Download ADVANCES IN AGRICULTURAL SCIENCES XIV (1–2)

WEST POMERANIAN UNIVERSITY OF TECHNOLOGY, SZCZECIN
ADVANCES IN AGRICULTURAL SCIENCES
XIV (1–2)
SZCZECIN – 2011 – POLAND
Editor-in-Chief
Antoni J. Furowicz DVM PhD Prof.
Editorial Advisory Board
Maciej Gajęcki (Olsztyn), Stefania Giedrys-Kalemba (Szczecin),
Antoni J. Furowicz (Szczecin) – przewodniczący, Antoni Jakubczak (Łomża),
Krzysztof Janus (Szczecin), Józef Kur (Gdańsk), Jerzy Molenda (Wrocław),
Zofia Rotkiewicz (Olsztyn), Jolanta Karakulska (Szczecin), Paweł Nawrotek (Szczecin)
Approved for publication by the Rector of the West Pomeranian University of Technology, Szczecin
ISSN 1230-1353
© Copyright by Wydawnictwo Uczelniane Zachodniopomorskiego Uniwersytetu Technologicznego
w Szczecinie, Szczecin 2011
Publisher: Wydawnictwo Uczelniane Zachodniopomorskiego Uniwersytetu Technologicznego
w Szczecinie, al. Piastów 50, 70-311 Szczecin, Poland, e-mail: [email protected]
Head Editor: Antoni J. Furowicz
Druk: PPH „Zapol” Dmochowski, Sobczyk, Sp.j., 71-062 Szczecin, al. Piastów 42,
e-mail: [email protected]
Wydanie I. Nakład 200 egz. Ark. wyd. 11,5, format B-5
Motto: Lucundi acti labores
Cicero
CONTENTS
SPIS TREŚCI
FOREWORD ......................................................................................................................................
7
I. REVIEW ARTICLES
Joanna Działo, Jarosław Poleszczuk, Wiesław Deptuła ..........................................................
11
Important yet little known biological phenomena
Ciekawe i ważne zjawiska biologiczne
Antoni Jakubczak, Milena A. Stachelska, Renata Świsłocka, Monika Kalinowska,
Włodzimierz Lewandowski .........................................................................................................
19
Combination of bacteriocins with various factors as a way of natural food preservation
Zastosowanie bakteriocyn w połączeniu z innymi czynnikami w biokonserwacji żywności
Paulina Niedźwiedzka-Rystwej, Agata Mękal, Jarosław Poleszczuk, Wiesław Deptuła ......
35
TLR receptors – selected data
Receptory TLR – wybrane dane
Paulina Niedźwiedzka-Rystwej, Wiesław Deptuła ....................................................................
45
Haematological and biochemical factors in selected species of reptiles, rodents and animals
from artiodactyls order
Wskaźniki hematologiczne i biochemiczne u wybranych gatunków gadów, gryzoni i zwierząt
z rzędu parzystokopytnych
Antoni Jakubczak, Milena Alicja Stachelska ............................................................................
53
Health benefits resulting from probiotic bacteria consumption
Korzyści zdrowotne wynikające z konsumpcji bakterii probiotycznych
Joanna Śliwa-Dominiak, Arkadiusz Zupok, Wiesław Deptuła ................................................
65
Viruses of Archaea
Wirusy bakterii Archea
Joanna Śliwa-Dominiak, Wiesław Deptuła ................................................................................
71
The characteristics of selected environmental bacteria
Charakterystyka wybranych bakterii środowiskowych
Małgorzata Pawlikowska, Wiesław Deptuła ..............................................................................
79
RNA viruses infecting fish marine – selected data
Wirusy RNA infekujące ryby morskie – wybrane dane
Małgorzata Pawlikowska, Wiesław Deptuła ..............................................................................
85
RNA regulation of bacterial virulence – selected data
Regulowanie wirulencji bakterii przez RNA – wybrane dane
Agata Mękal, Beata Tokarz-Deptuła, Alicja Trzeciak-Ryczek, Wiesław Deptuła ...................
Complement and properdin, element of non-specyfic humoral immunity – important element
of innate (natural) immunity
Dopełniacz i properdyna, elementy nieswoistej odporności humoralnej – ważne składniki
odporności wrodzonej (naturalnej)
91
II. ORIGINAL ARTICLES
Paweł Nawrotek, Karol Fijałkowski, Danuta Czernomysy-Furowicz, Ewelina Michałek,
Alicja Solecka .............................................................................................................................. 107
Identification and differentiation of zoonotic Escherichia coli strains isolated from healthy
sheep, by molecular methods
Identyfikacja i różnicowanie odzwierzęcych szczepów Escherichia coli wyizolowanych
od zdrowych owiec, z użyciem metod molekularnych
Beata Hukowska-Szematowicz, Aleksandra Manelska, Beata Tokarz-Deptuła,
Wiesław Deptuła .......................................................................................................................... 115
Comparative analysis of the gene encoding VP60 capsid protein in various strains
of the RHD (rabbit haemorrhagic disease) virus
Analiza porównawcza genu kodującego białko kapsydu VP60 u różnych szczepów
wirusa RHD (rabbit haemorrhagic disease)
Karol Fijałkowski, Anna Silecka, Danuta Czernomysy-Furowicz ........................................... 125
Serum protein fractions in healthy sows during perinatal period
Analiza frakcji białkowych surowicy zdrowych macior w okresie okołoporodowym
Anna Silecka, Karol Fijałkowski, Danuta Czernomysy-Furowicz ........................................... 131
Serum protein fractions in piglets – comparison of two types of agarose carriers
Analiza obrazu elektroforetycznego surowicy klinicznie zdrowych prosiąt z zastosowaniem
dwóch typów nośnika agarozowego
Beata Tokarz-Deptuła, Bartłomiej Pejsak, Wiesław Deptuła ................................................... 135
White and red blood cell indices in healthy rabbits
Wskaźniki biało i czerwonokrwinkowe u królików zdrowych
Jolanta Karakulska, Anita Stępień, Karol Fijałkowski, Danuta Czernomysy-Furowicz ....... 147
The effect of penicillin and amoxicillin/clavulanic acid on the induction of L form
of staphylococci isolated from mastitic milk of cows
Wpływ penicyliny i amoksycyliny z kwasem klawulanowym na indukcję form L gronkowców
wyizolowanych z mleka mastitowego krów
Karol Fijałkowski, Danuta Czernomysy-Furowicz, Paweł Nawrotek ...................................... 155
Influence of Staphylococcus aureus exosecretions isolated from bovine mastitis on leukocyte
morphology in vitro
Ocena wpływu egzogennych czynników wirulencji wytwarzanych przez szczepy S. aureus
izolowane od krów z objawami mastitis na morfologię leukocytów w hodowlach in vitro
Milena A. Stachelska, Antoni Jakubczak .................................................................................. 165
Microbiological activity of salts of coumaric and cinnamic acids against Escherichia coli
O157:H7 and Staphylococcus aureus in vitro
Aktywność mikrobiologiczna soli kwasu kumarowego i cynamonowego w stosunku
do Escherichia coli O157:H7 i Staphylococcus aureus in vitro
Karol Fijałkowski, Paweł Nawrotek, Danuta Czernomysy-Furowicz ...................................... 173
Usefulness of neutral red uptake method for investigation of the bovine leukocyte viability
and lymphocyte proliferation
Ocena możliwości zastosowania testu wychwytu i gromadzenia czerwieni obojętnej w badaniach
żywotności i proliferacji leukocytów bydlęcych in vitro
FOREWORD
The presented volume XIV (1–2) Advances in Agricultural Sciences contains review articles
and original papers prepared by scientists from different Polish universities. These articles deal
with many problems, especially in the fields of bacteriology, immunology, virology, epidemiology,
epizootiology and human and veterinary medicine. Presented issues are not broadly known
and they are crucial for immunotherapy and prophylaxis of animal and human diseases.
