic DNA was isolated individually
from each tick by the method of alkaline hydrolysis according to Guy and Stanek (1991).
PCR detection of 18S rRNA of Babesia spp. was performed according to Casati et al.
(2006). The primers IGSa and IGSb were used to amplify a fragment of the 5S-23S (rrfArrlB) intergenic spacer of the spirochetes from B. burgdorferi s.l. complex. Identification
of Borrelia genospecies was performed by the Tru1 restriction fragment length
polymorphism (RFLP) assay (Derdáková et al., 2003). The molecular detection of
A. phagocytophilum was done by Courtney et al. (2004).
Results and Discussion: A total of 395 Ixodes ricinus and 585Dermacentor
reticulatusticks were studied for the presence of tick-borne pathogens. B. burgdorferi s.l.
was detected in questing I. ricinus ticks from all studied sites. The prevalence of B.
burgdorferi s.l. in I. ricinus ticks was 26.8%: 14.7% infected adults and 12.1% nymphs.
We identified four genospecies: B. afzelii in 24.1% (52 adults, 43 nymphs), B. garinii in
2.3% (5 adults, 4 nymphs), B. burgdorferi s.s. 0.2% (1 male) and B. valaisiana 0.2% (1
nymph). These genospecies are considered to be the most abundant in Europe (Rizolli et
al., 2014). The overall mean prevalence of Borrelia spp. in questing ticks in Europe is
around 13.7% (from 0 up to 49.1%) (Rizzoli et al., 2011).This is the first case of molecular
identification of B. burgdorferi s.s. and B. valaisiana from hard ticks from Ukraine.
A. phagocytophilum was also found in all studied sites. The prevalence of infection in
I. ricinuswas 9.1% (36 ticks). D. reticulatus samples have only 1 infected tick (0.2%
prevalence) from Kytaivs'ki stavky, Holosiivskyi National Park. That’s an interesting
result, because previous study of questing D. reticulatus ticks from the Chernobyl
exclusion zone showed of 25.4% prevalence of A. phagocytophilum (Karbowiak et al.
2014).
A total of 4% I. ricinus (16 ticks) and 6% D. reticulatus (35 ticks) samples were confirmed
to contain DNA of Babesia spp.The infection rate of Babesia spp. in I. ricinuswas 2.5% for
adults and 1.5% for nymphs. D. reticulatus ticks showed 1.9% for males and 4.1% for
females. All positive samples will be further sequenced to identity of amplified DNA of
Babesia spp..
Conclusion: We have confirmed that hard ticks I. ricinus and D. reticulatus from urban
parks of the capital of Ukraine Kyiv are infected with tick-borne pathogens: B. afzelii,
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B. garinii, B. burgdorferi s.s., B. valaisiana, A. phagocytophilum and Babesia spp..Mostly,
they are in high risk for humans that indicate an increasing need for the medical and
veterinary attention to prevent infection of people and animals who are visiting city green
areas.
This study was financially supported by the National Scholarship Program for the Support
of Mobility of Students, PhD students, University Teachers, Researchers and Artists
(SAIA) in 2015-2016 and project APVV-14-0274.
Literature
1.
Rizzoli, A., Silaghi, C., Obiegala, A., Rudolf, I., Hubálek, Z., Földvári, G., Plantard, O., VayssierTaussat, M., Bonnet, S., Špitalská, E., Kazimírová, M., 2014. Ixodes ricinus and its transmitted
pathogens in urban and peri-urban areas in Europe: new hazards and relevance for public health.
Frontiers in Pub. Health. 2, 251.
2.
Pangrácová, L., Derdáková, M., Pekárik, L., Hviščová, I., Víchová, B., Stanko, M., Hlavatá, H., Peťko,
B. 2013. Ixodes ricinus abundance and its infection with the tick-borne pathogens in urban and suburban
areas of Eastern Slovakia. Parasit. Vectors. 6, 238–246.
3.
Schorn, S., Pfister, K., Reulen, H., Mahling, M., Silaghi, C., 2011. Occurrence of Babesia spp.,
Rickettsia spp. and Bartonella spp. in Ixodes ricinus in Bavarian public parks, Germany. Parasit.
Vectors. 4, 135.
4.
Akimov, I.A., Nebogatkin, I.V. 2002. Ticks of Kyiv – Urbozoological and Epyzootological Aspects.
Vestnik zoologii, 36(1): 91—95.
5.
Karbowiak, G., Víchová, B., Slivinska, K., Werszko, J., Didyk, J., Pet’ko, B., Stanko, M., Akimov, I.,
2014. The infection of questing Dermacentor reticulatus ticks with Babesia canis and Anaplasma
phagocytophilum in the Chernobyl exclusion zone. Vet. Parasitol. 204 (3-4), 372–375.
6.
Emchuk E.M. 1960. Fauna of Ukraine. Ixodid ticks. Academy of Sciences of Ukraine, Kyiv.
7.
Guy, E.C., Stanek, G., 1991. Detection of Borrelia burgdorferi in patients with Lyme disease by the
polymerase chain reaction. J. Clin. Pathol. 44, 610–611.
8.
Casati, S., Sager, H., Gern, L., Piffaretti, J.C., 2006. Presence of potentially pathogenic Babesia sp. for
human in Ixodes ricinus in Switzerland. Ann. Agric. Environ. Med. 13, 65–70.
9.
Derdáková, M., Beati, L., Peťko, B., Stanko, M., Fish, D., 2003. Genetic variability within Borrelia
burgdorferi sensu lato genospecies established by PCR-single-strand conformation polymorphism
analysis of the rrfA-rrlB intergenic spacer in Ixodes ricinus ticks from the Czech Republic. Appl. and
Env. Microbiol. 69 (1), 509–516.
10. Courtney, J.W.,Kostelnik, L.M., Zeidner, N.S., Massung, R.F.2004. Multiplex Real-Time PCR for
Detection of Anaplasmaphagocytophilum and Borrelia burgdorferi J. Clin. Microbiol. 42 (7), 3164–
3168.
11. Rizzoli A., Hauffe H. C., Carpi G., Vourc’h G. I., Neteler M., Rosà R. 2011. Lyme borreliosis in Europe.
Euro. Surveill.16 (27), 19906.
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TRICHINELLA (NEMATODA, TRICHINELLIDAE) AMONGST WILD AND
DOMESTIC MAMMALS IN UKRAINE
Didyk Yu. M., Akimov I. A.
Schmalhausen Institute of Zoology NAS of Ukraine, B. Khmelnytskogo Str., 15, Kyiv-30,
01030, Ukraine,
[email protected]
Introduction: Trichinellosis is one of the most dangerous helminthic diseases common to
humans and animals. It is caused by the nematodes from the genus Trichinella Railliet,
1895. Trichinella infections have been reported for almost all mammal species, as well as
for reptiles and birds (Pozio &Murrell, 2006). In Ukraine Trichinella infection was
detected in domestic pigs and humans (Shelemba, 1999; Derbal, 1997; Veterinary
Reports). Over the past 30 years more than 1,200 cases of human trichinellosis were found
in Ukraine (Sanitary-Epidemiological Service Reports). Infected pork used to be the main
source of human Trichinella infection in past, but the consumption of infected game
caused most recent human trichinellosis (Artemenko, 1997, Shelemba, 1999, Didyk, 2006).
The aim of the studywas to study the prevalence and species composition of Trichinella
among wild animals in Ukraine.
Methodology: Materials were collected from 2002 to 2015 during the hunting seasons in
all regions of Ukraine. The muscle tissue samples from ungulates (wild boar, roe) and
carnivores (bear, lynx, wolf, fox, marten, badger) were studied. The samples were
examined following the standard protocol of ITRC. The identification of parasites was
carried out by multiplex PCR analyses (Borsuk et al., 2003; Pozio & La Rosa, 2003).
Results and Discussion: Trichinella has been found in all regions of Ukraine. Larvae were
detected in 3% investigated wild boars, 15.5% wolves, 16% red foxes, 12% martens, 10%
badgers. Wolves and foxes were found to be the main reservoir of Trichinella in a sylvatic
cycle. Our studies demonstrate the presence of tree species of Trichinella in Ukraine.
T. britovi (ITRC codes: ISS1590, 1591, 1592, 1593) is found in wild boars, wolves, foxes
and martens from the Carpathians (the Transcarpathian region), and also from the Volyn,
Zhitomir, Kyiv and Novgorod-Seversky Polesie (Akimov et al., 2005). T. spiralis
(ISS1594) is found in the domestic pigs from the Kyiv Polesie. At the same time, T. nativa
(ISS1595) is found only in the foxes from the Chernihiv Polesie.Mixed infections
T. britovi-T. spiralis were found in southern regions.T. britovi (found in the majority of
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infected wolves and red foxes) is a dominant species in the sylvatic cycle in Ukraine. The
intensity of infection was the highest in animals from the Ukrainian Carpathians (350-380
larvae/10 g) and lowest in Polesie (4-50 larvae/10 g). The infestation extent of wild
mammals’ trichinellosis is on its raise over the past 30 years in Ukraine: 3.5% (Kondratyev
et al., 1976), 8.8% (Kulikova, 1989) and 15% (our data). Probably, it’s caused by high
densities of predators’ populations that correspondingly lead to increased levels of
scavenging and cannibalism, and also humans have big influence on Trichinella
prevalence, because hunters leave animal carcasses in forests like “baits” or throw it away
as garbage near villages.
Conclusion: Tree species of Trichinella (T. spiralis, T. britovi and T. nativa) and mixed
infections are found in Ukraine amongst wild and domestic animals.The infestation extent
of wild mammals’ trichinellosis is on its raise over the last years. This could increase
Trichinella prevalence amongst wild animals in sylvatic cycles and make a risk for
infection animals in domestic cycles and for humans.
This study was financially supported by the project № 289 (17.04.2015)NAS of Ukraine.
Literature
1.
Pozio, E., Murrell, K.D. 2006. Advances in Parasitology. 63, 367–439.
2.
Shelemba, I.Y. 1999. Med. Parasit. and Parasit. Dis. 1, 8-10.
3.
Derbal, M.Yu. 1997. Veterinary Med. of Ukraine. 9, 25.
4.
Reporst of the Central Laboratory of Veterinary Medicine of Ukraine (for 1995-2015 years).
5.
Reporst of the Sanitary-Epidemiological Service of Ukraine (for 1970-2015 years).
6.
Artemenko, Y., Sinitsyn, V., Derbal, M. 1997. Veterinary Med. 2, 24-26.
7.
Didyk, Yu. M. 2006. Vestnik Zoologii. 40 (5), 457-461.
8.
Borsuk, P., Moskwa, B., Pastusiak, K., Cabaj, W. 2003. Parasit. Research. 91, 374-377.
9.
Pozio, E., La Rosa, G. 2003. Methods Mol. Biol. 216, 299–309.
10. Akimov, I.A., Didyk, Yu.M., Pastusiak, K., Cabaj, W. 2005. Vestnik Zoologii. 19, 24-25.
11. Kondratyev, I.A., Gavrilyuk, N.D., Denisenko V. 1977. Med. Parasit. and Parasit. Dis. 5, 622-624.
12. Kulikova, N.A. 1989. Med. Parasit. and Parasit. Dis. 6, 51-54.
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CHARACTERIZATION OF CRONOBACTER STRAINS ISOLATED FROM
DIFFERENT SOURCES
Drahovská H.,Kajsík M., Belanová V., Šoltýs K., Szemes T., Turňa J.
Department Molecular Biology, Faculty of Natural Sciences, Mlynská dolina B-2,
84215 Bratislava, Slovakia
[email protected]
Introduction: Cronobacter spp. are Gram-negative, rod shaped bacteria from the family
Enterobacteriaceae. Currently genus Cronobacter contains seven species, strains
belonging to three species (C. sakazakii, C. malonaticus, and C. turicensis) are
predominantly isolated from human infections and some clones with enhanced virulence
potential have also been described within these species (1, 2). Cronobacter is an
opportunistic pathogen that can cause serious infections in neonates, including meningitis,
necrotising enterocolitis and sepsis with low frequency, but high lethality rate (2).
Infections in adults also have been reported, in particular among the elderly and
immunocompromised patients (3). Whereas Cronobacter is ubiquitous and has been
isolated from various foods and environments the main vehicle for its transmission in
neonatal infections is rehydrated powdered infant formula (PIF) (4, 5, 6). In comparison
with other Enterobacteriaceae, Cronobacter is considerably resistant to desiccation and
this property contributes to its survival in PIF (7). Thermal tolerance is another important
factor for Cronobacter survival in food (8).
The aim of the study: This work was aimed at occurrence and diversity of Cronobacter
spp. in several sources covering food of animal and plant origin, powered infant meals and
samples isolated from patients and healthy volunteers to compare variability of
Cronobacter strains of food and clinical origin.
Methodology: Food products and powdered infant meals were analyzed in in the State
Veterinary and Food Institute Bratislava and in Public Health Authority of the Slovak
Republic for the presence of Cronobacter spp. using currently available standard
microbiological method ISO/TS 22964: 2006 (5, 6). Throat swabs were cultivated
overnight in BPW and Cronobacter strains were isolated by inoculation on DFI medium.
Presumptive Cronobacter strains were confirmed by genus specific PCR (9, 10). MLST
and PCR-serotyping of strains were prepared according to published protocols (11, 12).
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Results and Discussion: In our study, various food, powered infant meals and clinical
samples were analyzed for the presence of Cronobacter (Table 1). Out of 604 food
analysed samples, 71 (12 %) were found to be positive. We confirmed that food of plant
origin were more frequently positive (31 %) comparing to animal products (6%). The
highest contamination was observed in desiccated foods, e. g. spices, teas, chocolate, nuts
and pastries as well as in vegetables. It is also interesting that the majority of positive
samples of animal origin contained plant ingredients, namely herbs and spices. Six
Cronobacter species were observed in samples, C. sakazakii was the most prevalent with
55 (77 %) strains, followed by C. malonaticus with 9 (12 %), C. turicensis, C. dublinensis
and C. condimenti were isolated only rarely. In further study powdered infant formulae and
follow-on formulae products available on Slovak market were analyzed and eight of 916
(0.9 %) samples were detected to be Cronobacter positive; two of them were powdered
infant milk products intended for the newborns and infants younger than six months of age
and six samples belonged to other milk-based and cereal-based follow-on powdered infant
foods for the children older than six months (Table 1). Overall 13 isolates were obtained by
repeated analysis of positive formulae, which were identified as C. sakazakii (12 strains)
and C. dublinensis (one isolate). The MLST method was used for molecular typing an the
isolates were assigned into ten different sequence types. Only one analyzed strain was
assigned to C. sakazakii ST4, the highly virulent sequence type responsible for the
majority of Cronobacter meningitidis cases. Two strains which were isolated from
different powdered infant formulae were assigned to another highly prevalent sequence
type ST1. Cronobacter strains from food were compared with human isolates. No positive
samples were detected by analysis of 26 throat swabs from healthy volunteers. However,
14 from 211 (7 %) analyzed throat swabs isolated from
hospital patients were
Cronobacter positive. Twelve strains were identified as C. sakazakii belonging to MLST
clonal complex CC4 (8 strains) and to sequence type ST8, two other strains belonged to C.
malonaticus ST7. All MLST types of clinical strains detected in our study belonged to
clones which are frequently isolated from infant (ST4) or adult (ST7 and ST8) patients (2,
3).
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Table 1: Number of positive/analysed samples for Cronobacter spp. in different
sources
Origin
Food
products
Powdered
infant meals
Humans
Sample
category
No.
analysed
samples
No. positive
samples
Plant
131
41
Animal
471
29
398
2
C. sakazakii, C. dublinensis
518
6
C. sakazakii
211
14
C. sakazakii, C. malonaticus
26
0
Powdered
infant formula
Follow on
formulae
Clinical
samples
Healthy
volunteers
Species
C. sakazakii, C. malonaticus, C.
turicensis, C. dublinensis, C.
muytjensii
C. sakazakii, C. malonaticus, C.
dublinensis, C. muytjensii, C.
condimenti
Conclusion: Cronobacter strains were isolated with high frequency from food, especially
from that of plant origin. However, the Cronobacter presence in powdered infant meals
was low due to strict hygienic measures in production facilities. Strains isolated from food
and powdered infant meals differed from clinical strains in species distribution and virulent
MLST types were less frequent in food samples comparing to clinical strains.
Acknowledgement: This work is a result of implementation of the project Development of
Competence
center
for
research
and
development
in
molecular
medicine,
ITMS:26240220071 supported by the Research & Development Operational Programme
funded by the ERDF.
Literature
1.
Joseph, S., Cetinkaya, E., Drahovska, H., Levican, A., Figueras, M.J., Forsythe, S.J., 2012.. Int. J. Syst.
Evol. Microbiol. 62, 1277-1283.
2.
Holy, O., Forsythe, S., 2014. J. Hosp. Inf. 86, 169-177.
3.
Alsonosi, A., Hariri, S., Kajsik, M., Orieskova, M., Hanulik, V., Roderova, M., Petrzelova, J., Kollarova,
H., Drahovska, H., Forsythe, S., Holy, O., 2015. Eur. J. Clin. Microbiol. Inf. Dis. 34, 1979.
4.
Yan, Q.Q., Condell, O., Power, K., Butler, F., Tall, B.D., Fanning, S., 2012. J. Appl. Microbiol. 113, 115.
5.
Turcovsky, I., Kunikova, K., Drahovska, H., Kaclikova, E., 2011. Antonie van Leeuwenhoek 99, 257269.
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6.
18th-20th October 2016
Gicova, A., Orieskova, M., Oslanecova, L., Drahovska, H., Kaclikova, E., 2014. Identification and
characterization of Cronobacter strains isolated from powdered infant foods. Let. Appl. Microbiol. 58,
242-247.
7.
Jaradat, Z.W., Al Mousa, W., Elbetieha, A., Al Nabulsi, A., Tall, B.D., 2014.. J. Med. Microbiol. 63,
1023-1037.
8.
Orieskova, M., Kajsik, M., Szemes, T., Holy, O., Forsythe, S., Turna, J., Drahovska, H., 2016. Antonie
van Leeuwenhoek. 109, 405-414.
9.
Lehner, A., Nitzsche, S., Breeuwer, P., Diep, B., Thelen, K., Stephan, R., 2006. BMC Microbiol. 6, 15.
10. Mohan Nair, M.K., Venkitanarayanan, K.S., 2006. Appl. Environm. Microbiol. 72, 2539-2546.
11. Blazkova, M., Javurkova, B., Vlach, J., Goselova, S., Karamonova, L., Ogrodzki, P., Forsythe, S., Fukal,
L., 2015. Appl. Environm. Microbiol. 81, 5574-5582.
12. Joseph, S., Forsythe, S.J., 2012. Frontiers Microbiol. 3, 397.
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SIMULTANEOUS DETECTION AND DIFFERENTIATION OF FLAVIVIRUSES
BY PCR AND REVERSE LINE BLOT (RLB)
P. Drzewnioková, T. Csank, E. Schréterová, J. Pistl
Department of microbiology and immunology, The University of veterinary medicine and
pharmacy in Košice
[email protected], [email protected]
Introduction: Arboviruses (an acronym for “arthropod-borne viruses”) form an ecological
but not taxonomic grouping and there are about 500 of them worldwide, belonging to nine
families 1. Tick-borne encephalitis virus (TBEV), West Nile virus (WNV) and Usutu
virus (USUV) are viruses belonging to family Flaviviridae (Flavivirus), which have the
greatest medical and veterinary importance in Central Europe. Reverse Line Blot
Hybridization assay (RLB) allows the detection of high number of molecular targets in
several samples using multiplex PCR (mPCR) reaction followed by probe hybridization
on a nylon membrane. Probes are 5' amine modified to allow fixation to the membrane.
Primers are 5' biotin modified which allows detection of hybridized PCR products using
streptavidin-peroxidase and a chemiluminescent substrate via photosensitive film or CCD
chip. With low setup and consumable costs, this technique is relatively inexpensive, high
throughput and has a short turnaround time [2].
Methodology: Eight bird brains and one tick pool have been used as clinical samples. As
positive controls cells infected with WNV 578/10 (kindly provided by Doc. Bakonyi
Tamás, University of St. Stephen, Budapest), TBEV Hypr (kindly provided by RNDr.
Boris Klempa, DSc., Institute of Virology SAS, Bratislava) and USUV 939/01 (kindly
provided by prof. Nowotny Norbert, University of veterinary medicine Vienna) have been
used. Uninfected Vero cells and destilled DEPC-treated water have been used as negative
controls. Isolation of viral RNA was done by using commercially manufactured kit
QIAamp Cador Pathogen Mini Kit (QIAGEN) according to the manufacturer's manual.
Maximum 5 micrograms of RNA were reverse-transcribedinto cDNAusing random
hexamers and reverse transcriptase (RevertAid H Minus, Thermo Scientific)according to
manufacturer's protocol. In PCR(DreamTaq master mix, Thermo Scientific) 5´ biotynilated
forwardgeneric primer PanFlavi-NS5-F [3] and non-modified cFD2-REV [4] reverse
primer in a concentration of 2.5 mM has been used. The oligonucleotides flank a 599 bp
sequence of the NS5 gene. The thermal profile was as follows: 1 × 95 °C for 1 min; 40 x
95 °C 45 sec, 50 °C 40 sec, 72 °C for 45 sec, 72 °C for 5 minutes. Result was checked by
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electrophoresis (1.5% agarose, 90 V / 35 min). Nylon membrane Biodyne C was activated
by 16% EDAC solution at room temperature for 10 min and subsequently rinsed in
deionized water. Probes were 5' amine modified to allow fixation to the membrane. Probe
WNVinNS5-Probe4 (5'-CCC ATC ATG ATG TTG TAR CAR GTG T-3'), has been
proposed as a specific probe for the detection of WNV within the PCR product. For
detection of TBEV two probes were designed, TBEVinNS5-Probe1 (5'-GCT CTC ATG
ATG ACC TTT GTG CCA GGC TG-3') and degenerated TBEVinNS5-Probe1 D (5'-GCT
CTC ATG ATG ACYYTY GTR CCR GGC TG-3'). For USUV detection one probe was
designed, USUVinNS5-Probe1 (5'-CCT CTT AAA CTC TTC CCT GGT GCA-3'). All
probes were diluted in 0.5 M NaHCO3 to a concentration of 10 pM, 5 pM, 2,5 pM and 1
pM and immobilized on the activated membrane. First line, last line and lines in between
probes were filled with 0.5 M NaHCO3. After 5 minute of incubation at room temperature
the membrane was washed with these solutions in the following order: 0.1 M NaOH at
room temperature for 9 minutes, 2×SSPE at room temperature for 30 sec and finally 2×
SSPE / 0.1% SDS for 5 min at 60 °C. Twenty microlitres of PCR product was mixed with
150 μl of 2× SSPE / 0.1% SDS and denatured for 10 minutes at 100 °C. Subsequently,the
lines perpendicular to probes were filled with denatured PCR product mixes. First and last
line were filled with 2× SSPE / 0.1% SDS. Membrane was then incubated for 60 min at 60
°C and then washed twice with pre-warmed 2× SSPE / 0.5% SDS for 10 min at 60 °C.
After washing, the membrane was incubated with streptavidin-peroxidase conjugate for 60
min at 42 °C. After a two times washingwith 2× SSPE / 0.5% SDS for 10 min at 42 °C and
2× SSPE for 5 minutes at room temperature, the reaction was visualized using a
chemiluminescent solution (Amersham ECL Western Blotting Detection Reagent, GE
Healthcare Life Sciences).The light emission was recorded on a CCD chip (LI-COR
Biosciences).The membrane was then washed twice in 1% SDS for 30 min at 80 °C and
once in 20 mM EDTA for 15 min at room temperature. After regeneration the membrane
was stored in plastic bag or box with 20 mM EDTA and placed in fridge at 4 °C for
subsequent re-use.
Results and Discussion: TBEV, WNV and USUV viruses belong to the genus Flavivirus.
Selected primers are targeting 599 bp fragment of NS5 gene. NS5 gene codes RNAdependent RNA polymerase, which is conserved among flaviviruses and allows design of
generic primers. Generic primers amplified flavivirus RNA what was proved by
electrophoresis(Fig. 1). Specific probes have been designto detect WNV, USUV and
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TBEV.For TBEV detection degenerated probe made stronger signal. We determined the
most appropriate concentration of the probes to be 5 pM, because it is the lowest
concentration at which we can still see a sufficient signal.The specificity of reaction is
improved by higher hybridization temperature. All probes we have designed were specific
and were able to detect targetvirus sequences also in clinical samples at 60 °Chybridization
temperature (Fig. 2).
1 2
3 4
5 6 7
Legend: 1, 15 – 100 bp Ladder, 2 –
TBEV Hypr, 3 – USUV 939/10, 4 –
WNV 578/10, 5 – clinical sample (CS)
1, 6 – CS 2, 7 – CS 3, 8 – CS 4, 9 –
CS 5, 10 – CS 6, 11 – CS 7, 12 –CS 8,
13 – VERO cells, 14 – negative
control of PCR. Clinical samples 2, 3
and 5 are WNV positive. Rest of
clinical samples are negative for
presence of targeted fragment of NS5
gene.
8 9 10 11 12 13 14 15
Figure 1: PCR result employing generic PanFlavi-NS5-BTN-F andcFD2-R primers
Legend: A, O – 2 x
SSPE / 0,1 % SDS, B –
WNV 578/10, C –clinical
sample(CS) 2, D – CS 3,
E – CS 5, F – CS 1, G –
CS 4, H – CS 7, I – TBEV
Hypr, J – CS 6, K – CS 8,
L – USUV 939/01, M –
Legend: A, O – 2 x SSPE /
0,1 % SDS, B – WNV
578/10, C – CS 2, D – CS
3, E – CS 5, F – CS 1, G –
CS 4, H – CS 7, I – TBEV
Hypr, J – CS 6, K – CS 8,
L – USUV 939/01, M –
VERO cells, N – Negative
control of PCR. Spots,
which are not located in
place were probes are
bounded, are smaller in
size and have irregular
shape, are probably the
result
of
insufficient
washing.
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Figure 2: Result of RLB with specific probes
The RLB method permits simultaneous detection of a large number of PCR amplicons.
The advantages of RLB include a relatively low cost, high throughput and short turnaround
time and also re-use of the membrane with bounded probes [2]. On one membrane multiple
probes can be bound, so not only various genes of viruses, but also their different lines or
subtypes can be determined. Probes we have currently designed are located within the NS5
gene, but design of probes in other Flavivirus genes are needed to increase specificity of
the assay. In case of various probes in multiple genes, mPCR would be necessary. The
combination of mPCR and RLB assay has proved its strength in detection of multiple virus
species [5].
The very next step will be to design specific probes to differentiate individual lines, or
subtypes and to determine the sensitivity of these reactions. The disadvantages of RLB
assay are relatively complicated manipulation and possible miss-interpretation of results,
because of relatively difficult orientation on membrane. However, these issues may be
solved by cutting a corner of the membrane and optimizing the set-up of positive controls,
thus improve the reading of results.
Acknowledgement: This work was supported by VEGA 1/0729/16, ITMS 26220120002
(INFEKTZOON) a ITMS 26220220185 (MediPark).
Literature
1.
Hubálek, Z., Rudolf, I., Nowotny, N. (2014). Chapter Five - Arboviruses Pathogenic for Domestic and
Wild Animals, In: Karl Maramorosch and Frederick A. Murphy, Editor(s), Advances in Virus Research,
Academic
Press
(89),
Pages
201-275,
ISSN
0065-3527,
ISBN
9780128001721,
http://dx.doi.org/10.1016/B978-0-12-800172-1.00005-7.
2.
O'Sullivan, M.V.N., Zhou, F., Sintchenko, V., Kong, F., Gilbert, G.L. (2011). Multiplex PCR and
Reverse Line Blot Hybridization Assay (mPCR/RLB). J. Vis. Exp. (54), e2781, doi:10.3791/2781
3.
Csank, T.; Bhide, K.; Bencúrová, E.; Dolinská, S.; Drzewnioková, P.; Major, P.; Korytár, Ľ.; Bocková,
E.; Bhide, M. a Pistl, J. (2016).Detection of West Nile virus and Tick-borne encephalitis virus in birds in
Slovakia with universal primer set.Archives of Virology, 161(6). 1679–1683. doi:10.1007/s00705-0162828-5
4.
Scaramozzino, N., Crance, J.-M., Jouan, A., DeBriel, D. A., Stoll, F., Garin, D. (2001). Comparison
of Flavivirus Universal Primer Pairs and Development of a Rapid, Highly Sensitive Heminested Reverse
Transcription-PCR Assay for Detection of Flaviviruses Targeted to a Conserved Region of the NS5
Gene Sequences. Journal of Clinical Microbiology, 39(5), 1922–1927. doi: 10.1128/JCM.39.5.19221927.2001
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Korimbocus J., Scaramozzino N., Lacroix, B., Crance, J. M., Garin, D., Vernet, G. (2005). DNA Probe
Array for the Simultaneous Identification of Herpesviruses, Enteroviruses, and Flaviviruses. J. Clin.
Microbiol: 43(8), 3779-3787. doi: 10.1128/JCM.43.8.3779-3787.2005
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ALIMENTARY INTOXICATIONS CAUSED BY FOODSTUFF CONTAINING CN CHEMICAL GROUP
R. Eckerová, M. Toropila, D. Toropilová, M. Tomko, M. Vargová, K. Veszelits –
Laktičová, F. Zigo
University of Veterinary Medicine and Pharmacy in Košice, 040 01, Slovak Republic
Department of biology, zoology and radiobiology
[email protected]
Introduction: Cyanide is a chemical group consisting of one atom of carbon connected to
one atom of nitrogen by three molecular bonds (C≡N) and cyanides are compounds
(substances formed by the joining of two or more atoms) that contain a cyanide group
(typically shown as CN). Cyanides can both occur naturally or be man-made and many are
powerful and rapid-acting poisons. Hydrogen cyanide (HCN), which is a gas, and the
simple cyanide salts (sodium cyanide and potassium cyanide) are common examples of
cyanide compounds. Certain bacteria, fungi, and algae can produce cyanide, and cyanide is
found in a number of foods and plants. In certain plant foods, including almonds, millet
sprouts, lima beans, soy, spinach, bamboo shoots, and cassava roots (which are a major
source of food in tropical countries), cyanides occur naturally as part of sugars or other
naturally-occurring compounds (ARSDR, 2006).
Aim of study: Cyanide occurs naturally as cyanogenic glycosides in at least 2000 plants.
Amygdalin (D-mandelonitrile-β-D-glucoside-6-β-D-glucoside) has been found in about
1000 species of plants, including cassava (tapioca, manioc), sweet potato, corn, cabbage,
linseed, millet, and bamboo, in pits of stone fruits, such as cherries, peaches, and apricots,
and in apple seeds (JECFA, 1993; Sharma, 1993; Padmaja, 1995). It is also present in
bitter almonds and American white lima beans (Ermans et al., 1972).
Figure
1
Hydrolysis
of
amygdalin
Many edible plants contain cyanogenic glycosides, whose concentrations can vary widely
as a result of genetic and environmental factors, location, season, and soil types (JECFA,
1993).
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The roots of cassava are rich in carbohydrates, mainly starch. According to Food and
Agriculture Organization, cassava is the third most important source of calories in the
tropics, after rice and corn. Cassava is consumed in a variety of ways, including eaten as
whole root, grated root or root chips. Cassava tubers vary widely in their cyanogenic
glycoside content, although most varieties contain 15– 400 mg cyanide/kg fresh weight.
Occasionally varieties of cassava tubers contain 1300–2000 mg cyanide/kg fresh weight,
and cassava leaves contain 1000–2000 mg cyanogenic glucosides/kg on a dry matter basis
(Padmaja, 1995).
Fermentation of cassava pulp for 96 h during gari production reduced the hydrogen
cyanide content by 50%; soaking of sliced cassava for 24 h, 40%; and sun-drying, some
15% (Kendirim et al., 1995).
Table 1 Cyanide concentrations in food products
Type of product
Cereal grains and their products
Soy protein products
Soybean hulls
Apricot pits, wet weight
Home-made cherry juice from pitted
fruits
Home-made cherry juice containing
100% crushed pits
Commercial fruit juices
Cherry
Apricot
Prune
Tropical foodstuff
Cassava (bitter) / dried root cortex
Cassava (bitter) / leaves
Cassava (bitter) / whole tubers
Cassava (sweet) / leaves
Cassava (sweet) / whole tubers
Gari flour (Nigeria)
Sorghum / whole immature plant
Bamboo / immature shoot tip
Lima beans from Java (coloured)
Lima beans from Puerto Rico (black)
Lima beans from Burma (white)
Cyanide concentration (in mg/kg or mg/litre)
0.001-0.45
0.07-0.3
1.24
89-2170
5.1
23
4.6
2.2
1.9
2360
300
380
451
445
10.6-22.1
2400
7700
3000
2900
2000
Methodology: In humans, the clinical signs of acute cyanide intoxication include rapid
respiration, drop in blood pressure, rapid pulse, dizziness, headache, stomach pain,
vomiting, diarrhoea, mental confusion, twitching and convulsions. Death due to cyanide
poisoning can occur when the cyanide level exceeds the limit an individual is able to
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detoxify. The acute lethal dose of hydrogen cyanide for humans is reported to be 0.5 to 3.5
mg per kilogram of body weight. Children are particularly at risk because of their smaller
body size.Chronic cyanide intoxication may lead to the development of certain conditions
including disturbance of thyroid function and neurological disorders. It tends to affect
those individuals who have regular long-term consumption of cassava with poor nutrition
status.
Results and Discussion: Although acute cassava poisoning — sometimes leading to the
death of whole families - has been occasionally reported after the consumption of
inadequately processed cassava (Cliff & Countinho, 1995), a much larger literature is
available on the effects of long-term exposure to food containing cyanogenic glycosides.
Accidental poisonings have been reported in children (and, exceptionally, in adults) who
had ingested apricot kernels or seeds or candy made from apricot kernels containing D,Lamygdalin, which, after hydrolysis, yields cyanide (Suchard et al., 1998). Presumably
because of lower body weight, children are especially vulnerable, with several fatal
poisonings occurring after they had consumed apricot seeds. It has been estimated that,
depending on the total cyanogenic potential of apricot seeds, 10 or more seeds could be
fatal to a child (Nahrstedt, 1993).
Accidental choke cherry poisonings (attributed to D,L-amygdalin) have also been reported
(Pentore et al., 1996). Pentore et al. (1996) described a case of a 56-year-old woman in
Italy who was accidentally poisoned when she ingested choke cherries whose pulp
contained cyanide (amygdalin).
Conclusion: Given the state of the science, the pressure on the food supply and the
development of new products, the FDA has performed admirably in protecting the
consumer from exposure to toxins in food with its judicious use of warning labels, action
levels, tolerances, specifications, prohibitions and the ability conferred by Congress to
declare substances “unsafe” or “unfit for food.” However, the FDA cannot protect
consumers absolutely from exposure to toxins normally present in foods. At normal levels
of food consumption, there is little potential for toxicity from natural food toxins.
Nevertheless, there is always the possibility of an idiosyncratic response or undetected
contamination (Laurie et al., 2010).
Literature
1.
Agency for Toxic Substances and Disease Registry. Toxic Substances Portal - Cyanide.2006.
http://www.atsdr.cdc.gov/PHS/PHS.asp?id=70&tid=19
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Cliff J, Coutinho J. Acute intoxication from newlyintroduced cassava during drought in Mozambique.
1995. Tropical Doctor, 25:193.
3.
Ermans AM, Delange F, Van Der Velden M, Kinthaert S. Possible role of cyanide and thiocyanate in the
etiology of endemic cretinism. 1972. Advances in Experimental Medicine and Biology, 30:455–486.
4.
JECFA. Cyanogenic glycosides. In: Toxicological evaluation of certain food additives and naturally
occurring toxicants.1993. Geneva, World Health Organization, 39th Meeting of the Joint FAO/WHO
Expert
Committee
on
Food
Additives
(WHO
Food
Additives
Series
30).
http://www.inchem.org/documents/jecfa/jecmono/v30je18.htm.
5.
Kendirim OC, Chukwu OA, Achinewhu SC. Effect of traditional processing of cassava on the cyanide
content of gari and cassava flour. 1995. Plant Food for Human Nutrition, 48:335–339.
6.
Laurie CD, Matulka RA, George AB. Naturally Occurring Food Toxins. Toxins (Basel). 2010 Sep; 2(9):
2289–2332.
Published
online
2010
Sep
20. doi: 10.3390/toxins2092289.
ttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC3153292/
7.
Nahrstedt AF. Cyanogenesis and food plants. In: van Beek TA, Breteler H, eds. 1993. Proceedings of the
International Symposium on Phytochemistry and Agriculture, 22–24 April 1992, Wageningen. Oxford,
Oxford University Press, pp. 107– 129.
8.
Padmaja G. Cyanide detoxification in cassava for food and feed use. 1995. Critical Reviews in Food
Sciences and Nutrition, 35:259–339.
9.
Pentore R, Venneri A, Nichelli P. Accidental choke cherry poisoning: early symptoms and neurological
sequelae of an unusual case of cyanide intoxication.1996. Italian Journal of Neurological Science,
17:233–235.
10. Sharma RP. Cyanide containing foods and potential for fetal malformations. In: Sharma RP, ed. Dietary
factors and birth defects. 1993. San Francisco, CA, American Association for the Advancement of
Science, Pacific Division, pp. 332–348.
11. Suchard JR, Wallace KL, Gerkina D. Acute cyanide toxicity caused by apricot kernel ingestion. 1998.
Annals of Emergency Medicine, 32:742–744.
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PREVALENCE OF CHLAMYDIA FELIS INFECTION ACROSS VARIOUS
POPULATIONS OF CATS AND ITS IMPACT FOR PUBLIC HEALTH
Halánová M., Kalinová Z., Čechová L., Babinská I., Čisláková L.
Pavol Jozef Šafárik University, Faculty of Medicine, Department of Epidemiology,
Šrobárova 2, 041 80 Košice, Slovakia
[email protected]
Introduction: The interaction between human and animal health is not a new
phenomenon. However, the scope, scale, and world-wide impact of zoonoses we are facing
today have no historical precedent. The ever increasing impact of globalization,
industrialization, restructuring of agricultural systems, as well as significant changes in
climatic and environmental conditions, rapid adaptability of microorganisms and their
movement within different ecosystems, animal migration, travel, deforestation, population
growth, but also increase the use of antibiotics and overcoming barriers between man and
animal kingdoms have resulted in a continuous increase in the number of zoonoses, which
are a serious problem not only in developing but also in developed countries. Increasing
trend in occurrence of diseases with zoonotic potential is currently recorded also due the
knowledge that the germs that were previously considered exclusively as animal can cause
diseases in humans.
At present, out of the 1,415 identified infectious pathogens for human has zoonotic
character 868 (61%), (6). The rapid detection and response to diagnostic diseases is crucial,
because many times there is unobserved spreading and infiltration of diseases to new
territorial units especially in the case of emerging, respectively re-emerging diseases.
One of the most common microorganisms in nature are also chlamydiae from the family
Chlamydiacea.
Chlamydiaceae only has a single genus Chlamydia that comprises 12 species (4, 7). One of
them, Chlamydia felis can cause infection which may be associated with conjunctivitis
and/or respiratory tract disease, particularly in kittens, but could be the cause of the disease
in adult cats as well. Infection is more common in multi-cat environments, particularly
breeding catteries.
The aim of the study: To investigate the various populations of cats in Slovakia for the
presence of chlamydia from conjunctival swabs, and in the case of positive result to
examine the identity of the obtained sequences.
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Methodology: Samples. In our study we have examined 127 cats of various breeds from
Košice region in Slovakia. The age of the cats varied from 1 month to 10 years.
Conjunctival samples were obtained from 59 clinically healthy cats (46.5%) and 68 cats
(53.5%) with clinical signs of conjunctivitis and upper respiratory tract impairment. Cats
were divided into 4 groups according to breeding and the type of environment in which
they lived. In the 1st group were cats kept inside only, with no possible contact with other
cats and other animals, with the exception of the time of regular deworming and
vaccination (n=22). The 2nd group includes free-roaming cats, which are characterized by
free movement and contact with other cats, or animals generally (n=49). The 3rd group
comprised stray cats, taken from the streets and brought to the clinic for examination
(n=28). The 4th group included cats kept in shelters or deposit devices, for which is
characteristic a high concentration of animals (n=28).
Molecular analysis.For isolation of the DNA were used samples resulting from mixing
150μl samples from both eyes. DNA isolation was carried out according to the
manufacturer's instructions using a commercial diagnostic kit DNA-Sorb/AM (Nucleic
Acid Extraction Kit, AmpliSens, Federal State Institution of Science, Moscow, Russia). In
the case were the amplification could not been carried out immediately, the samples were
stored at -20 °C until their next use.For the detection of Chlamydiaceae spp. cPCR
amplification method using a commercial test AmpliSens-55-R Chlamydiaceae CHLAKOM (Federal State Institution of Science, Moscow, Russia) was used.Positive specimens
for Chlamydiaceae spp. were subsequently amplified using a set of primers that amplify
domain I of 23S rRNA. Identification was made by direct sequence identification of 600bp
PCR product amplification using specific oligonucleotides U 23F and 23 SIGR (IDT,
Integrated DNA Technologies, USA). The reaction mixture for one reaction was prepared
by mixing 0.2 ml 10 pm of primer number 1, 0.2 ml 10 pm of primer number 2, 9.6 ml of
purified water, 0.2 ml of hot start Taq polymerase buffer containing MgCl2 (Solis
BioDyne, Estonia) and 5 ml of template tempered to 60 °C (isolated DNA).