For example, there are emphasized matters such as the identification and differentiation
of zoonotic Escherichia coli strains isolated from healthy sheep by molecular methods, the effect
of penicillin and amoxicillin/clavulanic acid on the induction of L form of staphylococci isolated
from mastitic milk of cows, as well as the RNA regulation of bacterial virulence – selected data.
The original and review articles were prepared in English with Polish abstracts.
Hope the presented articles trigger the interests of the readers in the raised topics.
Antoni J. Furowicz
Jolanta Karakulska
Paweł Nawrotek
I. REVIEW ARTICLES
ADVANCES IN AGRICULTURAL SCIENCES
Adv. Agric. Sci. 2011, XIV (1–2), 11–18
Joanna Działo1, Jarosław Poleszczuk2, Wiesław Deptuła1
IMPORTANT YET LITTLE KNOWN BIOLOGICAL PHENOMENA
1
Department of Microbiology and Immunology, University of Szczecin,
Felczaka 3c, 71-412 Szczecin, Poland, phone: 91 444 1605, fax. 91 444 1606,
email: [email protected]
2
Provincial Hospital in Kalisz, Department of Neurosurgery,
Poznańska 79, 62-800 Kalisz, Poland
Abstract. This paper presents the results of the studies from recent years. The topic of the impact of age
and stress onto the immune system has been discussed. Promising results have been presented
as regards the application of protein kinase inhibitors as new generation anti-inflammatory drugs. Changes
to mitochondrial genome were discussed, which impact on tumour generation. Furthermore, positive
impact of bee venom components onto the body was described.
Key words: aging, immunological system, stress hormones, protein kinases, mitochondrial genome,
bee venom
In the 21st century, many studies in the biological science move people around the world,
as they have high cognitive potential. However, there are also works, which really electrify
the public, as they refer to basic elements conditioning human life.
IMMUNOLOGICAL SYSTEM – CAN ONE STOP THE TIME?
Human population worldwide is aging. In 2000, people above 60 constituted 10% of global
population. According to forecasts, their share in 2025 will amount to 15%, while in 2050 – as
much as 22%. Hence, recently, immunological studies in medicine and geriatrics have been
dynamically developing (Dorshkind et al. 2009). It has been evidenced that together with age,
human immunological system ages, which results in greater susceptibility to: contagious, autoimmunological and allergic diseases, including lower capacity of the body response to e.g.
vaccination (Boraschi et al. 2010). It was evidenced that in elderly people, their immunological
system is characterised with: reduced number of T and B lymphocytes and unfavourable
changes to their functioning, which is manifested with the fact that B lymphocytes in the elderly
produce antibodies with lowered affinity to antigens, while T CD8+ (cytotoxic and suppressor)
lymphocytes reveal lower capacity to respond to new antigens, such as new strands
12
J. Działo, J. Poleszczuk, W. Deptuła
of the influenza virus (Prelog 2006). Researchers search for effective methods not only to stop
the processes of immunological system’s aging, but also try to “rejuvenate" it. A perfect method
to keep the immunological system “young” is to limit the number of calories consumed. Diet
with the number of calories reduced by 30–50% prolongs life in many species, although
the detailed mechanisms of its impact on the immunological systems are yet unknown
(Dorshkind et al. 2009). Exclusively in reference to primates, it was determined that reduction
of calories consumed increases the number of virgin T cells and increases diversity among
subpopulations of T cells (Messaoudi et al. 2006). It must be, however, remembered that
limitation of calories consumed at an elderly age may also lead to increased mortality as
a result of infection, which is testified to by studies on mice (Ritz and Gardner 2006). This way
of rejuvenation of the immunological system still requires many findings, e.g. as regards
the duration of low-calorie diet. Another considered method of impacting on the youth
of the immunological system involves application of hormones and cytokines (Dorshkind et al.
2009). The main objective of this type of treatment is to rejuvenate the disappearing thymus,
as well as many cells of the immunological system (Dorshkind et al. 2009). At present, there
are pre-clinical and clinical trials which use for that purpose e.g.: IL-7, sex hormones, growth
hormone (GH), or keratinocyte growth factor (KGF, also referred to as FGF7), which bring
promising results (Dorshkind et al. 2009). However, treatments with this type of substances still
require many studies in order to determine possible side effects (Dorshkind et al. 2009).
STRESS HORMONES AND THE IMMUNE SYSTEM
Stress defined as a threat to homeostasis, occurring as a result of impact of unfavourable
factors, referred to as stressors (Kyrou and Tsigos 2009). Various types of stress, including
physical and psychological stress, cause the release of neuroendocrine signals from the brain
(Webster-Marketon and Glaser 2008). Two main neuroendocrine pathways activated by stress
have been described, which impact on the immune system. One of them is the HPA axis
(hypothalamic- pituitary- arenal), in the course of which glucocorticoids are released (Aguilera
2010). The other runs in the sympathetic nervous system, resulting in catecholamine release
(epinephrine and norepinephrine) (Kyrou and Tsigos 2009). Furthermore, there is a number
of other neuroendocrine factors released during stress (luteotropin, growth factor, nerve growth
factor – NGF), which also impact on the immune system (Webster-Marketon and Glaser 2008).
HPA axis is the main mechanism via which the brain can control the immune system.
As a result of stimulation with stress factors, corticotrophin-releasing hormone (CRH)
is secreted from the paraventricular nucleus of hypothalamus that stimulates adenohypophysis
to release adrenocorticotropic hormone (ACTH) (Aguilera 2010). In turn, ACTH stimulates
adrenal glands to synthesise and secrete glucocorticoids, which in physiological concentrations
Important yet little known biological phenomena
13
have broad immunostimulating properties, while their elevated level as a result of stress
causes immunosuppressive action (Webster-Marketon and Glaser 2008, Aguilera 2010).
Stress is one of the factors activating sympathetic nervous system, as it was evidenced that its
activation results in release of acetylcholine by pregangiolic sympathetic fibres in the adrenal
medulla, which in turn induces epinephrine secretion to blood (Kyrou and Tsigos 2009).
Norepinephrine is released by nerve terminals in the vicinity of immune system cells, and both
these catecholamins indicate broad immunomodulating effect on the immunological system
(Webster-Marketon and Glaser 2008, Kyrou and Tsigos 2009). Also, the level of luteotropin
secreted by adenohypophysis and many places other than hypophysis, including by immune
system cells, increases as a result of stress, and it is suggested that its immunostimulating
effect is adversative to the effect of corticosteroids (Dorshkind and Horseman 2001). Also the
growth hormone (GH) is produced in greater quantities under the impact of stress. It was
described that the hormone, similarly as luteotropin, is not only secreted by the adenohypophysis,
but may also be secreted by immunological system organs, revealing immunomodulating effect
onto the immunological system, and it is assumed that its effect is adversative to glucocorticosteroids (Dorshkind and Horseman 2001). Moreover, the hormone seems to acts as cytokine,
promoting the progress of cell cycle in the lymphoid line cells, while its effect on tissues often
occurs via insulin-like growth factor 1 (IGF-1), which is induced with growth hormone (WebsterMarketon and Glaser 2008). Also, nerve growth factor (NGF) is a neurotropic hormone capable
of regulating immunological response. Its secretion increases under stress conditions, and it
may have double effect. First, it may function via hypothalamus and thus activate the HPA axis,
and it may also act as autocrine and paracrine factor, regulating growth and activity of immune
system cells (Webster-Marketon and Glaser 2008). It was determined that NGF stimulates
proliferation and differentiation of T and B lymphocytes, and is a survival factor for B memory
lymphocytes (Webster-Marketon and Glaser 2008). Hence, to conclude, it may be stated that
stress has an unfavourable effect onto immunological system, causing e.g. changes to
lymphocyte population, and increases e.g. the number and activity of NK cells, cripples
immunological response with participation of antibodies, and leads to activation of latent viral
infections (Webster-Marketon and Glaser 2008). A lot of attention is also devoted to the impact
of stress and hormones released under its influence on health. One of the major
consequences of stressful life to health is the increased morbidity with tumours, and their
quicker development. In view of such information, results of studies pointing to relaxation
techniques that may prevent and reverse negative effects of stress onto immunological
system, which are deemed ever-present in humans of the 21st century, might prove comforting
(Webster-Marketon and Glaser 2008).
14
J. Działo, J. Poleszczuk, W. Deptuła
PROTEIN KINASE INHIBITORS – NEW ANTI-INFLAMMATORY SUBSTANCES?
Protein kinases are a large group of enzymes with capacity of protein phosphorylation,
which makes them key components of practically all signal pathways, including energy
pathways, occurring in cells. One should know that many diseases are accompanied with
chronic inflammation which, aside significant discomfort to the patient, brings about severe
consequences to the body’s functioning. Symptoms of the inflammation are fought against with
various chemotherapeutic agents, including non-steroidal anti- inflammatory drugs, glucocorticosteroids, cytostatic drugs, immunosuppressive drugs, or specific agents against proinflammatory cytokines, such as tumour necrosis factor (TNF), or interleukin-1 (IL-1) (Gaestel
et al. 2009). Despite the existence of drugs bringing relief in chronic inflammations, there is still
a need to search for new methods of treatment for such diseases. The increasing progress
in learning about molecular mechanisms of inflammatory response made protein kinases
considered as a perfect target of new anti-inflammatory drugs (Gaestel et al. 2009). There are
many indications that low-molecular inhibitors of such kinases will become new generation
anti-inflammatory drugs in the future. This is indicated not only by the vast number of such
molecules, but also the possibility of checking their selectivity on the panel of approx.
70 various kinases and observation of their effect in animals with induced chronic inflammation
(Bain et al. 2007). At present, there are ongoing clinical trials of several such preparations,
which do not reveal unfavourable effect in experimental animals, and bring the desired
medicinal effect (Gaestel et al. 2009). It was evidenced that they inhibit the effect of kinase
p38, which takes part in signal cascades controlling cell response to cytokines and stress,
Janus kinase – transmitting signals via cytokines via the JAK-STAT pathway, or SYK kinase
(spleen tyrosine kinase), which transmits signals from surface receptors of B and T lymphocytes
(Gaestel et al. 2009). Furthermore, experimental studies indicate that low-molecular inhibitors
of other kinases (IRAK- interleukin-1 receptor-associated kinase, MK2- MAPK-activated protein
kinase 2, MK3- MAPK-activated protein kinase 3, TPL2- tumor progresion locus 2, MEKK3MAPK-ERK kinase kinase 3), may bring progress to treatment of inflammations and do not
cause harmful side effects (Gaestel et al. 2009). For example, IRAK kinase has the capacity
of binding to activated interleukin-1 receptor (IL1R), through which it impacts on the IL-1-induced
regulation of nuclear transcription factor NFκB (Gaestel et al. 2009). In turn, MK2 and MK3
kinases, activated by p38 kinase, cooperate with one another in stimulation of TNF biosynthesis,
regulation of tritrestapolin (TTP) function, and stabilisation of p38 kinase (Ronkina et al. 2010).
It was evidenced that MEKK3 kinase has the capacity of regulating the activity of other kinases
in the MAPK cascade, such as MKK3, MKK5/6/7, which control the progress of cell cycle and
cell divisions (Gaestel et al. 2009). Finally, TPL2 kinase is necessary for activation of ERK
Important yet little known biological phenomena
15
kinase that regulates extracellular signals and signals for production of TNF-α and PGE2
(prostaglandin E) by activated macrophytes (Gaestel et al. 2009).
MITOCHONDRIAL GENOME AND CARCINOGENESIS
Mitochondria are organelles present in almost all eucariotic cells the role of which
is to generate energy in the form of highly-energetic ATP compound (adenosine triphosphate)
and where genetic material is present (Higuchi 2007). Mitochondrial dysfunction related
to mutations in mitochondrial DNA (mtDNA) have been described for many diseases, such as:
epilepsy, ataxia, cortical blindness, dystonia, cardiomyopathy (Tuppen et al. 2010). What
is interesting is the fact that mitochondrial dysfunction and mutations of mitochondrial genome
are related to carcinogenesis, and are recorded in various tumours, although their role
in carcinogenesis is still unexplained (Higuchi 2007, Verma and Kumar 2007, Lu et al. 2009).
It was evidenced that the genome of mammal mitochondria is of the size of approx.
16.5 kilobase pairs (kbp) and is formed of double-stranded, round DNA. The genome contains
37 genes, encoding 13 peptides of oxidative phosphorylation apparatus, as well as 22 types
of tRNA and 2 types of rRNA necessary for protein synthesis within such organelle (Verma
and Kumar 2007, Tuppen et al. 2010). Apart from encoding regions in the mitochondrial
genome, there is also D-loop, which contains elements regulating replication and transcription
of mitochondrial genes (Verma and Kumar 2007, Tuppen et al. 2010). In eukaryotic cells, there
is a high number of copies of the genome, reaching several thousand (Verma and Kumar 2007).