Electrophoresis was carried out for 20 minutes at 300V. Positive products Chlamydiaceae
spp. were compared against the positive control for the length of 300bp under UV light at a
wavelength of 254nm. Products of PCR amplification were sent for sequence analysis and
then the species were identifying in the gene bank (http://www.ncbi.nlm.nih.gov) using
BLAST searches.
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Results and Discussion: Of the total number of 127 examined cats, 23 showed positivity
for chlamydial infection, which represented 18.1% positivity.After sequencing,
consecutive evaluation and comparison of sequences with BLAST software,all positive
samples were identifying as Chlamydia felis.
The average age of infected cats was 11.5 months, with a range from 1 month to 10 years.
Out of the 23 positive cats, the highest positivity was detected in the population of stray
cats – 9 positive (32%) and shelter cats – 9 positive (32%). In group of free-roaming cats
were positive 5 cats, which represented 10% positivity. No positive animal has been
detected in the group of cats kept inside only.
C. felis is endemically spread between domestic cats worldwide, and its prevalence was
published in several studies (1, 2, 5). And although is feline chlamydiosis one of the most
common diseases in cats, there is very little information regarding the prevalence of the
disease in Slovakia. As showed our results the positivity for C. felis infection in our study
was more than 18%, with the highest prevalence in group of stray and shelter cats. These
cats may then pose a risk to people around them. C. felis infection in humanshas been
related particularly with reproductive disease (abortion, neonatal mortality and infertility),
(3), but generally the zoonotic potential of C. felis appears low, and exposure to this
microorganism is possible by handling the affected cats, by contact with their aerosol and
also via fomites. Generally at present, diseases with zoonotic potential (including
chlamydial infection) have increasing trend not only in developing but also in developed
countries. The ecological changes caused due to destruction of the natural environment
increase the exposure of animals and humans to the vectors and reservoirs of unknown
pathogenic microorganisms. In addition to this, weak public health system, other natural
disasters, absence of better surveillance and controlling systems against the disease and
unhygienic living conditions are the main factors for the emergence and re-emergence of
the zoonotic diseases in many countries of the world.
Conclusion: The results obtained suggest that C. felis is largely involved in causing
diseases of the conjunctiva and respiratory tract in cats in Slovakia. In terms of chlamydial
infection are the highest risks group of stray cats and shelter population around the age of
three months.
Acknowledgements: The study was supported by the Slovak Grant Committee VEGA,
Grant No. 1/0390/12 and partially supported by the Research and Development Support
Agency, contract No. APVV-15-0134.
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Literature
1.
Cai Y, Fukushi H, Koyasu S, Kuroda E, Yamaguchi T, Hirai K. An etiological investigation of domestic
cats with conjunctivitis and upper respiratory tract disease in Japan. J Vet Med Sci. 2002; 64(3): 215–
219.
2.
Di Francesco A, Carelle MS, Baldelli R. Feline chlamydiosis in Italian stary cat homes. Vet Rec. 2003;
153 (8):244–245.
3.
Pointon AM, Nicholls JM, Neville S. Chlamydia infection among breeding catteries in South Australia.
Aust Vet Practit. 1991; 21:58–63.
4.
Sachse K, Bavoil PM, Kaltenboeck B, Stephens RS, Kuo Ch, RossellóMóra R, et al. Emendation of the
family Chlamydiaceae: Proposal of a single genus, Chlamydia, to include all currently recognized
species. Sys App Micro. 2015; 38: 99–103.
5.
Sykes EJ. Feline Upper Respiratory Tract Pathogens: Chlamydophila felis. Compendium on Continuing
Education for the Practicing Veterinarian. 2001; 23(3): 231–241.
6.
Taylor, L.H., Latham, S.M., Woolhouse, M.E.J. Risk factors for human disease emergence. In Phil.
Trans. R. Soc. Lond. B, 2001. 356, p. 983-989.
7. Vorimore F, Hsia RC, Huot-Creasy H, Bastian S, Deruyter L, Passet A, et al. Isolation of a new
Chlamydia species from the Feral Sacred Ibis (Threskiornis Aethiopicus): Chlamydia ibidis. PLoS One.
2013; 8(9): e74823.
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MOLECULAR CHARACTERISTICS OF GENOTYPES AND SUBTYPES OF
CRYPTOSPORIDIUM HOMINIS IN SLOVAKIA
Hatalová, E.1, Valenčáková, A.1, Danišová, O.1, Kalinová, J.1, Luptáková, L.1, Ondriska,
F.2, Boldiš, V.2
1
University of Veterinary Medicine and Pharmacy, Komenského 73, 04181 Košice, 2HPL
spol. s.r.o. Istrijská 20, 841 07, Bratislava, Slovak Republic
[email protected]
Introduction: Cryptosporidiosis is a protozoan infection caused by obligate intracellular
extracytoplasmatic parasites from the genus Cryptosporidium. These parasites infect
numerous vertebrates, including humans1. Cryptosporidiosis in humans has been
recognized since 19762, and to this date, at least eight species (C. hominis, C. parvum, C.
meleagridis, C. canis, C. felis, C. suis, C.muris, C. andersoni) and seven genotypes (C.
hominis monkey genotype, C. parvum mouse genotype, C. cervine chipmunk genotype I,
rabbit and horse genotype) have been detected in humans3. Although cryptosporidia are
referred as host specific pathogens, numerous studies have confirmed their zoonotic
potential. Various subtypes of C. hominis were described in numerous species of mammals
(equidae4,primates5,and small mammals6).Cryptosporidia cause gastrointestinal infections
with typical symptoms, including watery diarrhea, nausea, vomiting, abdominal pain and
fever. In an immunocompetent host the diarrhea is self-limiting and lasting two to three
weeks. In an immunosupressed host, the infection may lead to chronic diarrhea,
dehydration, weight loss, malabsorption and death7. Cryptosporidiosis occurs worldwide
and is often associated with contaminated water supply, socioeconomic status and age of
humans8. Accurate identification of parasites on a species and genotype level has a great
importance in human and veterinary parasitology, for diagnostics, therapy and prevention
of infections. Molecular methods for species identification of Cryptosporidium spp. are
based on the amplification of the SSU rRNA region. Subtyping of C. hominis is based on
the analysis of the 60 kDa glycoprotein, which has tandem repeats of the serine-coding
trinucleotides TCA, TCG or TCT. The name of GP60 subtypes starts with the subtype
family designation (Ia-Ig), based on the dominant trinucleotide repeat, followed by the
number of TCA (represented by the letter A), TCG (represented by the letter G), or TCT
(represented by the letter T) repeats. In the C. hominis Ia subtype family, the subtypes can
be
further
identified
by
the
copy
a
15
bp
repetitive
sequence
AA/GGACGGTGGTAAGG-3` (the last copy is 13 bp AAA/GACGGTGAAGT)9.
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The aim of the study: Our objective was to detect species and genotype representations of
Cryptosporidium in humans with PCR analysis targeting the SSU rRNA region (species
identification) and GP60 gene (genotype determination), and a comparison with their
occurrence of genotype families in neighboring states and small mammals to show on the
possibility of their transmission between countries and numerous host species.
Methodology: Collected samples were divided into 3 groups. The first group consists of a
total of 91 human faecal samples examined for the presence of the Cryptosporidium spp.,
collected in years 2012-2015.The population study is described in detail by Petrincová et
al., 201510.In the second group, additional 4 samples were processed in 2016, 3 patients
were immunocompetent with symptoms, one patient was hospitalized with a diagnosed
oncological illness.In the third group, during years 2012-2013, a total of 262 faecal
samples from four species of rodents (Myodes glareolus, Apodemus agrarius, Apodemus
flavicollis, Rattus norvegicus) were examined for the presence of Cryptosporidia. The
population study is described in detail in Danišová et al.11.DNA isolation and PCR
amplification for species and genotype determination was performed according to protocol
by Hasajová et al., 201412. For genotype determination in the second group, we used
a modified protocol, with annealing temperatures for primers GP60 F1/R1 55°C and for
GP60 F2/R2 58°C.Final sequences were compared with homologous sequences from
GenBank using Blast.
Results and discussion: From the collected samples, six were identified as C. hominis,
from which five were confirmed in humans and one inApodemus agrarius11. Subtyping
based on the GP60 gene fragment and its specific tandem repeats was carried out by the
rules of subtyping9 and shown every sample as a different subtype. We have identified
families and subgenotypes: C. hominis IaA11G2R8 in a patient after kidney
transplantation, C. hominis IbA10G2T1 in an immunocompetent patient with clinical signs
and C. hominis IbA11G2 in an immunocompetent patient without symptoms10.Additionaly,
C. hominis IeA12G3T3 and C. hominis IbA10G2 were detected at our department. C.
hominis IeA12G3T3 was identified in an immunocompromised patient with a diagnosed
oncologicaldisease in Slovakia, and was found also in China13, Australia14and
a America15.C. hominis IbA10G2 was identified in a patient with diarrhea in Slovakia, and
the occurrence of this subtype was also confirmed in, in Spain, in the UK16 and
Netherlands17. Although C. hominis IbA10G2 was never confirmed in rodents, it was
identified in a European hedgehog (Erinaceus europaneus) in Netherlands6. Studies shown
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the prevalence of the same subtype families worldwide. Subtype families Ia were
confirmed in Spain, in the United Kingdom, Pakistan, Egypt and Kenya16, although their
subgenotypes had different main tandem repeats as the ones in this study. Subtype families
Ib in Europe were confirmed in Spain, Portugal and the UK16. Families Ib were also
confirmed in Asia16America18, Australia16 and Africa19. Cryptosporidia belonging to the
family Ie were diagnosed in Pakistan16. This confirms the occurrence of different subtypes
of C. hominis is not strictly geographically localized. Studies also show the presence of C.
hominis in other mammals, as was confirmed at our department12. This proves that
zoonotic transmission of C. hominis cannot be excluded.
Conclusion: The prevalence of cryptosporidiosis worldwide ranges from 0.1% to 31.5%.
This wide range is due to the economical development, geographical location and climatic
conditions of a country20. Similarly to other countries in Central Europe, in Slovakia
cryptosporidiosis is a very rare infection. The first human infection was reported in 201321.
We have confirmed the presence of various genotypes and subtypes of C. hominis in
Slovakia. Molecular methods prove to be a useful tool for species determination, and also
genotype and subtype identification of various cryptosporidian parasites.
Acknowledgement: This work was supported by VEGA MŠ SR No. 1/0061/16 and
APVV No. 15/0134
Literature
1.
Santin, M. Clinical and subclinical infections with Cryptosporidium in animals. N Z Vet J. 2013;61(1):110
2.
Nime, FA., et al. Acute enterocolitis in a human being infected with the protozoan Cryptosporidium.
Gastroenterol 1976;70:592-598
3.
Ajjampur, SS, et al. Molecular and spatial epidemiology of cryptosporidiosis in children in a semiurban
community in South India. J Clin Microbiol 2007;45:915-92.
4.
Jian F., et al. Common occurrence of Cryptosporidium hominis in horses and donkeys. Infect Genet
Evol. 2016;43:261-266.
5.
Parson MB., et al. Epidemiology and molecular characterization of Cryptosporidium spp. in humans,
wild primates and domesticated animals in the Greater Gombe Ecosystem, Tanzania. Trop Dis
2005;9(2):e0003529.
6.
Krawczyk AI, et al.Presence of zoonotic agents in engorged ticks and hedgehog faeces from Erinaceus
europaeus in (sub) urban areas. Parasit Vector,. 2015;8:210.
7.
Fayer R., Xiao L. (2008): Cryptosporidium and cryptosporidiosis. CRC Press 2008, IBSN 978-1-42005226-8, s. 10.
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8.
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Leach CHT, et al. Prevalence of Cryptosporidium parvum infection in children along the Texas-Mexico
border and associated risk factors. Am J Trop Med Hyg, 2000;62(5):656-661.
9.
Xiao L. Molecular epidemiology of cryptosporidiosis: an update. Exp Parasitol. 2010; 124(1):80-9
10. 10.Petrincová A., et al. Molecular characterization and first report of Cryptosporidium genotypes in
human population in the Slovak Republic.Electrophoresis. 2015;36(23):2925-30.
11. Danišová, O, et al. Human genotypes of zoonotic pathogens Cryptosporidium spp. identified in rodents
in Slovakia. 2016
12. Hasajová A et al. Significantly higher occurrence of Cryptosporidium infection in Roma children
compared with non-Roma children in Slovakia. Eur J Clin Microbiol Infect Dis. 2014 Aug;33(8):1401-6.
13. Feng Y. Cryptosporidium genotype and subtype distribution in raw wastewater in Shanghai, China:
evidence for possible unique Cryptosporidium hominis transmission.J Clin Microbiol. 2009;47(1):153-7.
14. Koehler, AV, et al. First genetic analysis of Cryptosporidium from humans from Tasmania, and
identification of a new genotype from a traveller to Bali. Electrophoresis. 2014;35(18):2600-2607
15. Chalmers, RM et al. Direct comparison of selected methods for genetic categorisation of
Cryptosporidium parvum and Cryptosporidium hominis species. Int J Parasitol., 2005;35397-410.
16. Jex AR, Gasser RB. Analysis of the genetic diversity within Cryptosporidium hominis and
Cryptosporidium parvum from imported and autochtonous cases of human cryptosporidiosis by mutation
scanning. Electrophoresis. 2008;29:4119–4129.
17. Wielinga, PR, et al. Molecular epidemiology of Cryptosporidium in humans and cattle. Int J parasitol.
2008;38(7):809-817
18. Zhou L, et al. Molecular surveillance of Cryptosporidium spp. in raw wastewater in Milwaukee:
implications for understanding outbreak occurrence and transmission dynamics. J Clin Microbiol.
2003;41:5254–5257.
19. Leav BA, et al. Analysis of sequence diversity at the highly polymorphic Cpgp40/15 locus among
Cryptosporidium isolates from human immunodeficiency virus-infected children in South Africa. Infect.
Immun. 2002;70:3881–3890.
20. Fayer R. Cryptosporidium: a water-borne zoonotic parasite. Vet Parasitol. 2004;126(1-2): 37-56.
21. Ondriska F., et al. The first reported cases of human cryptosporidiosis caused by Cryptosporidium
hominis in Slovak Republic. Folia Microbiologica. 2013;58(1): 69-73.
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APPLICATION OF MULTILOCUS SEQUENCE TYPING IN THE ANALYSIS
OF BORRELIA BURGDORFERI SENSU LATO
Chvostáč M.1, Margos G 2, Derdáková 1
1
Istitute of Zoology, Slovak Academy of Sciences, Dúbravská cesta 9, 84506 Bratislava,
Slovakia, [email protected]
2
Bavarian Health and Food Safety Authority, Veterinärstrase 2, 85764, Oberschleissheim,
Germany
Background: Ixodes ricinus (Linnaeus 1758) is the most widespread tick species in
Europe. It inhabits the sylvatic, as well as urban localities, such as city parks and gardens,
which increases the probability of contact with humans and domestic animals. It is vector
of the most common tick-borne bacterial disease in Europe-Lyme borreliosis caused by the
spirochetes of the Borrelia burgdorferi sensu lato complex.
Reported mean prevalence of B. burgdorferi s.l. in ticks in Europe is 13,7 % (1). The
Borrelia prevalence in Slovakia ranges from 0% (2) to 39 % (3). B. burgdorferi s.l.
currently encompasses 21 known genospecies. Genetic variability within and between the
genospecies is linked to different clinical symptomatics as well as their different
association to reservoir hosts.
Intraspecific variability within single genospecies with ecological, clinical and
epidemiological relevance has been recorded as well. Multilocus sequence typing (MLST)
and multilocus sequence analysis (MLSA) are methods for typing of multiple loci in
microbial strains for population genetic studies. By this techniquenew borrelial species,
such as B. bavariensis and B. kurtenbachii had been identified recently (4; 5).
MLST of B. burgdorferi s.l. consists of amplification and sequencing of 8 house-keeping
genes. Every allele is compared to MLST database and the allelic number is allocated.
Connection of 8 allelic numbers defines the allelic profile and consequently the sequence
type. In case of new allelic number or allelic profile, the sequence is sent to the MLST
curator and set into the database. The main advantages of MLST are portability,
comparability of data and high precision of phylogenetic studies and population analysis
(6). It is also able to detect all of known B. burgdorferi s.l. genospecies, compared to other
methods like SSCP or RFLP.
Aim of the study: The aim of the study was to compare the prevalence, genospecies
distribution and genetic variability of B. burgdorferi sensu lato between different
geographical regions in Europe (Slovakia, Finland) and to clarify the host association, and
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ecology of B. lusitaniae in natural focus on Martinské hole.
Methodology: Questing I. ricinus ticks were collected in Bratislava (SAS campus),
Slovakia and near Helsinki, Finland. The DNA was isolated from ticks individually by
alcaline-hydrolysis method and the samples were stored at -20 °C. To verify the succes of
the DNA extraction tick mitochondrial gene cytochrome b (620bp) was amplified from
each sample.For detecting the prevalence of B. burgdorferi s.l., the amplification of 222255bp fragment of 5S-23S rRNA intergenic spacer was performed (7). The PCR products
were electrophoresed on 1.5% agarose gel.Positive samples were further typed to Borrelial
genospecies by Restriction Fragment Length Polymorphism (RFLP) (7). Representative
samples were tested using MLST. Nested PCR was performed for
single genes
individually (6). PCR products were purified using commercial DNA-purifying kit and
sent for sequencing. The sequences were processed by SeqMan pro (DNASTAR). MLST
trees were constructed in program MEGA (Maximum likelyhood, Boostrap:1000).
Results and discussion: B.burgdorferi s.l. prevalence in 543 questing I. ricinus ticks in
Bratislava was 6.8% and it was represented by 6 genospecies with the dominance of birdassociated genospecies B. garinii and B. valaisiana (35.1%, 24.3%). Rodent-associated B.
afzeliiwas present in 16.2% of questing I. ricinus, followed by B. lusitaniae (10.8%), B.
burgdorferi s.s. (5.4%) and B. spielmanii (2.7%). Mixed infections were found in 5.4% of
samples. The total prevalence, discovered in our study is relatively low, as compared to
mean prevalence of Borrelia in Europe. Rauter and Hartung (1) state, that the mean
prevalence of B. burgdorferi s.l. in Ixodes ricinus ticks in Europe is 13.7%, whilemean
prevalence for Slovakia is 18%.We assume, that the low prevalence of B. burgdorferi s.l. is
due to the low abundance of rodents on our study site (8).
14.7% of 150 ticks were infected with B. burgdorferi s.l in the sylvatic habitat of
Martinske hole mountains. The most prevalent genospecies was B. lusitaniae (45.5%),
followed by B. afzelii (40.9%), B. burgdorferi s.s. (9.1%) and B. garinii (4.5%).
Discovered prevalence of B. burgdorferi s.l. is comparable to the mean borrelia prevalence
in Europe, as described above. However the rate of infected ticks is considerably variable
on this site and varies between 18.9-46.1% (3).The prevalence of B. lusitaniaedetected in I.
ricinusis lower, compared to the infection rate of 62.8%, found by Tarageľová (3).
In Finland, 12.3% of 760 collected I. ricinus were Borrelia-positive, represented by 3
genospecies: B. afzelii (59.5%), B. garinii (33%), B. valaisiana (2.5%) and mixed
infections (5%). Our results are comparable to the findings of (9), defining the B.
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burgdorferis.l. prevalence in southwest Finland at the level of 17.4% with the dominance
of B. afzelii (53.7%).
By comparing of three B. afzelii samples from Finland to B. afzelii sequences from MLST
database, we have found it´s relation to samples from Europe. B. garinii isolate from
Finland is located in cluster with B. garinii from Austria. (pubmlst.org). B. afzelii from
Glis glis ear lobes from Slovakia clusters with B. afzelii from rodents from Europe.B.
lusitaniae from Martinské hole are genetically more related to Serbian isolates of this
genospecies than to the Portuguese ones. Based on the analysis of concatenated sequences
of clpX, recG and uvrA genes, two B. lusitaniae samples from Martinské hole represent
isolated genotypes.
Conclusion: Prevalence of B. burgdorferi s.l. on our study sites shows considerable
variations. The lowest prevalence (6.8%) was found in Bratislava, while higher borreliainfection rate (14.7%) was discovered on Martinské hole. Also the genospecies spectrum
varies between the study sites. In Bratislava, mainly bird-associated genospecies are
present (B. garinii, B. valaisiana), while in Finland, rodents-associated B. afzelii is the
most abundant. The most prevalent borrelia genospecies on Martinské hole is B. lusitaniae,
associated with lizards. Using MLST we have found a new borrelia genotype on this study
site. For that reason we would like to focus on this locality in our next work. B. afzelii
isolates from G. glis ear lobes are genetically related to B. afzelii sequences from rodents
from Europe and don‘t show higher relations to dormice-associated B. spielmanii. Hereby
we have approved the reservoir competence of dormice for B. afzelii.
This work was supperted by grant APVV-0274
References
1.
.Rauter C, Hartung T. Prevalence of Borrelia burgdorferi sensu lato genospecies in Ixodes ricinus ticks
in Europe: a metaanalysis. Appl Environ Microbiol. 2005;71(11):7203–16.
2.
Pangrácová L, Derdáková M, Pekárik L, Hviščová I, Víchová B, Stanko M, et al. Ixodes ricinus
abundance and it´s infection with the tick-borne pathogens in urban and suburban areas of Eastern
Slovakia. Parasit Vector. 2013;6(1):1.
3.
Rusňáková-Tarageľová V, Mahríková L, Selyemová D, Václav R, Derdáková M. Natural foci of
Borrelia lusitaniae in a mountain region of Central Europe. Tick Tick Borne Dis. 2016;7(2):350-6.
4.
Margos G, Vollmer SA, Cornet M, Garnier M, Fingerle V, Wilske B et al. A new Borrelia species
defined by multilocus sequence analysis of housekeeping genes. Appl Environ Microbiol.2009;75(16):
5410-5416.
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Margos G, Hojgaard A, Lane RS, Cornet M, Fingerle V, Rudenko N, et al. Multilocus sequence analysis
of Borrelia bissettii strains from north America reveals a new Borrelia species, Borrelia
kurtenbachii.Ticks Tick Borne Dis. 2010;1(4):151-8.
6.
Margos G, Gatewood AG, Aenensen DM, Hanincova K, Terekhova D, Vollmer SA, et al.MLST of
housekeeping genes captures geographic population structure and suggests a European origin of Borrelia
burgdorferi. Proceeding of the national academy of sciences of the United States of America.
2008;105(25):8730-5.
7.
Derdáková M, Beati L, Peťko B, Stanko M, Fish D. Genetic variability within Borrelia burgdorferi
sensu lato genospecies established by PCR-single-strand conformation polymorphism analysis of the
rrfA-rrlB intergenic spacer in Ixodes ricinus ticks from Czech republic. Appl Environ Microbiol.
2003;69(1):509–16.
8.
Svitálková Z, Haruštiaková D, Mahríková L, Berthová L, Slovák M, Kocianová E, et al. Anaplasma
phagocytophilum prevalence in ticks and rodents in an urban and natural habitat in South-Western
Slovakia. Parasite Vector. 2015;8:276.
9.
Sormunen JJ, Klemola T, Vesterinen EJ, Vuorinen I, Hytonen J, Hanninen J, et al. Assessing the
abundance, seasonal questing activity and Borrelia and tick-borne encephalitis virus (TBEV) prevalence
of Ixodes ricinus ticks in a Lyme borreliosis endemic ares in Southwest Finland. Tick Tick Borne Dis.
2016;7(1):208-15.
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LIVER TREMATODES IN WILD RUMINANTS
Iglódyová A.1, Lazar P.1, Čurlík J.1, Karolová R.1, Ciberej J.1, Bocková E.2, Štrkolcová G.2
1
Institute of Game and Fish Breeding and Diseases, 2 Institute of Parasitology;
University of Veterinary Medicine and Pharmacy in Košice, Komenského 73, 041 81
Košice, Slovak Republic
[email protected]
Introduction: Wild ruminants are often infected by wide spectrum of parasites. Various
helminths cause severe health problems and also they have zoonotic potential. Liver flukes
in wild ruminants, occuring in Slovakia, are important parasites belonging to the genera
Fasciola, Fascioloides and Dicrocoelium.
In the definitive host, adult Fasciola hepatica occurs in the bile ducts of the liver. Natural
infections are associated mainly with domestic ruminants accompained by marked clinical
signs (weight loss, diarrhoea, death). The presence of this parasite in cervids generally are
not associated with clinical signs or significant pathology. Human fasciolosis is known in
several world regions. Most cases are found randomly during surgery or autopsy. In some
cases, infection with one or two flukes may result in severe reactions with difficult
treatment. Clinical signs can occur when larvae are migrating to and within the liver.
Human infections are associated with the consumption of emergent vegetation
contaminated with viable metacercariae. Infections of F. magna occurs primarily in cervids
and bovids with formation of thin-walled fibrous capsules in liver parenchyma containing
adult flukes. Most of infections are subclinical, occasionaly with sympthoms such as
lethargy, anorexia, depression and weight loss. Infections in humans have not been
reported [7]. Dicrocoelium dendriticum occurs in the bile ducts of a wide range of
domestic and wild mammals including species of ovids, bovids, suids, equids, cervids,
rodents, lagomorphs, primates, and camelids [3], particularly sheep and mouflon. A few
cases of verminous hepatitis due to infection with lancet fluke in humans have been
reported [1,3,5,8]. Zoonotic potential exists from ingestion of infected ants or water
containing infected ants. In addition, eggs of D. dendriticum may occur in human faeces
for a few days after ingestion of infected liver, but these people are not infected [1,7].
Liver fluke development is very dependent of the environmental characteristics. F.
hepatica and F. magna are found mainly in aquatic and moist environments, suitable for
the survival of their hosts (freshwater snails belonging to the family Planorbidae and
Lymnaeidae). By contrast, the habitat of the small liver fluke Dicrocoelium dendriticum
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(aslo known as „lancet fluke“) is in dry lowland or mountain pastures representing
adequate conditions for development of terrestrial snails and ants [4,6].
The aim of the study: The aim of the study was monitoring liver flukes in wild ruminants
including red deer (Cervus elaphus), roe deer (Capreolus capreolus), fallow deer (Dama
dama) and mouflon (Ovis musimon) and find out the risk of cross infection from wild to
domestic ruminants in selected regions of Slovakia.
Material and Methods: Between 2014-2016 641 faecal samples from wild ruminants
(463 red deer - Cervus elaphus, 67 roe deer - Capreolus capreolus, 37 fallow deer - Dama
dama and 74 mouflons - Ovis musimon) we examined. Samples were taken from 3 regions
of Slovakia (Prešov, Košice and Žilina). For liver flukes detection we used coprological
methods (sedimentation method and faecal sieving- staining method by Kleimann [2]. For
ELISA detection of Fasciola hepatica coproantigen the commercial BIO K 201/2 Fasciola
hepatica Elisa Kit (Bio-X Diagnostics, Belgium) was performed.
Results and discussion: No F. hepatica and F. magna eggs were detected in the faeces of
wild ruminants. In mouflons and red deer we isolated characteristic D. dendriticum eggs
(overall prevalence 6,86 %). The most of positive samples were from mouflons (39 from
44 faecal samples – 88,64 %). 5 positive samples were from red deer (11,36 %). Positive
samples came from Prešov and Košice region.
Environmental conditions in Slovakia are ideal not only for the development of native
species, as well as the introduced, as recorded by our previous findings - 14,03%
prevalence of fascioloidosis in the south of the Slovakia and D. dendriticum infections in
mouflons (prevalence 3,74%). No F. hepatica cases in wild ruminants were recorded,
although infections in cattle are known especially in the northern regions of Slovakia and
pastures shared with wild ruminants.
Conclusion: The results of previous study revealed areas with favorable environmental
conditions for F. magna development situated along the Danube river in the south of
Slovakia. Although no infection of F. hepatica in wild ruminants was confirmed but cases
of fasciolosis in domestic ruminants are found. D. dendriticum is fixed mainly on domestic
and wild ovids. Depence of liver fluke incidence from weather factors indicate, that
climate change have a marked influence on their evolution. Regions with the presence of
F. hepatica and D. dendriticum are risk areas not only for domestic and free living animals,
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but also for people. Therefore, it is necessary to continue with monitoring the occurence of
liver flukes in the territory of Slovakia.
Acknowledgement: This study was supported by the Science Grant Agency VEGA No.
1/0791/14
References
1.
Cengiz, Z. T., Yilmaz, H., Dülger, A. C., Çiçek, M. Human infection with Dicrocoelium dendriticum in
Turkey. In Annals of Saudi Medicine. 2010, vol. 30, no. 2, pp. 159–161.
2.
Kleiman, F., Pietrokovsky, S., Gil, S., Wisnivesky-Colli, C.: Comparison of two coprological methods
for the veterinary diagnosis of fasciolosis. In Arquivo Brasileiro de Medicina Veterinária e Zootecnia.
2005, vol.57, no.2, p. 181 – 185.
3.
Mapes, C.R.: Studies on the biology of Dicrocoelium dendriticum (Rudolphi, 1819) Looss, 1899
(Trematoda: Dicrocoeliidae), including its relation to the intermediate host, Cionella lubrica (Muller). I.
A study of Dicrocoelium dendriticum and Dicrocoelium infection. In Cornell Veterinarian. 1951, vol.
41, pp. 382–432.
4.
Mas-Coma, S., Bargues, M. D., Valero, M. A. Fascioliasis and other plant-borne trematode zoonoses. In
Int. J. Parasitol. 2005, vol. 35, no.11–12, pp. 1255–1278.
5.
Ondriska F., Sobota K., Janosek J., Joklová E. A rare case of human autochthonous dicroceliasis in
Czechoslovakia. In Bratislavské lekárske listy. 1989; vol. 90, pp. 467–469
6.
Otranto, D., Traversa, D.: Dicrocoeliosis of ruminants: a little known fluke disease. In Trends in
parasitology. 2003, vol. 19, no. 1, p. 12-15.
7.
Samuel W.M., Pybus M.J., Kocan A.A. Parasitic Diseases of Wild Animals. Iowa State University press,
2001, pp. 121-142.
8. Sing A., Tybus K., Fackler I. Acute urticaria associated with Dicrocoelium dendriticum infestation. In
Indian J Med Microbiol. 2008, vol 26, pp 97–98.
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THE INCIDENCE OF HEPATITIS E VIRUS IN DIFFERENT AGE CATEGORIES
OF DOMESTIC PIGS
Jackova, A., Mandelik, R., Vlasakova, M., Vilcek, S.
University of Veterinary Medicine and Pharmacy in Kosice, Slovakia
[email protected]
Introduction : Hepatitis E virus (HEV) is the causative agent of hepatitis E - an emerging
zoonotic disease distributed worldwide. HEV is non-enveloped virus with single-stranded
positive sense RNA genome approximately 7.2 kb composed of a short 5´untranslated
region (UTR), three partially overlapping open reading frames (ORFs) and a short 3´end
terminated by a poly(A) tail. ORF1 encodes non-structural polyprotein, ORF2 encodes the
capsid protein and ORF3 encodes the cytoskeleton-associated phosphoprotein (Meng et al.,
2002). The virus belonging to the family Hepeviridae, genus Hepevirus, is subdivided into
four genotypes (King et al., 2012). Two of genotypes (HEV-1 and HEV-2) contain strains
infecting only human population. The other two genotypes HEV-3 and HEV-4 can be
isolated from humans, pigs and other animal’s species (Schlauder and Mushahwar, 2001).
The aim of the study: The aim of this study was to investigate the incidence of HEV in
different age categories and to compare two groups of healthy and suffering pigs.
Methodology:
Samples
Clinical samples (rectal swabs, n=442) were obtained from eleven domestic pig farms.
First group of pigs (n=173), which was named suffering group, showed enteric problems
associated with diarrhea, other problems of digestive system, respiratory problems or
growth retardation. Second group (n=269) was healthy without any clinical signs. Both
groups were divided into the following three different age categories: suckling piglets
(animals before weaning < 28 days, n=59), weaned (28 – 70 days, n=204) and fattening (>
70 days, n=179) pigs. The health status of pigs was evaluated by qualified veterinarians
directly on the each farm.
Isolation of RNA and synthesis cDNA
The rectal swabs were processed in the laboratory by elution into 1 ml of 0.01 mol/l PBS
(Merck Millipore Corp., USA) for 30 min. The eluted solution was vortexed at 2000 rev.
min-1 for 3 min and then centrifuged at 17 500 x g for 5 min. Total RNA was isolated using
TRIzol Reagent (Life Technologies, USA) from 200 µl of sample according to the
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manufacturer’s instruction and dissolved in 20 µl of molecular biology grade water
(Merck, GmbH, Germany). Samples with aliquots of isolated RNA were stored at -800C.
The cDNA was synthesized in a 20 µl reaction mixture comprising 5 µl of isolated RNA, 5
µM of random hexamers (Invitrogen, USA), 50 µl dNTPs (Thermo Fisher Scientific, Inc.,
USA), 200 U RevertAid Premium reverse transcriptase with 1xRT buffer (Thermo Fisher
Scientific, Inc., USA), 20 U RNase inhibitor (Takara Bio, Inc., Japan) and molecular
biology grade water (Merck, GmbH, Germany). The mixture was incubated at 650C for 5
min and then chilled on ice to destroy RNA secondary structure. Subsequently, the mixture
was incubated at 250C for 10 min, then at 500C for 30 min to synthesise cDNA and at 850C
for 5 min to terminate reaction.
Detection of HEV by nested RT-PCR
The detection of partial HEV genome was based on the amplification of a 242 bp fragment
of ORF1 gene using outer and inner primers by Erker et al. (1999). The PCR reaction
mixture (25 µl) was composed of 1x ThermoPol reaction buffer (New England Biolabs,
Inc., USA), 200 µM dNTPs (Thermo Fisher Scientific, Inc., USA), 0.3 µM of outer
primers, 0.5 U Taq DNA polymerase (New England Biolabs, Inc., USA), 2 µl cDNA and
molecular grade water (Merck, GmbH, Germany). The first PCR was run with the
following thermal profile: 1 cycle at 950C for 1 min, and 35 cycles with denaturation at
950C for 30 s, annealing at 550C for 1 min, and extension at 680C for 1 min and final
extension at 680C for 5 min. In the second PCR inner primers and similar thermal profile
were used. The size of PCR products was checked by electrophoresis in 2% agarose gel
after staining with GelRedTM (Biotum, Inc., USA) and visualization by Gel Doc EZ imager
(Bio-Rad Laboratories, Inc., USA).
Sequencing of DNA and phylogenetic analysis of HEV.
Purified PCR products were sequenced using automatic sequencer ABI PRISM. The
sequences from both strands of the PCR products were determined with the same primers
as used for the nested PCR amplification. Partial ORF1 sequences (242nt) were assembled
using MEGA 4.0 software.
Results and Discussion: The results of the incidence of hepatitis E virus in domestic pigs
are summarized in Table 1. The RT-PCR analysis of 442 enteric samples revealed that 72
pigs (16.3%) of all age categories together were positive for HEV RNA. Out of the virus
positive pigs, 32 (11.9%) were clinically healthy and 40 (23.1%) were suffering animals.
When looking across age categories HEV RNA was the most often detected in age
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categories – fattening in both groups healthy (14.8%) and suffering (26.3%). Lower
prevalence was detected in age category – weaned pigs, in healthy group 12.5% and
suffering group 23.9%. HEV RNA was not at all observed in healthy group of suckling
piglets. We assume that the influence of maternal antibodies in this age categories has high
protective effect.
Table 1
Incidence of hepatitis E virus in different age categories of healthy and
sufferingdomestic pigs
Age
Number
category
Healthy (n=269)
Suffering (n=173)
Total (n=442)
Posit/Negat
Posit/Negat
Posit/Negat
Posit (%)
Posit (%)
Posit (%)
Suckling
59
0/35
0.0
3/21
12.5
3/56
5.1
Weaned
204
14/98
12.5
22/70
23.9
36/168
17.6
Fattening
179
18/104
14.8
15/42
26.3
33/146
18.4
Total
442
32/237
11.9
40/133
23.1
72/370
16.3
The nucleotide sequences of ten HEV isolates were aligned and compared to each other
and to selected strains deposited in GenBank. The comparison of nucleotide sequences
identity Slovak isolates to each other showed 81.0-100 %. By comparison of nucleotide
sequences neighbouring countries, Czech Republic and Hungary, was observed identity
81.8-86.8% and 80.6-86.8%, respectively. By phylogenetic analysis of partial ORF1 we
found out that Slovak isolates clustered into two genetic groups. All Slovak HEV isolates
were typed as genotype 3
which is common genotype of animal and human isolates.This is considered as important
character of the possible source of people infection. In the near future we would like to
intend analysis for another pig farms in Slovakia and more HEV isolates.
Conclusion: In summary, this study showed differences of prevalence HEV in age
categories. As HEV RNA was not at all observed in healthy group of suckling piglets, we
assume high protective effect of maternal antibodies in this age category.Results of the
study did not demonstrate a clear relationship between the prevalence of HEV in suffering
pigs and healthy animals.
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Acknowledgements: This work was supported by scientific project VEGA 1/0342/14 and
project INFEKTZOON, ITMS: 26220120002 supported by the EU.
Literature
1.
Meng et al. (2002) J Clin Microbiol, 40, 117-122
2.
King et al. (2012) Virus Taxonomy. Ninth Report ICTV, Elsevier, 2012.
3.
Schlauder and Mushahwar (2001) J Med Virol, 65, 282-292.
4.
Erker et al. (1999) J Virol Methods, 81, 109-113.
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TAIL FIBER ASSOCIATED DEPOLYMERASES AS A POTENTIAL BIOFILM
DEGRADATION AGENT
M. Kajsik., V. Belanova, H. Drahovska, J. Turna
Comenius University Science Park, Ilkovičova 8, 841 04 Bratislava, Slovakia
[email protected]
Introduction: Cronobacter spp. is opportunistic food-borne pathogen responsible for
serious infections as necrotizing enterocolitis, septicaemia and meningitis. Bacteria infect
mainly newborns, elderly and immunodeficient individuals. Powdered infant milk formula
has been implicated as the vehicle of transmission.Prevalence of these gram-negative
bacteria in food is caused by their high resistance to long-term drying, high temperatures
and osmotic stress [1-3]. Moreover Cronobacter spp. strains are able to form biofilms on
glass, stainless steel, polyvinyl chloride (PVC), polycarbonate, silicone, and enteral
feeding tubes in different media. These features markedly increases risk of food-bourne
infections [4]. Using bacteriophages as asanitary or medical product for treatment and
prevention is one possible solution. Phages are viruses capable to infect bacteria, but are
harmless to human. Bacteriophages are ubiquitous and could be purified from many
ecological environments, even from samples of human microflora[5, 6]. Phages are
extremely host specific. Host specificity of bacteriophages is determined mainly by the
structure of their receptor binding proteins. Some of these proteins (depolymerases) have
an enzymatic activity responsible for the degradation of bacterial capsular polysaccharides
(EPS), which are essential for bacterial survival in harsh conditions. Their activity enables
the phages to access the bacterial surface. They occur as tail-associated structures in
phages of various hosts. Soluble depolymerases may also be isolated from phage
lysatesand are responsible for formation of translucent, expanding haloes around phage
plaques. After application of depolymerases, pathogen wasn´t able to survive due to
removal of the protective EPS layer, exposing bacterial cells to defense reactions and
environmental influences[7, 8].
The aim of the study: This work was aimed at searching for phage tail fiber associated
depolymerases, their overexpression in bacterial expression model and characterization of
their antibiofilm and antimicrobial activity in vitro. In addition we focused our work on the
comparison of bacteriophage and phage tail fiber antimicrobial activity.
Methodology: Bacteriophages Dev-CT-57 and Dev-CD-23823 were isolated and
characterized at the Department of Molecular biology, Comenius University in Bratislava.
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Whole genomic sequences were obtained using next-gen sequencing (MiSeq, Illumina),
and assembled using CLC Genomic Workbench software. Putative tail fiber proteins were
specified using RAST annotation tool [9]. DNA sequences of candidate proteins were
amplified by specific PCRand inserted into T7 expression system. The proteins CT57-gp40
and CD23-gp41 were expressed in the T7 expression system of E. coli BL21 and isolated
in soluble form.Acquired proteins were applied in antimicrobial activity tests using the
agar diffusion test against Cronobacter spp. and Enterobacter strains.
Results and Discussion: In our study, whole genome sequences of seven bacteriophages
were analyzed for the presence of tail fiber with depolymerase domains. We found putative
tail fibers in phages Dev2 [10], Dev-CT-57 and Dev-CD-23823. Last two phages were
selected by reason of wide host specificity. The subjects of this study were the receptorbinding proteins CT57-gp40 and CD23-gp41. Amino-acid sequences of studied proteins
were compared using BLASTp. Proteins had identical domain arrangement, but the
similarity was only 68%. WcaM domain involved in Colanic acid synthesis was observed
between 170th and 358th amino-acid of both proteins. Similar domain arrangement was
observed also in the case of closest neighbor cronophage vB_CskP_GAP227[11]. DNA
sequence of both putative tail fibers were amplified and cloned into T7 expression system.