Due to the increased contact of mitochondrial genome with reactive oxygen species (ROS),
generated during oxidative phosphorylation in such organelles, and lack of repair
and protective mechanisms for DNA in such organelles, mitochondrial DNA (mtDNA) showed
potentially increased level of mutation (Lu et al. 2009). In a normal situation, all mtDNA in the
cell are identical, in which case we deal with homoplasia, whereas when aside wild mtDNA,
there are also its mutated forms in the cell, we deal with heteroplasia (Verma and Kumar 2007,
Tuppen et al. 2010). Studies regarding correlation between mutation in mtDNA and the carcinogenic process revealed such correlations in very many different types of tumours and referred
to various location in the mitochondrial genome (Higuchi 2007, Verma and Kumar 2007, Lu
et al. 2009, Tuppen et al. 2010). Most changes are described in the non-encoding region
of the mitochondrial genome, namely in D-loop, and this is related to such cancers as e.g.:
breast cancer, bowel cancer, ovarian tumour, tumours of neck and head, liver, lungs, oral
cavity, stomach, thyroid gland, as well as digestive system (Lu et al. 2009). Furthermore,
a large number of mutation was recorded in genes encoding peptides of complex I in the oxidative
phosphorylation chain, which accompanied such cancers as e.g.: leukaemia, bowel cancer,
J. Działo, J. Poleszczuk, W. Deptuła
16
liver cancer and thyroid tumour (Lu et al. 2009). Equally many changes were described
for encoding genes tRNA and rRNA, which were related to e.g.: prostate cancer, liver cancer,
or thyroid tumour (Lu et al. 2009). To a lesser scale, changes were described to genes
of complex III (i.a. in the case of head and neck cancer, oral cavity cancer and thyroid
tumours), and in the genes of complex IV (including leukaemia, prostate and thyroid tumours),
as well as complex V (in the case of breast cancer, head and neck cancer, thyroid tumour)
of oxidative phosphorylation chain (Lu et al. 2009). It is assumed that the results of the studies
on the relation between changes to the mitochondrial genome and carcinogenic process
and the development of tumours will create further basis for discoveries in this area, which may
give the grounds for treatment of such severe and lethal diseases.
BEE VENOM
It is generally known that bee stinging may cause an anaphylactic reaction which, in the event
of lack of proper therapeutic procedure, may even end with death of the person stung. Bee
venom contains many chemical substances impacting on organisms entering in contact with
it to various extent. Fundamental groups of components of bee venom include peptides (e.g.:
melittin, apamin, adolapin MCD-peptyde, secapin), enzymes (phospholipase A2, hialuronidase,
acid phoshatase, glucosidase), active biological amines (e.g: histamine, procamin, serotonin,
norepinephrine, epinephrine) and non-protein substances (lipids, hydrocarbons and free
aminoacids) (Son et al. 2007). However, adverse properties of bee venom are principally
related to its three enzymes (phospholipase A2, hialuronidase, acid phosphatase) and peptide
– melittin (Pałgan and Bartuzi 2009).
Apart from adverse effects, bee venom may also have a healing effect onto human body,
which has long been used in eastern medicine for treatment of e.g.: arthritis and rheumatism,
backache, and even cancer and dermatological diseases, hence treatment with these products
is referred to as “bee venom therapy” (Son et al. 2007, Pałgan and Bartuzi 2009, Chen
and Lariviere 2010). It was evidenced that bee venom may modify functioning of immunological
system and increase cortisol production (anti-inflammatory substance) in the body and thus
alleviate the symptoms of e.g. arthritis or rheumatism (Pałgan and Bartuzi 2009). Also, other
mechanisms of anti-inflammatory effect of bee venom were described, which are conditioned
by its various components. It was evidenced that melittin, by reduction of the expression
of cyclooxygenase 2 (COX-2) and phospholipase A2, as well as concentration in ill tissues
of tumour necrosis factor (TNF-α), interleukin 1 and 6 (IL-1, IL-6) and reactive oxygen species
(ROS), including nitrogen oxide (NO), has a healing effect in arthritis (Son et al. 2007, Pałgan
and Bartuzi 2009). It was also discovered that melittin inhibits binding to DNA of transcription
Important yet little known biological phenomena
17
factor NFκB, which regulates expression of pro-inflammatory genes (Son et al. 2007). It was
recorded that adolapin peptide also has anti-inflammatory effect, as it blocks synthesis
of prostaglandins, while another peptide, apamin, inhibits histamine release from mast cell
of lung tissue, thus preventing allergic respiratory infection (Son et al. 2007). It was also
evidenced that MCD-peptide has an anti-allergenic effect by inhibition of histamine release
from mast cells, and binding to mast cell receptors, inhibiting the capacity of immunoglobulin E
binding to such receptors (Son et al. 2007). Bee venom also has the capacity of alleviating pain
caused with overheating, as well as coeliac pains, and pains caused by inflammation, hence
it is principally used to alleviate pains accompanying chronic diseases. Bioamins present
in the bee venom, such as histamine, procamine, serotonin, and norepinephrine, facilitate
nervous transmission and healing of nerve cells in various diseases of the system (Chen
and Lariviere 2010). Administration of bee venom also alleviates rheumatic pains and pains
related to arthritis (Son et al. 2007, Pałgan and Bartuzi 2009). Furthermore, it was determined
that bee venom may reveal anti-cancer properties owing to the capacity of killing cancer cells
(Son et al. 2007). It is assumed that this cytotoxic effect, as regards cancer cells, is due
to activation of phospholipase A2 by melittin (Son et al. 2007). It was determined that the latter
may also act on cells of kidney cancer, lung, liver, prostate bladder and breast cancer, as well
as leukemic cells (Son et al. 2007, Pałgan and Bartuzi 2009). At present, pharmaceutical
companies are carrying out intensive studies on the use of bee venom in anti-cancer treatment.
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expression and phosphorylation of tristetraprolin. Biochem. Pharmacol. 80, 1915–1920.
Son D.J., Lee J.W., Lee Y.H., Song H.S., Lee C.K., Hong J.T. 2007. Therapeutic application of anti-arthritis,
pain-releasing, and anti-cancer effects of bee venom and its constituent compounds; Pharmacol.
Ther. 115, 246–270.
Tuppen H.A.I., Blakely E.I., Turnbull D.M., Taylor R.W. 2010. Mitochondrial DNA mutations and human
disease. Biochim. Biophys. Acta, 1797, 113–128.
Verma M., Kumar D. 2007. Application of mitochondrial genome information In cancer epidemiology.
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Webster-Marketon J.I., Glaser R. 2008. Stress hormones and immune function; Cell. Immunol. 252, 16–26.
CIEKAWE I WAŻNE ZJAWISKA BIOLOGICZNE
Streszczenie. W niniejszej pracy przedstawiono wyniki badań z ostatnich lat. Poruszono temat wpływu
wieku oraz stresu na układ odpornościowy. Przedstawiono obiecujące wyniki badań zastosowania
inhibitorów kinaz białkowych jako leków przeciwzapalnych nowej generacji. Przybliżono zmiany w genomie mitochondrialnym, mające wpływ na powstawanie nowotworów. Nadto, opisano pozytywny wpływ
składników jadu pszczelego na organizm.
Słowa kluczowe: starzenie, układ odpornościowy, hormony stresu, kinazy białkowe, genom mitochondrialny, jad pszczeli
ADVANCES IN AGRICULTURAL SCIENCES
Adv. Agric. Sci. 2011, XIV (1–2), 19–34
Antoni Jakubczak1, Milena A. Stachelska1, Renata Świsłocka1,2,
Monika Kalinowska1,2, Włodzimierz Lewandowski1,2
COMBINATION OF BACTERIOCINS WITH VARIOUS FACTORS
AS A WAY OF NATURAL FOOD PRESERVATION
1
Food Technology and Nutrition Institute, The State College of Computer Science and Business
Administration in Łomża, Akademicka 14, 18-400 Łomża, Poland
2
Division of Chemistry, Bialystok University of Technology, Zamenhofa 29, 15-435 Białystok, Poland
Abstract. Lactic acid bacteria (LAB) and their metabolites having antimicrobial activity may be used as
natural food preservatives preventing the multiplication of spoilage as well as pathogenic microorganisms
thus prolonging the shelf-life of food products. Bacteriocins are peptides or proteins which are ribosomallysynthesized and may indicate quite broad spectrum of inhibition. There are some bacteriocins which might
be applied in food production and constitute a natural preservative. They enjoy a great popularity and are
an alternative for chemical preservatives and intensity of applied heat. Such methods enable to maintain
organoleptic and nutritional properties. Bacteriocins are bio-preservatives which may fulfill the expectations
of demanding customers in terms of keeping the food product safety. There are different bacteriocins
which might be used as bio-preservatives. They include nicsin, pediocin PA-1/AcH, lacticin 3147,
enterocin AS-48 and variacin. Some bacteriocins possess additive or synergistic effects when they are
applied together with chemical preservatives, natural phenolic compounds and certain other antimicrobial
proteins. When food is treated with bacteriocins and some physical factors including high pressure
or pulsed electric fields there is a much better chance to completely destroy not only vegetative forms
of spoilage and pathogenic bacteria but also bacterial endospores. The activity of bacteriocins is often
dependent on different environmental factors including pH, temperature and food composition. Food
products are regarded to be sophisticated ecosystems in which interactions between microorganisms
might have a huge impact on the microbial balance and ability of beneficial and harmful microorganisms to
growth and proliferate. The current discoveries in molecular microbial ecology enable to be more familiar
with the effects of bacteriocins in food ecosystems, and the investigation of bacterial genomes might help
to discover new sources of bacteriocins.
Key words: bacteriocins, biopreservation, lactic acid bacteria
INTRODUCTION
Currently the preservation of food products seems to be a debated matter both in developing
and developed countries. The food industry has got a few main aims including the reduction
of economic losses, the decrease in food processing costs and the avoidance of passing
of foodborne pathogens during the whole production process. Simultaneously, it focuses its
20
A. Jakubczak, M.A. Stachelska, R. Świsłocka, M. Kalinowska, W. Lewandowski
attention on fulfilling the growing expectations of consumers and makes all the attempts to produce
safe food products which are ready to eat, possess a fresh taste are nutritious and rich with
vitamins as well as minimally-processed. The production of safe foods require to take into
considerations the following problems: the appearance of new foodborne pathogens not known
previously, the capability of bacteria to adjust into new environmental conditions as well as the
application of preservation and packaging (Ross et al. 2002).
It is commonly known that the lactic acid bacteria (LAB) are able to secret a variety
of antimicrobial metabolites which include organic acids, diacetyl, acetoin, hydrogen peroxide,
reuterin, reutericyclin, antifungal peptides as well as bacteriocins (Holzapfel et al. 1995, El-Ziney
et al. 2000, Holtzel et al. 2000, Magnusson and Schnürer 2001). A very important group of such
metabolites consists of bacteriocins which are known as ribosomally synthesized peptides
or proteins having antimicrobial features (Jack et al. 1995). LAB are believed to release
different bacteriocins. Such bacteriocins have been classified by Klaenhammer (1993). Their
structure, biosynthesis, genetics and food application have been discovered lately (Cleveland
et al. 2001, O'Sullivan et al. 2002, Chen and Hoover 2003, Cotter et al. 2005, Fimland et al. 2005,
Deegan et al. 2006, Drider et al. 2006). The bacteriocins released by LAB possess certain
significant features which make them to be applied in food bio-preservation. Such features
include their safety, they do not show the activity and toxicity towards eukaryotic cells, they are
not destroyed by digestive proteases, they do not have the destructive impact on the gut
microbiota, are resistant to pH and heat, they possess quite a wide antimicrobial spectrum
against many food-borne pathogens and spoilage bacteria, they influence on the bacterial
cytoplasmic membrane, their genetic determinants are plasmid-encoded which enables
genetic manipulation (Gálvez et al. 2007).
There were many researches carried out to estimate the benefits coming from the usage
of bacteriocins in food preservation (Thomas et al. 2000). They are able to prolong shelf life,
guarantee extra protection during temperature abuse conditions, reduce a risk for passage
of foodborne pathogens during the production process, minimize the economic losses related
to food spoilage, decrease the addition of chemical preservatives, enable less intense heat
treatments which makes positive impact on preservation of food nutrients and vitamins and
organoleptic features of food products, enable to produce foods containing less acidic, less salt
content and more water content, fulfill the expectations of more and more demanding
consumers (Robertson et al. 2004). The current paper will try to characterize various aspects
associated with bio-preservation by bacteriocins with relation to food systems, hurdle technology,
Combination of bacteriocins…
21
and the influence of newest advances in molecular biology as well as the analysis of bacterial
genomem.
APPLICATION OF BACTERIOCINES IN FOOD INDUSTRY
There two ways of bio-preservation of food products with bacteriocins. They can be added
in the form of preparations, or food may be inoculated with bacteriocins releasing LAB in
favourable environmental conditions (Schillinger et al. 1996, Stiles 1996). At first case, bacteriocins
are released by cultivation of LAB strains in a special fermentor, then they are recovered
and purified and might be added by in a form of bacteriocin concentrates approved from
the legislative point of view. Nowadays, only nisin is accepted to be applied in the food industry
(E234). At the second case, it is very important to choose the suitable bacteriocinogenic
cultures for food application (Ross et al. 2000, Työppönen et al. 2003, Peláez and Requena
2005, Foulquié Moreno et al. 2006, Leroy et al. 2006). It means a very careful selection
of strains which will easily adjust to the particular food environment to which they will be added
and multiply in order to release a high enough number of bacteriocins to destroy possibile foodborn pathogens and spoilage bacteria in food (Rodríguez et al. 2003, Zhou et al. 2006).
Bacteriocinogenic strains may be applied either directly as starter cultures or as protective
cultures (Todorov et al. 1999). What is more bacteriocin-released strains may function as an
adjunct culture and may be addend in the combination with starter culture. For example,
inoculation of milk with an enterocin AS-48 producer enterococcal strain having a role
of adjunct culture in combination with a commercial starter culture in cheese production had
no negative influence on multiplication of the starter and the physicochemical properties
of the produced cheese. A high enough number of bacteriocins was release in the cheese to
prevent Bacillus cereus from growth (Muñoz et al. 2004).