The proteins CT57-gp40 and CD23-gp41 were expressed in the T7 expression system of E.
coli BL21. Different expression conditions were used for increasing amount of soluble
form of proteins. Highest concentration of soluble form was reached using temperature
30°C and 0.25mM concentration of IPTG. The second part of our work was aimed at
characterization of depolymerase antimicrobial activity. The agar diffusion test was used to
set antimicrobial activity on different bacterial strains. The proteins CT57-gp40 and CD23gp41 and phages Dev-CT-57 and Dev-CD-23823 showed identical ability of reduce
bacterial exopolysaccharides. Proteins were able to reduce growth of 10 from 11 tested
Cronobacter strains capable to produce exopolysaccharide capsule. C. dublinensis, strain
140807/04 wasn´t sensitive for neither phage nor depolymerase activity. Proteins and
phages were able to infect both tested Enterobacter strains capable to produce capsule.
Strains incapable of capsule production were used as a negative control. On the basis of
our results, we propose that the proteins CT57-gp40 and CD23-gp41 degrade
capsularexopolysaccharides, probably colanic acid, produced by Cronobacter spp.
Conclusion: The subjects of this study were the receptor-binding proteins of the phages
Dev-CT-57 and Dev-CD-23823, which infect a broad host range of Cronobacter spp. We
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prepared expression vectors containing sequences of these phage receptor molecules. We
also confirmed the enzymatic activity of the proteins using the agar diffusion test. Both
proteins were active on 10 out of 14 strains of Cronobacter and on 2 out of 9 strains of
Enterobacter. We observed a correlation between the activity of CT57-gp40 and CD23gp41, host-specificity of the phages Dev-CT-57 and Dev-CD-23823 and the ability of the
tested strains to produce capsules. On the basis of our results, we propose that the proteins
CT57-gp40 and CD23-gp41 degrade colanic acid, a capsular exopolysaccharide produced
by Cronobacter spp.
Literature
1.
Orieskova, M., et al., Contribution of the thermotolerance genomic island to increased thermal tolerance
in Cronobacter strains. Antonie Van Leeuwenhoek, 2016. 109(3): p. 405-14.
2.
Hariri, S., S. Joseph, and S.J. Forsythe, Cronobacter sakazakii ST4 strains and neonatal meningitis,
United States. Emerg Infect Dis, 2013. 19(1): p. 175-7.
3.
Jaradat, Z.W., et al., Cronobacter spp. - opportunistic food-borne pathogens. A review of their virulence
and environmental-adaptive traits. J Med Microbiol, 2014. 63: p. 1023-1037.
4.
Hartmann, I., et al., Genes involved in Cronobacter sakazakii biofilm formation. Appl Environ
Microbiol, 2010. 76(7): p. 2251-61.
5.
Minot, S., et al., Rapid evolution of the human gut virome. Proc Natl Acad Sci U S A, 2013. 110(30): p.
12450-5.
6.
García, P., et al., Food biopreservation: promising strategies using bacteriocins, bacteriophages and
endolysins. Trends in Food Science & Technology, 2010. 21(8): p. 373-382.
7.
Born, Y., et al., The tail-associated depolymerase of Erwinia amylovora phage L1 mediates host cell
adsorption and enzymatic capsule removal, which can enhance infection by other phage. Environ
Microbiol, 2014. 16(7): p. 2168-80.
8.
Flachowsky, H., et al., Transgenic expression of a viral EPS-depolymeraseis potentially useful to induce
fire blight resistance in apple. Ann Appl Biol, 2008. 153.
9.
Aziz, R.K., et al., The RAST Server: rapid annotations using subsystems technology. BMC Genomics,
2008. 9: p. 75.
10. Kajsik, M., et al., Characterization and genome sequence of Dev2, a new T7-like bacteriophage
infecting Cronobacter turicensis. Arch Virol, 2014. 159(11): p. 3013-9.
11. Abbasifar, R., et al., The Genome of Cronobacter sakazakii Bacteriophage vB_CsaP_GAP227 Suggests
a New Genus within the Autographivirinae. Genome Announc, 2013. 1(1).
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SEROPREVALENCE OF ANAPLASMOSIS IN HUMANS
FROM EASTERN SLOVAKIA
Kalinová Z., Halánová M., Čisláková L., Čechová L., Babinská I.
Pavol Jozef Šafárik University, Faculty of Medicine, Department of Epidemiology,
Šrobárova 2, 041 80 Košice, Slovakia
[email protected]
Introduction: Human granulocytic anaplasmosis (HGA) is an emerging tick-borne
infectious disease caused by Anaplasma phagocytophilum ((formerly Ehrlichia
phagocytophila, E. equi and human granulocytic ehrlichiosis – HGE agent) [3]. A.
phagocytophilum has a tropism for granulocytic cells, where the bacteria replicates within
the host cell vacuoles to form microcolonies called morulae [2]. Anaplasmosis is usually
transmitted to human and animals through the bite of an infected tick. In Europe, including
Slovakia, the main vector of A. phagocytophilum is the common tick, Ixodes ricinus, which
can also transmit other pathogens, such as the tick-borne encephalitis (TBE) virus, the
bacteria Borrelia burgdorferi, and the spotted-fever group of bacteria Rickettsia [5, 7]. In
Europe, the first serological evidence of HGA was described in 1995 in Switzerland [1],
and first clinical case was confirmed in 1997 in Slovenia [9]. In Slovakia, the first case of
HGA was confirmed in a 54-year old hunter from northern Slovakia [8].
The aim of the study: The aim of this work was to study the occurrence of IgG A.
phagocytophilum antibodies in the blood sera in the general population and people at risk
of infection.
Methodology: Samples. A total 448 human serum samples were examined for the
presence
of anti-A. phagocytophilum IgG antibodies. The
samples of blood sera were obtained from patients with suspected Lyme borreliosis (214
samples), from the staff of University of Veterinary Medicine and Pharmacy (53 samples),
from patients with suspected tick-borne encephalitis (113 samples) and from medical
students (68 samples).
Methods. Anti-A. phagocytophilum IgG antibodies were detected using an Indirect
Immunofluorescence Antibody (IFA) IgG test from Focus Diagnostics (California, USA).
The IFA assay is a two stage “sandwich” procedure. In the first stage, the patient serum is
diluted in PBS. The diluted serum is placed on the slide in contact with the substrate, and
incubated. Following incubation, the slide is washed in PBS, which removes unbound
serum antibodies. In the second stage, each antigen well is overlaid with fluorescein204
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labelled antibody to human IgG. The slide is incubated allowing antigen antibody
complexes to react with the fluorescein-labelled anti-human IgG. After the slide is washed,
dried, and mounted, it is examined using fluorescence microscopy.
Positive reactions appear as apple-green fluorescence of the morulae. Semi-quantitative
endpoint titters are obtained by testing serial dilutions of positive specimens. The serum
screening dilution was 1:64, according to the test producer.
Results and Discussion: Of the total number of 448 blood sera examined, 63 (14.1%)
were positive. From 214 examined patients with suspected Lyme borreliosis the IgG
antibodies against
A. phagocytophilum were detected in 7%. Two cases of the co-
infection B. burgdorferi withA. phagocytophilum, which equals 0.9% of the 214 examined
patients, were found. IgG antibodies against A. phagocytophilum were detected in 16
(30.2%) person out of 53 examined employees of the University of Veterinary Medicine
and Pharmacy. Of the total number of 113 examined sera of patients with suspected tickborne encephalitis were positive 22 (19.5%). Of the 68 examined blood serum samples
obtained from students of Medical Faculty reacted positively 10, which represented 14.7%
positivity.
HGA is tick-borne zoonosis. In Europe, Ixodes ricinus is the principal vector of
A. phagocytophilum. This tick is a known vector of several microorganisms such as
Borrelia burgdorferi, tick-borne encephalitis (TBE) virus, Coxiella burneti, spotted fever
group Rickettsiae. TBE and Lyme borreliosis (LB) are the most familiar tick-borne
diseases in Slovakia [5, 7, 10]. The real infection rate of HGA in Europe is still hard to
establish. Seroprevalence rates range from zero to up to 28 % [11]. No official
epidemiological data on the prevalence of this infection in the human population are
available in Slovakia. Only a few studies have been published relating to anaplasmosis,
with results of prevalence ranging from 7% to 25% [4, 6].
Conclusion: The total prevalence of A. phagocytophilum antibodies in our sample (14.1%)
corresponds with the findings of similar studies. The results indicate that the disease occurs
also in our country, especially in some exposed population groups.
Acknowledgements: The study was supported by the Slovak Grant Committee VEGA,
Grant No. 1/0390/12 and partially supported by the Research and Development Support
Agency, contract No. APVV-15-0134.
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Literature
1.
Brouqui P, Dumler JS, Lienhard R, Brossard M, Raoult D. Human granulocytic ehrlichiosis in Europe.
Lancet 1995; 346: 782–783.
2.
Carlyon JA, Fikrig E. Invasion and survival strategies of Anaplasma phagocytophilum. Cell
Microbiol.2003; 5: 743–754.
3.
Chen S, Dumler JS, Bakken JS, Walker AR. Identification of a granulocytotropic Ehrlichia species as
the etiological agent of human disease. J Clin Microbiol.1994; 32: 589–595.
4.
Kalinová Z, Halánová M, Čisláková L, Sulinová Z, Jarčuška P. Occurrence of IgG antibodies to
Anaplasma phagocytophilum in humans suspected of Lyme borreliosis in eastern Slovakia. Ann. Agric.
Environ. Med. 2009; 16: 285–288.
5.
Kmety E, Rehacek J, Vyrostekova V, Gurycova D. Infestation of ticks with Borrelia burgdorferi and
Francisella tularensis in Slovakia. Bratisl. Lek. Listy 1990; 91: 251–256.
6.
Kocianová E, Košťanová Z, Štefanidesová K, Špitalská E, Boldiš V, Hučková D, Stanek G. Serologic
evidence of Anaplasma phagocytophilum infections in patients with a history of tick bite in central
Slovakia. Wien. Klin. Wochenschr. 2008; 120: 427–431.
7.
Kožuch O, Labuda M, Lysy J, Weismann P, Krippel E. Longitudinal study of natural foci of Central
European encephalitis virus in West Slovakia. Acta Virol. 1990; 34: 537–544.
8.
Nováková M, Víchová B, Majláthová V, Lesňáková A, Pochybová M, Peťko B. First case of human
granulocytic anaplasmosis from Slovakia. Ann. Agric. Environ. Med. 2010; 17: 129–133.
9.
Petrovec M, Lotrič-Furlan S, Zupanc TA, Strle F, Brouqui P, Roux V, Dumler JS. Human disease in
Europe caused by a granulocytic Ehrlichia species. J. Clin. Microbiol. 1997; 35: 1556–1559.
10. Řeháček J, Úrvolgyi J, Kocianová E, Sekeyová Z, Vavreková M, Kováčová E: Extensive examination of
different tick species for the infestation with C. burnetii in Slovakia. Eur J Epid 1991, 7, 299-303.
11. Strle F. Human granulocytic ehrlichiosis in Europe. Int. J. Med. Microbiol. 2004; 293: Suppl 37, 27–35.
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ANTIBACTERIAL EFFECTS OF HERBAL ESSENTIAL OILS AGAINST
SELECTED FOOD-BORNE PATHOGENS
Koščová, J., Demjanová, S.
University of Veterinary Medicine and Pharmacy in Košice
Department of Microbiology and Immunology, Komenského 73, 041 81 Košice, Slovakia
[email protected]
Introduction: Food-borne pathogens are mostly bacteria, rarely viruses or parasites that
can cause food-born illneses (also known as food-borne infections or food-borne
poisoning). The most important food-borne germs are known as members of the genus
Escherichia, Salmonella, Shigella, Yersinia, Campylobacter,Clostridium,Listeriaor Vibrio.
Viruses that can be transferred by food are mostly noroviruses, rotaviruses and also
hepatitis A virus. Microscopic parasites from the genus Cyclosporaor Giardia can be also
found as food contaminant. The food can be contaminated during slaughter by contact of
the meat with small amounts of intestinal content, wrere the bacteria are normally present
as intestinal microflora of healthy animal. Technologies of food preservation include
nonthermal inactivation, such as ionization radiation, high hydrostatic pressure, and pulsed
electric fields, modified atmosphere and active packaging, biopreservation and natural
antimicrobial compounds [1]. Plants are a source of bioactive molecules and have been
widely used both traditionally and commercially to increase the shelf-life and safety of
foods [2]. Biological properties of herbal essential oils (EOs) and their antimicrobial
activity have been attributed to phenolic compounds, such as the carvacrol, thymol or
eugenol. These compounds have hydrophobic characteristics and interact with different
sites of microbial cells (e.g., cell wall and cytoplasmic membrane), causing loss of cellular
constituents, collapse of membrane structure, and cell death [3].
The aim of the study: The aim of the study was to determine in vitro antibacterial activity
of four plant EOs againstselected food-borne pathogens (2 Gram-positive and 2 Gramnegative bacteria) by agar disc diffusion method and to determine the minimum inhibitory
concentration (MIC).
Methodology: Essentail oils.The plant essential oils (oreganum, thyme, clove and
cinnamon) were obtained as pure natural substances (Calendula, Nová Ľubovňa, SVK).
Stock solutions were prepared at 3 different concentrations (1:2; 1:5 and 1:10) with
dimethylsulfoxid (DMSO).
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Bacterial strains. Bacterial reference strains used for this in vitro assay were as follows:
Staphylococcus aureusCCM 3953, BacilluscereusCCM 2010, Salmonella Enteritidis CCM
4420 and Escherichia coliCCM 7372.
Disc diffusion method. This assay were done to detect antibacterial activity of all 4 EOs.
Mueller-Hinton agar (MHA) was inoculated with 0.1 ml of overnight bacterial suspension
of tested bacteria with counts determined previously in meat-peptone broth (1×108
CFU/ml). After absorption of the inoculum, the blank paper discs with 6 mm in diameter
(Becton Dickinson, USA) were placed on the surface and 10 µl of appropriate EOs and
concentration were inoculated on it. Streptomycin antibiotic disc (50 mg/ml) served as
positive control and disc impregnatd with sterile DMSO as negative one. Plates were
incubated aerobically at 37°C for 24 hours. After incubation the inhibition zones around
the discs were measures in mm. All tests were 3 times repeated and statistically evaluated.
Minimum inhibitory concentration. MIC was evaluated after preparing two-fold dilutions
of all 4 EOs in five different concentrations (1%; 0.5%; 0.25%; 0.125% and 0.0625%) in
MHA with addition of 0.5% Tween 20 (Sigma Aldrich, Germany). The surface of
cultivation media in Petri dishes was inoculated as drop with 2 µl of 18 hour bacterial
culture. MHA agar without EOs was inoculated by same procedure and served as positive
control. Plates were incubated aerobically at 37°C for 24 hours. For statistical evaluation
was each assay three times repeated under the same conditions. MIC was determined as the
lowest concentration of EO that inhibited growth of selected pathogens on agar plates.
Results for disc diffusion method were processed as mean value ± standard deviation in
mm. Positive results were evaluated in case of diameter ˃ 6 mm. For analysis, statistical
programme GraphPad Prismversion 3.0 was used. The results of MIC determination were
evaluated as presense or absence of pathogen growth on the plate surface.
Results and Discussion: Disc diffusion method. All selected bacteria showed sensitivity
against streptomycin. Its antibacterial effect increased in order: S. aureus<E. coli<B.
cereus<S. Enteritidis (Tab. 1).
Table 1 Inhibtion zones around streptomycin disc
Inhibition zones (mm)
G-
Reference control
Streptomycin
50mg/ml
G+
Sal. Enteritidis
E. coli
S. aureus
B. cereus
19
13,5
13
16
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In general, the strongest antibacterial activity against bacteria showed oregano a thyme
EOs. The most resistant against effects of essential oils in all 3 tested concentrations was
Salmonella Enteritidis, and on the contrary the most sensitive was Bacillus cereus (Tab. 24).
Table 2 Antibacterial activity of EOs tested at concentration 1:2 (n=3)
Inhibition zones (mm)
EO
1:2
Oregano
Thyme
Cinnamon
Clove
GSal. Enteritidis
35,33 ± 3,09 ***
14,67 ± 0,47
11,67 ± 1,7
14,67 ± 2,62
G+
E. coli
24,67 ± 2,87
23,33 ± 6,6
15 ± 0,81
16,67 ± 0,47
S. aureus
23,17 ± 4,09 *
23,33 ± 4,92 *
12 ± 0,82
11 ± 0,82
B. cereus
25,67 ± 1,89 ***
25,67 ± 1,89 ***
14,33 ± 0,47
14,33 ± 1,25
*** significant difference (P<0,001), * significant difference (P<0,05)
Table 3 Antibacterial activity of EOs tested at concentration 1:5 (n=3)
Inhibition zones (mm)
EO
1:5
Oregano
Thyme
Cinnamon
Clove
GSal. Enteritidis
11 ± 2,16
11,33 ± 0,94
11,33 ± 1,25
12,33 ± 1,25
G+
E. coli
28,67 ± 0,47 ***
16 ± 1,41
15,33 ± 1,7
15,33 ± 0,94
S. aureus
26 ± 0,82 ***
23 ± 1,41 ***
12 ± 0,82
12,67 ± 0,47
B. cereus
29 ± 2,45 **
18 ± 3,56
15,67 ± 1,25
16,33 ± 0,47
*** significant difference (P<0,001), * significant difference (P<0,01)
Table 4 Antibacterial activity of EOs tested at concentration 1:10 (n=3)
Inhibition zones (mm)
EO
1:10
Oregano
Thyme
Cinnamon
Clove
GSal. Enteritidis
11,33 ± 0,47 **
8,67 ± 0,47 *
9,67 ± 0,47
10,67 ± 0,47
G+
E. coli
12,33 ± 0,94 *
11 ± 0
8,67 ± 0,94
11,33 ± 1,7
S. aureus
18,33 ± 2,05 ***
9±0
10,67 ± 1,25
10 ± 1,41
B. cereus
18 ± 5,1
11,33 ± 2,87
11,33 ± 0,47
10,67 ± 0,94
*** significant difference (P<0,001), ** significant difference (P<0,01), * significant difference (P<0,05)
Figure 1: Inhibition zones of thyme essential oil in concentrations 1:2; 1:5 a 1:10 (from
left)
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In disc diffusion assay the best antibacterial effects showed oregano and thyme EO at both
concentrations 1:2 and 1:5 (Fig. 1). The inhibition zones were even wider in comparison to
streptomycin disc. Clove and cinnamon oils at the same concentrations showed much
weaker antibacterial activity. However, at concentration 1:10 the differences between EOs
were not so significant. Differences in antibacterial activity of essential oils is strictly
attributed to their chemical structure. EOs that consist mainly from phenolic compounds
such are carvacrol, thymol or eugenol, are the most effective [4]. These compounds are the
major parts of oregano and thyme EOs, that is the reason why they are so effective. In
cinnamon EO, the main compound is trans-cinnamaldehyd, in clove it is eugenol. And it is
well known that not only major compounds play an important role in effectivity of EOs,
but also minor compounds are very important, by influencing the antibacterial effect of
EOs by synergic effects [5]. In our study we also confirmed that Gram-negative bacteria
were more resistant than Gram-positive Chyba! Nenašiel sa žiaden zdroj odkazov..
Conclusion: The use of essential oils in food industry has a place and importance not only
as flavoring ingredients but also as antibacterial components. However, it must be taken
into account the characteristics of the food to which will be added. Its composition may
also positively or negatively influence the effects of EOs. Therefore, more research is
needed in this field focused not only on the actual antibacterial effects of EOs but also on
their combinations and the effect of food as a whole.
Aknowledgement: This work was supported by the Slovak Research and Development
Agency under the contract No. APVV-15-0377 and project ITMS 26220220185
(MediPark).
Literature
1.
Devlieghere, F., Vermeiren, L., and Debevere. J.: New preservation technologies: possibilities and
limitations. Int Dairy J, 2004; 14: 273–285.
2.
Sasidharan, S., Zuraini, Z., Yoga Latha, L., Sngetha, S., and Suryani, S.: Antimicrobial activities of
Psophocarpus tetragonolobus (L.) DC extracts. Food Pathog Dis, 2008; 5:303–309.
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3.
18th-20th October 2016
Burt, S.: Essential oils: their antibacterial properties and potential applications in foods-a review. Int J
Food Microbiol, 2004; 94: 233–253.
4.
Sokovic et al.: Antibacterial Effects of the Essential Oils of Commonly Consumed Medicinal Herbs
Using an in vitro Model. In Plant Research International, 2010; 15 (11): 7532-46.
5. Paster, N. et al.: Antifungal Activity of Oregano and Thyme Essential Oils Applied as Fumigants
Against Fungi Attacking Stored Grain. Journal of Food Protection. 1995; 58:81-85.
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LABORATORY DIAGNOSTICS OF LEGIONELLOSIS AND TULAREMIA –
SEROLOGICAL TESTING AND PCR
Kotrbancová, M., Výrosteková, V., Guryčová, D., Špaleková, M.
Institute of Epidemiology, Faculty of Medicine, Comenius University, Bratislava
[email protected]
Introduction: Legionellae and francisellae are closely related gram-negative bacteria
(Proteobacteria) with some ecological, biological and pathogenic similarities. Both germs
could be found in natural water habitats and in wet soil often with amoebae. Both
pathogens are invasive intracellular parasites. Currently 58 species of legionellae (most
common is Legionella pneumophila – L.p.) with 30 species pathogenic for humans [1].At
least 4 subspecies of
Francisella
tularensis (F.t.) are recognised, as
F. t. subsp.
tularensis,F. t. subsp. holarctica, F. t subsp. mediasiatica and antigenically different less
pathogenic F. t. subsp. novicida. F. philomiragia is considered as an opportunistic
pathogen [2]. Both infections occur worldwide. While legionellosis is considered as
sapronosis, tularemia is a zoonosis with natural focality transmitted by several ways.
Clinical manifestation of atypical pneumonia could be similar [3] following inhalation of
either contaminated water aerosol resulting in severe legionnaires´ disease (LD) or
contaminated dust or aerosols in 5-20% cases of tularemia [4]. Both undiagnosed or late
treated infections could be fatal in more than 20% [4, 5], therefore specific diagnostics is
of special importance. Laboratory diagnosis of both infections relies mainly on detection of
specific antibodies and also cultivation in LD, but these methods are too slow and
demanding to be clinically useful, thus new molecular PCR/RT-PCR tests started to be
introduced [2].
Both infections are of actual medical and epidemiological importance in Europe
(EU/EEA), with increasing incidence of LD (rate 1.4/105 in 2015) and tularemia with
incidence 0.20/105 in 2012 [6, 7].Both infections are notified for several decades in
Slovakia, occurring in sporadic intensity (LD), tularemia also in epidemics. F.t. subsp.
holarctica is endemic but also F.t. subsp. tularensis near Bratislava in 80-ties of the 20th
century was isolated from animal reservoirs and vectors (8). Slovakia reported low
incidence of LD, in average 3.9 cases yearly with overall 117 patients in the years19852015. The highest incidence of LD 0.26/ 100 000 (15 patients) was reported in 2014 [6, 9].
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The aim of the study: The study was focused on comparative evaluation of standard
agglutination tests commonly used in diagnostics of legionellosis and tularemia and on of
PCR methodfor detection of DNA of both pathogens in patients sera.
Methodology
1. Comparative serologicals studies in agglutination tests (AG)
1.1. Study with rabbit immune sera prepared by formolized (F), live (L), F-L antigens
from:
- 23 strains of L. pneumophila 1-15(incl. 10 subtypes of L.p.1) and 5 species of Legionella
like organisms - LLO (L.bozemanii 1, L.dumoffii, L.micdadei, L.gormanii, L.longbeachae
1)
- F. tularensis subspecies tularensis (strain Schu), F.t. subsp. holarctica (strain 503), F. t.
subsp. novicida (strain U112) and F. philomiragia (strain 309-L)
Antigens used in AG tests:
- 28 heated legionella antigens (10 subtypes L.p.1, 13 L.p.sg 2 - 14, 5 LLO - L.bozemanii
1, L.micdadei, L.dumoffii, L.gormanii, L.longbeachae 1)
- 4 formolized francisella antigens from strains of F.t. (Schu, 503, U112), F.ph. (309-L)
1.2. Study with human sera with 21 legionella antigens and commercial F. tularensis
antigen:
- 48 patients with LD caused by L.p. sg 1 (19), other L.p. sg. (18) and LLO (11 cases).
- 34 patients with antibodies against F.t. from endemic Nitra district
2. Comparative detection of DNA of legionellae and F.t. by PCR in patients sera with
LD and tularemia – using Thermocycler G-storm GS1 (Gene Technologies, Ltd.)
- by legionella DNA „in house“ PCR targeted to mip gen [10]. Performance: denaturation
at 94°C/1 min, amplification – 40 cycles
at 62°C/1 min, 72°C/2 min, 95°C/1 min.
Extraction of DNA from sera - Qiagen-DNeasy Blood and Tissue kit (Germany).
- by commercial GenPak® DNA Francisella tularensis (Gentech, Russia). Performance:
denaturation at 95°C/1 min., amplification - 45 cycles at 95°C/40 s, 60°C/40 s, 70°C/40 s,
70°C/1 min. Extraction of DNA from sera - High Pure PCR Template Preparation kit
(Roche).
Results and Discussion: Comparative studies with rabbit hyperimmune sera against
francisellae antigens (table 1) did not reveal any cross - reactions with heterologous
legionella antigens, except only low reactivity (1:16) of sera against F.t. subsp. novicida
prepared by F-L antigens with some L.p.sg 1 subtypes (Pontiac subgroup), L.p.sg 11 and L.
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bozemanii (table 1). No cross-reaction was observed in testing hyperimmune sera against
legionellae with francisellae antigens.
Table 1. Cross-agglutination of immune rabbit sera against francisellae and
legionellae with homologous and heterologous antigens
Antigens
Immune sera
against
L.p.1 - 10subtypes
L. p. 2 – 14
-F
-F-L
-L
-F
-F-L
-L
-F
-F-L
-L
-F-L
-F-L
LLO – 5 species
-F-L
F. t.
F. n.
F. ph.
francisellae
F.t.
F.n.
1:2560
1:20
1:2560
1:20
1:2560
1:40
1:320
1:160
1:1280
1:20
1:160
1:40
1:40
-
F.ph.
1:320
1:160
1:160
-
L.p.1*1
1:16
1:16
1:16
1:16
1:4096 -16400
1:32-128*4
-
-
-
legionellae
L.p.2-14
1:16*2
LLO *
1:16*3
-
-
1:32-256*4
1:1024 – 8192
-
-
1:4096 –
8192
Antigens - F- formolized, L-live, F-L formolized and live antigens
F.t. – F. tularensis subsp. tularensis, holarctica, F.n.-F. t. subsp. novicida, F.ph.- Francisella philomiragia,
L.p.- Legionella pneumophila, LLO- Legionella like organisms- 5 species
*1 reactivity with only some subtypes of L.p.1-Philadephia, Knoxville, Benidorm, Heysham, Bellingham
2 reactivity - L.p.sg 11
3 reactivity - L. bozemanii
4 reactivity - L.p.sg 1,4,6,7,11,12
Comparative studies of 48 sera from patients with LD (table 2) caused by L. p. sg 1 and
other serogroups of L. p. and LLO showed only once low reactivity (1:20) with F. t.
antigen.
Table 2. Patients with legionellosis tested with F. tularensis in agglutination test
No.
19
1
3
6
1
1
6
11
48
Tested sera with LD
antibodies against legionellae
L.p. 1
L.p.2
L.p. 3
L.p. 4
L.p. 6
L.p.10
L.p.12
L.boz.(6), L.mic.(2), L.gorm.(1), L.long.(2)
Titres
1:256 - 8192
1:2048
1:256 - 1024
1:256
1:256
1:512
1:256 - 1:512
1:256-1:2048
1:256 - 1:8192
214
Reactivity with F.tularensis
No.
Titres
1
1:20
1
1:20
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Table 3. Patients with antibodies against F.t. tested with legionellae in agglutination
test
Tested sera
Ab against
No.
F.t.-titres
5
1:1 280
6
1:640
3
1:320
6
1:160
7
1:80
7
1:40
34
1:40 -1:1280
Reactivity in titres 1:32 with legionellae antigens
No.
2
1
0
2
4
5
14
L.p.1 -subtypes
2
2
3
2
B(1), C(1)
B(1), C(2),Bl(1),F(1)
P(2), C(1), F(1)
C(2),Ox(1)
9
L.p.12
L.p.15
LLO
+(1)
+(1)
2
+ (1)
+ (2)
+ (3)
6
L.b., L.m.(1)
L.b., L.d.(1)
L.b.(2)
L.d.(1)
L.d.(1), L.m. L.g.(1)
7
C - Camperdown, B - Benidorm, Bl - Bellingham, F - France, P - Philadelphia, Ox -Oxford,
L.b.-
L.bozemaniii, L.d.- L.dumoffii, L.m. - L. micdadei, L.g. - L.gormanii
Testing 34 sera of patients with tularemia (table 3) revealed low reactions in titres 1:32 in
14 sera with some subtypes of L.p.1 (9), L.p. 12 (2), L.p.15 (6) and some LLO (7).
Limitations in both culture and serologic testing in diagnostics of LD and tularemia have
led to development of new diagnostic techniques and their clinical application.Some rapid
assays (ELISA, antigen detections, PCR/RT-PCR) started to replace the agglutination tests
[2, 8, 11].
Table 4. Reactivity of patients with Legionnaires disease with F. tularensis antigens
in AG tests and specificity of PCR- legionella-mip/Phoresis and PCR – F.t.
No of patientsLegionnaires ´
disease
1.
time from
onset-serum
sampling
8 weeks
2.
3 weeks
3.
1 week
4.
5.
6.
7.
NK
5 weeks
2 weeks
5 weeks
8.
1 week
9.
10 weeks
10.
Total 10
9 weeks
1-10 weeks
Diagnostics of
Legionnaires´disease
L.micd. 1:64
cultivation,
L.p.1 (K,Ben) 1:32
L.p. 4 1:128,
L.boz 1:64
L.micd. 1:128
L.p.1,6,8 1:256
L.p.1 (K,P) 1:2 048
L.p.2 1:4 096
cultivation, URT,
L.p.6, 8 1:128
L.p.11, L.dum 1:256,
URT
L.p.1 (K,P) 1:256
NK-not known, URT –urinary test
215
PCR-mip –
DNA/ Phoresis
Legionella
+
PCR –
Francisella
tularensis
-
+
-
+
+
+
+
+
+
-
+
-
+
-
+
Posit 10/10
Negat 9/10
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Preliminary results from testing 10 sera with confirmed LD by cultivation, urinary test and
anti-legionella antibodies (table 4) revealed that all were positive in detecting legionella
DNA by PCR-legionella-mip for a long time from onset of the disease. All sera of patients
with LD, except one, were negative in PCR – F. tularensis.
Table 5. Reactivity of patients tested for tularemia with legionella antigens in AG test
and in PCR –F. tularensis and PCR – legionella-mip
2
2
3
1
2
Ab against
F. tularensis
negat.
1:10
1:20
1:40
1:80
7
1:160
No. of patients
2
1:1280
1
1:2560
Total 20 posit/total
1:10 -1:2560
% posit.
18/18
Ab – antibodies, ND-not done
Ab against
legionellae
2/2 ND
0/2
0/3
0/1
0/2
0/5
2/2 ND
2/2 ND
1/1 ND
7 x ND
0/13
PCRF. tularensis
2/2
2/2
3/3
1/1
2/2
5/7
0/2
2/2
1/1
18/20
90%
PCRLegionella-mip
0/2
0/2
0/3
0/1
0/2
0/6
1/1 ND
1/2
0/1
1/19
5,2%
Preliminary results from testing 20 sera examined for F. tularensis infection by PCR (table
5)showed positivity of DNA F.t. in 90% sera, even in two serologically negative patients.
One serum of patient with tularemia was also positive by PCR–legionella-mip and this coreactivity needs further testing for possible dual infection.
Conclusion: Comparative testing of standard agglutination tests detecting antibodies
against legionellae and francisellae in immune sera and patients sera confirmed specificity
and reliability of the tests commonly used in diagnostics of legionella infection and
tularemia. Preliminary results from testing PCR methods in detection of DNA of both
pathogens in patients sera revealed no substantial co-reactivity and could be very helpful as
rapid test for early diagnosis of both infections.
Literature
1.
Cunha, B., Burillo, A., Bouza, E. Legionnaires´disease. Lancet, 2016; 387: 375-85
2.
Lai, X.H., Zhao, L.F., Chen, X.M., Ren, Y. Rapid Identification and Characterization of Francisella by
Molecular Biology and Other Techniques. Open Microbiol J, 2016; 10: 64–77.
3.
Westerman, E.L., McDonald, J. Tularemia pneumonia mimicking Legionnaires´ Disease: Isolation of
organism on CYE agar and successful treatment with Erythromycin. Southern Med. J, 1983; 76
(9):1169-1170
4.
Bossi, P., Teqnell, A., Baka, A. et al. Bichat guidelines for the clinical management of tularaemia and
bioterrorism-related tularaemia. Euro Surveill. 2004; 9(12):9-10.
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5.
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Phin, N., Parry-Ford, F., Harrison, T. et al. Epidemiology and clinical management of Legionnaires´
disease. Lancet Infect Dis, 2014; 14:1011-21.
6.
ECDC Surveillance Report. European Centre for Disease Prevention and Control. Legionnaires’ disease
in Europe, 2014. Stockholm: ECDC; Stockholm, 2016; 36 s.
7.
ECDC Surveillance Report. Annual epidemiological report Food and water borne diseases and zoonoses
2014, Stockholm, 2014; 103s..
8.
Guryčová, D., Maďarová, L., Výrosteková, V. et al. Molekulárne biologické metódy –PCR v diagnostike
tularémie. Zborník III. Labudove dni Eds.E.Kocianová et al , 2013; s.18-21.
9.
Špaleková, M., Kotrbancová, M. Diagnostika a epidemiológia legionelóz na Slovensku. 26.Pečenkovy
epidemiologické dny, Luhačovice, 2014; s.91.
10. Lindsay, D.S.J., Abraham, W.H., Fallon, R.J. Detection of mip Gene by PCR for Diagnosis of
Legionnaires´Disease. J Clin Microbiol, 1994; 32(12):3068 – 3069
11. Khodr, A., Kay, E., Gomez-Valero, L., et al. Molecular epidemiology, phylogeny and evolution of
Legionella. Infection, Genetics and Evolution, 2016; 43:108–122.
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CHARACTERIZATION OF ESCHERICHIA COLI O157:H7 STRAINS ISOLATED
FROM CATTLE HIDES AT SLAUGHTER
Kramarenko, T.1,2, Roasto, M.1, Mäesaar, M.1,2, Maugliani, A.3, Tozzoli, R.3
1
Estonian University of Life Sciences, Kreutzwaldi 56/3, 51014 Tartu, Estonia
2
3
Veterinary and Food Laboratory, Kreutzwaldi 30, 51006, Tartu, Estonia
Istitituto Superiore di Sanita, Viale Regina Elena 299, 00161 Rome, Italy
[email protected]
Introduction: Shiga toxin-producing Escherichia coli (STEC), which are also referred as
verocytotoxin-producingE. coli (VTEC), are a group of E. coli that are characterised by
theircapability to elaborate toxins closely related with the Shiga-toxin produced by
Shigella dysenteriae[1].Nowadays a considerable number of STEC serotypes have been
recognised as a causative agent of human disease, but severe infections and outbreaks
reported have been mostly associated with the O157 serogroup [2].Contaminated bovine
meat is considered to be a major source of foodborne STEC infections in humans,
particularly by E. coli O157:H7 [3]. Sampling cattle hides at slaughterhouse for STEC
monitoring is advisable, since the highest prevalence is expected at this stage of the meat
production and hides represent a major source of carcass contamination by E. coli O157
[4], [5].The results reflect the possibility of further contamination of carcasses and meat,
and represent a tool to evaluate the efficacy of good hygiene practices as a control measure
of carcass contamination [6].
The aim of the study: The aim of the study was to evaluate the prevalence of STEC
O157:H7 in cattle at slaughter in Estonia by investigating the contamination of cattle hides,
which can be an important source of these pathogens in the first steps of beef processing.
The isolates were further characterized by identifying the presence of genes of main
virulence factors, the subtypes of the Stx-coding genes and determining the antimicrobial
susceptibility and genetic relationship among the isolates.
Methods: During the period from January 2011 to December 2013 sponge samples of
cattle hides were collected from slaughterhouses in Estonia. Starting with the
slaughterhouses of largest throughput, those covering at least 80% of the national
throughput were included into the monitoring programme. Animals to be sampled were
chosen randomly throughout the year and only one animal per slaughterhouse was sampled
per selected day. Furthermore, the prerequisite that the animals to be sampled had to
originate from different herds was followed. Samples were analysed for the presence of E.
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coli O157 using the procedure described by standard method ISO 16654 [7] followed by
the conventional serotyping and detection of stx1, stx2 and eae genes in compliance with
ISO/TS 13136 [8]. Identifying of sxt1 and stx2 genes subtypes was performed as described
by Scheutz and colleagues [9]. The genetic relationship among isolates was investigated by
PFGE in accordance with the standard operating procedure of EFSA external scientific
report [10] and analyses were performed by European Union Reference Laboratory for
E.coli.The susceptibility to antimicrobials was determined by minimal inhibitory
concentration (MIC) microdilution method using VetMIC GN panel.Based on
epidemiological cut-off values, the results were categorized as susceptible or resistant
according to the guideline of European Committee on Antimicrobial Susceptibility Testing
and EU Reference Laboratory for Antimicrobial Resistance [11].
Results and discussion: Shiga toxin-producing Escherichia coli (STEC) O157:H7 was
isolated from 30 (4%, CI952.8% - 5.7%) of 744 cattle hide swab samples collected at
Estonian slaughterhouses within a three year monitoring programme. All isolates were
motile, possessed H7 antigen and showed enterohaemolytic phenotype. Twelve isolates
(40%) showed resistance to at least one of the 14 antimicrobials tested and the isolates
were predominantly resistant to streptomycin, sulfamethoxasole and ampicillin. Multiple
drug resistance, which was defined as resistance to three or more unrelated antimicrobials,
was detected in eight (27%)isolates although no extended spectrum beta-lactamase
(ESBL)-producing isolates were ascertained.Seventeen isolates carried bothstx1 and stx2
genes and the presence of stx2 gene was solely detected in case of 13 isolates. None of the
isolates carried genes of other stx1 and stx2 subtypes. The most frequently detected
subtype was stx2c, occurring alone or in combination with subtype stx1a.None of the
isolates carried genes of other stx1 and stx2 subtypes.All isolates harboured the intimincoding eae gene. Comparison of PFGE profiles showed that the isolates presented an
overall similarity more than75%. In total, 20 different XbaI PFGE patterns were
differentiated among the 30 isolates, of which 14 grouped into 4 clusters based on a genetic
relatedness criterion of 100%.
Conclusions: The current study describes for the first timeE. coli O157:H7 strains
circulating amongst Estonian cattle population, including their virulence gene profiles and
stx gene subtypes. Indistinguishable PFGE patterns of isolates originated from different
regions and time-points refer that some persistent clones are present in Estonian beef
production chain. The high level of multiple drug resistance among isolates raises the
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question of the appropriate use of antimicrobials in the Estonian cattle herds. Until today,
theyearly state monitoring programmes of STEC in Estonia has been episodic and focused
only to serogroup O157, but the importance of other STEC serogroups cannot be
underestimated in STEC epidemiology and these should also be included into national
monitoring programmes.
Literature
1.
VKM, Vitenskapskomiteen for mattrygghet. Norwegian Scientific Committee for Food Safety, Panel on
Biological Hazards. (2007). A risk assessment of shiga toxin-producing Escherichia coli (STEC) in the
Norwegian meat chain with emphasis on dry-cured sausages. VKM, pp. 74.
2.
EFSA Panel on Biological Hazards (BIOHAZ). Scientific Opinion on VTEC-seropathotype and
scientific criteria regarding pathogenicity assessment. EFSA Journal 2013;11(4):1-106.
3.
Farrock C, Jordan K, Auvray F, Glass K et al. Review of Shiga-toxin-producing Escherichia coli
(STEC) and their significance in dairy production. Int J Food Microbiol 2013;162:190-212.
4.
Keen JE, Elder RO. Isolation of Shiga-toxigenic Escherichia coli O157 from hides surfaces and oral
cavity of finished beef feedlot cattle. J Am Vet Med Assoc 2002;220:756-763.
5.
O`Brien S, Duffy G, Carney E, Sheridan JJ et al. Prevalence and numbers of Escherichia coli O157 on
bovine hides at a slaughter plant. J Food Prot 2005;68:660-665.
6.
EFSA, European Food Safety Authority. (2009). Scientific Report of EFSA. Technical specifications for
the monitoring and reporting of verotoxigenic Escherichia coli (VTEC) on animals and food (VTEC
surveys on animals and food). EFSA Journal 2009, 7(11),1366. doi: 10.2903/j.efsa.2009.1366.
7.
ISO, International Organization for Standardization. International standard 16654. Microbiology of food
and animal feeding stuffs – Horizontal method for the detection of Escherichia coli O157. International
Organization for Standardization, Geneva, Switzerland. ISO 2001.
8.
ISO, International Organization for Standardization. Technical specification 13136. Microbiology of
food and animal feed – real-time polymerase chain reaction (PCR) based method for the detection of
food-borne pathogens – Horizontal method for the detection of Shiga toxin-producing Escherichia coli
(STEC) and determination of O157, O111, O26, O103, O145. International Organization for
Standardization, Geneva, Switzerland. ISO 2012.
9.
Scheutz F, Teel LD, Beutin L, Pierard D et al. Multicenter evaluation of a sequence-based protocol for
subtyping Shiga toxins and standardizing Stx nomenclature. J Clin Microbiol 2012;50:2951-2963.
10. Caprioli A, Maugliani A, Michelacci V, Morabito S. Molecular typing of Verocytotoxin-producing E.
coli (VTEC) strains isolated from food, feed and animals: state of play and standard operating
procedures for pulsed field gel electrophoresis (PFGE) typing, profiles interpretation and curation. EFSA
supporting publication 2014;704:1-55.
11. EURL-AR, European Union Reference Laboratory for Antimicrobial Resistance. Cut-off values
recommended by the EU Reference Laboratory for Antimicrobial Resistance (EURL-AR). EURL-AR
2013:1-2.
Retrieved
10.02.2015,
from
recommended%20cut%20off%20values-29-11-2013.pdf
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SAPROLEGNIA PARASITICA – DANGEROUS FISH PARASITE
Luptáková, L.1, Valenčáková A.1, Danišová O.1, Petrovová E.2
1
University of Veterinary Medicine and Pharmacy, Department of Biology and Genetics,
Komenského 73, 041 81 Kosice, Slovakia
2
University of Veterinary Medicine and Pharmacy, Department of Anatomy, Histology
and Physiology, Komenského 73, 041 81 Kosice, Slovakia
[email protected]
Introduction: Oomycetes represent a ubiquitous group of eukaryotic organisms. They are
common inhabitants of terrestrial and aquatic environments and include saprophytes as
well as pathogens living mainly in wet soils or freshwater environments. Oomycetes
represent a ubiquitous group of eukaryotic organisms. They are common inhabitants of
terrestrial and aquatic environments and include saprophytes as well as pathogens living
mainly in wet soils or freshwater environments. Oomycetes have been traditionally
considered as fungi because of their morphology (mycelial growth), ecology and they also
share the same nutritional mode. Several species have developed parasitic lifestyles and
belong to the most devastating organisms on farmed and wildlife population of plant and
aquatic animals.
One representatives of parasitic water mold with the impact on
aquaculture and animal health is Saprolegnia parasitica. Saprolegnia parasitica belonging
to genus Saprolegnia and order Saprolegniales is mostly reported from areas with
temperature climate, such as Northwest Europe, Chile, Japan and Canada, where they have
a large impact on salmon forming (1).Between the years 2009-2011 abnormally high
mortality among fish was observed in rivers Doubs and Sorne. The most affected fish were
trouts (Salmo trutta) and graylings (Thymallus thymallus). Initial findings isolated a
species of the genus Saprolegnia from lesions of diseased fishes mainly Thymallus
thymallus.
The aim of the study: The aim of our study was focused on the detection and
identification of unknown fish pathogen using 18S rRNA gene region by nested PCR.
Methodology: Isolation of Saprolegnia spp from infected fish tissues and water and
culturing
Saprolegnia spp. strains were isolated both from diseased fishes (Salmo trutta, Thymallus
thymallus) and baiting methods (2). These samples were placed in sterilised Petri dishes
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containing a V8 juice medium, for routine growth and maintenance. Cultures were grown
at 25°C in a dark incubator, and transferred to new Petri dishes every four weeks.
Genomic DNA extractionand 18S sequencing.
DNA was isolated from mycelia using the guanidinium thiocyanate protocol described in
Chomczynski and Sacchi, 2006 (3).
For the 18S a nested PRC protocol was applied. For the first amplification, eukaryotic
domain-specific primers EK-42F and EK-1520F (4) were used, for the second
amplification, 20 ng of DNA obtained from the first amplification were used with EK 82F
and EK1498R primers (5). The two PCR programs were identical with the following
cycling profile: initial denaturation of 95°C for 5 min, followed by 40 cycles of 95°C for
15 s, 58°C for 15 s and 72°C for 90 s, with a final elongation of 72°C for 7 min.
The consensus sequences were edited and assembled using Bioedit and aligned using
Alignment ClustalW2 (6) with all available sequences on Genbank. Trees were constructed
by maximum likelihood using the program TREEFINDER (7), applying a GTR+G+I
model of nucleotide substitution (8). All necessary parameters were estimated from the
data sets. Bootstrap values were calculated from 1,000 replicates. In addition, we built a
tree using the program RAxML (9).
Results and Discussion:
18S rRNA
All sequences obtained from diseased fish and some sequences obtained by baiting from
river water belong to genus Saprolegnia.
Based on the 18S rRNA gene’s sequence, isolates were divided into two different groups.
Group 1, a larger group contains majority of strains (19/25) which were sampled from
diseased fish and they correspond to complex Saprolegnia parasitica s.l. The spread of
isolates is demonstrated by their presence in two different rivers (Doubs and Sorne). Based
on the 18S phylogenetic tree it is obvious that species belonging to Saprolegnia parasitica
s.l. are very close to each other with the low level of genetic variability.
Group 2, a minor group is represented by 6 isolated strains, one from diseased fish (brown
trout) and five strains (from river water of Doubs (baiting) and they are corresponding to
Saprolegnia sp.
18S rRNA
According to 18S phylogenetic tree our strains belong to two groups. Group 1
(Saprolegnia parasitica s.l.) contains strains all obtained from diseased fish and they show
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a very low degree of genetic variability. These strains are in the branch with other
reference strains representing Saprolegnia parasitica with bootstrap support 0.63. Based
on the low level of genetic variability it could be considered that these strains were
introduced to these rivers recently. And because of high mortality of infected fish these
strains probably possess a high pathogenic potential. Very interesting is that strains came
from three different fish species (grayling, brown trout and stone loach) belonging to two
different fish families (Salmonidae and Balitoridae) and it suggests that the pathogen is
independent on fish species.
Group 2 (Saprolegnia sp.) is represented by strains obtained from river water and one from
diseased fish. These branch supported by bootstrap 0.76 contains the strains which don’t
belong to one species (such as Saprolegnia parasitica), they are also other strains (such as
Saprolegnia ferax), therefore this group we called Saprolegnia sp. Based on the fact that
these strains were obtained from river water (only one from diseased fish) these strains
could be considered as non-pathogenic and native.
Acknowledgement: The paper is a result of research work done within the frames of grant
projects VEGA of the Ministry of Education of the Slovak Republic No. 1/0061/16 and
APVV-14-0274.
Literature
1.
Van den Berg AH, McLaggan D, Diéguez-Uribeondo J, Van West P, 2013: The impact of the water
moulds Saprolegnia diclina and Saprolegnia parasitica on natural ecosystems and the aquaculture
industry. Fungal Biology Reviews, 27, 33-42.
2.
Johnson Jr, TW, Seymour, RL and Padgett, DE (2002) Biology and systematics of the Saprolegniaceae.
On-line publication: http://www.ilumina-dlib.org. 1028p.
3.
Chomczynski P, Sacchi N (2006) The single-step method of RNA isolation by acid guanidinium
thiocyanate-phenol-chloroform extraction: twenty-something years on. Nat Protoc. 1(2):581-5.
4.
López-García P, Rodriguez-Valera F, Pedrós-Alió C, Moreira D (2001) Unexpected diversity of small
eukaryotes in deep-sea Antarctic plankton. Nature 409:603-607.
5.
Medlin L, Elwood HJ, Stickel S, Sogin ML (1988) The characterization of enzymatically amplified
eukaryotic 16S-like rRNA coding regions. Gene 71:491–499Osman HM, Solman WE, Noor El Deen
AE, Mohamed LA, 2008: Induction of Saprolegniosis in Oreochromis niloticus with Special Reference
to its Biological Control. Global Veterinaria Vol 2 (1), 01-45.
6.
Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace
IM, Wilm A, Lopez R, Thompson JD, Gibson TJ and Higgins DG. (2007) Clustal W and Clustal X
version 2.0. Bioinformatics 2007 23(21): 2947-2948
7.
Jobb G, von Haeseler A, Strimmer K (2004) TREEFINDER: a powerful graphical analysis environment
for molecular phylogenetics. BMC Evol Biol 4:18
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18th-20th October 2016
Rodriguez F, Oliver JL, Marin A, Medina JR (1990) The general stochastic-model of nucleotide
substitution. J Theor Biol 142:485–501
9.
Stamatakis, A., Hoover, P. and Rougemont, J., (2008). A Rapid Bootstrap Algorithm for the
The phylogeny tree can be seen on poster.
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THE CURRENT EPIZOOTIOLOGICAL AND EPIDEMIOLOGICAL SITUATION
IN OCCURENCE OF RABIES IN SLOVAKIA
Mandelík R.1, Ondrejková A.1, Ondrejka R.1, Smrčo P.1, Chvojka D.2,
Korytár Ľ.2, Koščo J.2, Oravec M.3,
1
University of Veterinary Medicine and Pharmacy in Košice
Department of infectious and parasitic diseases
Komenského 73, 041 81 Košice, Slovakia
[email protected]
2
University of Veterinary Medicine and Pharmacy in Košice
Department of the environment, veterinary legislation and economy
Komenského 73, 041 81 Košice, Slovakia
3
Veterinary and Food Institute in Košice
Hlinkova 1/A, 043 65 Košice, Slovakia
Introduction: Rabies is an acute progressing lyssavirus disease of warm-blooded animals
transmissible to humans - zooantroponosis. Infectious agent is a virus of the family
Rhabdoviridae, genus Lyssavirus, type Rabiesvirus, a neurotrophic agent spread from the
wound site along the nerve pathways to the central nervous system [1].
Rabies is transmitted through the saliva of an infected animal. Infection occurs primarily
via bite wounds, or infected saliva entering an open cut or wound or mucous membrane,
such as those in the mouth, nasal cavity or eyes. Infection through inhalation of the virus
has been documented, for example, in the environment of a densely populated bat cave [2].
The disease has a long incubation period (six months) and symptoms may take several
weeks to appear after infection. However, once symptoms appear, rabies is always fatal
[2].
Clinical signs of rabies in animals vary depending on the effect of the virus on the brain.
Typical signs include sudden behavioural changes and progressive paralysis leading to
death. In some cases, however, an animal may die rapidly without demonstrating
significant clinical signs [2].
In humans, early signs can include fever or headache. As the disease progresses, symptoms
may include confusion, depression, sleepiness, agitation or paralysis of the face, throat and
neck. Death generally results from progressive paralysis [2].
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Rabies is a disease listed in the World Organization for Animal Health (OIE) Terrestrial
Animal Health Code and must be reported to the OIE (OIE Terrestrial Animal Health
Code) [2, 3].
The aim of study: The aim of our study was to prepare a review about the current
epizootiological and epidemiological situation of rabies in Slovakia.
Methodology: We have collected these data: a chronological overview of rabies cases in
Slovakia, the incidence of positive rabies cases in Slovakia from 2001 to 2015, the results
of the examinations of animals for rabies in Slovakia from 2000 to 2014.
We obtained these information from the sources that are published on the official website
of the State Veterinary and Food Administration [4].
We investigated these data: the number of diseases in humans to rabies for the calendar
year, the number of reported threats of rabies in each calendar year after contact persons
with the animal rabid or suspected of rabies, the number of vaccinated persons after
exposure of the animal, reported threats by rabies according to individual regions in
Slovakia, threatening cases of rabies in different age groups, an overview of the species
and number of animals, that exposed humans, the site of injury of human induced by
animal, the method of vaccination and kind of vaccine.
We obtained these information from the annual reports on the activities of Public Health
Authorities in Slovakia, which are published on the official website of the Public Health
Authority of the Slovak Republic since 2006 [5].
Results and Discussion: Rabies is found almost worldwide. The greatest incidence of
rabid animals on the territory of Slovakia was recorded in period from 2000 to 2005. The
most common reservoir of rabies was the fox.
Thanks to the full-area oral vaccination of foxes in spring and autumn campaign, Slovakia
has achieved positive results in the control of rabies in the fox population. Seven new cases
of rabies (4 foxes, 2 dogs, 1 marten) were confirmed from January 2013 to June 2013 in
the Slovak Republic near the border with Poland in the district Bardejov. In 2015, 5
positive cases of rabies were confirmed in foxes in districts of Stará Ľubovňa and Poprad.
In 2003 was recorded the most positive cases of rabies in foxes and other wildlife animals.
The total number of positive cases of rabies was 308 and 46 positive cases of rabies in
dogs, cats and other domestic animals. No positive case of rabies has been confirmed from
2007 to 2012.
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From 2000 to 2014, total of 47778 animals were tested for rabies. Overall 1052 animals
were positive. The most numerous group of positive animals were foxes – 295 specimens
and other wildlife animals - 13 animals were recorded in 2003, since 2003 has recorded a
decrease of rabies in the population of foxes and other wildlife animals. The most
numerous rabid dogs and cats were recorded in 2000 - 66 specimens, then in 2003 - 43
specimens. Since 2003 is registered the reduction in the incidence of rabies in the
population of dogs and cats.
From 1966 to 2015, three men from the districts of Medzilaborce, Spišská Nová Ves and
Rožňava died of rabies. Two of them were shepherds, in one case employment was not
mentioned. Injuries were caused by rabid wolf, fox and feral cat. The latest death of a
person caused by rabies was recorded in 1990 in the village Dlhá Ves, in the district of
Rožňava. From 1991 to 2015 we had no rabies disease in humans [6].
From 2006 to 2015 a total of 9287 cases of contact between man and rabid or rabies
suspect animal were reported in the Slovak Republic (an average of 928,7 persons/ per
year). During monitored period (2006 – 2015) a total of 7687 people (an average of 768,6
persons/ per year) were completely and incompletely vaccinated.
The threats of rabies were reported from all regions of Slovakia, with a peak incidence in
the Banská Bystrica region from 2006 to 2009, then in the years 2011 – 2013 and most
recently in 2015, in the Košice region in 2010 and in Bratislava region in 2014.
Children at the age of 5 to 9 years are in the highest risk group, followed by the group of
children at the age of 10 to 14 years.
From 2006 to 2015 a bite was the most common type of injury in human by animal in
Slovakia (8576 cases; 92,68 % of all cases), this type of injury is followed by scratching
(480 cases; 5,18 % of all cases), and salivation of the wound (28 cases; 0,30 % of all
cases).
Conclusion: An important measures in prevention of rabies: legally required preventive
vaccination and revaccination of carnivores against rabies over three months of age using
the vaccination scheme declared by manufacturer of vaccine, indicated preventive
vaccination of cattle, sheep and goats in outbreak of rabies, monitoring of rabies in
indicated cases at suspicion for disease, veterinary examination of animals of susceptible
species which exposed a human or animal (contact type II or III) according to the WHO
classification, population reduction of foxes by their yearlong shot, an oral vaccination of
foxes against rabies in the spring and autumn campaign with subsequent monitoring of its
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effectiveness (checking income bait and determine the titre of antibodies against rabies)
[7].
Acknowledgement: This work was financially supported by VEGA grant No. 1/0591/15.
Literature
1.
Chvojka, D., Oravec, M., Saladiová, D., Nagyová, A. 2011: Aktuálne problémy besnoty. In Slovenský
veterinársky časopis. ISSN 1335-0099, 2011, roč. XXXVI, č. 4, s. 197.
2.
http://www.oie.int/fileadmin/Home/eng/Media_Center/docs/pdf/Disease_cards/RABIES-EN.pdf
3.
http://www.oie.int/animal-health-in-the-world/oie-listed-diseases-2016/
4.
http://www.svssr.sk/zvierata/choroby_besnota.asp /
5.
http://www.uvzsr.sk/index.php?option=com_content&view=category&id=25:vyronasprava&layout=blog&Itemid=34&layout=default./
6.
Čisláková a kol. 2001: Epidemiológia vybraných nákaz. Vyskoškolské učebné texty Lekárska fakulta
Univerzity P.J. Šafárika v Košiciach. Str. 59.
7.
http://www.svssr.sk/dokumenty/zvierata/NPP2016bes.pdf[cit. 2016-06-29].
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MOLECULAR CHARACTERIZATION AND TRACING OF PERSISTENT
LISTERIA MONOCYTOGENES STRAINS IN THE PRODUCTION CHAIN OF
TRADITIONAL MEAT PROCESSING FACILITY IN SLOVAKIA
Kaclíková E.1, Véghová A.1, Minarovičová J.1, Drahovská H.2
1
Department of Microbiology, Molecular Biology and Biotechnology, National
Agricultural and Food Centre - Food Research Institute - Priemyselná 4, 82475
Bratislava, Slovakia
2
Department Molecular Biology, Faculty of Natural Sciences, Mlynská dolina B-2,
84215 Bratislava, Slovakia
[email protected], [email protected]
Introduction: Listeria monocytogenes is a foodborne pathogen responsible for listeriosis,
representing a significant health hazard, mainly in high-risk groups of newborns, elderly,
immuno-compromised individuals, and pregnant women (1). Listeriosis is associated with
reported fatality rate of 15.6% in 2013 (2), the highest among foodborne pathogens, while
the case-fatality rate is usually as high as 20-30 %. Food contaminated with
L. monocytogenes is considered the primary source of transmission to humans and most
foodborne listeriosis cases are caused by ready-to-eat products contaminated during or
after processing as the pathogen is able to survive at production stress conditions and
persist in the food processing environment (3). All these facts and data emphasize the
importance of environmental monitoring to identify potential contamination sources and
transmission routes in food production chain, particularly of persistent L. monocytogenes
strains.Persistence of specific L. monocytogenes PFGE types over long time periods has
been documented despite of regularfood processing environments and equipment cleaning
and disinfection (4).
The aim of the study: This work was aimed at occurrence and diversity of Listeria
monocytogenes in production chain of traditional meat processing facility in Slovakia to
trace the sources and transmission routes, as well as to reveal the potentially persistent
contamination by the pathogen.
Methodology: A total of 268 samples, including 196 samples of meat processing
environment, with 106 and 90 food-contact and non-food-contact samples, respectively,
and 72 food samples including raw meat, semi-products and ready-to-eat final products
were collected during the four year period from November 2010 to December 2014. The
sampling program was designed to include sites of difficult sanitation, thus most likely to
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harbour L. monocytogenes, and focused on those previously detected positive.
Environmental swabs were collected using sampling kit, to analyse raw materials and meat
products, amounts of 25 g were collected. Immediately after sampling, each sponge and
samples were placed into sterile bags, kept refrigerated during transportation and analysed
within 24 h. The detection of L. monocytogenes was performed using the conventional
culture method according to the currently valid standard procedure ISO 11290-1:1996.
Isolated strains were maintained in 20 % glycerol or freeze-dried for long-term storage at 18 °C. To confirm L. monocytogenes identity, all isolates were analysed by the speciesspecific duplex TaqMan real-time PCR targeting actA gene (5). Isolates identified as
L. monocytogenes were subjected to sub-species molecular characterization. Multiplex gelbased PCR targeting serotype-specific marker genes according to Kérouanton et al. (6),
was used in order to cluster L. monocytogenes strains into five molecular serogroups IIa,
IIb, IIc, IVa and IVb. All L. monocytogenes isolates were analysed using PFGE protocol
with AscI/ApaI restriction enzymes according to the last updated Standard Operating
Procedure for PulseNet PFGE of Listeria monocytogenes (7). The AscI/ApaI-PFGE of
individual L. monocytogenes isolates were analysed by BioNumerics software (Applied
Maths, Kortrijk, Belgium). Similarity levelsbetween PFGE patterns were calculated using
Dice´s coefficient with band tolerance of 1.0% and cluster analysis was performed by
UPGMA. The MLST scheme of amplification and sequencing of seven gene fragments
according to the Protocol PF8 Genotyping of Pathogens and Public Health Platform,Institut
Pasteurwas used to subtype representative L. monocytogenes strains (8). Detection of
genetic markers for epidemic clones ECI, ECII and ECIII was performed by multiplex
PCR described by Chen and Knabel (9).
Results and Discussion: Out of 268 analysed samples, 70 (26.1 %) were found to be
positive for the presence of L. monocytogenes when the positivity was associated mainly
with environmental samples (85.7 %) (Table 1). From 70 positive samples, 77 individual
L. monocytogenes isolates were obtained. Molecular serotyping by using multiplex PCR
classified these strains into four serogroups, with the majority of 34 (44.1 %) strains in
serogroup IIa, followed by 22 (28.6 %) strains in IVb, 15 (19.5 %) strains in serogroup IIc,
and 6 (7.8 %) strains in serogroup IIb.
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Table 1: Number of positive/analysed samples for L. monocytogenes in sampling
period from 11-2010 to 12-2014 in meat-processing facility
Sampling year
2010
Environmental samples
1/9
Food contact
0/2
Non-food contact
1 / 11
Subtotal
Food samples
0/4
Final products
0/0
Raw meat products
0/4
Subtotal
1 / 15
Total
2011
2012
2013
2014
Number of samples positive/analysed
Total
1 / 20
1/7
2 / 27
1 / 22
1/7
2 / 29
5 / 12
13 / 16
18 / 28
8 / 43
29 / 58
37 / 101
16 / 106
44 / 90
60 / 196
1 / 13
0/0
1 / 13
3 / 40
0/8
0/0
0/8
2 / 37
0/6
0/4
0 / 10
18 / 38
4 / 25
5 / 12
9 / 37
46 / 138
5 / 56
5 / 16
10 / 72
70 / 268
Molecular markers specific for three epidemic clones (ECI–ECIII) of respective
L. monocytogenes serogroups were investigated with positive results for ECIII in 18 strains
of serogroup IIa particularly associated with a single AscI/ApaI-PFGE profile. To evaluate
the type diversity, molecular subtyping by PFGE of all isolated strains and MLST for
selected strains was applied. Cluster analysis of AscI/ApaI-PFGE patterns resulted in strain
discrimination into 18 PFGE profiles at 100 % similarity. Three of them, AscI-PFGE
patterns (MLST) classified in our study as 1 (ST 9), 2 (ST 14) and 9 (ST 2) for 14, 18 and
17 strains, respectively, were predominant (63.6 %) and considered persistent, being
isolated repeatedly from at least five successive samplings during more than one year,
despite of regular cleaning and sanitation. Distribution of the remaining 29 sporadically
isolated L. monocytogenes strains to 15 profiles showed their high diversity.
Conclusion: Molecular subtyping by PFGE was confirmed to be a reliable tool to identify
the contamination routes and niches in food-processing facilities and, complemented with
molecular characterization of the virulence potential and persistence, can contribute to the
improvement of L. monocytogenes control programs in food processing. In meatprocessing facilities, most attention should be paid to hygiene of the production chain and
to development of improved process control strategies focused on effective prevention, not
only to final products.
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Acknowledgement: This work is a result of implementation of the project ITMS
26240220089 “Effective control methods for safety foods“ of the Agency for the Structural
Funds of the European Union, Ministry of Education, Science, Research and Sport of the
Slovak Republic.
Literature
1.
VAZQUEZ-BOLAND, J.A, KUHN, M., BERCHE, P., CHAKRABORTY, T., et al. 2001. Listeria
pathogenesis and molecular virulence determinants. Clin. Microbiol. Rev, 14, 584-640.
2.
EFSA 2015. The European Union summary report on trends and sources of zoonoses, zoonotic agents
and food-borne outbreaks in 2013. In: EFSA Journal [online], 13 (1).
3.
THÉVENOT, D., DELIGNETTE-MULLER, M.L., CHRISTIEANS, S., LEROY, et al. 2006.
Serological and molecular ecology of Listeria monocytogenes isolates collected from 13 French pork
meat salting-curing plants and their products. Int. J. Food Microbiol., 112, 153-161.
4.
DI CICCIO, P., MELONI, D., FESTINO, A.R., CONTER, M., et al. 2012. Longitudinal study on the
sources of Listeria monocytogenes contamination in cold-smoked salmon and its processing environment
in Italy. Int. J. Food Microbiol., 158, 79-84.
5.
ORAVCOVÁ, K., KACLÍKOVÁ, E., KRASCSENICSOVÁ, K., PANGALLO, D., et al. 2006.
Detection and quantification of Listeria monocytogenes by 5'-nuclease polymerase chain reaction
targeting the actA gene. Lett. Appl. Microbiol., 42, 15-18.
6.
KÉROUANTON, A., MARAULT, M., PETIT, L., GROUT, L., et al. 2010. Evaluation of a multiplex
PCR assay as an alternative method for Listeria monocytogenes serotyping. J. Microbiol. Methods, 80,
134-137.
7.
Standard Operating Procedure for PulseNet PFGE of Listeria monocytogenes (PNL04, last update April
2014), 1-11. (http://www.cdc.gov/pulsenet/PDF/listeria-pfge-protocol-508c.pdf).
8.
Protocol
PF8
Genotyping
of
Pathogens
and
Public
Health
Platform,Institut
Pasteur
(http://www.pasteur.fr/recherche/genopole/PF8/mlst/primersLmono.html).
9.
CHEN, Y. and KNABEL, S.J. 2007. Multiplex PCR for Simultaneous Detection of Bacteria of the
Genus Listeria, Listeria monocytogenes, and Major Serotypes and Epidemic Clones of L.
monocytogenes. Appl. Environ. Microbiol., 73, 6299-6304.
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OCCURRENCE OF ESCHERICHIA COLI PRODUCING ESBL-, AMPC- IN
POULTRY, PORK AND BEEF SAMPLES TAKEN FROM THE RETAIL.
Mojžišová, A., Čuvalová Z.
State Veterinary and Food Institute – Veterinary and Food Institute in Dolny Kubin,
Janoskova 1611/58, 026 01 Dolny Kubin, Slovakia
[email protected]
Introduction: Codex Alimentarius Commision adopted, during its 34th session in Geneva,
the Guidelines for the Risk Analysis of Foodborne Antimicrobial Resistance which
highlight AMR as a major global public health concern and a food safety issue. The use of
antimicrobial agents in food –producing animals and crops is a potentially important risk
factor for the selection and dissemination of AMR microorganisms and determinants from
animals and food crops to human via the consumption of food. Therefore in the
Communication of 15 November 2011 from the Commission to the European Parliament
and the Council – Action Plan against the rising threats from Antimicrobial Resistance, the
Commission have proposed to put in place a five-year action plan to fight against AMR
based on 12 key actions, including strengthened surveillance systems on AMR. One
component of the Action Plan is also Decision 2013/652/EU on the monitoring and
reporting of antimicrobial resistance in zoonotic and commensal bacteria. This Decision
lays down details rules for the harmonised monitoring and reporting of AMR in to be
carried out by member States in accordance with Article 7 and 9 of Directive 2003/99/EC
and Annex II (B) and Annex IV.
The aim of the study: The objective of this study was detection of ESBL- or AmpCproducing Escherichia coli from samples of fresh meat of broilers, pig and bovine meat
gathered at retail according to Decision 2013/652/EU1.
Methodology: Slovakia shall collect at retail random 150 samples of fresh meat of broilers
from 15 RVFI (10 samples per each RVFI) and 150 samples of pig and 150 samples of
bovine meat from 5 RVFI (30 samples per each RVFI). Samples should be without any
additives and without pre-selecting samples based on the origin of the food. Subject to
harmonized monitoring in 20152 was pork and beef, poultry meatin 20163.
For the detection of ESBL- or AmpC producing E.coli the method starts by a preenrichment step, followed by inoculation on MacConkey agar containing a third generation
cephalosporin in a selective concentration according to the most recent version of the
detailed protocol for standardisation of the EURL for Antimicrobial Resistance4. The
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microbial species E. coli shall be identified using appropriated method. ISO 20776-1:20065
- microdilution method was performed to determine atb profile. One presumptive ESBLor AmpC- E. coli isolate obtained from each meat sample is tested on the first panel of
antimicrobials in accordance with Table 1 in Decision and further submitted to extended
susceptibility testing if they are resistant to cefotaxime or ceftazidime or meropenem based
on the interpretative criteria – epidemiological cut-off values. These resistant isolates are
futher tested with a second panel of antimicrobial substances in accordance Table 2.This
panel includes cefoxitin, cefepime and clavulanate synergy test in combination with
cefotaxime and ceftazidime for detection of ESBL and AmpC production. In addition the
second panel also contains imipenem, meropenem and ertapenem to phenotypically verify
the presumptive carbapenemase-producers.
Results and Discussion: Poultry meat: monitoring in poultry meat is still ongoing,
therefore the data is used to 31.7.2016.
47 samples (63%) out of 75 sampled poultry meat were positive for presence of ESBL- or
AmpC- producing Escherichia coli. 15 isolate showed resistance to Cefoxitin, no synergy
test with clavulanic acid for ceftazidime nor synergy test with clavulanic acid for
cefotaxime and according to EFSA criteria for interpretation of ESBL-, AmpC-,
carbapenemase producing Escherichia coli, was phenotypically included to the group of
presumptive AmpC- producer. 25 isolates were sensitive to Cefoxitin, showed synergy test
with clavulanic acid for ceftazidime and / or synergy test with clavulanic acid for
cefotaxime and were phenotypically included to the group of presumptive ESBLproducers. 7 isolatesshowed resistance to Cefoxitin, synergy test with clavulanic acid for
ceftazidime and / or synergy test with clavulanic acid for cefotaxime was phenotypically
included to the group of presumptive ESBL + AmpC- producer.
Origin of positive samples were Slovakia, Czech Republic, Romania and Hungry. Poland ś
retail poultry meat were not sampled yet.
Bovine meat:5 samples (3%) out of 150 sampled bovine meat were positive for presence of
ESBL- or AmpC- producing Escherichia coli. All 5 isolates showed resistance to
Cefoxitin, no synergy test with clavulanic acid for ceftazidime nor synergy test with
clavulanic acid for cefotaxime and according to EFSA criteria for interpretation of ESBL-,
AmpC-, carbapenemase producing Escherichia coli, and was phenotypically included to
the group of presumptive AmpC- producer.
Origin of positive samples were Slovakia and Poland.
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Pork meat: 5 samples (3%) out of 150 sampled pork meat were positive for presence of
ESBL- or AmpC- producing Escherichia coli. 1 isolate showed resistance to Cefoxitin, no
synergy test with clavulanic acid for ceftazidime nor synergy test with clavulanic acid for
cefotaxime and according to EFSA criteria for interpretation of ESBL-, AmpC-,
carbapenemase producing Escherichia coli, and was phenotypically included to the group
of presumptive AmpC- producer. 3 isolates showedisolates were sensitive to Cefoxitin,
showed synergy test with clavulanic acid for ceftazidime and / or synergy test with
clavulanic acid for cefotaxime, were phenotypically included to the group of presumptive
ESBL- producers.1 isolate showed resistance to Cefoxitin, synergy test with clavulanic
acid for ceftazidime and / or synergy test with clavulanic acid for cefotaxime, was
phenotypically included to the group of presumptive ESBL + AmpC- producer. Origin of
positive samples were Slovakia and Poland as well.
This is the first study from Slovakia reporting high rates of ESBLs in Escherichia coli
isolated especially from retail chicken meat. These results raise serious concerns about
public health and food safety as retail meat could serve as a reservoir for these resistant
bacteria which could be transferred to humans through the food chain.Good work and hand
hygiene can be used to prevent ESBL infections related to cooking. ESBL bacteria can by
destroyed in food by means of heating, meaning that cooked meat and other products do
not contain ESBL bacteria, provide that the contamination of these foods has been
prevented after cooking.
Literature
1.
Decision of 12 November 2013 on the monitoring and reporting of antimicrobial resistance in zoonotic
and commensal bacteria (2013/652/EU)
2.
Instruction the State Veterinary and Food Administration of Slovak Republic No.1848 / 2015-370:
Monitoring of antimicrobial resistance in 2015
3.
Instruction the State Veterinary and Food Administration of Slovak Republic No.675/16-370:
Monitoring of antimicrobial resistance in 2016
4.
Laboratory Protocol: Isolation of ESBL-, AmpC- and carbapenemase – producing E. coli from fresh
meat.
October
2015,
version
3
http://eurl-
ar.eu/data/images/protocols/esbl_ampc_cpeprotocol_version_meat_october2015_ version3.pdf .
5.
EN ISO 20776-1:Clinical laboratory testing and in vitro diagnostic test systems -- Susceptibility testing
of infectious agents and evaluation of performance of antimicrobial susceptibility test devices -- Part 1:
Reference method for testing the in vitro activity of antimicrobial agents against rapidly growing aerobic
bacteria involved in infectious diseases
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ENVIRONMENT IN LOW HYGIENIC STANDARDS SETTLEMENT IN PREŠOV
REGION – A POTENTIAL SOURCE OF ZOONOSES
Papajová, I.1, Pipiková, J.1, Šoltýs, J.1, Sasáková, N.2, Takáčová, D.2, Gregová, G.2
1
Institute of Parasitology Slovak Academy of Sciences, Hlinkova 3, 040 01 Košice,
Slovakia [email protected]
2
University of Veterinary Medicine and Pharmacy in Košice, Komenského 73, 041 81
Košice, Slovakia
Introduction: Parasitic infections, as well as related bacterial, fungal and viral infections
affect hundreds of millions people living in low- and middle-income countries. Giardia
intestinalis and amoebae, together with Cryptosporidium spp. belong to the most
commonly detected intestinal protozoa. From a group of helminths there are Ascaris
lumbricoides, Trichuris trichiura, Enterobius vermicularis, hookworms, and tapeworms
Taenia spp. [1].
Due to the rapid growth of human population, intestinal parasitic infections represent a
health problem not only in developing but also for industrialised countries. It is well
known that the origin of these parasitic infections is very often endemic and easily
transmitted at the places with poor sanitation and crowded living conditions. In Slovakia,
such places are largely represented by the Roma settlements and housing.
The aim of the study: The aims of this study was to monitor the level of soil
contamination with propagative stages of intestinal parasites at different public places in
low hygienic standards settlement in Prešov region and to determine the incidence of
parasites in dog’s faecal samples.
Methodology: 65 soil samples from the low hygienic standards settlement in Prešov region
were investigated according to Kazacos [2]. A total of 197 faecal samples of unknown
dogs were collected at random from the public areas of the settlement. After collection,
faecal samples were stored at 4°C and examined for the presence of propagative stages of
endoparasites as soon as possible. A flotation method with the Shaeter’s flotation solution
(specific gravity 1.3 g.ml-1) was used for coprological examination.
Results and Discussion: The objective of this study was to determine the possibility of
soil contamination with propagative stages of intestinal parasites at different public places
in low hygienic standards settlement in Prešov region. In a one-year-study, totally 65 soil
samples were examined for the presence of parasitic germs. Parasitic germs have been
confirmed in soil samples from each colletction, while predominate findings were the eggs
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of Ascaris spp. (51.5%), strongyle-type eggs (48.5%) and Toxocara spp. (36.4%). Soil near
dwellings where dogs move and children play is highly contaminated with eggs of
parasites and serve as a medium for spreading the infection.
The presence of large populations of untreated animals or stray dogs and cats establish a
permanent reservoir for the infection in Roma settlement.The animals from settlement have
free movement without restriction and they can contaminate public places with
excrements. The main sources of endoparasitic germs within environment are faeces of
infected animals. Additionally 197 dog’s faecal samples were examined. The average
occurrence of endoparasites in dogs’ faecal samples during the whole studied period was
70.2%. A total of 12 taxa of intestinal parasites were detected and the most prevalent were
the eggs from the family Ancylostomatidae, Ascaris spp. eggs and Toxocara spp. eggs.
Problems with uncontrolled and untreated dogs lead to a heavily contaminated
environments which under warm climatic conditions could provide good transmission
opportunities for zoonotic infections. The presence of parasites found in soil and dogs
excrements demonstrates a possible risk also of human infection.
Conclusion: The source of soil contamination in studied low hygienic standards settlement
in Prešov region are dogs, which in addition to its own parasite fauna passage through
digestive system germs of human internal parasites. Children probably participate in soil
pollution, as the village lacks sewerage and sanitary facilities and children who lack basic
hygiene habits, carry out their physiological needs close to dwellings. Contaminated soil in
this locality is a risk factor contributing to the transmission of endoparasitic infections
(including those of zoogenous character) in children.
Acknowledgement: The study was supported by the Project VEGA No. 2/0140/13.
Literature
1.
Rudohradská, P., Halánová, M., Ravaszová, P., Goldová, M., Valenčáková, A., Halán, M., Papajová, I.,
Pohorencová, A., Valko, J., Čisláková, L., Juriš, P. 2012. Prevalence of intestinal parasites in children
from minority group with low hygienic standards in Slovakia. In Helminthologia, Vol. 49, Iss. 2, p. 63 –
66.
2.
Kazacos, K. R. 1983. Improved method for recovering ascarid and other helminth eggs from soil
associated with epizootics and during survey studies. In American Journal of Veterinary Research, Vol.
44, p. 896-900.
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LEPTOSPIROSES IN SLOVAKIA IN 2006 – 2015
Perželová J., Macháčová M., Bakoss P., Špaleková M., Jareková J.
Institute of Epidemiology, Faculty of Medicine Comenius University, Špitálska 24, 813 72
Bratislava, Slovakia
[email protected]
Introduction: Leptospiroses are worldwide occurring diseases transmissible to humans
from wild and domestic animals. Their occurrence in the world shows significant regional
differences related to different natural and social factors. The incidence of this disease
is highest in humid tropical and subtropical regions, particularly in developing countries
(with incidence
>10/100,000
population),
in temperate
areas
reported
incidence
is 0.1 ‒ 1/100,000 (1). Over the years, there are changes in economic and social conditions
of community affecting the occurrence and epidemiological features of many infectious
diseases including leptospiroses.
The aim of the study: Presentation of epidemiological characteristics of leptospiroses
in Slovakia in 2006 ‒ 2015.
Methodology:
2006 ‒ 2011
Data
were
of the human
obtained
leptospiral
diseases
from published
in Slovakia
for the years
records
Surveillance
of zoonoses ‒ leptospiroses and for the years 2012 ‒ 2015 from the Reports of zoonoses,
alimentary infections and water-borne infections in Slovakia (2, 3). Data were analyzed
according to conventional epidemiological indicators.
Results and Discussion: During the studied period the incidence of leptospiroses
in Slovakia ranged from 0.07/105 to 0.4/105, with the average rate 0.27/105 (Tab. 1).
Compared to period 1997 ‒ 2006 (4) this represents more than 2-fold decrease, and this
downward trend is long-term, the same is also observed in other developed countries (1).
Tab. 1 Incidence of leptospiroses in Slovakia.
years
2006 2007 2008 2009 2010 2011 2012 2013 2014 2015
incidence rate per
0.4
0.4
0.3
0.4
0.4 0.15 0.17 0.07 0.22 0.17
105 people
This decrease of incidence is probably due to several social and natural factors (lower
engagement of workers in agriculture, improving housing conditions, environmental
and climate changes) and not least also by the fact that currently leptospiroses are
sometimes forgotten in differential diagnosis and reported incidence could be clearly
undervalued.
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Fig. 1. Distribution of leptospiroses by etiology in Slovakia in 2006 – 2015.
SJ – Sejroe
group,
BA ‒ Bratislava/Jalna
GT – Grippotyphosa,
PO – Pomona,
IC – Icterohaemorrhagiae/Copenhageni,
Total 146 human leptospiroses were diagnosed in Slovakia in the studied period
2006 ‒ 2015. The highest proportion of diseases accounted for Weil's disease caused
by leptospires of serovar Icterohaemorrhagiae/Copenhageni (35%), followed by field fever
(18%)
and leptospirosis
sejroe
(16%).
Leptospiroses
pomona,
bratislava/jalna
and the group of other serovars accounted for 10% and 11%, respectively (Fig. 1).
It appears that in comparison to the period 1997 ‒ 2006 (4), the proportion of Weil's
disease increased, while proportion of field fever decreased.
0,5
Incidence rate per 100 000
0,45
0,4
0,35
0,3
0,25
0,2
0,15
0,1
0,05
0
5-9
10-14
IC,CO
GT
15-19
20-29
SJ
30-39 40-49
Age groups
PO
BA
50-59
60-69
other
70+
total
Fig. 2. Incidence of leptospiroses by age and etiology in Slovakia in 2006 – 2015.
SJ – Sejroe
group,
BA ‒ Bratislava/Jalna
GT – Grippotyphosa,
PO – Pomona,
IC – Icterohaemorrhagiae/Copenhageni,
Leptospiroses were notified in population older than 10 years and in elderly people.
Overall incidence shows increasing rate with growing age. The highest incidence was
in the age group of 50 – 59 years. Similarly, etiologic variability of leptospiroses was
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broader with increasing age. While Weil's disease and field fever occurred in 10 – 19 years
old children (in the age group 15 – 19 years also leptospirosis sejroe), in the higher age
groups also other types of leptospiroses were detected (Fig. 2).
90
76.8
80
70
60
%
50
40
30
28.1
23.3
13.7
20
4.1 10.3 5.5 7.5
6.8
10
2.8
8.9
8.2
2.7
1.3
0
M
F
M
IC
F
GT
M
F
SJ
M
F
PO
M
F
BA
M
F
other
M
F
total
Fig. 3. Distribution of leptospiroses by gender and etiology in Slovakia in 2006 – 2015.
SJ – Sejroe
group,
GT – Grippotyphosa,
PO – Pomona,
IC – Icterohaemorrhagiae/Copenhageni,
BA ‒ Bratislava/Jalna, M – males, F – females
The occurrence of leptospiroses was 3.3-times higher in men compared to women (112 vs.
34 cases). Leptospiroses caused by all types predominated in men, as was also observed
in the past (4). The greatest gender differences (Fig. 3) were found in Weil's disease
(4.1 times more cases among men), field fever (3.3 times more) and leptospirosis
bratislava/jalna (3 times more), whereas the lowest long-term sex difference has been
observed in leptospirosis sejroe (1.9 times more).
Comparing proportional occurrence of leptospiroses according to occupation and social
groups (Fig. 4) a quarter of diseases occurred in the group of pensioners and housewives.