There are also some bacteriocinogenic protective strains which are able to inhibit and even
destroy spoilage and food-born pathogens during the shelf life of non-fermented food products.
Such strains can even grow and release bacteriocins during refrigeration storage of the food.
The strains should not influence on phisicochemical and organoleptic properties of the food
and at the same time should protect food products from the growth of unwanted microflora
(Holzapfel et al. 1995). There are some important factors which may have a positive influence
on the production of bacteriocins. Much higher effectiveness of bacteriocins towards
the destruction of unwanted microflora in food can be observed in case of production the m
in culture media in comparison to food systems (Schillinger et al. 1996). Moreover, the efficacy
of bacteriocins in foods will be also highly dependent on a number of food-related factors which
22
A. Jakubczak, M.A. Stachelska, R. Świsłocka, M. Kalinowska, W. Lewandowski
mainly involve interaction with food components, precipitation, inactivation, or uneven distribution of bacteriocin molecules in the food matrix. The proof for such a statement is the fact
that application of nisin in meat products meet diferent limitations such as its interaction with
phospholipids, emulsifiers and some other food constituents (Henning et al. 1986, Jung et al.
1992, Aasen et al. 2003), their poor solubility at pH above 6.0, and inactivation by formation
of a nisinglutathione adduct (Rose et al. 2003).
There is a long list of factors which influence negatively on the antimicrobial activity
of bacteriocins. They include: food-related factors such as food processing conditions, food
storage temperature, food pH, bacteriocin unstability in pH changes, inactivation by food enzymes,
interaction with food additives/ingredients, bacteriocin adsorption to food components, low
solubility and uneven distribution in the food matrix, limited stability of bacteriocin during food
shelf life as well as the food microbiota factors including microbial load, microbial diversity,
bacteriocin sensitivity, microbial interactions in the food system, bacteriocin sensitivity (Gramtype, genus, species, strains), physiological stage (growing, resting, starving or viable but nonculturable cells, stressed or sub-lethally injured cells, endospores), protection by physicochemical barriers (microcolonies, biofilms, slime), development of resistance/adaptation lower
in cooked meats due to the loss of free sulphydryl groupsduring cooking as a result of the
reaction of glutathione with proteins (Stergiou et al. 2006).
Food products are considered to be complex ecosystems for a variety of different microbial
strains. Foods appear in a form of sterile to raw and fermented ones. In the production
of commercially sterile foods, there is a high risk of post-process contamination because there
is an absence of competitors for spoilage mikroflora. In such circumstances, the effectiveness
of bacteriocin destructive activity will be dependent on the microbial load of the contaminantion
mikroflora, which means that a much higher bacteriocin concentration will be needed to destroy
a high number of spolilage and pathogenic mikroflora. In case of raw foods, there is a possiblity
that the autochthonous microbiota can counteract development of potential pathogens.
On the other hand, a complex food microbiota might lead to a decrease in the activity
of bacteriocins because resistant bacteria (such as Gram-negatives) capable of producing
the inactivating enzymes (such as proteases) are present. In case of processed foods, bacteria
can appear in different physiological stages, which have a huge impact on bacteriocin sensitivity.
Cells which are not able to grow easily, could show a bigger resistance to bacteriocins,
and a rate of their destruction is relatively low (Kumar and Anand 1998). Different variations
in the sensitivities of strains can be observed. Such a development of strain resistance constitutes
a big worry for application of bacteriocins. It was found that some strains of L. monocytogenes
show the same sensitivity towards antilisterial bacteriocins (Martínez et al. 2005).
Combination of bacteriocins…
23
What is more, it can be said that the resistance to one bacteriocin can also cause protection
against other bacteriocins, and the observed cross-resistance between pediocin-like bacteriocins
was attributed to a general mechanism of resistance. It can be concluded that there is a strong
demand for discovering new bacteriocins which do not show cross resistance with existing
ones. It is very important to find such bacteriocinogenic cultures which produce bacteriocins
with a wide spectrum of antimicrobial activity towards microflora contaminating food products.
Such bacteriocinogenic strains should within a more or less complex microbial population
in the food ecosystem and their bacteriocin production will be highly dependent on factors such
as microbial load and diversity, the microbial interactions in the food during shelflife including
the competitiveness over nutrients as well as physiological state of the bacteria (Holzapfel
et al. 1995, Rodríguez et al. 2003, Devlieghere et al. 2004). Such factors have a huge impact
on the growth of bacteriocinogenic strains which must dominate the food environment through
exploiting nutrients and production of metabolites as well as alteration of the food microbial
balance due to their ability to release bacteriocins.
There are different factors which may influence the bacteriocin production. The addition
of sodium chloride and pepper reduced production by 16-fold. The temperature and pH had
a huge impact on the enterocin production. The optimal temperature was between 25 and 35ºC,
and at pH between 6.0 to 7.5 (Aymerich et al. 2000). Bavaricin A production was deteriorated
when the concentration of NaCl was 3% or higher, while cells were still multiplying (Larsen
et al. 1993). In case of Lactobacillus sake CTC 494 its multiplication and sakacin production
in meat sausages was significantly decreased by salting and curing with nitrite and manganese
limitation (Leroy and De Vuyst 1999, 2005). The multiplication of Lactobacillus curvatus
LTH 1174 and its curvacin A production were negatively influence by nitrite (at a very low
concentration of 10 ppm) and sodium chloride (Verluyten et al. 2003, 2004a).
APPLICATION OF BACTERIOCINS IN COMBINATION WITH OTHER FACTORS
Bacteriocins and hurdle technology
The hurdle technology is definied as the combination of bacteriocin activity with other factors
contributing to the decrease in a number of undesired bacteria in food products. There were
some researches carried out on food which was treated with different antimicrobial factors.
The results of such investigations showed a significant reduction of food microflora after
treatment with salt, reduced pH, reduced water activity, heat treatment and packaging (Leistner
1978, Leistner and Gorris 1995, Leistner 2000). The survival of bacteria after treating them with
a single antimicrobial factor or factors staying in combination with themselves depends
24
A. Jakubczak, M.A. Stachelska, R. Świsłocka, M. Kalinowska, W. Lewandowski
on the intensity of treatment. Some fraction of the population may be given a lethal dose
of the antimicrobial factor causing the cell death. The rest of bacteria may still remain alive
thanks to the reasons such as being given a sub-lethal dose, having a big resistance due
to the physiological state, and some cells show a natural resistance towards some antimicrobial
factors. There are some possibilities for bacteria which are sub-lethally injured or are characterised
by a relatively high resistance towards different antimicrobial factors that they might create
mechanisms of resistance or adaptation that might lead to them being absolutely resistant
towards these antimicrobial factors.