Another major groups include unemployed and homeless people (16.4%) and very
heterogeneous group “others” (15.1%). The lowest proportion of leptospiroses was
reported among abattoir workers (3.4%) and agriculture workers (6.2%). The same
as in the period 1997 ‒ 2006 (4), continuing decline in professional leptospiroses was
observed while the proportion of diseases associated with frequent recreational outdoor
activities increased. These changes may be related to improved work conditions or with
reduction of some professions associated with risk of leptospiroses and on the other side,
with emergence of some new risk groups in the context of social changes (unemployed,
homeless people).
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IC
GT
SJ
PO
BA
18th-20th October 2016
other
10
25.3
8
16.4
13.0
6
15.1
9.6
%
4
6.2
11.0
2
3.4
0
A
B
C
D
E
occupation / social groups
F
G
H
Figure 4.Proportion of leptospiroses by occupation/social groups in Slovakia, 2006 2015.
SJ – Sejroe
group,
GT – Grippotyphosa,
PO – Pomona,
IC – Icterohaemorrhagiae/Copenhageni,
BA ‒ Bratislava/Jalna, A – farmers, B – workers, C – abattoir workers, D – field workers, E – pupils,
students, F – housewives, pensioners, G – unemployed people, homeless, H – other
Leptospiroses occurred with typical seasonality in summer, with more than 51%
of the diseases from June to September.
Conclusion: In Slovakia decreasing incidence of human leptospiroses has continued.
Changes in the epidemiological indicators of the disease (etiological structure, age-specific
incidence and particularly occupation/social groups distribution) are associated with risk
of exposure especially reflecting changes in social and economic conditions observed
in our country in the last decades.
Literature
1.
Picardeau M.: Diagnosis and epidemiology of leptospirosis. Med Mal Infect, 2013; 43(1), 1-9
2.
Surveillance of zoonoses. Slovak Republic. Leptospiroses (2006 – 2011, yearly issues), State Veterinary
and Food Administration of the Slovak Republic, Bratislava
3.
Report of zoonoses, alimentary infections and infections from water in Slovakia (2012 – 2015, yearly
issues), Ministry of Agriculture and Rural Development of the Slovak Republic, Martin
4. Bakoss, P., Macháčová,, E., Jareková, J.: Long-term trends in the epidemiology of human leptospirosis
(Slovak Republic, 1954 ‒ 2006), European Journal of Clinical Microbiology & Infectious Diseases,
2012; 31(9), 2167-2176
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DETECTION OF GENE ENCODING ENTEROTOXINS IN COAGULASENEGATIVE STAPHYLOCOCCI ISOLATED FROM FOODS OF ANIMAL
ORIGIN
Regecová I., Pipová M., Jevinová P., Valenčáková A., Danišová O., Mačanga J.
Department of Food Hygiene and Technology, University of Veterinary Medicine and
Pharmacy in Košice, Slovak Republic
[email protected]
Introduction: Epidemiological studies have identified Staphylococcus aureus as the most
common agent involved in food poisoning. However, current research highlights the
importance of toxigenic coagulase-negative staphylococci (CoNS) isolated from food [6].
S.aureus is the most common agent involved in food poisoning, there is current concern in
the scientific community regarding CoNS, which have been recognized as opportunistic
pathogens in human and animal infections, allied to risks of toxigenic lineages in cases of
food poisoning in humans [4].DNA amplification methods (polymerase chain reaction,
PCR) can show the presence of enterotoxigenic strains of Staphylococcus spp. before the
expression of enterotoxins on the base of specific gene sequences and in this way detect
the potential source of staphylococcal enterotoxicosis[5].Moreover, no studies aimed at
production enterotoxin of CoNS isolates from marine fish and wild rabbit are available.
Therefore, this study is focused on both species identification and detection of gene sea,
seb, sec, sed, see in coagulase-negative staphylococci isolated from the meat of alaska
pollock, atlantic herring and wild rabbit.
The aim of the study: The aim of this study was to isolate staphylococci from samples of
meat of marine fish and samples from thigh muscle of wild rabbit. Isolated staphylococci
were then subjected to identification by MALDI-TOF mass spectrometry. Consequently, in
the identified coagulase-negative staphylococci, the genes responsible for the production of
enterotoxins A, B, C, D and E were detected by multiplex PCR. The aim was also to
confirm that coagulase-negative staphylococci can be a potential source of staphylococcal
enterotoxicosis in meat of marine fish and wild rabbit.
Methodology: Staphylococci were isolated from samples of meat of Alaska Pollack
(Theragra chalcogramma), meat of herring (Clupea harengus) and thigh muscle of wild
rabbit (Oryctolagus cuniculus). From the collected samples, staphylococci were isolated
according to the instructions [8] Species identification was performed by MALDI-TOF
mass spectrometry [1]. Subsequently, in identified staphylococcal isolates the presence of
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genes coding for the production of staphylococcal enterotoxin (sea, seb, sec, sed and see)
were detected using multiplex PCR [7]. Following standard strains of Staphylococcus
aureus (CSM Brno, Czech Republic) were used as a positive control for the multiplex
PCR: CCM 5756 (sea gene), CCM 5757 (seb gene), CCM 5984 (sec gene), CCM 5973
(sed gene) a CCM 5972 (see gene).
Results and Discussion: Many authors present in their works the incidence of coagulasenegative staphylococci isolated from food of marine fish and game. These claims are also
confirmed by our study. 90 isolates of coagulase-negative staphylococci were gained by
cultivation microbiological examination of samples of marine fish meat and wild rabbits
meat. Isolates were classified in individual species using MALDI-TOF.
Table 1, Numbers of the species of staphylococci identified by MALDI-TOF
Species (number of isolates) Alaska Pollack Herring Wild rabbit
S. warneri(n=64)
S. epidermidis (n=54)
S. pasteuri
(n=18)
S. haemolyticus (n=10)
S. xylosus
(n=8)
S. hominis
(n=5)
S. cohnii
(n=1)
S. capitis
(n=1)
S. sciuri
(n=1)
12
13
1
1
16
2
2
5
1
1
-
14
2
14
5
1
-
In this study, all strains of staphylococci were isolated from samples of frozen meat of
Alaska Pollock, herring and samples of meat of wild rabbit and all of them werecoagulasenegative as shown in Table 1. Strains of CoNS can cause serious nosocomial infections.
Suchstrains
frequently
possessvarious
virulence
factors
including
enterotoxins,
antimicrobial resistance and the ability to form a biofilm. Therefore, correct identification
and typing of CoNS is important from both clinical and epidemiological points of view[3].
The identified isolates of staphylococci were then assayed by PCR method for the presence
of genes coding the production of enterotoxin sea, seb, sec, sed and see. In all
staphylococcal isolates, only the presence of seb and sed gene (in 9 strains; 8,1 %) was
confirmed (Table 2).
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Table 2, The presence of seb and sed genes in different species of staphylococci
Alaska Pollock
(na=3)
Herring
(na =2)
Wild rabbit
(na= 5)
Species
S. warneri
S. pasteuri
seb gene (nb= 5) sed gene (nc= 4)
2
1
-
S. warneri
1
1
S. pasteuri
-
2
S. warneri
S. xylosus
-
1
S. epidermidis
1
-
a
n – the number of strains, in which at least one gene coding the production entrotoxínu was detected
nb – the number of strains, in which presence of seb gene was detected
nc – the number of strains, in which presence of sed gene was detected
Our results correlate with the study of Mello et al. [6], where seventy-six (41.9%) of the
181 coagulase-negative staphylococci were positive for at least one of the genes studied.
The seb gene was detected in 14 (7.7%) and sed in 1 (0.5%) of the isolates. The sed gene
was detected in only 0.5% of the strains studied, in an S. epidermidis isolate. This gene was
not detected by Calsolari et al. [2] in toxigenic coagulase-negative isolates, with only one
S. aureus strain being positive. However, in our study we confirmed the presence of the
sed gene in two strains of S. pasteuri, in one strain of S. xylosus and S. epidermidis.
Presence of seb gene was confirmed in 3 strains of S. warneri, in one strain of S. pasteuri
andS. epidermidis.The presence of both enterotoxin genes at the same time was not
recorded even in one strain.
Conclusion:The presence of genes encoding enterotoxin B and D was confirmed in the
coagulase-negative staphylococci, which indicates their easy spreading among strains of
Staphylococcus spp.Limitation of this study was in the fact that we have not done the
analysis of gene expression, because the presence of enterotoxin genes in isolates of
Staphylococcus spp. does not mean incidence of staphylococcal enterotoxicosis. But
presented results contribute to the argument that coagulase-negative staphylococci are the
carriers of genetic information needed for production of staphylococcal enterotoxins and
thus, they can be considered as a potential risk of staphylococcal enterotoxicosis in food of
animal origin.
Acknowledgement: This study was supported by grants VEGA 1/0705/16 and APVV-150134.
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Literature
1.
BRUKER DALTONICS, MALDI Biotyper 2.0. Software for microorganism identification and
classification user manual. 2008.
2.
CALSOLARI, R.A.O.; PEREIRA, V.C.P.; JÚNIOR, J.P.A.; CUNHA, M.L.R.S. Determination of
toxigenic capacity by RT-PCR in coagulase-negative staphylococci and Staphylococcus aureus isolated
from newborns in Brazil. In Microbiol. Immunol. 2011, vol. 55, 394–407.
3.
CHOMOUCKÁ J. Typizace fenotypově obtížně odlišitelných izolátů koaguláza negativních druhů
stafylokoků [Graduation theses]. Czech Republic: Masaryk University of Brno.2009, 73pp. Available
from: Ústav experimentální biologie - Biologická sekce - Přírodovědecká fakulta.
4.
CUNHA, M.L.R.S.; PERESI, E.; CALSOLARI, R.A.O.; JÚNIOR, J.P.A. Detection of enterotoxins
genes on coagulase-negative staphylococci isolated from foods. In Braz. J. Microbiol. 2006, vol. 37, 70–
74.
5.
HOLECKOVÁ, B.; HOLODA, E.; FOTTA, M.; KALINACOVÁ, V.; GONDOL, J.; GROLMUS, J.
Occurrence of Enterotoxigenic Staphylococcus aureus in Food. In Ann Agric Environ Med.2002, vol. 9,
179-182.
6.
MELLO,
P.L.; RIBOLI, D.F.M.; PINHEIRO, L.; MARTINS, L.A.; BRITO M.A.V.P.; CUNHA,
M.L.R.S. Detection of Enterotoxigenic Potential and Determination of Clonal Profile in Staphylococcus
aureus and Coagulase-Negative Staphylococci Isolated from Bovine Subclinical Mastitis in Different
Brazilian States. In Toxins. 2016, vol. 8, no. 104; doi:10.3390.
7.
SHARMA, N.K.; REES, C.E.; DODD, C.E. Development of a single-reaction multiplex PCR toxin
typing assay for Staphylococcus aureus strains. In Appl. Environ. Microbiol. 2000, vol. 66, no. 4, 13471353.
8. STN EN ISO 6888-1/A1. Mikrobiológia potravín a krmív. Horizontálna metóda stanovenia počtu
koaguláza-pozitívnych stafylokokov (Staphylococcus aureus a ďalšie druhy). Časť 1: Metóda s použitím
Bairdovho-Parkerovho agarového média. Bratislava, SR: SÚTN. 2004, 14.
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DETECTION OF TICK-BORNE PATHOGENS IN DOG – FEEDING TICKS IN
BRATISLAVA (SLOVAK REPUBLIC)
D. Selyemováa, V. Rusňáková Tarageľováa, E. Špitalskáb, M. Derdákováa
a
Institute of Zoology, Slovak Academy of Sciences, Dúbravská cesta 9, 845 06 Bratislava,
Slovakia, [email protected]
b
Institute of Virology, Biomedical Research Center, Slovak Academy of Sciences,
Dúbravská cesta 9, 845 05 Bratislava, Slovakia
Introduction: Ixodes ricinus is the most abundant tick species in Slovakia and the most
important vector of many pathogens.
Lyme disease in dogs was described for the first time in the USA in 1984 (1). Limp,
arthritis, fever, anorexia, fatigue, lethargy are the most frequently clinical signs of Lyme
disease. Both, dogs and humans have the similar clinical signs, but diseases in dogs is more
difficult identifiable.
A. phagocytophilum causes canine granulocytic anaplasmosis with the range of clinical
symptoms, like fever, lethargy, inappetence, vomiting and diarrhea.
Seroprevalence of Rickettsia spp. in dogs varies from 26% to 60% in the endemic areas.
Dogs are considered as “sentinels” in the Mediterranean with the occurrence of MSF in
humans for the determination of this disease. Dogs may have a role as reservoir of
Rickettsia conorii, agent of MSF. Occurrence of this rickettsia in Slovakia is questionable.
Infestation of dogs by Rhipicephalus ticks that are the vectors of MSF is possible when
traveling to Mediterranean area. Symptoms of MSF are similar to influenza and with
petechiae. Symptoms in dogs were described after experimental infection by R. conorii and
include erythema, ache and edema in the injection site. Clinical manifestation of this
disease in dogs is very little studied in Europe (2).
Candidatus N. mikurensis, is a newly emerging tick-borne bacterium associated mainly
with human cases in immunosuppressed people described in last decades. Fatal cases were
also recorded. Vectors of Candidatus N. mikurensis are ticks of Ixodes genus. First cases
of Candidatus N. mikurensis infection in a chronically neutropenic dog from Germany was
confirmed by DNA sequencing in 2007 (3). The signs are asymptomatic in dogs, despite
persistent hematological abnormalities.
Babesiosis is caused by intracellular protozoa transmitted by ticks. The causative agent of
canine babesiosis in our area is Babesia canis. Dermacentor reticulatus tick is an important
vector of canine babesiosis. Infection by this protozoan parasite causes destruction of
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erythrocytes
which
leads
to
anemia,
18th-20th October 2016
haemoglobinanaemia,
haemoglobinuria,
trombocytopenia. Massive infection may lead to multiple organ dysfunction and to death
(4).
The aim of the study: The aim of this study was to investigate the presence of different
tick-borne pathogens in ticks from dogs and the role of dogs in the epidemiology of tickborne diseases. Following agents were screened: Borrelia burgdorferi sensu lato (s.l.),
Babesia spp., Anaplasma phagocytophilum, Rickettsia spp.and Candidatus Neoehrlichia
mikurensis
Methodology: Dog-feeding ticks in various stages of engorgements (nymphs and adult
ticks) were collected in 2011-2014 and tested by PCR methods. Most of dogs came from
western Slovakia (from Bratislava region), but we have obtained ticks from veterinary
clinics in norther Slovakia (Liptovský Mikuláš) as well. Ticks were classified to
developmental stage and sex and placed in 70% ethanol until the DNA extraction.
Genomic DNA was extracted from ticks using a commercial isolation kit (DNeasy blood
and tissue kit, Qiagen, Hilden, Germany).
Ticks were tested for the presence of B. burgdorferi s.l., A. phagocytophilum, Rickettsia
spp.,Candidatus N. mikurensis and Babesia spp. by PCR or realtime PCR. For detection of
each pathogen were used genus-specific primers. A 620-bp fragment of the tick
mitochondrial cytochrome b gene was amplified to confirm the presence of tick (5).
Borrelia, rickettsia and babesia were screened by conventional PCR using previously
described primers and protocols (6, 7, 8). The PCR products were analyzed by
electrophoresis in a 1% agarose gel, stained with GelRed (Biotium, Hilden, Germany) and
visualized with the UV transilluminator. Selected positive PCR products were purified
using a purification kit Wizard SV Genomic DNA Purification System (Promega,
Madison, USA) according to the manufacturer protocol. Further genotyping of Borrelia
was done by RFLP of 5S-23S rRNA and sequencing.
The presence of Candidatus Neoehrlichia mikurensis and A. phagocytophilum was
detected by RT-PCR (9, 10).
Results and discussion: 527 feeding ticks belonging to four species in various stages of
engorgements were collected from dogs.
Female Ixodes ricinus ticks were the most
commonly recorded (86%). Nymphs and males of I. ricinus were collected too. The
additional species belonged to Ixodes hexagonus (1.9%), Haemaphysalis concinna (1.1%)
and Dermacentor reticulatus (5.5%).
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The most prevalent tick-borne pathogens in dogs were A.phagocytophilum (13.3%) and B.
burgdorferi s.l. (12.7%). Borrelia afzelii was the most dominant genospecies (61.1%).
7.3% of feeding ticks (n=451) were infected with Rickettsia spp.
For Babesia spp. and Candidatus Neoehrlichia mikurensis 3.0% and 0.9% of ticks were
PCR-positive, respectively.
In the Czech republic, 8.5% I. ricinus ticks collected from dogs were infected with A.
phagocytophilum and 6.8% with Borrelia spp. The most prevalent was B. garinii (11). In
Belgium, 19.5% and 10.1% of Ixodes ticks collected from dogs and cats, were positive for
A. phagocytophilum and Borrelia spp., respectively (12). In Germany, Borrelia spp. were
detected in 11.6% of I. ricinus and Rickettsia spp. in 61% of. I.ricinus. A. phagocytophilum
and CandidatusN. mikurensis have been reported in I. ricinus 6.5% and 4.3% respectively.
Babesia spp. was found in 2.5% of I. ricinus (13).
During the last decade the growing incidence of the babesiosis in dogs in Slovakia was
recorded (14, 15). Significantly higher prevalence of B. canis in D. reticulatus was
recorded in southeastern Slovakia (14.7%), while the prevalence in southwest was lower
(2.3%) (14). In our study, the prevalence of B. canis in D. reticulatus was 20.1% from all
collected D. reticulatus. Prevalence 35.6% of B. canis in D. reticulatus was found by
Majláthová et al. in the southern part of Slovakia (15).
Conclusion: Higher prevalence of A. phagocytophilum in feeding I. ricinus collected from
dogs than in questing I. ricinus points out on possible reservoir role of dogs in circulation
of A. phagocytophilum in urban areas. In the presented study dogs were used as a sentinel
animals to express the epidemiological risk of being bitten by infected tick for human
population.
Acknowledgements: This study was financially supported by the projects VEGA
no.2/0108/13, VEGA no.2/0061/13, by ITMS: 26240220044 by the Research &
Development Operational Programme founded by the ERDF.
Literature
1.
Lissman, B.A., Bosler, E.M., Camay, H., Ormiston, B.G., Benach, J.L. Spirochete-associated arthritis
(Lyme diseases) in a dog. Journal of the American Veterinary Medical Association 1984, 185: 219-220
2.
Solano-Galeno, L., Trotta, M., Caldin, M. Molecular survey of Rickettsia spp. In sick dogs in Italy.
Zoon. Pub. Health 2008, 55: 521-525
3.
Diniz, P. P., Schulz, B. S., Hartmann, K., Breitschwedt, E.B. “Candidatus Neoehrlichia mikurensis”
infection in a dog from Germany. Journal of Clinical Microbiology 2011, 49(5): 2059-2062
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Zoonoses - Protection of Public and Animal Health
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4.
18th-20th October 2016
Zygner, W., Gójska, O., Rapacka, G., Jaros, D., Wedrychowicz, H. Hematological changes during the
course of canine babesiosis caused by large Babesia in domestic dogs in Warsaw (Poland). Veterinary
Parasitology 2007, 145: 146-151
5.
Black, W. C., IV, and R. L. Roehrdanz. Mitochondrial gene order is not conserved in arthropods:
prostriate and metastriate tick mitochondrial genomes. Mol. Biol. Evol., 1998, 15:1772–1785.
6.
Derdáková, M., Beati, L., Peťko B., Stanko, M., Fish, D. Genetic variability within Borrelia burgdorferi
sensu lato genospecies established by PCR–single-strand conformation polymorphism analysis of the
rrfA-rrlB intergenic spacer in Ixodes ricinus ticks from the Czech republic. Appl Environ Microbiol
2003, 69: 509–516
7.
Regnery R.L, Spruill C.L, Plikaytis B.D. Genotypic identification of rickettsiae and estimation of
intraspecies sequence divergence for portions of two rickettsial genes. J Bacteriol. 1991, 173(5):1576–
1589.
8.
Casati, S., Sager, H., Gern, L., Piffaretti, J-C.: Presence of potentially pathogenic Babesia sp. for human
in Ixodes ricinus in Switzerland. Ann. Agric. Environ. Med, 2006, 13, 65-70.
9.
Jahfari, S., Fonville, M., Hengeveld, P., Reusken, C., Scholte, E.J., Takken, W. et al. Prevalence of
Neoehrlichia mikurensis in ticks and rodents from north-west Europe. Parasit Vectors 2012, 5: 74
10. Courtney, J. W., Kostelník, L. M., Zeidner, N. S., Massung, R.F. Multiplex real-time PCR for detection
of Anaplasma phagocytophilum and Borrelia burgdorferi. J Clin Microbiol., 2004, 42: 3164-3168
11. Kybicová, K, Schánilec, P., Hulínská, D., Spejchalová, S. Detection of Anaplasma phagocytophilum and
Borrelia burgdorferi s.l. in dogs in the Czech republic. Vector-borne and Zoonotic Diseases, 2009, 9(6):
655-661
12. Claerebout, E., Losson, B., Coches, Ch., Casaert, S., Dalemans, A. C., De Cat, A., Madder, M.,
Saegerman, C., Heyman, P., Lempereur, L. 2013: Ticks and associated pathogens collected from dogs
and cats in Belgium. Parasites and vectors 2013, 6: 183.
13. Schreiber, C., Krücken, J., Beck, S., Maaz, D., Pachnicke, S., Krieger, K., Gross, M., Kohn, B., von
Samson-Himmelstjema, G., 2014: Pathogens in ticks collected from dogs in Berlin/Brandenburg,
Germany. Parasites and Vectors 2014, 7: 535
14. Kubelová, M., Tkadlec, E., Bednář, M., Roubalová, E., Široký, P., 2011: West-to-east differences of
Babesia canis canis prevalence in Dermacentor reticulatus ticks in Slovakia. Veterinary parasitology
180 (2011): 191-196
15. Majláthová, V., Majláth, I., Víchová, B., Guľová, I., Derdáková, M., Sesztáková, E., Peťko, B.:
Polymerase chain reaction Confirmation of Babesia canis canis and Anaplasma phagocytophilum in
Dogs Suspected of Babesiosis in Slovakia. Vector-borne and Zoonotic diseases 2011, 11: 1447-1451
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MALASSEZIOSIS IN HUMANS AND ANIMALS
Sihelská Z., Čonková E.
Department of Pharmacology and Toxicology, University of Veterinary Medicine and
Pharmacy in Košice, Komenského 73, 04181 Košice, The Slovak Republic
[email protected]
Introduction: Yeasts belonging to the genus Malassezia are common members of the
normal microflora of the human skin and inhabit the skin of a variety of animal species
(Guého et al., 1998).However, these yeasts are associated with many dermatological
disorders of the human skin, such as; atopic dermatitis, dandruff, folliculitis, pityriasis
versicolor (PV) or seborrheic dermatitis, and intravascular catheter-acquired infections
(Marcon and Powell, 1992). They have participated in different skin disorders in animals,
especially otitis externa and dermatitis (Crespo et al., 2002).Currently, the genus
Malassezia includes 16 species:M. globosa, M. restricta, M. slooffiae, M. obtusa, M.
furfur, M. sympodialis, M. japonica, M. yamatoensis, M. dermatis, M. pachydermatis, M.
caprae, M. equina, M. nana, M. cuniculi, M. brasiliensis and M. psittaci. They can be
divided into three groups according to the hosting organism: Malassezia occurring only in
animals (M. brasiliensis, M. psittaci, M. caprae, M. equina, M. cuniculi and M. nana),
Malassezia occurring primarily in humans (M. dermatis, M. japonica, M. obtusa, M.
restricta, M. yamatoensis) and Malassezia occurring in animals and humans (M. furfur, M.
globosa, M. slooffiae, M. sympodialis and M. pachydermatis) (Cabañes a kol.,
2011;Cabañes a kol., 2016).
The aim of the study: The purpose of this work was to determine the presenceof
Malasseziayeasts on the skin and the occurrence of the malasseziosis − in a random group
of humans and animals.
Methodology: The survey was carried out on 42 persons and 210 animals (cows – 50,
horses – 30, pigs – 30, sheeps – 30, dogs – 70).The samples were collected from back and
hair area of head in humans and from external ear canals and perineuminanimalsbyusing
sterile cotton swabs (Fungi-Quick, Dispolab).
The samples were inoculated on specific media for the culturing of Malassezia: Modified
Leeming & Notman agar medium, Sabouraud´s dextrose agar with chloramphenicol and
Modified Candida-Chrom agar with Tween 40. Culture media were incubated at 32ºC for
14 days. The identification of yeasts was based both on macroscopic appearance of
colonies and microscopic cell morphology. Each sample was stained by Gram staining and
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Zoonoses - Protection of Public and Animal Health
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examined by microscopy for the presence of typical Malassezia yeast cells. More detailed
identification of Malassezia species in animals was performed according to Kaneko et al.
(2007). In humans were not identified the accurate Malassezia species.
Results and Discussion: Thirty out of 42 persons scored positive for Malassezia spp.
(71,4 %) and these yeasts occurred on the skin of the back. Sixteen persons had Malassezia
yeasts on the skin of the back and also of the head (Table 1).
Malassezia positive culture were obtained from 52 animals (24,8 %), mostly in dogs (62,9
%) and pigs (23,3 %). In dogs were diagnosed 43 isolates M. pachydermatis and one
isolate M. furfur.In the group of pigs wereobtained five isolates M. sympodialis and two
isolates M. slooffiae. Malassezia species were not present in horses and sheeps. Only one
Malassezia isolate (M. pachydermatis) was diagnosed in the cow (Table 2). More than 70
colony-forming units per sample might be indicative of infection (Velegraki et al., 2015).
Despite the this number of colony-forming units per sample, people and animals didn´t
show clinical symptoms of Malassezia diseases.
Table 1 Data of human samples
No. of
persons
42
Number of individuals with
confirmed Malassezia spp.
Number of Malassezia Number of Malassezia
isolates from the back isolates from the heat
30 (71,4 %)
30 (71,4 %)
16 (38,1 %)
Table 2 Data of samples from animals
Animal
species
Number of examined
individuals
Number of individuals
with confirmed
Malassezia spp.
Malassezia species −
number of isolates
M. pachydermatis −1
−
M. sympodialis− 5
M. slooffiae − 2
30
0 (0 %)
−
Sheep
70
44 (62,9 %)
M. pachydermatis −43
Dog
M. furfur −1
The lipophilic yeasts Malassezia are part of the normal flora of the skin and mucosa in
Cow
Horse
Pig
50
30
30
1 (2 %)
0 (0 %)
7 (23,3 %)
warm-blooded animals and humans. At the same time these can act as opportunistic
organisms that under certain conditions can become pathogenic and they may result in
various diseases of skin or fungaemia. The pathophysiology of Malassezia-caused or
Malassezia-exacerbated skin conditions is largely unknown (Velegraki et al., 2015). A
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range of skin microenvironmental factors, such as the bacterial microbiota present, pH,
salts, immune responses, biochemistry, and physiology, may play a role in adherence and
growth of Malassezia species, favouring distinct genotypes depending on the geographical
area and/or the skin sites (Gaitanis et al., 2012).
Currently, the results of scientific studies on Malassezia yeasts in humans and animals
vary. The most Malassezia affected animal is dog (Nardoni et al., 2004) ant the main
colonizing yeast in healthy and also in diseased dogs is M. pachydermatis. These were
confirmed in a group of examined animals.The high incidence of Malassezia yeasts was
recorded in the group of pigs and diagnosed species were M. sympodialis and M. slooffiae.
In pigs were diagnosed M. furfur, M. sympodialis, M. slooffiae and also M.
pachydermatis(Garau et al., 2005). In examined groups of horses and sheeps were not
identified Malassezia yeasts. Typical genus in horses are M. equina, M. caprae and M.
sympodialis (Cabañes et al., 2005; Cabañes et al., 2007) and in sheepM. caprae, M.
sympodialisand M. slooffiae. (Cabañes et al., 2005; Cabañes et al., 2007; Uzal et al., 2007).
In livestock Malassezia are mostly isolated from healthy individuals (Crespo et al., 2002).
Conclusion: Lipophilic Malassezia yeasts were normally present in a large amounton the
skin of examined humans and animals. In humnas Malassezia occurred most commonly on
the back. In animals were the most affected dogs and pigs.In the random group of humans
and animalswere not record malasseziosis.
Acknowledgement: This work was supported by the Slovak Research and Development
Agency under the contract No. APVV-15-0377.
Literature
1.
Guého, E., Boekhout, T., Ashbee, H. R., Guillot, J., Van Belkum, A., Faergemann, J. The role of
Malassezia species in the ecology of human skin and as pathogens. Med Mycol 1998,36, 220−229.
2.
Marcon, M. J., Powell, D. A. Human infections due to Malassezia spp. J Clin Microbiol 1992,5,
101−119.
3.
Crespo, M. J., Abarca, M. L., Cabañes, F. J. Occurrence of Malassezia spp. in the external ear canals of
dogs and cats with and without otitis externa. Med Mycol 2002, 40, 115−121.
4.
Cabañes, F. J., Vega, S., Castellá, G. Malassezia cuniculi sp. nov., a novel yeast species isolated from
rabbit skin. Med Mycol 2011, 49, 40−48.
5.
Cabañes, F. J., Coutinho, S. D., Puig, L., Bragulat, M. R., Castellá, G. New lipid-dependent Malassezia
species from parrots.Rev Iberoam Micol 2016, 33, 92−99.
6.
Kaneko J., Makimura K., Abe, Shiota R., Nakamura Y., Kano R., Hasegawa A., Sugita T., Shibuya S.,
Watanabe S., Yamaguchi H., Abe S., Okamura N. Revised culture-based system for identification of
Malassezia species. J Clin Microbiol 2007, 45, 3737−3742.
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Velegraki A., Cafarchia C., Gaitanis G., Iatta R., Boekhout T. Malassezia infections in humans and
animals: pathophysiology, detection, and treatment.PLOS Pathog 2015, 11, 1−6.
8.
Gaitanis G., Magiatis P., Hantschke M., Bassukas I. D., Velegraki A. The Malassezia genus in skin and
systemic diseases. Clin Microbiol Rev 2012, 25, 106–141
9.
Nardoni, S. Mancianti, F., Corazza, M, Rum, A. Occurrence of Malassezia species in healthy and
dermatologically diseased dogs. Mycopath2004, 157, 383–388.
10. Garau, M., Del Palacio, A., García, J. Prevalence of Malassezia spp. in healthy pigs. Mycoses 2005, 48,
17–20.
11. Cabañes, F. J., Hernández, J. J., Castellá, G. Molecular analysis of Malassezia sympodialis-related
strains from domestic animals. J Clin Microbiol 2005, 4, 277–283.
12. Cabañes, F. J., Theelen, B., Castellá, G., Boekhout, T. Two new lipid-dependent Malassezia species
from domestic animals. FEMS Yeast Res 2007, 7, 1064–1076.
13. Uzal, F. A., Paulson, D., Eigenheer, A. L., Walker, R. L. Malassezia slooffiae-associated dermatitis in a
goat. Vet Dermatol 2007, 18, 348–352.
14. Crespo, M. J., Abarca, M. L., Cabañes, F. J. Occurrence of Malassezia spp. in horses and domestic
ruminants. Mycoses 2002, 45, 333–337.
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PREVALENCE OF SALMONELLA IN POULTRY GALLUS GALLUS IN THE
SLOVAK REPUBLIC IN THE YEARS 2011 - 2015.
A. Skarkova, G. Gaspar, M. Sasik, L. Mikula
State Veterinary and Food Institute, Botanicka 15, 842 52 Bratislava
Introduction: Salmonellosis is still one of the main causes of infectious foodborne
gastroenteritis in humans. Laying hens and broilers poultry Gallus gallus are an important
source of salmonellosis through the medium of eggs and poultry meat.
The aim of the study: Analysis of prevalence and trends of Salmonella serovars in all the
categories of poultry Gallus gallus in Slovakia in the last five years.
Methodology: The sampling frame was fixed in the National Salmonella control program
in Slovak Republic (poultry). Laboratory analysis was carried out applying ISO 6579-2002
Annex D. The Salmonella isolates were serotyped following the White - Kauffmann - Le
Minor scheme.
Results: Analysis of data demonstrates the number of examined flocks and percentage of
positive flocks of poultry in the years 2011 – 2015.
Prevalence of Salmonella in breeding flocks was sporadic. There were recorded a
variations among poultry categories (laying hens, broilers) and Salmonella species
prevalence and Salmonella serovar distribution. S. Enteritidis was the predominant serovar
detected in poultry Gallus gallus in Slovakia for years. Results indicate that prevalence of
S. Infantis has increased significantly in poultry category broilers in the years 2011 – 2015.
S. Enteritidis is still the most frequent serovar in breeding flocks and laying hens. The
prevalence of other serovars has varied. Data of the prevalence of Salmonella species and
of the antimicrobial resistance of the most frequent serovars are presented by Figures.
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3500
3000
2500
2000
1500
1000
500
0
2011
2012
2013
breeding flocks
2014
flocks of laying hens
2015
flocks of broilers
Figure 1 Number of flocks examined in the monitored categories of poultry (Gallus Gallus) in
Slovakia in the years 2011 - 2015.
100
90
80
70
60
50
40
30
20
10
0
2011
2012
breeding flocks
2013
flocks of laying hens
2014
2015
flocks of broilers
Figure 2 Prevalence of Salmonellapositive flocks of poultry (Gallus Gallus) in Slovakia in the
years 2011 - 2015.
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100,0
90,0
80,0
70,0
60,0
50,0
40,0
30,0
20,0
10,0
0,0
2011
2012
S. Enteritidis
2013
S. Typhimurium
2014
S. Infantis
2015
others
Figure 3 Prevalence of Salmonella serovars in flocks of laying hens (Gallus gallus) in
Slovakia in the years 2011 - 2015.
100,0
90,0
80,0
70,0
60,0
50,0
40,0
30,0
20,0
10,0
0,0
2011
2012
S. Enteritidis
2013
S. Typhimurium
2014
S. Infantis
2015
others
Figure 4 Prevalence of Salmonella serovars in flocks of broilers (Gallus gallus) in
Slovakia in the years 2011 - 2015.
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40
35
30
25
% 20
2011-(19)
15
2012-(51)
10
2013-(16)
5
2014-(25)
0
2015-(15)
Figure 5 Prevalence of antimicrobial resistance among S. Enteritidis isolates from poultry
and poultry meat (Gallus Gallus) in Slovakia in the years 2011 - 2015.
100
80
60
2011-(27)
%
2012-(35)
40
2013-(41)
20
2014-(59)
0
2015-(10)
Figure 6 Prevalence of antimicrobial resistance among S. Infantis isolates from poultry and
poultry meat (Gallus Gallus) in Slovakia in the years 2011 - 2015.
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Conclusion: S. Enteritidis is still the most prevalent serovar isolated from poultry Gallus
gallus. Prevalence of S. Infantis has increased significantly in poultry category broilers and
in food category broiler meat in the last years.
References
1.
STN ISO 6579:2002 Microbiology of food and animal feeding stuffs, Horizontal method for the
detection of Salmonella spp., Annex D.
2.
WHO Collaborating Centre for Reference and Research on Salmonella, Patrick A.D.Grimont, FrancoisXavier Weill, White – Kauffmann-Le Minor scheme : Antigenic formulae of the SALMONELLA
Serovars, 2007
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IXODES RICINUS TICK, ITS PATHOGENS AND HUMAN
E. Špitalská1, V. Rusňáková Tarageľová2, M. Chvostáč2, E. Kocianová1, K. Štefanidesová1
1
Institute of Virology, Biomedical Research Center, Slovak Academy of Sciences,
Dúbravska cesta 9, 845 05 Bratislava, Slovakia [email protected]
2
Institute of Zoology, Slovak Academy of Sciences, Dúbravska cesta 9, 845 06 Bratislava,
Slovakia
Introduction: Ticks can induce severe local lesions (irritations, allergy) or systemic
effects (paralyses, toxicoses). The European tick Ixodes ricinus is a vector of several
pathogens of humans and animals. The medically and veterinary important of pathogens
are the TBE virus (TBEV) causing tick-borne encephalitis, Rickettsiaspecies causing
rickettsial diseases, the Borrelia burgdorferi sensu lato (s.l.) spirochaetes causing Lyme
disease, Anaplasma phagocytophilum(causative agent of granulocytic anaplasmosis in
humans, tick-borne fever in sheep and canine ehrlichiosis in dogs), Babesia
divergens(causative agent of babesiosis in humans and redwater fever in cattle), Babesia
venatorumand Babesia microti(causative agents of babesiosis in humans), occasionally
Francisella tularensis(1-3).Ixodes ricinus is a three-host tick so to complete its life cycle,
which consists of three active stages (larva, nymph and adult), it must ingest blood from a
vertebrate host in each one of the three stages. Humans are occasional hosts for each one of
these stages. The duration of tick feeding, including salivation and blood ingestion, is
important for transmission of pathogens. The virions of TBEV-infected ticks will begin to
be transmitted with the tick’s saliva to the vertebrate host almost instantaneously after tick
attachment (< 1 hour), (4). In contrast, B. burgdorferi s.l. spirochaetes are not transmitted
immediately, but the risk increases with the prolongation of tick feeding (5, 6). The risk
may, in fact, already be present during the first 24 hours of tick feeding (6). Unfed nymphs
and unfed adult Amblyomma aureolatum ticks had to be attached to the host for >10 hours
to transmit Rickettsia rickettsii. In contrast, fed ticks needed a minimum of 10 minutes of
attachment to transmit R. rickettsii to hosts (7).
The aim of the study: The present study aimed to identify and characterize tick-borne
pathogens of ticks attached to humans and domestic animals as a potential risk of their
diseases in Slovakia.
Methodology: Ticks were collected from humans and domestic animals in Slovakia during
2008–2016. All collected ticks were stored in 1.5 ml tubes filled with 70 % ethanol and
identified to species and developmental stage according to the taxonomic key of Siuda (8).
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Estimated feeding time of nymphs and adult ticks based on scutal and coxal indices was
calculated as previously described (9). Ticks were rinsed in sterile water, and dried on
sterile filter paper. The DNA was extracted from ticks individually by alkaline hydrolysis
method (10). To verify the success of the DNA extraction tick mitochondrial gene
cytochrome b was amplified from each sample (11). Rickettsial DNA was detected by PCR
amplifying fragment of the citrate synthase gene (gltA) of Rickettsia spp. (12).Rickettsiapositive tick samples were screened for the presence of R. helvetica using TagMan PCR
assay targeting a 65-bp fragment of the 23S rRNA gene (13). The presence of C. burnetii
DNA in samples was detected by PCR assay using CBCOS and CBCOE primers targeting
fragment of the outer membrane protein com1 gene of C. burnetii (14). The presence of
Anaplasma phagocytophilum was tested by Taqman PCR targeting a 77-bp fragment of
msp2 gene (15). The presence of Candidatus N. mikurensis was detected by nested PCR of
the specific fragment of 16S rRNA gene (16). Detection of Babesia spp. was performed
using the genus-specific primers to amplify fragment of the 18S rRNA gene (17). R.
helvetica-negative samples by TagMan PCR assay and amplicons of 18S rRNA were
purified and analysed by sequencing both DNA strands by Macrogen Inc., (Amsterdam,
The Netherlands). The DNA sequences were compared with those available in GenBank
using the Basic Local Alignment Search Tool on http://blast.ncbi.nlm.nih.gov/. The
presence of B. burgdorferi s.l. was detected by TagMan PCR assay targeting 23S rRNA
gene (15) or by the amplification of rrfA-rrlB intergenic spacer. Positive samples were
further typed to Borrelial genospecies by RFLP, (18).
Results and Discussion: A total of 418 attached ticks were removed mainly from humans,
but also from dogs and cats. Ixodes ricinus was dominant tick species attached on host
(26.2% females, 0.3% male, 66.4% nymphs and 7.1% larvae). During 2012–2016, the
proportion of ticks was significantly different on men (55.1%) and women (44.9%, p =
0.037). Feeding ticks originated from all parts of Slovakia, the Czech Republic (n = 7),
Austria (n = 3), Hungary (n = 1) and Croatia (n = 1). Tick bites were recorded from March
to December. The legs were the major location of ticks’ attachment (35.3%), followed by
upper torso with arms (21.5%), the head/neck area (19.0%) and lower torso (15.5%). Based
on time of tick feeding in relation to the participants self-estimated time of tick attachment,
54.4% ticks had been attached < 24 hours, 32.3% > 24 hours and 13.3% did not know to
estimate the feeding times of attached ticks. But self-estimated time of tick attachment was
identical with calculated times based on the scutal and coxal indices only in 38.5% (10/26)
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man. Rickettsia sp. was present in 19.6% of ticks, B. burgdorferi s.l. in 22%, Babesia spp.
in 8.4%, A. phagocytophilum in 6.8% and Cand. N. mikurensis in 1.6% of attached ticks.
Rickettsia helvetica. R. monacensis, Borrelia afzelii, B. valaisiana, B. garinii, Babesia
microti and B. divergens are species identified by the next analyses.
In the present study, only few larval ticks were removed. However, this tick stage is
considered to be a much less important vector of B. burgdorferi s.l. and TBEV infections
to humans; the unfed larva is almost never infected with LB-causing bacteria (19), nor with
TBEV (20). The majority of the I. ricinus ticks removed were nymphs, the most important
stage in the transmission of borreliae. For transmission of rickettsia are important all
developmental stages.
The majority of the ticks had attached to the legs of the participants, which is
approximately within the same height above the ground where nymphs and adults of I.
ricinus quest in the vegetation (21). The presence of various pathogens in ticks indicates
their epidemiological significance in Slovakia.
Conclusion: The present study showed that the dominant species feeding on humans in
Slovakia is I. ricinus, mainly nymphs, more often on men. Tick bites were recorded from
March to December. The majority of the ticks were attached to the legs. Ticks were
infected with pathogenic microorganisms
–
R. helvetica. R. monacensis, A.
phagocytophilum, Cand. N. mikurensis, B. afzelii, B. valaisiana, B. garinii, B. microti and
B. divergens.
Acknowledgements: This study was financially supported by the projects VEGA
nos.2/0061/13 and 2/0108/13, and SRDA-0280-12. This contribution is also the result of
using infrastructure acquired by the project implementation (code ITMS: 26240220044),
supported by the Research & Development Operational Programme funded by the ERDF.
Literature
1.
Jongejan F, Uilenberg G. The global importance of ticks. Parasitol 2004,129:S3–S14
2.
Stanek G. Pandora‘s Box: pathogens in Ixodes ricinus ticks in Central Europe. Wien Klin Wochenschr
2009,121:673–83
3.
Gray J, Zintl A, Hildebrandt A, Hunfeld KP, Weiss L. Zoonotic babesiosis: overview of the disease and
novel aspects of pathogen identity. Ticks Tick Borne Dis 2010,1:3–10
4.
Alekseev AN, Burenkova LA, Vasilieva IS, Dubinina HV, Chunikhin SP. Preliminary studies on virus and
spirochete accumulation in the cement plug of ixodid ticks. Exp Appl Acarol 1996,20:713–23
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Crippa M, Rais O, Gern L. Investigations on the mode and dynamics of transmission and infectivity of
Borrelia burgdorferi sensu stricto and Borrelia afzelii in Ixodes ricinus ticks. Vector Borne Zoonot Dis
2002,2:3–9
6.
Kahl O, Janetzki-Mittmann C, Gray JS, Jonas R, Stein J, de Boer R. Risk of infection with Borrelia
burgdorferi sensu lato for a host in relation to the duration of nymphal Ixodes ricinus feeding and the method
of tick removal. Zentralbl Bakteriol 1998,287:41–52
7.
Saraiva DG, Soares SS, Soares JF, Labruna MB. Feeding Period Required by Amblyomma aureolatum ticks
for transmission of Rickettsia rickettsii to vertebrate hosts. Emerg Infect Dis 2014,20:1504-10
8.
Siuda K. Ticks (Acari: Ixodida) of Poland. Part II. Taxonomy and Distribution. Warszawa: PTP; 1993.
9.
Gray J, Stanek G, Kundi M, Kocianova E. Dimensions of engorging Ixodes ricinus as a measure of feeding
duration. Int J Med Microbiol 2005,295:567-72
10. Guy EC, Stanek G. Detection of Borrelia burgdorferi in patients with Lyme disease by the polymerase
chain-reaction. J Clin Pathol 1991,44:610–1
11. Black WC, Roehrdanz RL. Mitochondrial gene order is not conserved in arthropods: prostriate and
metastriate tick mitochondrial genomes. Mol Biol Evol 1998,15:177285
12. Regnery RL, Spruill CL, Plikaytis BD. Genotypic identification of rickettsiae and estimation of intraspecies
sequence divergence for portions of two rickettsial genes. J Bacteriol 1991,173:1576–89
13. Boretti FS, Perreten A, Meli MM, Cattori V, Willi B, Wengi N, Hornok S,Honegger H, Hegglin D, Woelfel
R, Reusch CE, Lutz H, Hofmann-LehmannR. Molecular investigation of Rickettsia helvetica infection in
dogs, foxes,humans, and Ixodes ticks. Appl Environ Microbiol 2009,75:3230–7
14. Špitalská E, Kocianová E. Detection of Coxiella burnetii in ticks collected in Slovakia and Hungary. Eur J
Epidemiol. 2003,18:263-6
15. Courtney JW, Kostelnik LM, Zeidner NS, Massung RF. Multiplex real-time PCR for detection of Anaplasma
phagocytophilum and Borrelia burgdorferi. J Clin Microbiol 2004,42:3164–8
16. Kawahara M, Rikihisa Y, Isogai E, Takahashi M, Misumi H, Suto C, Shibata S, Zhang C, Tsuji M.
Ultrastructure and phylogenetic analysis of “Candidatus Neoehrlichia mikurensis” in the family
Anaplasmataceae, isolated from wild rats and found in Ixodes ovatus ticks. Int J Syst Evol Microbiol
2004,54:1837–43
17. Casati S, Sager H, Gern L, Piffaretti JC. Presence of potentially pathogenic Babesia sp. for human in Ixodes
ricinus in Switzerland. Ann Agric Environ Med 2006,13:65–70
18. Derdáková M, Beati L, Peťko B, Stanko M, Fish D. Genetic variability within Borrelia burgdorferi sensu
lato genospecies established by PCR-single-strand conformation polymorphism analysis of the rrfA-rrlB
intergenic spacer in Ixodes ricinus ticks from Czech republic. Appl Environ Microbiol 2003,69:509-16
19. Richter D, Debski A, Hubalek Z, Matuschka FR. Absence of Lyme disease spirochetes in larval Ixodes
ricinus ticks. Vector Borne Zoonot Dis 2012,12:21–7
20. Pinter R, Madai M, Vadkerti E, Nemeth V, Oldal M, Kemenesi G, Dallos B, Gyuranecz M, Kiss G, Banyai
K, et al. Identification of tick-borne encephalitis virus in ticks collected in southeastern Hungary. Ticks Tick
Borne Dis 2013,4:427–31
21. Mejlon HA, Jaenson TGT. : Questing behaviour of Ixodes ricinus ticks (Acari: Ixodidae). Exp Appl Acarol
1997,21:747–54
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ANAPLASMA PHAGOCYTOPHILUM – A PATHOGEN CIRCULATING IN WILD
ANIMALS IN NATURAL FOCI OF WESTERN SLOVAKIA
Štefanidesová, K.1, Hamšíková, Z.2, Krkoš, I.3, Smetanová, E.3, Špitalská, E.1
1
Institute of Virology BMC, SAS, Dúbravská cesta 9, 845 05 Bratislava,
[email protected]
2
Institute of Zoology, SAS, Dúbravská cesta 9, 845 05 Bratislava
3
Tekov Museum Levice, Sv. Michala 40, 934 69 Levice
Introduction: Anaplasma phagocytophilum is an obligate intracellular bacterium,
etiologic agent of zoonotic disease known as tick-borne fever of domestic ruminants or
canine, equine and human granulocytic anaplasmosis. In Europe it is transmitted mainly by
Ixodes ricinus ticks. Despite many animals such as rodents, deer, wild boars, hedgehogs
being recognised as hosts and possible reservoirs of A. phagocytophilum, its epidemiology
is not yet completely elucidated. (Stuen et al. 2013, Dugat et al. 2015)
The aim of the study: The aim of this study was to follow up the prevalence of
Anaplasma phagocytophilum and other tick borne bacteria in wild ungulates, mainly roe
deer from three localities of western Slovakia.
Methodology: Wild ungulates came from three hunting areas: Nitrianske Rudno –
Pravenec (district of Prievidza), Levice – Kalinčiakovo (district of Levice), and Želiezovce
– Lontov (district of Levice). Spleen samples of wild animals were stored at – 20°C or in
96% ethanol. DNeasy Blood & Tissue Kit (Qiagen) was used to extract DNA. The
presence of A. phagocytophilum in samples from 2009 was detected by nested PCR with
primers HS43 and HS45, GEHS1 and EHS6 targeting groESL operon (Sumner et al. 2000,
Bjoersdorff et al. 2002). Samples from 2010-2014 were tested by real-time PCR using
primers ApMSP2f and ApMSP2r and the Taqman Probe ApMSP2p targeted 77 bp
fragment of msp2 gene (Courtney et al. 2004). The presence of Ehrlichia sp. DNA was
assessed by PCR with primers Ehr521 and Ehr 790 targeting 16S rDNA (Kolbert 1996).
Candidatus Neoehrlichia mikurensis was detected using primers IS58-62f and IS58-594r
amplifying part of 16S rDNA (Kawahara et al. 2004). The presence of Ehrlichia muris
DNA in samples was tested by PCR with primers EmCS638F and EmCS1349R targeting
gltA gene (Tamamoto et al. 2007). Rickettsiae were detected by primers RpCS.877p and
RpCS.1258n, and Rr190.70p and Rr190-701, amplifying parts of gltA and ompA genes
respectively (Regnery et al. 1991, Roux et al. 1996). Observed prevalences were compared
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by two-tailed Fisher exact probability test or Freeman –Halton extension of Fisher exact
test.
Results and Discussion: Spleen samples of 81 ungulates, namely 74 roe deer (Capreolus
capreolus), four red deer (Cervus elaphus), and three wild boars (Sus scrofa) hunted from
May 2009 to August 2014 were analysed. Forty-three animals (37 roe deer, 4 red deer, 2
wild boars) came from Nitrianske Rudno, 36 (35 roe deer, 1 wild boar) from Levice, and 2
(roe deer) from Želiezovce. Seventeen roe deer were females, 35 were males of first age
group (1 year old), 17 males of second age group (2-5 years old), and 5 males of third age
group (aged six or more). Two red deer were females, one male belonged to first age group
(2 years old), and one male to second age group (aged 3-5). Of three hunted wild boars,
one was female, and two males.
A. phagocytophilum was detected in spleen samples from all monitored localities and
years. This bacterium was present in 57 (77.02%) roe deer, in two female red deer and in
two male wild boars. Two male red deer were infected by Ehrlichia sp. Other abovementioned bacteria were not detected. Prevalence of A. phagocytophilum in roe deer from
Nitrianske Rudno (73%) was comparable to that observed in Levice (82.9%) (p=0.40). No
statistically significant difference was observed between the proportion of A.
phagocytophilum-infected females (82.4%) and A. phagocytophilum-infected males
(75.4%) (p=0.75). Twenty-eight (80%) roe deer of first age group, 13 (76.5%) of second
age group and two of third age group were infected by A. phagocytophilum. Difference in
prevalences between age groups was not statistically significant (p=0.19).
High prevalence of A. phagocytophilum in roe (50%) and red (53.1%) deer was previously
reported from region of central Slovakia, where also 25% seroloprevalence was observed
in patients with a history of recent tick bite and clinical symptoms indicating Lyme
borreliosis (Štefanidesová et al. 2008, Kocianová et al. 2008). Moreover, A.
phagocytophilum was detected in ectoparasite Lipoptena cervi removed from deer, and in
2.7% of wild boars from southern Slovakia (Víchová et al. 2011, Reiterová et al. 2016).
High prevalence rates of A. phagocytophilum in deer species were observed in several
European countries (Stuen et al. 2013).
Conclusion: The overall high prevalence of A. phagocytophilum in roe deer from
Nitrianske Rudno – Pravenec and Levice – Kalinčiakovo, high infection rate in animals of
first age group, presence of this bacterium in red deer and wild boars suggest that these
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hunting areas should be considered as natural focci where A. phagocytophilum is
continually maintained.
Acknowledgements: This study was financially supported by the projects VEGA nos.
2/0061/13 and 2/0106/16.
References
1.
Bjoersdorff A., Bagert B., Massung R.F. et al. (2002) Isolation and characterization of two European
strains of Ehrlichia phagocytophila of equine origin. Clin Diagn Lab Immunol. 9(2): 341-3
2.
Courtney J.W., Kostelnik L.M., Zeidner N.S., Massung R. F. (2004) Multiplex real-time PCR for
detection of Anaplasma phagocytophilum and Borrelia burgdorferi. J Clin Microbiol. 42:3164–8
3.
Dugat T., Lagree A.C., Maillard R. et al. (2015) Opening the black box of Anaplasma phagocytophilum
diversity: current situation and future perspectives. Front Cell Infect Microbiol. 5, Article Number: 61
4.
Kawahara M., Rikihisa Y., Isogai E. et al. (2004) Ultrastructure and phylogenetic analysis of
“Candidatus Neoehrlichia mikurensis” in the family Anaplasmataceae, isolated from wild rats and found
in Ixodes ovatus ticks. Int J Syst Evol Microbiol. 54:1837–43
5.
Kocianová E., et al. (2008) Serologic evidence of Anaplasma phagocytophilum infections in patients
with a history of tick bite in central Slovakia. Wien. Klin. Wochenschr. 120(13-14):427-31
6.
Kolbert C. (1996) Detection of the agent of human granulocytic ehrlichiosis by PCR. Detection of the
agent of human granulocytic ehrlichiosis by PCR. p. 106-111 In D. Persing (ed), PCR protocols for
emerging infectious diseases. ASM Press, Washington, D. C.
7.
Regnery R.L., Spruill C.L., Plikaytis B.D. (1991) Genotypic identification of rickettsiae and estimation
of intraspecies sequence divergence for portions of two rickettsial genes. J Bacteriol 173: 1576–89
8.
Reiterova K., Spilovska S., Blanarova L. et al. (2016) Wild boar (Sus scrofa) - reservoir host of
Toxoplasma gondii, Neospora caninum and Anaplasma phagocytophilum in Slovakia. Acta Parasit.
61(2): 255-60
9.
Roux V., Fournier P. E., Raoult D. (1996) Differentiation of spotted fever group rickettsiae by
sequencing and analysis of restriction fragment length polymorphism of PCR-amplified DNA of the
gene encoding the protein rOmpA. J Clin Microbiol. 34(9): 2058-65
10. Stefanidesova K. et al. (2008) Evidence of Anaplasma phagocytophilum and Rickettsia helvetica
infection in free-ranging ungulates in central Slovakia. Eur. J. Wildl. Res. 54(3): 519-24
11. Stuen S., Granquist E., Silaghi C. (2013) Anaplasma phagocytophilum-a widespread multi-host pathogen
with highly adaptive strategies. Front Cell Infect Microbiol. 3: Article Number: UNSP 31
12. Sumner J.W. et al. (2000) PCR amplification and phylogenetic analysis of groESL operon sequences
from Ehrlichia ewingii and Ehrlichia muris. J Clin Microbiol. 38(7):2746 - 49
13. Vichova B., Majlathova V., Novakova M. et al. (2011) PCR detection of re-emerging tick-borne
pathogen, Anaplasma phagocytophilum, in deer ked (Lipoptena cervi) a blood-sucking ectoparasite of
cervids. Biologia 66(6):1082-6
14. Tamamoto C., Seino N., Suzuki M. et al. (2007) Detection of Ehrlichia muris DNA from sika deer
(Cervus nippon yesoensis) in Hokkaido, Japan. Vet Parasitol. 150(4):370-3.
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SHEEP MILK AS A SOURCE OF HUMAN TOXOPLASMOSIS
Šťavová, Ľ., Luptáková, L., Valenčáková A., Danišová O.
University of Veterinary Medicine and Pharmacy, Department of Biology and Genetics,
Komenského 73, 041 81 Kosice, Slovakia
[email protected]
Introduction: Toxoplasmosis is one of the most common parasitic zoonoses of mammals
and birds transmissible to humans. The agent of the disease is Toxoplasma gondii (T.
gondii), the definitive hosts of which are the representatives of the family Felidae, infected
with oocysts from the environment and with tachyzoites and bradyzoites from intermediate
hosts. Intermediate hosts are all vertebrates including man (1). It is a panthropic
cosmopolite, a facultative heteroxenic coccidium. Sheep were in fact the first mammals in
which congenital toxoplasmosis were proven with abortions, dead-born fetuses and
frequent manifestations of infection including infertility. The first case of manifest
toxoplasmosis in sheep with symptoms of encephalomyelitis and tachycardia was
described by Olafson and Monlux (2). Sheep play a significant role in the economy of
many countries since they are important source of meat and milk products. Toxoplasmosis
is the major parasitic disease affecting sheep. It is important for veterinary medicine,
animal science and public health since it causes reproductive and economic losses in the
herd, as well as damaging human health due to consumption of contaminated meat and
milk, which can facilitate zoonotic transmission (3).
The aim of the study: The aim of our study was focused on the determination of the
occurrence of T. gondii DNA in blood and milk samples in relation to the phase of
infection of naturally infected sheep using real-time PCR.
Methodology: Samples: A total of 50 milk samples of adult sheep were examined by real
time PCR. Sheep originated from farm located 30 km of Kosice in eastern Slovakia. In the
farm proper sanitary conditions were respected and sheep were regularly pastured on
grassland where they could contamined with T. gondii. Theblood samples were obtained
from a jugular vein. Fromeach ewe, 5 ml of blood sample for serological
examination(coagulation at 22 ◦C for 30 min, centrifugation at3000 rpm for 10 min), then
for PCR (into tube with EDTA),and also 5 ml of milk sample were collected (into 10 ml
Vshaped tubes).
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Isolation of DNA: DNA was isolated from milk and blood samples by commercially
available kit (DNA-sorbB, Amplisens, Russia). DNA was isolated according to the
manufacturer´s protocol and stored at -20 °C for futher examination.
Detection of DNA by real time PCR: Amplification of the isolated DNA was carried out by
the real time PCR with SYBR green as a detection system using gene region TGR1E ofT.
gondii, which was amplified by specific primers TGR1E-1 and TGR1E-2 (4). In each
reaction, a melting was determined to differentiate specific and non-specific PCR products.
The reaction volume was 25 μl, which contained commercial FastStart Universal SYBR
Green Master (Roche, Germany) and 0.2 μM primers (TGR1-1 and TGR1E-2). Real-time
PCR was completed using a thermocycler Line GeneK with the software Line GeneK
Fluorescent Quantitative Detection system (BIOER Technology, China). After incubation
at 50°C for 2 minutes and initial denaturation at 95°C for 10 minutes, 40 amplification
cycles were performed (95°C for 15 s, 60°C for 1 minute). Melting analysis was carried
out at temperatures ranging from 60 °C to 95 °C, in which the temperature was gradually
increased by 0.5 °C and the period of measurement at individual steps was 15 s. Every
PCR run included a control without DNA (containing the reaction mix alone and nucleasefree water).
Results and Discussion: Using quantitative real-time PCR the presence of T. gondii DNA
was detected and the number of copies quantified in the 50 milk samples of sheep. Using
real-time PCR, T. gondii DNA was detected and quantified in 5 samples of milk. As SYBR
Green a fluorescent dye, was used as a detection system, a melting analysis was a part of
the real-time PCR to distinguish between specific and non-specific products. During the
melting analysis, the melting temperature (Tm) of a positive control and positive samples
was 84°C.Consumption of raw food, especially among immunocompromised persons and
pregnant women, poses a potential threat to public health. The milk of sheep and goats
represent risk factors as they are consumed raw (5). Consumption of unpasteurized goat,
sheep or cow milk or its products represents a risk because tachyzoites, the stage most
likely present in milk are thought to be immediately destroyed by the gastric juice (6).
However, evidence indicates that the part of ingested tachyzoites is not destroyed by
gastric juice and it can cause infection (7). Toxoplasmosis transmission by unpasteurized
or inadequately processed milk or fresh cheese, important food sources in rural areas, can
be a significant means of contamination by this agent (8).In the period of 1999 – 2002,
studies conducted in Sardinia analyzed 9639 samples of sera and 815 samples from
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abortions (670 aborted foetus and 145 placentas) obtained from 964 sheep and goat farms.
The sampled sera were examined for presence of IgG and IgM specific antibodies against
Toxoplasma gondii by means of indirect immunofluorescence method while foetus and
placenta were examined by PCR. Specific antibodies against IgG were diagnosed in 652
(9%) sheep. of the total number of 2471 samples of sheep foetuses including muscles,
liver, abomasums, spleen, brain and placenta, PCR proved positivity in 271 (11.1%) cases.
on the one hand, these results indicate relatively high seroprevalence and, on the other
hand point to the important role of PCR diagnostics related to sheep abortions (9).In
southern Italy, region Campania, 117 sheep flocks kept on pasture were examined.
Samples of blood and milk were taken from 10 adult sheep (> 18 months old) from each
flock. Thus altogether 1170 sheep were subjected to examination. Blood sera were
examined for the presence of IgG antibodies by indirect immunofluorescence. Of the
examined samples collected from 1170 sheep 333 samples were positive (28.5%) and PCR
proved presence of Toxoplasma gondii in 4 samples of milk, which represents 3.4% of the
total analyzed samples, demonstrating the transmission potential of ovine milk and dairy
products (10).
Conclusion: The tachyzoites presented in sheep or goat milk are generally not considered
as an important source of oral transmission of T. gondii because they are rapidly killed
outside the host and because they are considered sensitive to proteolytic enzymes (11). But
the contamination of milk should not be underestimated since it can represent a critical
point in food safety. Some local home-made cheeses deriving from mass-milk production
and destined to be consumed fresh can represent a risk factor for public health if they are
produced in small family-based farms without previous milk pasteurization. Based on the
results of the present survey and other studies, the possibility of Toxoplasma transmission
through consumption of raw milk and its unpasteurized derivatives can be hypothesized
and it could have an impact on public health.
Acknowledgement: The paper is a result of research work done within the frames of grant
projects VEGA of the Ministry of Education of the Slovak Republic No. 1/0061/16 and
APVV-14-0274.
Literature
1.
Frenkel J.K. 2000. Biology of Toxoplasma gondii. In: Ambroise-Thomas P, Peterse E. (ed): Congenital
toxoplasmosis: scientific background, clinical management and control. Springer-Verlag, Paris, pp. 9-25.
2.
Olafson, P., Monlux, W.S. 1942. Toxoplasma infections in animals. Cornell Veterinarian, 32, 16-190.
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3.
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Camossi, L.G., Greca-Júnior, H., Correa, A.P.F.L., Richini-Pereira, V.B., Silva, R.C., Da Silva, A.V.,
Langoni, H. 2011. Detection of Toxoplasma gondii DNA in the milk of naturally infected ewes.
Veterinary Parasitology, 177, 256-261.
4.
Cristina, N., Liaud, M.F., Santoro, F., Oury, B.,Ambroise-Thomas, P. 1991. A family of repeated
DNAsequences in Toxoplasma gondii: cloning, sequenceanalysis, and use in strain characterization.
ExperimentalParasitology, 1991, 73, 73-81
5.
Spisak, F., Turcekova, L., Reiterova, K., Spilovska, S., Dubinsky P. 2010. Prevalence estimation and
genotypization of Toxoplasma gondii in goats. Biologia, 65, 670-674.
6.
Dubey, J.P. 1998. Re-examination of resistance of Toxoplasma gondii tachyzoites and bradyzoites to
pepsin and trypsin digestion. Journal of Parasitology, 116, 43-50.
7.
Cook, A.J.C., Gilbert, R.E., Buffolano, W., Zufferey, J., Peterse, E., Jenum, P.A., Foulon, W., Semprini,
A.E., Dunn, D.T. 2000. Sources of Toxoplasma infection in pregnant women: European multicentre
case-control study. BMJ, 321, 142-147.
8.
Hiramoto, R.M., Mayrbaurl-Borges, M., Galisteo, A.J., Meireles, L.R., Macre, M.S., Andrade, Jr., H.F.
2001. Infectivity of cystis of the ME-49 Toxoplasma gondii strain in bovine milk and homemade cheese.
Revista de Saúde Pública, 35, 113-118.
9.
Masala G, Porcu R, Madau L, Tanda A, Ibba B, Satta G, Tola S. 2003. Survey of ovine and caprine
toxoplasmosis by IFAT and PCR assays in Sardinia, Italy. Veterinary Parasitology 117, 15-21.
10. Fusco G, Rinaldi L, Guarino A, Proroga YTR, Pesce A, Giuseppina DM, Cringoli G. 2007. Toxoplasma
gondii in sheep from the Campania region (Italy). Periodical Veterinary parasitology 149, 1-4.
11. Powell, C.C., Brewer, M., Lappin, M.R. 2001. Detection of Toxoplasma gondii in the milk of
experimentally infecting lacting cats. Veterinary Parasitology, 102, 29-33.
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OCCURRENCE OF STRONGYLOIDES STERCORALIS IN CHILDREN AND
DOGS IN EASTERN SLOVAKIA
G. Štrkolcová, M. Goldová
Department of Parasitology, University of Veterinary Medicine and Pharmacy in Košice,
Komenského 73, 041 81 Košice, Slovakia
[email protected]
Introduction: Strongyloidiasis is the intestinal endemic geohelminthosis that belongs to
ʺsoil transmitted helminthsʺ – STH. The causative agent of Strongyloides stercoralis is a
parasite of humans, dogs, cats, and primates, occurring more frequently in tropics and
subtropics, but also in the temperate zone. [1].S. stercoralis is endemic in tropical and
temperate zones, in areas where sanitary conditions are poor and where climate is warm
and humid, such as Central and South America, sub-Saharan Africa, and Southeast Asia,
but it was also found in southern Europe and south-eastern USA [2]. Strongyloides
stercoralis with the potential of autoinfection in a host can lead to systemic infections, and
as
the
opportunistic
pathogen,
may
have
fatal
consequences
especially
in
immunocompromised hosts [3].
The aim of the study: The aim of this study was to determine the presence of
Strongyloides stercoralis in Roma, non-Roma children and dogs in Eastern Slovakia. We
focused on the presence of Strongyloides stercoralis larvae, by direct evidence, in the
faeces of children and dogs.
Methodology: We examined faeces of Roma children and non-Roma children from
preschool and school facilities in Medzev. Roma children were divided into three groups
by age: pre-school children aged 1–5 years, younger schoolchildren aged 6–9 years, older
schoolchildren aged 10–14 years. The group of non-Roma children consisted of healthy
children from the majority group of population in Medzev. Examined non-Roma
schoolchildren were aged 1–17 years.We examined excrements of dogs in the Roma
settlement in Medzev and dogs from the shelter maintained by the Union of Mutual Aid of
Humans and Dogs in Haniska near Košice. The dogs were divided into 2 groups: younger
than 7 months and older than 7 months. For the purpose of detection of Strongyloides
stercoralis infection in children and dogs, we used a direct method – microscopy of stool
samples and an indirect method – serological examination of blood using the ELISA
method. Stool samples of children and dogs were subjected to a Koga agar plate (KAP)
culture and Baermann technique.
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Results and Discussion: Microscopy of stool samples from children from the Roma
settlement in Medzev and children from the majority population did not detect
Strongyloidesstercoralis larvae in any of the groups of children. The samples examined for
the qualitative evidence of specific IgG antibodies against Strongyloidesstercoralis
applying the indirect immunological and enzymatic reaction (ELISA) showed the 33.3 %
serum prevalence in Roma children and 23.8 % in the majority group. Seroprevalence was
38.1 % among preschool children aged less than 5 years, 26.3 % among schoolage children aged 6–9 years, and 35.0 % among older pupils aged 10–14 years.
Seroprevalence in the majority group of children was 23.8 % (5/21). We confirmed
rhabditiform larvae in 13.3 % of dogs from the settlement and in 10 % of dogs from the
shelter applying the KAP method. Applying the Baermann method, we did not detect the
presence of larvae. Strongyloidiasis was first observed by in three patients on the territory
of Slovakia, in former Subcarpathian Ruthenia [4]. Later were confirmed cases of
strongyloidiasis in humans from southern area of Eastern Slovakia [5].
Conclusion: Occurrence of Strongyloides stercoralison the territory of Slovakia was first
reported almost 90 years ago [4]. All over the world are very little informations available
on the epidemiology of Strongyloides stercoralis, despite its significance. The diagnostics
is hindered by the low production of rhabditiform larvae and irregular excretion of them in
faeces. Majority of infected individuals are without any symptoms; however, eosinophilia
is always present. Our findings confirm that the occurrence of this helminth must be
expected also in the temperate zone.
Acknowledgments: This study was funded by the projects of the Scientific Grant Agency
of the Ministry of Education of the SR and the Slovak Academy of Sciences, VEGA
1/0455/15.
Literature
1.
Weller PF, Nutman TB (2012) Intestinal Nematode Infections. In: Longo DL, Fauci AS, Kasper DL,
Hauser SL, Jameson JL, Loscalzo J (ed) Harrison’s Principles of Internal Medicine 18th ed. McGraw
Hill Medical, New York, pp 1739–1744
2.
Agrawal V, Agrawal T, Ghoshal UC (2009) Intestinal strongyloidiasis: a diagnosis frequently missed in
the tropics. Trans R Soc Trop Med Hyg 103:242–246. doi: 10.1016/j.trstmh.2008.08.009
3.
Basile A, Simzar S, Bentow J, Antelo F, Shitabata P et al. (2010) Disseminated Strongyloides
stercoralis: hyperinfection during medical immunosuppression. J Am Acad Dermatol 63:896–902.doi:
10.1016/j.jaad.2009.09.037
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4.
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Dziuban M (1928) Contribution to the problem of the intestinal parasites in Subcarpathian Ruthenia. Čas
lék čes 67(42):1432–1434
5. Giboda M, Maloveská M (1982) Strongyloidosis in Eastern Slovakia Bratisl lek listy 77(2):197–201
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CIGUATERA FISH CONTAMINATION AND CONSEQUENCES OF
CIGUATOXIN CONTAMINATED FISH CONSUMPTION IN HUMANS
Toropilová D.*, Eckerová R., Tomko M., Toropila M., Zigo F., Veszelits Laktičová K.,
Vargová M.
Department of Biology, Zoology and Radiobiology, University of Veterinary Medicine and
Pharmacy in Košice, Komenského 73, 041 81 Košice, Slovakia, [email protected]
Introduction:
Ciguatoxin
is
derived
from
benthic
dinoflagellates
of
the
genus Gambierdiscus, (species Gambierdiscus toxicus, Gambierdiscus spp.) growing
mostly in association with macroalgae in coral reefs . These algae produce toxin that is
transferred through the food web and it is consumed by herbivorous fish, which are
consumed by carnivorous fish, which are in turn consumed by humans (1, 2) .
Ciguatera fish poisoning (CFP) is a bizarre poisoning syndrome with acute and sometimes
severe neurological (lingual and circumoral paresthesias, pruritus, arthralgias, myalgias,
weakness),
cardiovascular
(bradycardia,
hypotension,
pulmonary
edema)
and
gastrointestinal symptoms (abdominal pain, nausea, vomiting, diarrhea, painful defecation)
caused by eating tropical reef fish intoxicated ciguatera. CFP is the most common cause
of fish poisoning worldwide (3, 4). Ciguatoxin tends to accumulate in large predator fish
(weight over 2 Kg or about 4.5 lbs) and usually causes symptoms within a few minutes to
30 hours after eating contaminated fish, and occasionally it may take up to 6 hours. (4, 5).
Aim of study: Identify the source of the fish purchased or place of capture is important in
preventing additional cases that may occur when a large, recreationally caught fish is
shared or when a wholesaler may have shipped toxic fish to other areas for resale.
There are some important reasons: first global warming has contributed to the emergence
of dinoflagellate species in subtropical and even temperate regions that previously had
been constrained to tropical areas and second: in Europe globalization of fishing industry
and tourism has led to a progressive increase in the number of ciguatera cases and a lack of
awareness among medical personnel contributes to under-reporting (6). Other options are
f.e, Food and Waterborne Disease Surveillance Program with a website providing
information on CFP and other food borne diseases. This program contains a network of
regional epidemiologists which coordinate with county health departments, the Florida
Poison Information Center (FPIC), emergency departments and other health care providers
to facilitate case investigation, reporting and public health education (7).
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Methodology: Ciguatoxin (CTX) is the lipid-soluble polyether compounds made up of 13
or 14 rings fused into rigid ladder-like structures and cause increased nerve membrane
excitability through their action on voltage-gated sodium channels and depolarizes the
nerve cell (8). This depolarization of nerve cells is believed to cause the array of
neurological signs associated with CFP (9). CTX is concentrated in the fish liver,
intestines, heads, roe and sex organs. The toxins do not affect the taste, texture, or odour of
the fish and cannot be destroyed by cooking, smoking, freezing, salting or any other
method of food preparation (10, 4). The toxin may be found concentrated in large reef fish,
most commonly barracuda, grouper, red snapper, eel, amberjack, sea bass, and other.
Ciguatera fish poisoning (CFP) causes nausea, vomiting, diarrhea, muscle weakness, joint
aches, headache, dizziness, and low blood pressure. A characteristic symptom is "hot-cold
reversal"; hot items feel cold and cold items feel hot. Symptoms may begin within 15
minutes to 24 hours after eating affected fish. Most people are better in a few days, but in
some cases symptoms have lasted for months or even years (11).
Diagnosis of marine toxin poisoning is generally based on symptoms and a history of
recently eating a particular kind of seafood. Laboratory testing for the specific toxin in
patient samples is generally not necessary because this requires special techniques and
equipment available in only specialized laboratories (11).
The methodologies available for CTX detection, including those based on the
toxicological, biochemical, chemical, and pharmaceutical properties of CTXs. Selecting
any of these methodological approaches for routine monitoring of ciguatera may be
dependent upon the applicability of the method. However, identifying a reference
validation method for CTXs is a critical and urgent issue, and is dependent upon the
availability of certified CTX standards and the coordinated action of laboratories. Reports
of CFP cases in European hospitals have been described in several countries, and are
mostly due to travel to CFP endemic areas. Additionally, the recent detection of the CTXproducing tropical genus Gambierdiscus in the eastern Atlantic Ocean of the northern
hemisphere and in the Mediterranean Sea, as well as the confirmation of CFP in the Canary
Islands and possibly in Madeira, constitute other reasons to study the onset of CFP in
Europe (12).
The FDA fish testing procedure is a two-tiered protocol involving: 1) in vitro assay, i.e. a
high-throughput screen for toxicity consistent with ciguatoxin’s mode of action; and 2) an
analytical chemistry technique known as liquid chromatography-mass spectrometry (LCMS). In the first tier, suspect fish specimens are screened for the specific effects of CTX.
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Specifically, fish specimens are screened for voltage-gated sodium channel–specific
activating effects on cell membranes. The screening procedure used is the in vitro mouse
neuroblastoma (N2a) cell assay (American Type Culture Collection CCL-131), using a
ouabain-veratridine dependent method (13,14).
Results and Discussion: The epidemiology of ciguatera is complex and of central
importance to the management and future use of marine resources (15). The frequency of
CFP varies by region throughout the world. Eating ciguatera-contaminated tropical or
subtropical fish (Pacific and Indian Ocean regions, and in the tropical Caribbean) is the
main way that humans are exposed to the toxin. There are about 50,000 reported
poisonings worldwide per year, but rarely cause death; children have more severe
symptoms. There is no specific antitoxin available for ciguatera toxin. Treatment:
Currently some physicians recommend gastrointestinal decontamination with activated
charcoal. Activated charcoal may absorb the toxin if done 3 to 4 hours after ingestion.
Maintain hydration. Intravenous fluids may be necessary for uncontrollable nausea and
vomiting. Osmotic diuretics have been used to decrease symptoms (for example,mannitol
[Osmitrol]). Amitriptyline (Elavil, Endep) and gabapentin (Neurontin, Gralise, Horizant)
may help reduce neural pain symptoms. Diphenhydramine (Benadryl) and hydroxyzine
(Atarax, Vistaril) may help relieve itching. Avoid alcohol, fish, nuts, and nut oils after
exposure to ciguatera toxin because they may trigger recurrent symptoms (16).
Conclusion: The question of the possible contribution of climate change to the distribution
of toxin-producing microalgae and ciguateric fish is raised. The impact of ciguatera onset
on European Union (EU) policies will be discussed with respect to EU regulations on
marine toxins in seafood. Critical analysis and availability of methodologies for CTX
determination is required for a rapid response to suspected CFP cases and to conduct sound
CFP risk analysis. (12) The characteristic features of fatal ciguatera fish poisoning must be
recognized early. The public should be repeatedly reminded to avoid eating the most
ciguatoxic fish species and the CTX-rich parts of reef fish. To prevent mass poisoning in
gatherings and parties, the most ciguatoxic fish species and potentially toxic fish species
must be avoided. Particularly after hits by disastrous storms, it is important to monitor the
toxicity of reef fish and the incidence rates of ciguatera (17).
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1.
Lehane L. Ciguatera: recent advances but the risk remains. Int J Food Microbiol. 2000;61:91–125.
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2.
Pearn J. Neurology of ciguatera. J Neurol Neurosurg & Psych. 2001;70:4–8. [PubMed]
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Florida Health. Ciguatera Fish Poisoning. http://www.floridahealth.gov/environmental-health/aquatictoxins/ciguatera-fish-poisoning.html
8.
Lewis RJ., Holmes MJ. Origin and transfer of toxins involved in ciguatera.
9.
Comparative Biochemistry and Physiology Part C: Pharmacology, Toxicology and Endocrinology
Volume 106, Issue 3, November 1993, Pages 615-628
10. Nicholson GM. Ciguatoxins: cyclic polyether modulators of voltage-gated ion channel function. Mar
Drugs. 2006;4:88–118.
11. International Association for Medical Assistance to travellers (IAMAT). Ciguatera Fish Poisoning.
https://www.iamat.org/risks/ciguatera-fish-poisoning
12. Gould Soloway RA. Food Poisoning from Fish: Ciguatera and Scombroid What You Can't Smell, Can
Hurt Poison Control /National Capital Poison Center http://www.poison.org/articles/2009-jun/what-youcant-smell-can-hurt
13. Arias HR. Marine toxins targeting ion channels. Mar Drugs. 2006;4:37–69.
14. Dickey R. Monitoring brevetoxins during a Gymnodinium breve red tide: comparison of sodium channel
specific cytotoxicity assay and mouse bioassay for determination of neurotoxic shellfish toxins in
shellfish extracts. Nat Toxins. 1999;7:157–165. [PubMed]
15. Manger RL. Detection of sodium channel toxins: directed cytotoxicity assays of purified ciguatoxins,
brevetoxins, saxitoxins, and seafood extracts. J AOAC Int. 1995;78:521–527. [PubMed]
16. Lehane L., Lewis RJ. Ciguatera: recent advances but the risk remains. Int J Food Microbiol. 2000 Nov
1;61(2-3):91-125
17. Davis CHP. Ciguatera Fish Poisoning. Medically Reviewed by a Doctor on 3/26/2015,
http://www.emedicinehealth.com/wilderness_ciguatera_toxin/article_em.htm)
18. Chan TY. Characteristic Features and Contributory Factors in Fatal Ciguatera Fish Poisoning-Implications for Prevention and Public Education. Am J Trop Med Hyg. 2016 Apr;94(4):704-9
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TRANSMISSION AND INTERACTIONS BETWEEN BORRELIA AFZELII AND
RICKETTSIA SPP. IN IXODES RICINUS TICKS AND LABORATORY MICE
MODEL
Vaculová, T.1, Sallay, B.2, Špitalská, E.2, Derdáková, M.1, Rusňáková Tarageľová, V.1
1
Institute of Zoology, Slovak Academy of Sciences, Dúbravská cesta 9, 845 06 Bratislava,
Slovakia [email protected]
2
Institute of Virology, Biomedical Research Center, Slovak Academy of Sciences,
Dúbravská cesta 9, 845 05 Bratislava, Slovakia
Introduction: Tick-borne diseases which afflict humans and animals are caused by
infectious agents transmitted by ticks. Lyme disease and rickettsioses belong to the most
common tick-transmitted diseases [1, 2]. The sheep tick, Ixodes ricinus is a geographically
widespread species and the most common vector for Borrelia burgdorferi sensu lato (s.l.)
and Rickettsia spp.[3]. This tick feeds on three various hostsduring its life cycle therefore
probability of pathogen infection is very high [4]. Previous studies reveal that ticks and
their hosts areinfected with multiple pathogens, that is why they can interact and affect
eachother in natural cycle of vector-pathogen-host [5]. Until now the relationships between
Borrelia afzelii and Rickettsia helvetica in vectors and hosts have not been studied.
The aim of the study: Our interest focuses on the relationships between vectors and their
hosts, as well as among microorganisms in mixed infections, which play an important role
in the pathogen circulation in natural foci. The main goal of our experiment is to study the
interactions between two different bacterial pathogens under laboratory conditions with
laboratory mice as reservoir hosts and I. ricinus ticks as vector to better understand to
transmission efficiency and the potential influence between Borrelia and Rickettsia.
Methodology: The model host organism: 8-weekold pathogen-free female C3H/N mice
and Balb/c mice
Vector: Uninfected I. ricinus larvae from the laboratory colony (Institute of Zoology SAS,
Bratislava, Slovak republic and Institute of Parasitology, Biology centre CAS, České
Budejovice, Czech republic)
Bacterial pathogen: The CB-43 strain [6] of B. afzelii and R. helvetica [7] cultivated in
VERO cells.
Experimental infection of I. ricinus tickswith B. afzelii and R. helvetica:Four groups of
mice (1st group- mice inoculated by B. afzelii strain, 2nd group- mice inoculated by R.
helvetica, 3rd group- mice inoculated by B. afzelii strain and R. helvetica, 4th group- mice
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inoculated with saline as negative controls) were established. Bacteria were injected into
the miceinto peritoneum (ip) and in the dorsal thoracic midline with 103 spirochetes ofCB43 strain of B. afzelii (500 μl of suspension per mouse)and R. helvetica (500 μl of
suspension per mouse). Ear punch biopsy was taken at 2 weeks post-inoculation and tested
for the presence of pathogens using molecular methods mentioned below. Uninfected I.
ricinuslarvae were placed on each mouse (at 2nd and 14th day post-infection) and were
allowed to feed. Random samples of 10 fully engorged larvae per mouse were examinated
for the presence of pathogens. Remaining engorged larvae were allowed to moult into
nymphs. Random samples of 10 moulted nymphs per mouse will be individually tested for
the presence of B. afzelii spirochetes and R. helvetica. In this part of experiment the
transstadial transmission will be monitored. The mice were subsequently humanely
sacrificed. The selected internal organs were removedand kept for further investigation.