What is more, treatment of bacteria with a combination of antimicrobial factors may
contribute to their faster inactivation achieved within a shorter period of time depending on the
factor intensity. More then 60 potential antimicrobial factors called hurdles have been recognised
as contributing to the improvement of food stability and quality (Leistner 1999). The usage
of bacteriocins constituting part of advance hurdle technology has gained a lot of popularity
(Chen and Hoover 2003, Ross et al. 2003, Deegan et al. 2006). They might be applied with
other chosen antimicrobial hurdles to eliminate unwanted microflora from food. The choice
of hurdles will be dependent on the kind of food and its microbial composition. Such factor
combination has to be thought very consciously because various hurdles possess different
impact on the strains. It means that some aciduric bacteria may survive acidification
and endospore formers might survive heat treatment. Moreover, the inactivation of some strains
in the population might create the favourable conditions because of the lack of competition.
Combination of bacteriocins, natural antimicrobials and chemical preservatives
The results of some investigations proved that NaCl improved the activity of bacteriocins
like nisin, leucocin F10 and enterocin AS-48 (Harris et al. 1991, Thomas and Wimpenny 1996,
Mazzotta et al. 1997, Parente et al. 1998, Ananou et al. 2004). However, some researches
showed that NaCl diminished the antilisterial activity of acidocin CH5 (at 1–2%, Chumchalová
et al. 1998), lactocin 705 (at 5–7%, Vignolo et al. 1998), leucocins 4010 (at 2.5% NaCl,
Hornbæk et al. 2004), pediocin (at 6.5% NaCl, Jydegaard et al. 2000), curvacin (Verluyten
et al. 2002) and Carnobacterium piscicola A9b bacteriocin (at 2–4% NaCl, Himmelbloom et al.
2001). The protective impact of sodium chloride might result from a kind of interference with
ionic interactions between bacteriocin molecules and charged groups egaged in bacteriocin
binding to target cells (Bhunia et al. 1991). Sodium chloride can cause conformational changes
of bacteriocins (Lee et al. 1993) and alterations in the cell envelope of the target organisms
(Jydegaard et al. 2000).
Combination of bacteriocins…
25
The supplementation of food with bacteriocins enables to decrease an amount of nitrite added
to food. It was found that the application of nisin and nitrite caused a delay in botulinal toxin
formation in meat processing (Rayman et al. 1981, 1983, Taylor et al. 1985). The combination
of nitrite with nisin caused a higher the anti-listeria activity of bacteriocinogenic lactobacilli
in meat (Hugas et al. 1996) and increased an effectiveness of enterocin EJ97 against
L. monocytogenes, Bacillus coagulans and Bacillus macroides (García et al. 2003, 2004a, b)
and enterocin AS-48 against B. cereus (Abriouel et al. 2002). It was found that acidification
increases the antibacterial activity of both organic acids and bacteriocins (Jack et al. 1995,
Stiles 1996). Bacteriocins are better dissolved at low pH what enable diffusion of bacteriocin
molecules. The sensitivity of L. monocytogenes to nisin (400 IU/ml) increased significantly
when was used in combination with lactate. Nisin showed also more effective activity when
was applied together with sodium lactate (Scannell et al. 1997, Nykänen et al. 2000, Long and
Phillips 2003, Ukuku and Fett 2004). A complex of nisin and sorbate caused also a higher
activity against Listeria (Avery and Buncic 1997), and B. licheniformis (Mansour et al. 1998).
During the production of Ricotta cheese, the combination of nisin with acetic acid
and sorbate was used to eliminate the risk of L. monocytogenes growth for quite a long period
of time (70 days) at 6–8ºC (Davies et al. 1997). Lacticin 3147 effectiveness was enhanced
in combination with sodium lactate and sodium citrate (Scannell et al. 2000a, b). Pediocin AcH
effectiveness was also improved thanks to addition of sodium diacetate and sodium lactate
(Uhart et al. 2004). Lactic acid, sodium lactate and peracetic acid supported AS-48 activity
in the elimination of L. monocytogenes in sprouts (Cobo Molinos et al. 2005). The combination
of nisin and pediocin was less effective with chemical substances to destroy L. monocytogenes
then the combination of nisin with phytic acid (Bari et al. 2005).
A combination of nisin and sodium polyphosphate was highly effective against L. monocytogenes (Buncic et al. 1995). There were also some researchers carried out to check an
antimicrobial activity of ethanol which was used with nisin and effectively decreased the survival
of L. monocytogenes (Brewer et al. 2002). A high level of antimicrobial activity was observed in
case of L. monocytogenes, B. cereus (cells and spores), L. plantarum and Staphylococcus
aureus, but not against Gram-negative bacteria when they were treated with sucrose fatty acid
esters, sucrose palmitate, sucrose stearate and nisin (Thomas et al. 1998). Reuterin was found
to have a highly synergistic effect on L. monocytogenes and a poor impact on S. aureus when
it was used with nisin (100 IU/ml), but the antimicrobial effect of reuterin towards Gramnegative pathogens was not increased (Arqués et al. 2004).
Another very promising alternative is the application of essential oils and their active components
as well as the phenolic compounds constituting the natural preservatives (Burt 2004).
26
A. Jakubczak, M.A. Stachelska, R. Świsłocka, M. Kalinowska, W. Lewandowski
The combination of bacteriocins with the phenolic compounds make it possible to lower their
doses and thus decrease their influence on the food flavour and taste. Nisin had a better activity
in combination with carvacrol, eugenol and thymol against B. cereus and L. monocytogenes
(Pol and Smid 1999, Periago et al. 2001, Yamazaki et al. 2004). The application of nisin with
carvacrol, eugenol and thymol caused a better synergistic activity against B. subtilis
and Listeria innocua, while nisin and cinnamic acid indicated a highly sufficient activity against
L. innocua, but only additive against B. subtilis (Olasupo et al. 2004). Carvacrol (0.5 mM) was
applied to improve the effectiveness of nisin in combination with a pulsed electric field treatment
(PEF) towards vegetative forms of B. cereus in milk (Pol et al. 2001a,b). The combination
of nisin and cinnamon makes death of Salmonella typhimurium and Escherichia coli O157:H7
in apple juice possible (Yuste and Fung 2004). Nisin had also a positive antimicrobial influence
on L. monocytogenes and B. subtilis when it was used with thymol (Ettayebi et al. 2000).
Enterocin AS-48 against S. aureus in vegetable sauces was successfully used with the phenolic
compounds carvacrol, geraniol, eugenol, terpineol, caffeic acid, p-coumaric acid, citral
and hydrocinnamic acid (Grande et al. 2007). Different combinations of bacteriocins have also
been examined to check their effectiveness towards different unwanted bacteria. The simultaneous
application of nisin with pediocin AcH (Hanlin et al. 1993) or with leucocin F10 (Parente et al.
1998) as well as lactacin B or lactacin F with nisin or pediocin AcH, and lactacin 481/pediocin
AcH (Mulet-Powell et al. 1998) gives a much better antibacterial effectiveness than each
bacteriocin separately. The combination of a few bacteriocins might be very helpful not only to
decrease the bacteriocin doses, but also to prevent the regrowth of bacteriocin-resistant cells.