DNA was extracted from ticks and tissues by using a DNeasy Tissue Kit (Qiagen,
Valencia, CA). All samples were screened by RT-PCR for the presence of R. helvetica
(citrate synthase gen gltA) and B. burgdorferi s.l.(rrfA-rrlB intergenic spacer of 5S-23S
rDNA) respectively, using specific primers. The forward primerBb23Sf, the reverse primer
Bb23Sr and the TaqMan probe Bb23Sp-FAM were used for the detection of B. afzelii [8].
For detection of R. helvetica were used the forward primer CS-F, the reverse primer CS-R
and the probe CS-P [9].
Results and Discussion: Co-infection of several infectious agents of diseases can affect
various aspects of the their transmission, including the following [10, 11]:
1. Collaborative and competitive interactions between pathogens in reservoir host or in
vector
2. The effect of the transmission of pathogens from the host to vector or from the vector to
host
3. Increasing the seriousness of the disease in host.
In the first stage of our experiment we used Balb/c mice as hosts for pathogens. Infection
of Balb/c mice with B. afzelii was successful but not with R.helvetica. Thereforein the
second stage of our experiment specific strain C3H/N mice were used. In our research B.
afzelii was detected in ear biopsies from each infected C3H/N mice. Only one biopsy was
positive for the presence of R. helvetica. Larvae, which were placed on mice at second day
after inoculation of pathogens were negative for the presence of B. afzelii. R. helvetica was
confirmed only in the one larva from all tested ticks. First results suggest that selected
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strain of laboratory mice is not competent hosts for R. helvetica. After further investigation
we will be able to assess the host competence of the laboratory mice alternatively mutual
influence of seleted pathogens. Further results will be presented.
Conclusion: The relationship of microorganisms found in ticks and their potential role in
the transmission of disease are not yet fully understood. Ticks and hosts in natural
conditions can become infected with multiple pathogens. Co-infection can affect the
transmission of pathogens to the vectors and hosts. Within laboratory experiments we can
monitor the interactions of two selected tick-borne pathogens. Based on results from
laboratory experiments we can assess the impact of co-infection rate on transmission
between the vectors themselves and between the vectors and their hosts. The interactions
between tick-borne microorganisms are important from epidemiological point of view. In
accordance with increasing amount of informations not only diagnostic methods are beeing
improved, but also prevention of tick-borne diseases as well.
Acknowledgements: This study was financially supported by the projects VEGA
no.2/0108/13 and VEGA no.2/0061/13 and APVV 0267-10.
Literature
1.
Kurtenbach K., Hanincová K., Tsao J.I., Margos G., Fish D., Ogden N.H. 2006. Fundamental processes
in the evolutionary ecology of Lyme borreliosis. Nature Reviews Microbiology. 4: 660-669.
2.
Parola P., Paddock CH. D., Socolovschi C., Labruna M. B.,Mediannikov O.,Kernif T.,Abdad M. Y.,
Stenos J., Bitam I.,Fournier P. E., Raoult D. 2013. Update on tick-borne rickettsioses around the world:
a Geographic approach. Clinical Microbiology Review. 4: 657–702.
3.
Rizzoli A., Silaghi C.,Obiegala A., Rudolf I., Hubálek Z., Földvári G., Plantard O., Taussat M. V.,
Bonnet S., Špitalská E., Kazimírová M. 2014. Ixodes ricinus and its transmitted pathogens in urban and
peri-urban areas in Europe: new hazards and relevance for public health. Frontiers in Public Health. 2:
251.
4.
Gray J. S. 1984. Studies on the dynamics of active populations of the sheep tick, Ixodes ricinus L. in Co,
Wicklow, Ireland. Acarologia. 2: 167-178.
5.
Václav R., Ficová M., Prokop P., Betáková T. 2011. Associations between coinfection prevalence of
Borrelia lusitaniae, Anaplasma sp., and Rickettsia sp. in hard ticks feeding on reptile hosts. Microbial
Ecology. 2:245-253.
6.
Štěpánová-Tresová, G., Kopecký J., Kuthejlová M. 1999. Identification of Borrelia burgdorferi sensu
stricto, Borrelia garinii and Borrelia afzelii in Ixodes ricinus ticks from Southern Bohemia using
monoclonal antibodies.Zentralblatt für Bakteriologie . 289:797–806.
7.
Sekeyova Z., Mediannikov O., Subramanian G., Kowalczewska M., Quevedo-Diaz M., Kocianova E.,
Raoult D. 2012. Isolation of Rickettsia helvetica from ticks in Slovakia. Acta Virologica. 56: 247-252.
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8.
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Courtney J. W., Kostelnik L. M., Zeidner N.S., Massung R.F. 2004. Multiplex real-time PCR for
detection of Anaplasma phagocytophilum and Borrelia burgdorferi.Journal of Clinical Microbiology.
42: 3164–3168.
9.
Stenos J., Graves S. T., Unsworth N. B. 2005. A highly sensitive and specific real-time PCR assay for
the detection of spotted fever and typhus group rickettsiae. The American Journal of Tropical Medicine
and Hygiene. 73(6): 1083–1085.
10. Zeidner N. S., Dolan M. C., Massung R., Piesman J., Fish D. 2000. Coinfectionwith Borrelia
burgdorferiand the agent of human granulocytic ehrlichiosis suppresses IL-2 and IFN γ production and
promotes an IL-4 response in C3H/HeJ mice. Parasite Immunology. 22: 581-588.
11. Levin M. L. 2007. Effects of coinfection with Borrelia burgdorferi and Anaplasma phagocytophilum in
vector ticks and vertebrate hosts. van Nitch P (ed) Research onLyme disease. Nova, New York, pp 1–34
levin.
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DETECTION ZOONOTIC SPECIES ENTEROCYTOZOON BIENEUSI IN CALVES
Valenčáková, A., Danišová, O.
Department of Biology and Genetics, University of Veterinary Medicine and Pharmacy,
Komenského 73, 041 81 Košice, Slovak Republic
[email protected]
Introduction: Microsporidia spp. are obligate intracellular parasites infecting all major
animal groups. Over the past two decades, microsporidia have risen from obscure
organisms to well-recognized human and animal pathogens. Transmission is done by fecal
- oral route, where the sources of infection are infected humans, animals or contaminated
water and food.1The microsporidian species Enterocytozoon bieneusi is important
opportunistic pathogens for humans, causing localized or disseminated infections.He is the
most frequently diagnosed species of Microsporidia andfirst found in enterocytes of a
Haitian AIDS patient, the genus and species were named by Desportes et al. 19852 based
on electron microscopic morphology. Further morphological observations placed E.
bieneusi in the family Enterocytozoonidae.3 Nearly a decade passed before E. bieneusi
began to be recognized in domesticated and wild animals.4Mounting genetic data for E.
bieneusi specimens acquired from within and among host species clearly indicate
substantive differences suggesting that many species probably exist under the umbrella of
this single species name. Because most genetic data are based almost exclusively on the
internal transcribed spacer (ITS) sequence of the rRNA the status of E. bieneusi remains a
single species consisting of many genotypes. Over 90 such genotypes now comprise this
species, some associated with a single host species, others with two or more hosts.5
The aim of the study: Enterocytozoon bieneusi is the most common species infecting
immunocompromised and immunocompetent hosts. Various genotypes of E. bieneusi are
transmitted anthroponotic route or zoonotic route. Before our objective was to determine
the prevalence Microsporidiaspp. in rearing calves as a potential source of infection.
Methodology: Samples of faeces were collected from 66 calves over 1 month of age from
Zemplinská Teplica. Faecal samples were collected only from animals with clinical
symptoms (anorexia, diarrhoea, abdominal pain, weight loss to cachexia) indicating
suspect microsporidiosis.
Genomic DNA was extracted from the 100 mg stool samples using the DNA-Sorb-B
Nucleic acid Extraction kit (AmpliSence, Estónsko) according to the manufacturer’s
instructions. For real-time SYBR Green amplification we used the procedure with the use
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of specific primer pair PMP1/PMP2 to amplify a 450 bp fragment of the small subunit
rRNA gene. Positive PCR products were sequenced.6
Results and Discussion: Samples of faeces obtained from calves were analyzed for the
presence of microsporidia by molecular methods. The real-time SYBR Green PCR method
proved the presence of 5 microsporidia positives samples. Positive PCR products were sent
for sequencing and sequences were compared with sequences in GenBank database and
were identical with the sequence Enterocytozoon bieneusi in one calf and Microsporidium
sp. in four calves (3,3 %).
The our results of detection of pathogenic species E. bieneusi not only in calves show a
low host specificity and infected animals are a potential source of human microsporidial
infection. The detection of species E. bieneusi in immunocompetent calf increasing risk of
zoonotic transmissions and latent course of infection.7E. bieneusi in cattle was first
reported in three calves in a herd of 28 cattle with diarrhea in Germany.8Our prevalence
was higher than that reported for cattle in China (5%)9, Argentina (14.2%)10 and the Czech
Republic (14.5%).11In Korea, of 538 fecal samples from cattle approximately 15% were
positive; summer rates were the highest.12 In the first report of E. bieneusi in cattle in
North America fecal specimens were obtained from 413 dairy calves from 14 farms in
seven states in the United States.13 Microscopic examination revealed no spores but PCR
using generic primers for Microsporidia revealed 70 positive calves. When PCR analysis at
the SSU rRNA gene was conducted using specific primers for E. bieneusi, 13 positive
calves were identified from six farms in five states. DNA sequencing analysis of the
specimens confirmed the PCR results and indicated 96.8–99.8% similarity with E. bieneusi
sequences in GenBank. On most of the same farms cattle feces were examined on three
additional annual visits. Of 452 post-weaned, 3- to 8-month-old calves 13% were found
positive for E. bieneusi.14 Of 571 heifers, 12–24 months of age, 23% were positive for E.
bieneusi.15A similar prevalence was found in cattle in South Africa (18%).16Feces from
each of 30 dairy cattle on a farm in Maryland were examined for the presence of E.
bieneusi at weekly, bimonthly and then monthly intervals from 1 week to 24 months of
age.17 Of the 30 calves, none appeared ill but 100% became infected at some time over the
24 months. The cumulative prevalence of infection for calves 1–8 week of age, 3–12
months of age, and 13–24 months of age was 33%, 100%, and 80%, respectively. Therapy
of microsporidiosis is difficult and in most cases the infection can become permanent with
lifelong elimination of spores, which can have economic impact on the calf industry.
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Conclusion: This study examines the occurrence of microsporidia in calves in Slovak
republic and data suggest that human pathogenic microsporidia circulate in environment
and support the idea that they are zoonotic, so they should be considered as a potential
public health threat.
The study was supported by grants APVV-15-0134, MŠ SR VEGA 1/0061/16, VEGA
1/0196/15.
Literature
1. Izquierdo, F., Castro-Hermida, J.A., Fenoy, S., Mezo, M., Gonzalez-Warleta, M.: Detection of
microsporidia in drinking water, wastewater and recreational rivers. Water. Res. 45, 2011, pp. 4837–
4843.
2. Desportes I., Le Charpentier, Y., Galian, A., Bernard, F., Cochand-Priollet, B., Lavergne, A., Ravisse, P.,
Modigliani, R.: Occurrence of a new microsporidian: Enterocytozoon bieneusi n.g., n.sp., in the
enterocytes of a human patient with AIDS. Journal of Protozoology, 32, 1985, pp. 250–254.
3.
Cali, A., Owen, R. L.: Intracellular development of Enterocytozoon, a unique microsporidian found in
the intestine of AIDS patients. Journal of Protozoology, 37, 1990, pp. 145–155.
4.
Deplazes, P., Mathis, A., Muler, C., Weber, R.: Molecular epidemiology of Encephalitozoon
cuniculi and firsts detection of Entercytozoon bieneusi in faecal samples from pigs. Journal of
Eukaryotic Microbiology, 43, 1996, p. 93S.
5.
Breitenmoser, A. C., Mathis, A., Bürgi, E., Weber, R., Deplazes, P.: High prevalence
of Enterocytozoonbieneusi in swine with four genotypes that differ from those identified in humans.
Parasitology, 118, 1999, pp. 447–453.
6.
Malčeková, B., Valenčáková, A., Molnár, L., Kočišová, A., 2013. First detection and genotyping of
huma – associated microsporidia in wild waterfowl of Slovakia. Acta. Parasitol. Mar;58(1):13–7.
7.
Dengjel, B., Zahler, M., Hermanns, W., Heinritzi, K., Spillmann, T., Thomschke, A., Loscher, T., Gothe,
R., Rinder, H., 2001. Zoonotic potential of Enterocytozoonbieneusi. J. Clin. Microbiol. 39:4495–4499.
8.
Rinder, H., Thomschke, A., Dengjel, B., Gothe, R., Loscher, T., Zahler, M.:Close genotypic relationship
between Enterocytozoon bieneusi from humans and pigs and first detection in cattle. J. Parasitol., 86,
2000, pp. 185–188.
9.
Ma, J., Li, P., Zhao, X., Xu, H., Wu, W., Wang, Y., Guo, Y., Wang, L., Feng, Y., Xiao, L.: Occurrence
and molecular characterization of Cryptosporidium spp. and Enterocytozoon bieneusi in dairy cattle,
beef cattle and water buffaloes in China. Vet. Parasitol., 207, 2015, pp. 220–227.
10. Del Coco, V. F. et al.: First report of Enterocytozoon bieneusi from dairy cattle in Argentina. Vet.
Parasitol., 199, 2014, pp. 112–115.
11. Jurankova, J., Kamler, M., Kovarcik, K., Koudela, B.: Enterocytozoon bieneusi in bovine viral diarrhea
virus (BVDV) infected and noninfected cattle herds. Res. Vet. Sci., 94, 2013, pp. 100–104.
12. Lee, J. H.: Prevalence and molecular characteristics of Enterocytozoon bieneusi in cattle in Korea.
Parasitol. Res., 101, 2007, pp. 391–396.
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13. Fayer, R., Santin, M., Trout, J. M.: First detection of Microsporidia in dairy calves in North America.
Parasitol. Res., 90, 2003, pp. 383–386.
14. Santin, M., Trout, J. M., Fayer, R.: Prevalence of Enterocytozoon bieneusi in post-weaned dairy calves
in the eastern United States. Parasitol. Res., 93, 2004, pp. 287–289.
15. Santin, M., Trout, J. M., Fayer, R.: Enterocytozoon bieneusi genotypes in dairy cattle in the eastern
United States. Parasitol. Res., 97, 2005, pp. 535–538.
16. Abu Samra, N. et al.: Enterocytozoonbieneusi at the wildlife/livestock interface of the Kruger National
Park, South Africa. Vet. Parasitol., 190, 2012, pp. 587–590.
17. Santín, M., Fayer, R.: A longitudinal study of Enterocytozoon bieneusi in dairy cattle. Parasitology
Research, 105, 2009, pp. 141–144.
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USE OF ATP BIOLUMINESCENCE METHODS IN THE PREVENTION OF
FOODBORNE DISEASES
Vargová, M., Veszelits Laktičová, K., Hromada, R., Toropilová, D., Maľová, J.,
Výrostková, J., Eckerová, R
Institute of Animal Hygiene and Environment, University of Veterinary Medicine and
Pharmacy Košice, Komenského 73 041 81 Košice, Slovak Republic
[email protected]
Introduction: Food safety and quality isthe one of the main priorities of European
research.According toWHO, system HACCP firstly must ensure, wholesomeness of foods
and eliminate the impact of pathogenic microorganisms and their toxic metabolites, which
are among the main causes of alimentary disease (7). To ensure the quality of products in
food traffic according to requirements HACCP is a decisive factor the control of hygiene
and quality of cleaning places and quality finished products. Investigations by classical
microbiological methods in terms of speed of providing results do not meet the
requirements of practice, which requires quickly, evaluate the effectiveness of sanitation,
state production hygiene and quality of the finished product (4). For the production is
optimal combination of permanent monitoring hygiene standards to provide results of
laboratory tests even at a time which can be, if adverse findings make correction that
prevent debasement product and contamination of production areas and first of all
endanger the consumer health, eventuallyof foodborne diseases (5).
Aim of the work: The goal was to monitoring the level of hygiene of the premises and
equipment operation in slaughterhouse as well as compare and evaluate the results of
microbiological control the effectiveness of disinfection with results of bioluminescence
method.
Methods: Inspection of sanitation effectiveness in meat processing plant was carried out
by two methods, microbiological and bioluminescence. Swabs were taken from various
surfaces before, during and after disinfection from 3 sections of meat processing plant
(slaughter room, section room and production room). Microbiological swabs were taken
from the area of 10 cm² and evaluated for total bacterial counts, coliform germs and
moulds. Total bacterial counts were determined after cultivation on meat- peptone agar
(MPA) at 37 °C for 24 hours. Coliforms were cultivated on Endo agar for 24 hours at 37
°C. Bioluminescence was measured with the system HY–LITE NG, produced by Biotrace,
using a portable luminometer to measure emitted light produced in a reagent chamber of a
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sampling pen supplied by the manufacturer. By means of the sampling pens we took swabs
from the area of 100 cm². The system is based on reaction of ATP from the sample with
enzyme luciferase in a reagent chamber of the pen and the intensity of emitted light is
measured by the luminometer in relative luminescence units (RLU).
Results and discussion: Food industryin Slovak Republic due to the entry into European
Union had to adopt and implement a legal system HACCP. His strategy is to monitor and
implementation of measures which eliminating the microbial contamination and ensures
the production of safe food (6). Microbiological swabs and ATP swabs taken from the
monitored technological devices in the section room indicate that in the given room,
disinfection was performed sufficiently (Table No.1). Hygienic level of surfaces before
production was good. Depending on production, microbial counts increased during
production process.
Table No.1, Average numbers of microorganisms present on particular surfaces (CFU.10
cm‾²) and average numbers of ATP swabs (RLU.100cmˉ2) in the slaughter room
TMC
Moulds
ATP
TMC
Moulds
ATP
Coliform
germs
After disinfection
ATP
Coliform
germs
During production
Moulds
Desk
Cleaning
machine
Saw
Setae remover
Coliform
germs
Before production
5
0
0
0
0
7
0
0
125
12
8
1
10
1
over
5
1
10
0
0
0
0
0
0
0
10
0
0
3
2
0
0
98
250
5
2
5
3
1700
1200
5
2
0
0
1
0
0
0
TMC
Place of swabs
taking
In the section room, we observed an increased TMC and coliform microorganisms present
on the desk after disinfection (Table No.2). These values did not change even after the time
until the start of production elapsed. The total microbial counts increased during
production. The results obtained by determination of ATP indicate the contamination
during production. When is talking about hygiene and cleanliness, it cannot fail to mention
the phenomenon of biofilm, as a result of colonization of bacteria on surfaces of
technological equipment (2). The proof of the high resistance of microorganisms in the
biofilm barriers is the isolation of foreign bacteria even after rigorous cleaningand
disinfection process (1, 2, 3). By using ATP method that is beginning to introduce and used
to evaluate the effectiveness of sanitation, monitoring of compliance with the hygiene in
services advantage is especially the possibility of immediate correction.
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Table No.2, Average numbers of microorganisms present on technological devices
(CFU.10 cm‾²) and average numbers of ATP swabs (RLU.100cmˉ2) in the section room
ATP
TMC
Coliform
germs
Moulds
ATP
TMC
Coliform
germs
Moulds
ATP
After disinfection
Moulds
150
100
2
4
During production
Coliform
germs
Desk
Saw
Bolster
Scales
Before production
TMC
Place of
swabs
taking
0
0
0
0
0
0
0
0
over
over
0
0
260
298
15
14
8
15
3
1
0
8
2
1
over
over
900
700
0
75
0
1
2
0
0
0
0
1
0
0
over
900
0
700
Table No.3 shows results of microbiological and ATP swabs taken from technological
devices in the meat production room. Hygienic level of surfaces before production was
good. During production, we observed an increased TMC. ATP results correspond with
contamination during production. Based on the results, we can conclude that the
disinfection was effective.
Table No.3, Average numbers of microorganisms present on technological devices
(CFU.10 cm‾²) and average numbers of ATP swabs (RLU.100cmˉ2) in the production room
Moulds
ATP
TMC
Coliform
germs
Moulds
ATP
Coliform
germs
0
0
0
0
0
0
25
12
0
0
1
1
0
0
1
0
0
0
1
0
0
0
0
0
0
0
25
1
0
90
0
0
0
0
Coliform
germs
TMC
After disinfection
1
1
TMC
Desk
Stirring machine
No.1
Cutting machine
No.1
During production
ATP
Before
production
Moulds
Place of swabs
taking
Conclusion: Hygiene and sanitation has a leading position in meat processing plant. The
time demanding conventional microbiological methods do not fulfill requirements of food
hygiene practice that stress rapid obtaining of results when evaluating effectiveness of
sanitation. The bioluminescence methods based on determination of ATP are new modern
methods advantageous with regard to their rapidity, simplicity, low time, easy
manipulation. The results obtained in a meat processing plant showed that disinfection was
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effective in all sections with the exception of swabs taken from section room. In this
section we recorded on desk 2 coliform bacteria. Contamination of this part of premises
was supported by the ATP results. The mentioned counts changed after some time up to
the beginning of production and corrective measures were taken on the basis of ATP
swabs. Thus the practical use of bioluminescence method in food hygiene is justified as it
allows taking corrective measures.
Literature
1.
BAGGE, D., HJELM, M., JOHANSEN, CH., HUBER, I., GRAM, L.: Shewanella putrefaciens adhesion
and biofilm formation on food processing surfaces. App. Env. Microbiol., 67, 2001, s. 2319-2325.
2.
COSTERTON, J. W., LEWANDOWSKI, Z., CALDWELL, D. E., KORBER, D. R., LAPPIN-SCOTT,
H. M.: Microbial biofilms. Ann. Rev. Microbiol., 49, 1995, s. 711–745
3.
KUMAR, C. G., ANAND, S. K.: Significance of microbial biofilms in food industry: a review. Int. J.
Food Microbiol., 42, 1998, s. 9 - 27.
4.
VOJTAŠŠÁK, J.: Kontrola hygieny pomocou detektoru Lightning MVP. Maso, roč. 14, 2003, 5, s. 3335.
5.
LOPAŠOVSKÝ, Ľ, PAVLIČOVÁ, S., ŠIŠKA, B.:
Utilization method of bioluminiscence at the
monitoring at somefood processing facilities, Zborník z medzinárodnej konferencie, Rizikové faktory
potravového reťazca, Nitra, 2006, p. 210-21.
6.
MLYNARČÍKOVÁ, H., KOVALKOVIČOVÁ, N., LEGÁTH, J.: Pesticídy v potravinovom reťazci. In
Hygiena alimentorum XX. Košice: Štátna veterinárna správa Slovenskej republiky, ISBN 80- 7148- 0363, 1999, s. 162-166.
7.
PITZURA, O.: Microbial air monitoring in food industry. Laboratory Journal, 2, 2001, s. 62-64.
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MOLECULAR CHARACTERIZATION AND TRACING OF PERSISTENT
LISTERIA MONOCYTOGENES STRAINS IN THE PRODUCTION CHAIN OF
TRADITIONAL BRYNDZA CHEESE PROCESSING FACILITY IN SLOVAKIA
Véghová1, A. – Kaclíková1, E. – Minarovičová1, J. – Drahovská2, H.
1
National Agricultural and Food Centre – Food Research Institute, Priemyselná 4, 824 75
Bratislava, Slovakia
2
Faculty of Natural Sciences, Comenius University, Mlynská dolina B-2, 842 15
Bratislava, Slovakia
[email protected]
Introduction: Listeria monocytogenes is a bacterial pathogen capable of causing a life
threatening disease known as listeriosis with high fatality rate of 20-30 %. Listeriosis
mostly occurs after consumption of contaminated foods. This bacteria can contaminate
different food products, such as meat, milk, cheese, vegetables and ready-to-eat (RTE)
foods. Food contamination occurs most frequently in the equipment and the production
environment, where some strains of L. monocytogenes can persist over a long time period.
This requires monitoring of food processing factories environment to identify potential
contamination sources and transmission routes in the food production chain. The sources
and routes have been identified by characterization of L. monocytogenes strains by
molecular typing methods with high discriminatory power, such as pulsed-field gel
electrophoresis (PFGE), which allows identification of persistent contamination (1), or
multi-locus sequence typing (MLST) (2).
The aim of study: This work was aimed to the occurence, diversity and tracing of Listeria
monocytogenes strains in three small and medium-sized ewes´ milk processing factories in
Slovakia.
Methodology: A total of 639 samples were collected during years 2011-2014. From a total
of samples, 216 samples were collected from food-contact areas and 283 from non-foodcontact areas and equipment (including gloves, overshoes, floors, walls, cleaning rags,
feed, fly papers), and 140 food samples of milk, ewes´ lump cheese and final products
(bryndza cheese) were analysed. The samples were analysed using the current
microbiological standard according to ISO 11290-1:1996/Amd 1:2004. For the
identification of L. monocytogenes strains, real-time PCR targeting actA gene was used
(3). Strains identified as L. monocytogenes were characterized by molecular serotyping,
PFGE, MLST and detection of epidemic clones (EC). Serotyping was performed by
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multiplex PCR, which classified L. monocytogenes strains into five molecular serogroups
IIa, IIb, IIc, IVa and IVb (4). All L. monocytogenes strains were subtypedby pulsed-field
gel electrophoresis (PFGE) according to PulseNet protocol (5) using one restriction
enzyme AscI. The PFGE AscI-patterns of strains were analysed by BioNumerics software
(Applied Maths, Kortrijk, Belgium). Multi-locus sequence typing (MLST) of the
sequenced strains was performed according to the Protocol PF8 Genotyping of Pathogens
and Public Health Platform, Institut Pasteur (6). Detection of genetic markers for epidemic
clones ECI, ECII and ECIII was performed by multiplex PCR according to Chen and
Knabel (7).
Results and Discussion: Out of analysed samples, 20 (3.1 %) were positive for
L. monocytogenes. Among 20 positive samples, 18 samples were associated with
production environment and only two samples were food products (bryndza cheese and
ewes´ lump cheese). Molecular serotyping of 20 isolated L. monocytogenes strains
classified these strains into three serogroups IIa (80 %), IIc (10 %) and IVb (10 %). All
strains were analysed by PFGE in order to determine the diversity of these strains. The
L. monocytogenes strains were divided into 14 clusters at 100 % similarity and into 11
clusters at 90 % similarity. At 90 % similarity, two clusters of similar types (ST) of profiles
3 (four isolates) and 14 (three isolates) were identified. Most of strains were grouped into
one main cluster at 50 % similarity, only two strains of serogroup IVb and three identical
strains of serogroup IIa (profile 7) were outliers (Fig. 1). Results of MLST were in the
correlation with PFGE profiles, however shoved lower discrimination power. Genetic
markers for epidemic clones ECI, ECII and ECIII were negative for all analysed strains.
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Figure 1: Dendrogram of PFGE AscI patterns of 20 L. monocytogenes strains with
sample identification data and results of molecular serotyping and MLST.
Conclusion: To reveal the character, sources and transmission routes of contamination in
the food production environment, analytical methods with sufficient discrimination power,
such as molecular typing methods, need to be used. Based on the results, in all three
factories was observed only low and sporadic contamination with nonpersistent
L. monocytogenes strains originating from the external environment. By application of
PCR serotyping and PFGE typing, high strain diversity was determined.
Acknowledgement: This work is a result of implementation of the project ITMS
26240220089 “Effective control methods for safety foods“ of the Agency for the Structural
Funds of the European Union, Ministry of Education, Science, Research and Sport of the
Slovak Republic.
Literature
1.
BLATTER, S., GIEZENDANNER, N., STEPHAN, R., ZWEIFEL, C.: Phenotypic and molecular typing
of Listeria monocytogenes isolated from the processing environment and products of a sandwichproducing plant. Food Control, 2010, 21, 1519–1523.
2.
PARISI, A., LATORRE, L., NORMANNO, G., MICCOLUPO, A., FRACCALVIERI, R., LORUSSO,
V., SANTAGADA, G.: Amplified fragment length polymorphism and multi-locus sequence typing for
high-resolution genotyping of Listeria monocytogenes from foods and the environment. Food
Microbiol., 2010, 27, 101–108.
3.
ORAVCOVÁ, K., KACLÍKOVÁ, E., KRASCSENICSOVÁ, K., PANGALLO, D., BREŽNÁ, B.,
SIEKEL, P., KUCHTA, T.: Detection and quantification of Listeria monocytogenes by 5´-nuclease
polymerase chain reaction targeting the actA gene. Lett. Appl.Microbiol., 2006, 42, 15–18.
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4.
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KÉROUANTON, A., MARAULT, M., PETIT, L., GROUT, L., DAO, T. T., BRISABOIS, A.:
Evaluation of a multiplex PCR assay as an alternative method for Listeria monocytogenes serotyping. J.
Microbiol. Methods., 2010, 80, 134–137.
5.
Standard Operating Procedure for PulseNet PFGE of Listeria monocytogenes (PNL04, last update April
2014), 1-11. (http://www.cdc.gov/pulsenet/PDF/listeria-pfge-protocol-508c.pdf).
6.
Protocol
PF8
Genotyping
of
Pathogens
and
Public
Health
Platform,Institut
Pasteur
(http://www.pasteur.fr/recherche/genopole/PF8/mlst/primersLmono.html).
7.
CHEN, Y., KNABEL, S. J.: Multiplex PCR for Simultaneous Detection of Bacteria of the Genus
Listeria, Listeria monocytogenes, and Major Serotypes and Epidemic Clones of L. monocytogenes. Appl.
Environ. Microbiol., 2007, 73, 6299–6304.
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SANITATION AS AN IMPORTANT TOOL FOR MONITORING
ENVIRONMENTAL HYGIENE IN FOOD PROCESSING PLANTS
Katarína Veszelits Laktičová, Mária Vargová, Rudolf Hromada, Denisa Toropilová, Jana
Maľová, Jana Výrostková, Radka Eckerová
Institute of Animal Hygiene and Environment, University of Veterinary Medicine and
Pharmacy Košice, Komenského 73 041 81 Košice, Slovak Republic,
[email protected]
Introduction: A necessary condition for ensuring the healthy development of the body and
maintain health is to receive hygienically faultless and medically safe food. Share of
sanitation in the fulfillment of social tasks defined in the area of health and nutrition, as
well as the scope and complexity of the activities that are carried out within the sanitation
highlights the importance and need for a comprehensive security system. Food-borne
illnesses are a serious worldwide health and economic problems and their suppression
represents a significant share antiepidemiological activities. In order to prevent the spread
of infection of food is necessary to apply hygiene practices (6) and that is why carrying out
sanitation in food processing plants is justified. The sanitation system is a key element of
the control system HACCP. Cleaning and disinfection as part of everyday production
practice are indispensable for the proper functioning of the food production within the
legislative requirements.
The aim of the work: The aim of work was to monitor and evaluate the environmental
hygiene of the individual surfaces and technological equipment in the areas of production
puff pastry. We reviewed the quality of the air in the premises of production of puff pastry.
Methods: Inspection of sanitation effectiveness in the areas of production puff pastry was
carried out by microbiological method. Swabs were taken from various surfaces before,
during and after disinfection from surfaces and technological equipments. Microbiological
swabs were taken from the area of 10 cm² and evaluated for total bacterial counts, coliform
germs and moulds. Total bacterial counts were determined after cultivation on meatpeptone agar (MPA) at 37 °C for 24 hours. Coliforms were cultivated on Endo agar for 24
hours at 37 °C. Moulds were cultivated on Sabouradov agar for 3-5 days at 22 °C.
Results and discussion: Table 1 shows the microbial purity of Surface of production
technologies production of puff pastry. The results show that the disinfection surface
treatment the monitored technology was effective to the conveyor belt No.4, where we
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have detected 1 CFU coliform microorganisms and to start of production, the number of
coliform germs increased to 5 CFU. In terms of cleanliness it is important also hygiene of
staff. Food contamination during production, processing, distribution and preparation it is
able to cause the man himself. That is just in every food, whether raw or processed, a
particular risk for the creation of alimentary diseases (1). To ensure the production of
disabled safe foodstuffs each worker in food industry has to be knowledgeable with good
hygiene practices and his particular field of work (4).The results obtained by
microbiological controls the effectiveness of disinfection are used as basis forsearch
shortcomings in sanitation and re-create the possibility of their removal.
Table no.1: Average values of microorganisms on individual surfaces of production
technologies in the production of puff pastry
Moulds
TMC
0
12
0
6
1
0
0
Cutting knives
5
0
0
115
2
0
4
0
0
Guillotine
4
0
0
18
1
0
3
0
0
Box for margarine
0
0
0
19
1
0
2
0
0
Mixer
8
0
1
25
0
0
8
0
0
Conveyor belt č. 4
2
5
1
25
3
5
9
1
0
Conveyor output
1
0
0
14
1
0
1
0
0
Packing machine
1
0
0
154
0
1
1
0
0
germs
TMC
0
germs
Moulds
1
germs
Cutter
TMC
Moulds
After disinfection
Coliform
During production
Coliform
Before production
Coliform
Place of sampling
In the food industry, disinfectants are routinely used to sanitize and disinfect product
contact surfaces. These chemicals provide a necessary and required step to ensure that the
foods produced and consumed are as free as possible from microorganisms that can cause
foodborne illness. Table no. 2 shows the values of the monitored micro-organisms from the
various surfaces of other parts of production of puff pastry. The table shows that the
presence of the plate count, coliform bacteria and fungi on surfaces decreased after
disinfection performed. During production, we recorded the highest levels of pollution,
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pollution of the monitored surface is proportionally increased depending on the
manufacturing process.
Coliform germs
Moulds
35
0
0
52
0
1
10
0
0
Packing (floor)
110
0
1
155
1
3
45
0
1
Freezing box
15
0
0
30
0
0
5
0
0
10
0
2
21
1
0
2
0
0
TMC
Moulds
Moulds
Coliform germs
Coliform germs
Packing room (wall)
Place of sampling
TMC
TMC
Table no.2: The average value of microorganisms on individual surfaces in production of puff
pastry
Before production
During production
After disinfection
(wall)
Handling trucks
Evaluation of air microflora in the areas of production Puff is shown in Table No. 3.
During production increased numbers of microorganisms in direct proportion with running
technology. After disinfection, we recorded a low to negative counts of total numbers
colonies, coliform germs and fungi. Hygiene in food production is one of the key factors in
ensuring the production of high quality and hygienically flawless products (2).In the
process of manufacture and packaging of food products may be contaminated with dust
particles, which are often carriers of microorganisms in air traffic. It is therefore necessary
regular monitoring in the industry, which is done by settling of air on the cultivation
medium on Petri dishes (3).
Table no.3. The average value of airborne microorganisms in the area of operation puff pastry
Production hall
(beginning)
Production hall (middle)
Production hall (the
end)
Mixing room
Packing room
Moulds
Coliform
germs
After disinfection
TMC
Moulds
Coliform
germs
During production
TMC
Moulds
Coliform
germs
Before production
TMC
Place of sampling
0
0
0
11
0
1
0
0
0
5
4
0
0
2
2
10
52
0
0
1
2
1
0
0
0
0
1
0
2
0
0
1
4
10
12
1
0
1
4
0
1
0
0
0
2
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Conclusion: Sanitation system is one most important part of the control system HACCP
(5). Food processing facilities have to comply with high requirements on cleanness and
hygiene of all the equipment, premises, storage room and packaging materials. Failure to
comply with these requirements and insufficient cleaning and disinfection results have
negative impact on the final products.
Literature
1.
HUDECOVÁ, D., ŠIMKOVIČ, M. 2009. Mikrobiológia, Bratislava, 11-168 s.
2.
LACIAKOVÁ. A. 2004. Čistenie a dezinfekcia v potravinárskych prevádzkach. In: Slovenský
veterinárny časopis, XXIX, č. 4, , s. 17.
3.
PITZURA, O. 2001. Microbial air monitoring in food industry. Laboratory Journal, 2, p. 62-64.
4.
ROVNÝ, I. – FABIÁNOVÁ, E. – GAÁL, P. – NOVÁKOVÁ, J. – ONDREJKA, J. – MICHALUS, M.
1998. Hygiena 2. 1. vyd. Martin: Osveta, 1998, 158 – 172 s. ISBN 80-88824-42-7.
5.
VOJTAŠŠÁK, J. 2003: Kontrola hygieny pomocou detektoru Lightning MVP. Maso, roč. 14, 2003, 5, s.
33-35.
6.
ZELEŇÁKOVÁ,
L.,
PAUKOVÁ,
J.
2008.
Význam
protiepidemiologických
zariadeniachškolského stravovania. Potravinárstvo [online], 5.6.2008, 2 (2), 32-41 s.
296
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TULAREMIA IN SLOVAKIA - EPIDEMIOLOGIC AND EPIZOOTIC ISSUES,
2005 - 2014
Výrosteková, V.1, Guryčová, D.1, Gacíková, E.2, Strišková, K.2, Kocianová, E3.
1
Faculty of Medicine of the Comenius University in Bratislava, Institute of Epidemiology,
Špitálska 24, 813 72 Bratislava, Slovakia (e-mail: [email protected])
2
State Veterinary and Food Institute in Bratislava, Botanická 15, 842 52 Bratislava,
Slovakia
3
Institute of Virology, SAS, Dúbravská cesta 9, 845 05 Bratislava, Slovakia
Introduction: Tularemia is a zoonosis with natural focality, considered nowadays as
reemerging disease in Europe. In Slovakia, the bacteria Francisella tularensis subsp.
holarctica is endemic, mainly infecting hares and small mammals, with bloodsucking
arthropods as important live vectors. In the eighties of the last century, F. tularensis subsp.
tularensis was isolated in foci of the Danube river basin, indicating also the risk of
circulation of this highly pathogenic subspecies in ecological conditions of Central Europe
(1).
The aim of the study: Aim being to analyse epidemiologic and epizootic situation in the
occurrence of tularemia in Slovakia during the decade 2005 - 2014 with respect to
epidemiologically important issues.
Methodology: Data on reported cases in humans and on occurrence of natural foci were
from published results of tularemia surveillance in Slovakia (2), records of epidemiologic
examinations, research work and projects.
Results and Discussion: Trend of tularemia incidence has declined in Slovakia since
epidemics in the years 1995/1996
and 2002 (3.04/1.25 and 2.55 per 100 000 population, respectively) to 0.09/105 in 2011,
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Fig. 1 Incidence of tularemia in Slovakia, 1990 - 2014
followed by a slight increase to 0.24/105 in 2014 (Fig. 1), which in 2015 continued (4).
In the years 2005 - 2014, from a total of 179 cases reported, 92.8 % were from four
administrative regions of west Slovakia (Fig. 2), mainly from Nitra region (56.4 %) - each
year with 2 - 6 districts affected.
Fig. 2 Geographic occurrence of tularemia, Slovakia 2005 - 2014
The mean incidence rate of tularemia in 2005 - 2014 was 0.33 per 105 population on the
country level. In administrative regions (Fig. 2) it ranged from 0.03 in Košice (KI) and
Žilina (ZI), 0.05 and 0.08 in Prešov (PV) and Banská Bystrica (BC) respectively, 0.21 and
0.25 in Bratislava (BL) and Trenčín (TC), through 0.66 in Trnava (TA) up to 1.45 per 105
population in Nitra (NI) region. Most of cases occurred in the years 2005 - 2009 (70.95 %)
compared with 2010 - 2014 (29.05 %) five year period.
Mean incidence rates in 2005 - 2014 (Fig. 3) were generally higher in males compared
with females (0.43 and 0.24 per 105 in total), with age group of the 40-49 years old mostly
affected in both sexes (0.74 and 0.64 per 105 for males and females, respectively). Male to
female ratio was 1.67 :1 (112 men and 67 women).
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Fig. 3 Incidence of tularemia according to age groups and gender, Slovakia 2005 2014
Seasonal occurrence of tularemia in years 2005 - 2014 (Fig. 4) peaked in summer July/August (13.4/16.8 %). While in the first five years period (2005-2009) the peak was
inJanuary (16.5 %), in the second one (2010-2014) the peak was in August (23.1 %).
Fig. 4 Seasonality of tularemia, Slovakia 2005 - 2014
As sources of infection (Fig. 5) hares were identified in 12.3 % of cases, other animal
species, mainly rodents, in 45.3 %. Human cases transmitted by ticks and other arthropods
such as biting insects represented 20.1 % (10.6 and 9.5 %, respectively). Proportion of hare
sources of infection declined from 15.0 % in the years 2005-2009 to 5.8 % in 2010-2014,
rise in transmission by arthropod vectors from 17.3 % to 26.9 % was observed in these two
time periods.
Fig. 5 Tularemia according to sources of infection and transmission, Slovakia 2005 2014
Changes observed in the disease occurrence being associated namely with natural as well
as social factors influencing exposure (2).
Tularemia in hares in the years 2005 - 2014 (Tab. 1) was diagnosed by State Veterinary
and Food Institutes only in 2010 (from 2002 to 2009 zero reporting) with 2 hare tularemia
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foci identified in Bratislava and Trnava administrative regions, district Senec and Dunajská
Streda, respectively. Compared with years 1995-2004 and 81 hare foci indentified in five
regions (mostly Trnava and Nitra region), a longterm decline has been recorded.
Tab. 1 Hare tularemia foci according to administrative regions, Slovakia, 1995 - 2004 and 2005 -2014
Region
Bratislava
Trnava
Nitra
B. Bystrica
Košice
Total
1995-04
4
38
35
1
3
81
2005
0
0
0
0
0
0
2006
0
0
0
0
0
0
2007
0
0
0
0
0
0
2008
0
0
0
0
0
0
2009
0
0
0
0
0
0
2010
1
1
0
0
0
2
2011
0
0
0
0
0
0
2012
0
0
0
0
0
0
2013
0
0
0
0
0
0
2014
0
0
0
0
0
0
Serological positivity found in examined domestic animals (Tab. 2), reflecting possible
exposures to contaminated environment, infected arthropod vectors etc., pointed at
persistence and activity of endemic natural foci of tularemia in all regions of west Slovakia
also during the years of low disease occurrence in humans (2).
Tab. 2 Serological positivity against Francisella tularensis in domestic animals, west
Slovakia 2009-2014
Animals
horses
% posit.
dogs
% posit.
2009
22 / 94*
23.4 %
0
2010
0
2011
0
2012
0
34 / 296
11.5 %
22 / 273
8.1 %
35 / 241
14.5 %
2013
34 / 98
34.7 %
20 / 154
13.0 %
2014
62 / 210
29.5 %
5 / 42
11.9 %
*
number positive / number examined
Field studies conducted during the study period led in the years 2009 - 2011 to isolations
of
Francisella tularensis subsp. holarctica biovar II from small mammals, their
ectoparasites and ticks collected from vegetation in localities of Trnava and Bratislava
administrative regions.
In Trnava
region, district of Senica, in a natural focus under logterm surveillance,
tularemia epizootics were detected in 2009 - isolations from
rodents Clethrionomys
glareolus, and in 2011 - isolations from Apodemus flavicollis mice and from their
ectoparasites (Ixodes ricinus tick larvae, nymphae and from fleas), as well as from Sorex
araneus. Tick adults of Dermacentor reticulatus collected from vegetation were found
positive in 2009 - district of Senica, and in 2011 - district of Dunajská Streda.
In Bratislava region, F. tularensis was isolated from D. reticulatus adults collected in
2010 in district Bratislava V, and fed on experimental rabbits (3).
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Conclusion: Results
18th-20th October 2016
of epidemiologic and epizootiologic surveillance
pointed at
persistence and activity of endemic natural foci and continual risk of acquiring tularemia in
the west Slovakia endemic region.
Acknowledgement: Studies were supported by projects VEGA no.1/ 4281/07, 2/0142/10
and 1/0426/11.
Literature
1.
Guryčová, D.: First isolation of Francisella tularensis subsp. tularensis in Europe. Eur. J. Epidemiol.
1998, 14, 797-802.
2.
Guryčová, D., Výrosteková, V., Gacíková, E.: Surveillance of zoonoses. Tularemia.
Slovak Republic 2009-2012. Comenius University, Bratislava 2013. 113 p.
3.
Výrosteková, V., Guryčová, D., Kocianová, E., Bottková, E., Maďarová, L., Slovák, M.: Ticks in
Danube River basin area and risk of tularemia. Proceedings of a Conference III. Labudove dni,
Institute of Virology SAS, Bratislava 2013, 1-3.
4.
Report on zoonoses, food- and water-borne diseases in the Slovak Republic, 2015. Ministry of
Agriculture and Rural Development of the Slovak Republic, Bratislava 2016. 116 p.
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PROS AND CONS OF LYME DISEASE TESTS: AN AGREEMENT ANALYSIS
Waczulíková I.1, Mišenko P1., Matlahová K1,2., and Schwarzová K.3
1
Department of Nuclear Physics and Biophysics, Faculty of Mathematics, Physics and
Informatics, Mlynská dolina F1, 2Department of simulation and virtual medical education,
and3Institute of Microbiology, Faculty of Medicine, Sasinkova 4, Comenius University in
Bratislava, Slovakia
[email protected]
Introduction: Lyme borreliosis, or so-called lyme disease (LD), is a diagnosis based on
patient’s medical history, clinical symptoms and exposure to ticks (1, 2), however, it is
difficult to diagnose from the symptoms alone, because there are other conditions that
cause similar clinical picture. Laboratory tests can provide useful and necessary
information for the diagnostic process (2), but the question remains how to use them in the
most appropriate and cost effective manner.
Tests that are available for LD are mainly based on assays of antibodies against Borrelia
burgdorferi (B.B.) ˗ indirect fluorescent antibody (IFA), enzyme-linked immunosorbent
assay (ELISA), Western blotting (WB). Unfortunately these tests are not standardised and
the results obtained with different assays may not be sufficiently conclusive. The mainstay
of laboratory diagnosis for LD is two-tiered serological testing, in which a reactive firsttier ELISA is supplemented by separate IgM and IgG immunoblots(Figure 1). As a result,
patients with negative ELISA results, but affected with LD, might in fact be missed for
further investigation and intervention (3). Therefore, in this study we have analysed in
more detail the results obtained in a project where patients’ samples were investigated by
both commonly used serological tests, ELISA and WB. It is important to stress that firstand second-tier tests are not independent indicators of exposure to B.B. (4). ELISAs and
immunoblots are usually constructed with the same antigens, but they are processed
differently. Here, it is just fine to use the statistical methods and perform an agreement
analysis to evaluate the correspondence between two measurements on the same sample.
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Figure 1: Two-tiered testing for Lyme disease. Adapted from (2).
The aim of the study: To assess and discuss the degree of agreement between ELISA and
WB performed in a paired design on patients presenting symptoms potentially associated
with LD. More specifically, the aim was to identify possible groups of patients whose
ELISA and WB results disagree, and to discuss possible clinical scenarios.
Methodology: A total of 638 samples of patients with suspected LD were referred to the
microbiology laboratory. All patients gave informed consent regarding their care. Each
sample was then tested with ELISA with the recombinant variable surface antigen (VlsE),
and by WB also with the VlsE (reference test). The difference is that ELISA tests are using
whole-cell preparations ofB.B., whereas in a WB procedure the antigen extracts are
separated using a discontinuous gel electrophoresis (5, 6).
For analysis purposes, a dichotomised version of ELISA titre values based on the
epidemiological cut-off was used to represent patients’ results. Categorical results were
summarised in contingency tables. Agreement analysis (7) was used to evaluate the degree
of agreement and disagreement between ELISA and WB results over appropriate
categories in 2×2 tables. Cohen’s kappa, Maxwell’s chi-square and McNemar’s statistics
were used to test for agreement, disagreement and significant differences. In broad terms a
kappa below 0.2 indicates poor agreement and a kappa above 0.8 indicates very good
agreement beyond chance. Maxwell’s chi-square statistic tests for overall disagreement
between the two tests’ results. The general McNemar statistic tests for asymmetry in the
distribution of subjects about which the readings disagree, that means, disagreement more
over some categories of response than others. All tests were conducted at a significance
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level of 5% using StatsDirect® 2.8.0 software. Cross-classification tables were made using
Excel 2010.
Results: Patients demographic characteristics: gender male 238 (37.3%) with median age
of 52 years (min-Q1-Q3-max: 16-38-62-87); female 400 (62.7%), median age of 53 years
(min-Q1-Q3-max: 7-36-62-92). Male and female age distributions were not significantly
different.
The overall observed agreement between ELISA and WB was rather poor (69% for IgM
and 57% for IgG), even if it was significant (P<<0.001), and surprisingly better for females
(data not shown). The agreement was also related to age with decreasing agreement for
IgM and increasing agreement for IgG as it was expected in older individuals (Table 1).
Disagreement over any category and asymmetry of disagreement were also statistically
significant (P<<0.001 both) with higher false ELISA positive rates for IgM and higher
false ELISA negative rates for IgG with respect to findings from WB.
Table 1: Relationship of categorised age and degree of Cohen’s kappa agreement between
ELISA and WB.
Age category
mean kappa for IgM
mean kappa for IgG
up to 50
51-74
75 and above
From a practical point of view, however, a descriptive classification of patients into
respective WB categories according both ELISA test results is more informative than
statistical testing (Table 2). One can easily see and calculate (summarised under Table 2),
how many patients would be missed when proceeding according to the accepted testing
scheme (Figure 1), i.e. when a patient is tested with ELISA test on both, IgG and IgM, and
with WB as a confirmatory test only.
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Table 2: Cross-classification table for ELISA IgG with nested IgM categories classified according
the results from WB IgG with nested IgM categories. Each value in the table represents a matched
pair of ELISA(IgG;IgM) × WB(IgG;IgM) result.
WB IgG
WB IgM
Tot
al
Negative
ELISA IgG
ELISA IgM
Negative
Positive
Negative
Negative
120
8
Positive
37
Positive
Total
Grand
Total
Negative
Positive
128
123
27
150
278
11
48
49
47
96
144
157
19
176
172
74
246
422
Negative
15
2
17
71
17
88
105
Positive
9
3
12
49
50
99
111
Total
24
5
29
120
67
187
216
Grand Total
181
24
205
292
141
433
638
Total
Positive
Both, ELISA IgG and ELISA IgM negative / WB IgG or WB IgM positive (158 out of 278; 57%)
ELISA IgG negative, ELISA IgM positive / WB IgG positive (49 out of 144; 34%)
ELISA IgG positive, ELISA IgM negative / WB IgM positive (2 out of 105; 2%)
Discussion: Based on our results, there is a concern that sticking on the two-tiered testing
procedure might not be an optimal strategy, since it could lead to a high rate of missed
cases with acute or persistent/recurrent LD. It is important to note that a positive IgG
and/or IgM WB only implies exposure to B.B. and is not confirmatory for LD. But, even if
we cannot estimate proportions of missed cases due to a lack of data on definite diagnosis
and stage of disease, we can reason based on the observed rates of false positives and
negatives, and on the very low agreement between ELISA and WB represented by a
Cohen’s kappameasure. Some researchers state that when an ELISA is negative but an
immunoblot of the same sample is scored positive, it is probable that faint immunoblot
bands are being “over-read” (2). This may likely be true for ELISA IgM negative and WB
IgM positive cases (8 out 278; 3%), however it is less probable explanation of relatively
high rates of false negatives that we have found for other combinations of serology results
(Table 2). LD is a complicated clinical entity - it is known that the antibody tests are not
static over time, and a patient negative in the WB may seroconvert to a positive pattern
with treatment. Conversely, a patient could redevelop an IgM response, which is
suggestive of a persistent/recurrent (in other words chronic) infection, which is a real
diagnostic problem because the IgG response may be absent in more than 50% of the
patients. Thus, in addition to the IgG WB, an IgM WB should be used. The proper timing
of the investigation is an important consideration in the diagnosing LD. For example, a
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positive IgM result alone is not recommended for use in determining active disease in
persons with illness of longer than one month duration, because the likelihood of a falsepositive tests result is high for these individuals (8). The rationale for determining IgM and
IgG antibody profiles by WB is to learn whether a patient’s antibodies recognise proteins
of B.B. that have been established to be more predictive of LD than other components of
the bacteria (9).
Summarising Pros/cons for ELISA and WB:

ELISA tests provide an estimate of the magnitude of the IgG/IgM humoral antibody
response to all of the antigens that are expressed under the culture conditions used to
produce the whole-cell (corpuscular) antigen or to the recombinant or peptide antigens
used. ELISA results are objective and quantitative - they can be correlated with
antibody titres (2). ELISA method is simple, easy to perform (automated) and
relatively cheap.

The limitation is that ELISA test detects only free antibody, thus a negative test might
actually indicate a more serious infection. ELISA has low agreement/high
disagreement with WB, which implies low ELISA’s specificity (and potentially not
adequate sensitivity). ELISA may fail to detect antibodies from related strains of B.B.,
and is burdened with a considerable cross-reactivity to other spirochetes, heat-shock
proteins, and some viruses (8).

On the other hand,WB (with separated antigen extracts) is sufficiently specific and
sensitive. However, it is “manual” and so-called over-reading is a frequent concern. It
is also more expensive in comparison to ELISA.

Diagnostic performance of each single method could not be evaluated due to lack of an
accepted gold standard (1) by which we could compare diagnostic performance of
available tests.
Conclusion: A standard procedure is using an ELISA as the first test and immunoblots are
typically applied only when ELISA was positive, thus, many cases can be unrecognised.
However, our results support the necessity of Western blot confirmation for both positive
and negative ELISA. Antibody assays for lyme disease will improve when recombinant
antigens become available to the unique antigens of Borrelia burgdorferi making ELISA
assays highly sensitive (>95%) with acceptable specificity (>90%). At such a time, a twotiered testing procedure would make more sense. For the time being and based on our data,
it seem more reasonable to determine IgM and IgG antibody profiles in any new patient at
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any stage of disease by ELISA and Western blot to provide adequate support for clinical
evaluation. We believe that in a long-term horizon such a strategy could reduce health-care
spending.
Literature
1.
DeBiasi, R.L. (2014) A Concise Critical Analysis of Serologic Testing for the Diagnosis of Lyme
Disease. Curr Infect Dis Rep 16, 450. DOI 10.1007/s11908-014-0450-9
2.
Johnson, B.J.B. (2011) Laboratory Diagnostic Testing for Borrelia burgdorferi Infection. In: Lyme
Disease: An Evidence-based Approach (ed. J.J. Halperin). CAB International
73-88. ISBN
9781845938048. DOI 10.1079/9781845938048.0000
3.
Leeflang, M.M.G., Ang, C.W., Berkhout, J., Bijlmer, H.A., et al. (2016) The diagnostic accuracy of
serological tests for Lyme borreliosis in Europe: a systematic review and meta-analysis. Infectious
Diseases (2016) 16:140. DOI 10.1186/s12879-016-1468-4
4.
Wormser, G.P., Carbonaro, C., Miller, S., Nowakowski, J., Nadelman, R.B., Sivak, S. and AgueroRosenfeld, M.E. (2000) A limitation of 2-stage serological testing for Lyme disease: enzyme
immunoassay and immunoblot assay are not independent tests. Clin Infect Dis 30, 545–548.
5.
Vestník MZ SR, Vydanie 50-60/2013 [online] Bratislava [retrieved on the 22.08.2016]. Available at
http://www.zbierka.sk/
6.
Schwarzová, K., Holečková, K., and Kostanová Z. (2009) [Evidence of Borrelia burgdorferi spirochetes
in patients with early disseminated Lyme borreliosis]. Klinicka mikrobiologie a infekcni lekarstvi, 15(2),
40-43.
7.
Bland, J.M., Altman, D.G. (1986) Statistical methods for assessing agreement between two pairs of
clinical measurement. Lancet, 1(8476), 307–10.
8.
An Understanding of Laboratory Testing Journal of Spirochetal and Tickborne Diseases—Volume 5,
Spring/Summer (1998) [retrieved on the 22.08.2016]. Available at http://www.igenex.com/labtest.htm
1/14
9.
Engstrom, S.M., Shoop, E. and Johnson, R.C. (1995) Immunoblot interpretation criteria for
serodiagnosis of early Lyme disease. J Clin Microbiol 33, 419–427.
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IDENTIFICATION AND CHARACTERIZATION OF LISTERIA
MONOCYTOGENES ISOLATED FROM FISH IN POLAND
K. Wieczorek, J. Osek
National Veterinary Research Institute, Partyzantow 57, 24-100 Pulawy, Poland
[email protected]
Introduction: Listeriosis, caused by Listeria monocytogenes, is a disease in humans which
remains a major public health concern considering its high mortality rate (2, 5).Several
reports have shown that ready-to-eat (RTE) foods, including fish and fish products, are one
of the main sources of listeria for humans (2, 4, 7).According to the recent EFSA report, in
2014 a total of 2,161 human confirmed listeriosis cases were identifed in the EU, with the
notification rate 0.52 cases per 100,000 population (2). Furthermore, seven EU Member
States reported 210 deaths due to listeriosis. In the same year, in Poland 86 listeriosis
infections were identified, with the rates 0.23.L. monocytogenes has been differentiated
into 13 serotypes but only four of them (1/2a, 1/2b, 1/2c and 4b) have been isolated from
ca. 95% of human listeriosis cases (1).
L. monocytogenes has generally been considered susceptible to antimicrobials active
against Gram-positive bacteria and the effective treatment of choice for listeriosis are βlactam antibiotics (e.g. penicillin or ampicillin), alone or in combination with
anaminoglycosides (e.g. gentamicin) in case of immunocompromised patients (3).
However, the number of strains recovered from food, environment and sporadic cases of
human listeriosis resistant to one or more antibiotics used for treatment of the disease has
continually increased (8).Therefore, it is necessary to monitor the antibiotic resistance of L.
monocytogenesfrom food and environmental sources in different geographic areas in order
to understand the patterns of resistance and to plan the strategies for prevention of their
environmental spread.
The aim of the study: The aim of the present study was to determine: (i) the occurrence
and numbers of L. monocytogenes in raw and smoked fish at the retail level in Poland, (ii)
serotype distribution among the isolates, (iii) resistance to the several antimicrobials.
Methodology: A total of 301 fresh and smoked fish samples were purchased from
February 2014 to June 2016 from local fish and general stores located in the eastern part of
Poland.The samples were analyzed for detection and enumeration of L. monocytogenes
according to the EN ISO 11290-1:1999+A1:2005 and EN ISO 11290-2:2000/A1:2004
standards, respectively. Multiplex PCR for identification of the main L. monocytogenes
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serotypes was done as described previously (1, 9).The antimicrobial susceptibility test for
the L. monocytogenes isolates was performed by the minimal inhibitory concentration
(MIC) method (Trek Diagnostic Systems, USA) with the GPN3F plate (Trek) containing
the panel of 17 antimicrobials, including those used to treat human listeriosis (6).
Results and Discussion: The overall prevalence of L. monocytogenes in fresh and smoked
fish is presented in Table 1.
Table 1. Prevalence of L. monocytogenes in fish tested.
Fish species
No. of samples
tested
positive (%) for L. monocytogenes
fresh
smoked
fresh
smoked
Marine fish:
n = 102
n = 152
n = 18 (17.6)
n = 28 (18.4)
Salmon
25
74
8 (32.0)
25 (33.8)
Cod
22
21
7 (31.8)
1 (4.8)
Dorada
6
0
0
Turbot
6
0
0
Flounder
5
0
0
Halibut
4
12
0
1 (8.3)
Herring
4
14
0
0
Mackerel
4
16
1 (25.0)
1 (6.2)
Sardine
4
0
0
Mullet
3
0
0
Sea bass
3
0
0
Coalfish
2
0
0
Cock
2
0
0
Crimson
2
0
0
Moron
2
0
0
Swordfish
2
0
2 (100)
Sprat
1
10
0
0
Horse mackerel
0
3
0
Other
5
2
0
0
Freshwater fish:
n = 28
n = 19
n = 10 (35.7)
n = 1 (5.3)
Trout
15
8
4 (26.7)
1 (12.5)
Bream
4
11
2 (50.0)
0
Silver carp
3
0
3 (100)
Panga
3
0
1 (33.3)
Roach
2
0
0
Crucian carp
1
0
0
Total
130
171
28 (21.5)
29 (17.0)
A total of 57 isolates were recovered from 301 analyzed samples (18.9%). Among 102 and
152 marine fresh and smoked fish 17.6% and 18.4% were positive for these bacteria,
respectively. Freshwater fish were contaminated with L. monocytogenes at the levels of
35.7% (fresh fish) and 5.3% (smoked fish), respectively. The majority of the positive
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samples were identified in both fresh and smoked salmon (32.0% and 33.8%, respectively)
as well as in fresh cod (31.8%).
The number ofL. monocytogenes above the detection limit of the method used was
identified only in three samples of smoked salmon and the populations of the bacteria were
2.0×103, 7.0×103 and 7.6×103 CFU/g, respectively. The contamination level of the
remaining 54 fish samples was below 100 CFU/g.
Four serogroups were identified among 57 isolates tested. Serotype 1/2a (n = 40; 70.2%)
was the most prevalent, followed by serotype 1/2b (n = 14; 24.6%). Only three L.
monocytogenes strains were classified as 1/2c and 4b (one and two isolates, respectively).
The most common serotype 1/2a was present both in samples from marine (71.7% strains)
and freshwater fish (63.6% isolates). Similar prevalence was observed for L.
monocytogenes of 1/2b serogroup (23.9% and 27.3% positive samples of marine and
freshwater fish, respectively).
Most of theL. monocytogenesisolates tested were susceptible to the antimicrobials used in
the study Fig. 1). However, several strains displayed resistance to oxacillin (OXA; MIC ≥
4 mg/l; 33 isolates; 57.9%), ceftriaxone (AXO; MIC ≥ 32 mg/l; 18 strains; 31.6%) and
clindamycin (CLI; MIC ≥ 4; 5 isolates; 8.8%). Furthermore, some isolates showed
intermediate resistance to CLI (MIC 1 – 2 mg/l; 39 strains; 68.4%), AXO (MIC 16 mg/l;
20 isolates; 35.1%), ciprofloxacin (CIP; MIC 2 mg/l; 6 strains; 10.5%) or linezolid (LZD;
MIC 4 mg/l; 5 isolates; 8.8%). It was also found that two strains (3.5%) (both of serotype
1/2a, isolated from fresh cod and smoked salmon, respectively) showed a multiresistant
pattern since they were resistant to three classes of antimicrobials used, i.e. cephalosporins
(AXO)-penicillins (OXA)-lincosamides (CLI).
The isolates resistant to ceftraiaxone were identified in several fish species, mainly among
fresh and smoked salmon (5 isolates of each). L. monocytogenes resistant to oxacillin were
also originated from fresh (11 strains) and smoked (7 isolates) salmon as well as from fresh
cod (4 strains). The AXO- and OXA-resistant isolates were classified to serotypes 1/2a and
1/2b (24 and 8 strains, respectively). Furthermore, all 5 L. monocytogenes strains resistant
to CLI were of 1/2a serotype and were originated from fresh cod and bream (2 and 1
isolates, respectively) and from smoked salmon (2 strains). Several strains, displaying
intermediate resistance to this antimicrobial (n = 39), were isolated from the above as well
as other fish species.
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OXA
AXO
CLI
CIP
LZD
Others
0%
20%
40%
60%
80%
Percentage of L. monocytogenes isolates
Resistant
Intermediate
100%
Susceptible
Fig.1. Resistance (susceptibility) of L. monocytogenes isolated from fish. Antimicrobials: OXA:
oxacillin; AXO: ceftriaxone; CLI: clindamycin; CIP: ciprofloxacin; LZD: linezolid; Others: the
remaining 12 antimicrobials used in the study.
Conclusion: The high prevalence of this bacterial pathogen highlights the importance of
such food as potential vehicles for transmission of virulent L. monocytogenes. The
presence of the epidemiologically important serotypes 1/2a and 1/2b, which are associated
with human listeriosis, could be a major public health concern. Although most of L.
monocytogenes strains were susceptible to antimicrobials used in the study, a continued
investigation of antimicrobial resistance of this pathogen is important to ensure effective
treatment of human listeriosis and a possible spread of resistance genes. In spite of a
relatively limited number of the samples, the results provide useful information regarding
fresh and smoked fish contamination with potentially virulent L. monocytogenes. These
data have significant implications for epidemiological and public health studies of this
pathogen.
Literature
1. Doumith M. et al.: Journal of Clinical Microbiology, 2004, 42, 3819-3822.
2. EFSA/ECDC: EFSA Journal, 2015, 13, 4329.
3. Hof H.: Expert Opinion on Pharmacotherapy, 2004, 5, 1727-1735.
4. Kuzmanović J. et al.: Acta Veterinaria (Beograd), 20111, 61, 193-203.
5. Lomonaco S. et. al.: Infection, Genetic and Evolution, 2015, 35, 172-183.
6. Lyon S.A. et al.: Foodborne Pathogens and Disease, 2008, 5, 253-259.
7. Novoslavskij A. et. al.: Annals of Microbiology, 2016, 66, 1-15.
8. Swaminathan B., Gerner-Smidt P.: Microbes and Infection, 2007, 9, 1236-1243.
9. Wieczorek K. et al.: Foodborne Pathogens and Disease, 2012, 9, 681-685.
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MASTITIS PATHOGENS ISOLATED FROM SAMPLES OF MILK IN DAIRY
COWS AND THEIR RESISTANCE
F.Zigo,1 M. Chripková,2 M.Vasiľ, 1J.Elečko,1 D. Toropilová 1
1
2
University of Veterinary Medicine and Pharmacy in Košice, 040 01, Slovak Republic
Pavol Josef Šafarik University in Košice, Trieda SNP1, Košice, 041 80, Slovak Republic
[email protected]
Introduction: Mastitis is an inflammation of the mammary gland characterized by
physical, chemical, bacteriological and cytological changes in milk. Pathological changes
in glandular tissues of the udder and effects on the quality and quantity of milk have been
observed. This disease is mainly caused by microorganisms usually bacteria, including
gram-negative and gram-positive bacteria, mycoplasmas, yeasts and algae. The majority of
mastitis incidences are caused by only a few common bacterial pathogens involved: Staph.
spp. (Staph. aureus and Staph. epidermidis), Strep. spp. (Strep. agalactiae, Strep.
dysgalactiae, Strep. uberis and Strep. bovis), coliforms (mainly E. coli and Klebsiella
pneumoniae) and Actinomyces pyogenes (Sharma, 2010).
Antimicrobials are routinely used for treatment of dairy cattle affected with clinical and
subclinical infections. The use of antimicrobials have, over time, increased the number of
antimicrobial-resistant microbes globally, and any use of these agents will to some extent
benefit the development of resistant strains and also inappropriate usage of antimicrobials
such as wrong dose, drug or duration may contribute the most to the increase in
antimicrobial resistance without improving the outcome of treatment (Idriss et al., 2014).
The present work aimed to study the effectiveness of different antibiotics against isolated
microorganisms from milk samples of dairy cows.
Methodology: The study was conducted in herd of 320 Holstein dairy cows in east of
Slovakia. Dairy cows were kept freely housed in three cowsheds and milked twice a day in
the tandem milking shed Boumatic 2 x 10 Xpressway (Wisconsin, USA). A total of 1280
milk samples were collected from udder quarters and cultivated pathogenic bacteria
according Vasiľ et al. (2009) were examined and sensitivity of microorganisms against
antibiotics had been tested.
All strains of Staphylococcus spp. and Streptococcusspp. isolated through microbiological
procedures were subjected to antimicrobial susceptibility test by disc diffusion method to
identify the most effective drugs for mastitis treatment in the study area (Hameed, 2008).
The microbial sensitivity against twelve tested antibiotics were determined on Mueller
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Hinton agar as described by National Committee for Clinical Laboratory Standards
(NCCLS, 2002). The results were obtained by measuring the diameter of the growth
inhibition zone around the antibiotic disc for each isolated bacterial strain and recorded as
sensitive, intermediate and resistant.
Results and Discussion: A total of 1280 milk samples from udder quarters were
investigated, 242 (18.9 %) samples were positive. No pathogens were isolated from 1031
(80.5 %) milk samples as given in Table 1.
The study of the frequency of susceptibility of S. aureus (n = 18) to antibiotics has
revealed a higher sensitivity to the tetradelta (94.4 % to each), combinations of amoxicillin
plus clavulanat acid and cefallexin plus kanamycin (94.4 % to each) and ceftiotur (94.4 %).
A certain resistance has been noted to amoxicillin, streptomycin and penicillin (22.2 % to
each) (tab. 2).
Staphylococci were mostly susceptible to antimicrobials tested but, Muhamed et al. (2012)
found that S. aureus was resistant to penicillin and streptomycin (41.44 % and 25.65 %
respectively). Similar results were obtained by Sumathi et al. (2008) where
Staphylococcus and Streptococcus spp. were resistant to streptomycin and penicillin. Those
results are in accordance with our findings.
In contrast, CNS (n = 108) have been found to show a complete sensitivity to the tetradelta
(100%), and higher sensitivity to amoxicillin combination plus clavulanat acid, cefallexin
plus kanamycin and rifaximin (98.9% to each).
Foltys and Kirchnerová (2005) tested 60, 62 and 77 strains of Staph. aureus, Strep.
agalactiae and E. coli, respectively to various antibiotics and they reported that Staph.
aureus was sensitive to all antibiotics except lincomycin and streptomycin, whilst Strep.
agalactiae was 100 % sensitive to amoxicillin and ampicillin and resistant to streptomycin,
neomycin and tetracycillin and E. coli was resistant to all antibiotics.
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Table 1. Results of microbiological culture of milk samples collected from dairy cows
Total. No.
18
23
19
25
14
7
11
108
17
242
1031
7
1280
320
Isolated microorganisms
Staphylococcus aureus
Streptococcus agalactiae
Streptococcus uberis
E. coli
Enterococcus spp.
Corynebacterium pyogenes.
Pseudomonas aeruginosa
CNS*
Others ( bacteria and mould)
infected quarters
non-infected quarters
reject quarters
total quarters
total dairy cows in herd
%
1.4
1.0
1.5
3.2
1.1
0.5
0.9
8.4
1.3
18.9
80.5
0.5
100
Legend: Total No. - total number of isolate, % - percentage of bacteria, T.no. , CNS*- S. epidermidis, S.
chromogenes, S. schleiferi, S. intermedius, S. xylosus
Table 2. Frequency of susceptibility of Staphylococcus spp. and Streptococcus spp. to
antibiotics
Bacterial strains Staphylococcus
CNS
Streptococcus
Streptococcus
aureus (18)
(108)
agalactiae (23)
uberis (19)
Antibiotic agent
S (%)
R (%) S (%) R (%) S (%) R (%) S (%) R (%)
Amoxicillin
77.8
22.2
94.4
5.6
100
0
89.5
10.5
Amox. + clavulanat
94.4
5.6
98.9
1.1
100
0
100
0
Cephal. + kanamyc.
94.4
5.6
98.9
1.1
100
0
100
0
Ceftiofur
94.4
5.6
96.2
3.7
100
0
100
0
Cloxacillin
83.3
16.7
96.2
3.7
91.3
8.7
79
21
Enrofloxacin
88.9
11.1
95.4
4.6
100
0
100
0
Lincomycin
83.3
16.7
95.4
4.6
82.6
17.4
100
0
Nafpenzal
88.9
11.1
95.4
4.6
100
0
100
0
Penicillin
77.8
22.2
93.5
6.5
100
0
100
0
Rifaximin
88.9
11.1
98.9
1.1
47.8
52.2
100
0
Streptomycin
77.8
22.2
92.6
7.4
69.6
30.4
47.3
52.7
Tetradelta
94.4
5.6
100
0
100
0
100
0
Legend: Amox. - Amoxicillin, Amox. clavulanat - Amoxicillin clavulanat acid, Cephal. + kanamyc. Cephalexin + kanamycin, CNS- Coagulase negative staphylococci, n- number of bacteria strains, SSensitivity, R- Resistant.
Conclusion: Antibiotic susceptibility tests should be done to determine the effectiveness of
drug that can be used for successful treatment of diseases. Proper isolation and
identification of the causative organism play significant role in prevention and control of
the diseases. In our study a combinations of amoxicillin plus clavulanat acid, cefalexin plus
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kanamycin, enrofloxacin and tetradelta were the most effective antibiotics for control of
bovine mastitis.
Acknowledgements: This work was supported by project APVV-0679-10 and project
VEGA-1-0510-16.
Literature
1.
FOLTYS, V. – KIRCHRNEOVÁ, K. 2005: Development of mastitis pathogens occurrence and their
susceptibility to antibiotics in basic production of milk. Jour. of Farm Anim. Sci., vol. 38, 2005, p. 177180.
2.
HAMEED, S. – ARSHAD, M. – ASHRAF, M. – AVAIS, M. – SHAHID, M. A. 2008: Prevalence of
common mastitogens and their antibiotic susceptibility in tehsil Burewala, Pakistan. Pakistan Jour. of
Agricul. Sci., vol. 45 (2), 2008, p.182-183.
3.
IDRISS, SH. E. - FOLTYS, V. - TANČIN, V. - KIRCHNEROVÁ, K. - TANČINOVÁ, D. - ZAUJEC,
K. 2014: Mastitis pathogens and their resistance against antimicrobial agents in dairy cows in Nitra,
Slovakia. Slovak J. Anim. Sci., 47, 2014 (1): 33-38.
4.
MUHAMED, H. M. – DOSS, A. – VIJAYASANTHI, M. – VENKATASWAMY, R. 2012:
Antimicrobial drug susceptibility of Staphylococcus aureus from subclinical bovine mastitis in
Coimbatore, Tamilnadu, South India. Vet. World, vol. 5 (6), 2012,
5.
NATIONAL COMMITTEE FOR CLINICAL LABORATORY STANDARDS (NCCLS), 2002:
Performance Standards for Antimicrobial Disk and Dilution Susceptibility Tests for Bacteria Isolated
from Animals. Approved Standard. NCCLS Document M31-A2, Wayne, 2002, p. 352-355.
6.
SHARMA, D. K. – JALLEWAR, P. K. – SHARMA, K. K. 2010: Antibiogram of bacteria isolated from
bovine subclinical mastitis. Ind. Vet. Jour., vol. 87 (4), 2010, p. 407.
7.
SUMATHI, B. R. – VEERAGOWDA, B. – AMITHA, R. G. 2008: Prevalence and antibiogram profile
of bacterial isolates from clinical bovine mastitis. Vet. World, vol. 1 (8), 2008, p. 237-238.
8.
VASIĽ, M. 2009: Etiology, course and reduction of incidence of environment of dairy cows. Slovak
Jour. of Anim. Scien., vol. 42, 2009, p. 136-14.
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CHANGES IN THE COMPOSITION OF BACTERIAL MICROFLORA OF
PIGEONS DURING THE RACE SEASON
F. Zigo,1 M. Chripková,2 M.Vasiľ,1 J.Elečko ,1 R. Eckerová 1
1
2
University of Veterinary Medicine and Pharmacy in Košice, 040 01, Slovak Republic
Pavol Josef Šafarik University in Košice, Trieda SNP1, Košice, 041 80, Slovak Republic
[email protected]
Introduction: Pigeons belong among the first animals to be domesticated and are strongly
attached to the place where they live. Since the oldest times, pigeons attracted the interest
of people by their unbelievable sense of spatial orientation, flying capabilities,
monogamous behaviour and bond to their habitat. By successfully returning from the race
in a winning position, the pigeons demonstrate favourable factors, such as their inherent
qualities, health state, stress related to transport and hygiene conditions in their breeding
environment. Shortened flying times at competitions and more races per season indicate
the higher performance of these birds which is reflected in their health and short periods of
regeneration. The increasing stress is associated with weakened immunity, higher
susceptibility to diseases and the absence of their expected performance (Bergman, 2013).
The aim of this study was to characterize the most common diseases carrier pigeons and
compare isolated bacteria from swabs of cloaca, oropharynx, crop and infraorbital sinuses
during the race season.
Methodology: Before and after race season from holding of 80 pigeons were collected
faecal samples and swabs of the cloaca, oropharynx, crop and infraorbital sinuses. Clinical
examination of the health status was performed according to Kimpe et al. (2002).
The floatation technique was used for detecting coccidiosis and endoparasitosis from
faecal samples according to Dranzoa et al. (1999). Determination of trichomoniasis from
swabs of the oropharynx and crop as well as detection of ectoparasitosis was performed
according to Letkova et al. (1997).
Swabs of the cloaca, oropharynx, crop and infraorbital sinuses were cultivated onto blood
agar and suspect colonies Staphylococcus spp., Streptococcus spp. and Enterobacteriacae
spp. were isolated and identified biochemical using the STAPHYtest, STREPTOtest, ENTEROtest (Erba-Lachema, CZ) and identification by software TNW Pro 7.0 (ErbaLachema, CZ). The diagnostics for salmonella was performed after multiplication in
buffered peptone water and culturing on the Mac Conkey medium according to Stenzel et
al. (2013). For mycoplasma culture, were collected oropharynx, crop and infraorbital
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sinuses swabs. To culture mycoplasmas were used mycoplasma selective agar plates and
broths according to Turcsányi et al. (2012).
Results and Discussion: Little is known about the micrococcal flora in the intestines and
faeces of pigeons. In an earlier study, an enterococcal species which appeared to be
specific for pigeons was described as Enterococcus columbae
(Devriese et. al., 1993).
Enterococcus columbae is the predominant Gram-positive bacterial species in the pigeon
gastrointestinal tract. The prevalence of other enterococci, such as Ent. faecalisand Ent.
faecium, is very variable and depends on the health status and the use of antibiotics on the
holding (Baele et al. 2002).
In a study conducted Stenzel et al. (2013) from 683 pigeons in Poland were isolated from
cloacal swabs E. coli, S. faecalis and Strep. gallolyticus, which are considered the natural
digestive tract commensals. E. coli and Strep. gallolyticus are usually commensal but can
also act as an opportune pathogens. Several factors are needed for E. coli to cause disease
in pigeons, such as stress or adenoviral or herpesviral infection (Baele et al., 2002; Kimpe
et al., 2002).
Coagulase-negative staphylococci, Strep. gallolyticus and Stap. aureus were isolated in
swabs from cloaca, oropharynx and crop.Their increased incidence in the swabs from
oropharynx and crop may indicate to increase immunity in pigeons after the race season.
In our study before racing season were observed increased number of E. coli (66 pigeons),
Ent. columbae (51 pigeons) and Ent. faecalis (45 pigeons) compared to the number at the
beginning of the season (tab. 1).
According to Baele et al. (2002) Strep. gallolyticus does not appear to be an important
member of the intestinal flora. This species, identified at that time as Strep. bovis, was
considered a facultatively pathogenic agent belonging to the normal intestinal flora of
pigeons.
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Graph 1. Comparison of the most common diseases of pigeons in the monitored holding before and after race season (%)
34
35
30
25
19
20
15
10
11
9
7
7
3
5
before
7
2
1
0
2
after
0
Legend: * -
Endoparasitosiscaused by Ascaridia spp., Capillaria spp., Ectoparasitosis caused by
Columbicola columbae, Ceratophylus columbae, Res. infectious- Respiratory infectious – caused by
Mycoplasma spp.
Table 1. Species of bacteria isolated from swabs of the cloaca, oropharynx, crop and
infraorbital sinuses
Species of
bacteria
E. coli
E. columbae
E. faecalis
E. gallinarum
S. gallolyticus
S. faecalis
Mycoplasma spp.
S. aureus
S. intermedius
S. epidermidis
S. xylosux
S. schleiferi
S. simulans
S. sciuri
S. warneri
S. lentus
Total
n
B
66
51
45
10
19
12
1
1
7
4
4
4
2
0
1
0
227
cloaca
A
53
45
38
39
38
34
7
9
18
14
13
13
12
7
7
4
351
B
62
46
42
10
15
12
2
2
2
183
A
33
35
25
39
28
27
2
7
4
5
6
7
3
231
Oropharynx
+ crop
B
A
4
17
4
7
3
12
2
6
5
2
3
5
4
8
3
7
2
6
2
7
2
4
4
1
5
4
30
99
infraorbital
sinuses
B
A
3
1
3
1
2
4
2
1
5
1
2
1
3
1
3
2
1
2
7
28
Legend: B – before race season, A – after race season, n - the total number of isolated bacteria
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According to Bergman (2013), the most common diseases pigeons include coccidiosis,
trichomoniasis and respiratory infectious. After the race season was an increased incidence
of all common diseases in monitoring holding (graph 1).
Conclusion: Pigeon intestinal flora changes during racing season excessive performance
and short regeneration before the next race. Significant influence on the change of
intestinal microflora has an impact the farmer and the use of antibiotics in prophylactic
programs.
Literature
1.
BAELE, M. - DEVRIESE, L.A. - BUTAYE, P. - HAESEBROUCK, F. 2002. Composition of
enterococcal and streptococcal flora from pigeon intestines.Jour. Appl. Microbiol., Vol. 92, p. 348–351.
2.
BERGMANN, R. 2013. Sportovní výkony u holubu. Letu zdar, Vol. 23, No. 1, p. 2
3.
DEVRIESE, L.A. - POT, B. - COLLINS, M.D.1993. Phenotypic identification of the genus
Enterococcus and differentiation of phylogenetically distinct enterococcal species and species groups.
Jour. of Appl. Bacteriology, Vol.75, p.399–408.
4.
DRANZOA, C. - OCAIDO, M. - KATETE, P. 1999. The ecto, gastro-intestinal and haemo parasites of
live pigeons (Columba livia) in Kampala, Uganda. Avian Pat., Vol. 28, p. 119-124.
5.
KIMPE, A. - DECOSTERE, A. - MARTEL, A. - HAESEBROUCK, F. - DEVRIESE, L. A. 2002.
Prevalence of antimicrobial resistance among pigeon isolates of Streptococcus gallolyticus, Escherichia
coli and Salmonella enterica serotype Typhimurium. Avian Pathol, Vol. 31, p. 393-397.
6.
LETKOVÁ, V. - GOLDOVA, M. - CSIZMÁROVÁ, G. 1997. Laboratórna diagnostika vo veterinárskej
parazitológii. 1. edition, Datahelp, p. 71 - 82, ISBN 888867-12-6.
7.
PROSPERI, M. - VERAS, N. - AZARIAN, T. - RATHORE, M. et al. 2013. Molecular epidemiology of
community-associated Methicillin-Resistant Staphylococcus aureus in the genomicera: a cross-sectional
study. Sci. Rep.,Vol. 3, p. 1902.
8.
STENZEL, T. - BANCERZ-KISIEL, A. - TYKAŁOWSKI, B. - ŚMIAŁEK, M. - PESTKA, D. KONCICKI, A. 2013. Antimicrobial resistance in bacteria isolated from pigeon in Poland,Pol. Jour. of
Vet. Scie. Vol. 17, No. 1, p. 169–171.
9. TURCSÁNYI, I. - BISTYÁK, A. - MATIZ, K. et al. 2005. Isolation of Mycoplasma columbinasale from
pigeons in Hungary, Veterinary Record, Vol.157, p. 235-236.
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ISBN 978–80–89702–30–5
EAN 9788089702305
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