COMBINATION OF BACTERIOCINS AND HEAT TREATMENTS
It is highly recommended to use bacteriocins in order to decrease the intensity of heat
treatments in foods and simultaneously achieve an antimicrobial effect. Nisin and heat possess
a synergistical activity towards L. plantarum and L. monocytogenes (Mahadeo and Tatini 1994,
Ueckert et al. 1998). It was found that the heat resistance of L. monocytogenes in milk was
significantly decreased (Maisnier-Patin et al. 1995). The results of investigation showed that
nisin-resistant L. monocytogenes when was inoculated in the presence of nisin was significantly
more sensitive to heat at 55ºC than wild-type cells (Modi et al. 2000). The antimicrobial activity
of enterocin AS-48 was much higher on S. aureus cells which were sub-lethally damaged
by heat treatment (Ananou et al. 2004). Bacteriocins are considered to deliver an additional
protection during shelf-life and prevent from proliferation of endospores with simultaneous
application of heat treatments. The intensity of heat treatments towards bacterial endospores
Combination of bacteriocins…
27
might be reduced when it is in combination with nisin and with enterocin AS-48 (Beard et al.
1999, Wandling et al. 1999, Grande et al. 2006). Sub-lethal heat was found to make some
Gram-negative bacteria more sensitive to certain bacteriocins such as nisin or pediocin AcH
(Kalchayanand et al. 1992, Boziaris et al. 1998), enterocin AS-48 (Abriouel et al. 1998, Ananou
et al. 2005), and jenseniin G (Bakes et al. 2004).
COMBINATION OF BACTERIOCINS AND MODIFIED ATMOSPHERE PACKAGING
Modified atmosphere packaging (MAP) is often applied in the food industry to extend
the shelf life of food products. MAP can be defined as “the enclosure of food products in gasbarrier materials, in which the gaseous environment has been changed” (Young et al. 1988).
Prolongation of the shelf life of food by MAP is strongly associated with the prevention
of intrinsic food changes and reduction of spoilage. In MAP, the dissolved CO2 is able
to control the growth of microorganisms (Devlieghere et al. 1998). Gram-negative bacteria are
usually said to be more sensitive to CO2, and lactic acid bacteria show more resistance (Farber
1991, Church 1994). Gram-negative bacteria are known to be very resistant to bacteriocins, so
it is obvious that MAP and bacteriocins should be used to eliminate their growth. It was proved
that the multiplication of L. monocytogenes was totally stopped on pork immersed in 10 IU/ml
nisin and packed in 80% CO2/20% air during 30 days of storage at 4ºC. Activity of nisin
towards L. monocytogenes was much higher in cold smoked salmon when it was packaged
under vacuum as well as under a 100% CO2 atmosphere (Nilsson et al. 1997, Szabo
and Cahill 1999). Seven L. monocytogenes isolates were inhibited by nisin under a 100% CO2
atmosphere, but not under 100% N2, or 40% CO2/60% N2 (Szabo and Cahill 1998).
COMBINATION OF BACTERIOCINS AND PULSED ELECTRIC FIELDS
Pulsed electric field (PEF) technology enjoys a big popularity nowadays. It is known as
a non-thermal process where the destruction of microflora is made by application of highvoltage pulses between a set of electrodes (Vega-Mercado et al. 1997). The effects of PEF are
a kind of bacterial electroporation. A high intensity level of such treatment is responsible
for severe damage to the bacterial cell membrane. Such technology might only be used to
sterilize the pumpable food products. Its higher effectiveness may be achieved in combination
with bacteriocins. Bacteriocins have an influence on the bacterial cytoplasmic membrane,
and the combination of bacteriocins and PEF is used to increase antimicrobial activity. PEF
is capable of extending the antimicrobial spectrum of bacteriocins because PEF damages
the bacterial outer membrane and enables bacteriocins to reach the bacterial cytoplasmic
28
A. Jakubczak, M.A. Stachelska, R. Świsłocka, M. Kalinowska, W. Lewandowski
membrane target. The effectiveness of combination of PEF and bactericiocins is dependent
on different factors associated with the PEF treatment. They include field strength, number
of pulses, wave form, pulse duration, the food microbial load, composition and physiological
stage and the added bacteriocin (Wouters et al. 2001, Bendicho et al. 2002, Heinz et al. 2002).
Such factors have an impact on the numbers and types of bacteria which are able to survive
such treatments.
There were some investigations carried out on L. innocua. It was indicated that the combination
of bacteriocins and PEF treatments towards L. innocua present in liquid whole egg was very
effective and caused the inactivation of the microorganism (Calderón-Miranda et al. 1999a).
A synergistic activity was seen as the electric field intensity, number of pulses and nisin
concentration were increased both in liquid whole egg and in skim milk (Calderón-Miranda et al.
1999a, b). L. innocua which underwent the treatment of PEF-nisin in skimmed milk showed
an increase in the cell wall roughness, cytoplasmic clumping, leakage of cellular material,
and rupture of the cell walls and cell membranes (Calderón-Miranda et al. 1999c).
CONCLUSIONS
It was found that a huge number of bacteriocins releasing by LAB has got a great potential
which needs to by applied in the food industry for bio-preservation. Bacteriocins constitute
a variable group of proteinsand peptides possessing antimicrobial activity towards the spoilage
and pathogenic microflora contaminating food and posing a huge risk for food safety. They
show differentiated behaviour towards different bacteria and under different environmental
conditions. It was also found that the effectiveness of antimicrobial activity of bacteriocins might
be higher when they are applied in combination with other factors. There is a strong demand
for developing the conditions which will be the most helpful for their activity.
ACKNOWLEDGEMENTS
This work was supported by grant no. N N312 427639.
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ZASTOSOWANIE BAKTERIOCYN W POŁĄCZENIU Z INNYMI CZYNNIKAMI
W BIOKONSERWACJI ŻYWNOŚCI
Streszczenie. Bakterie kwasu mlekowego (LAB) i ich metabolity o aktywności przeciwbakteryjnej mogą
być stosowane jako naturalne konserwanty żywności zapobiegające namnażaniu mikroflory saprofitycznej,
jak również mikroorganizmów chorobotwórczych, przedłużając tym samym okres przydatności do spożycia
produktów spożywczych. Bakteriocyny to peptydy lub białka, które są syntetyzowane na rybosomach
i mogą wykazywać znaczną aktywność przeciwdrobnoustrojową. Istnieje kilka bakteriocyn, które mogą
być stosowane w produkcji żywności i stanowią naturalny środek konserwujący. Cieszą się dużą
popularnością i stanowią alternatywę dla chemicznych środków konserwujących i stosowanego ciepła.
Takie metody pozwalają zachować właściwości organoleptyczne oraz odżywcze. Bakteriocyny to biokonserwanty, które mogą spełnić oczekiwania wymagających klientów w zakresie utrzymania
bezpieczeństwa produktów spożywczych. Istnieją różne bakteriocyny, które mogą być stosowane jako
bio-konserwanty. Obejmują one nizynę, pediocynę PA-1/AcH, laktycynę 3147, enterocynę AS-48
i variacynę. Niektóre bakteriocyny posiadają dodatkowe synergiczne oddziaływanie gdy są stosowane
z konserwantami chemicznymi, naturalnymi związkami fenolowymi i niektórymi białkami o właściwościach
przeciwbakteryjnych. Potraktowanie żywności bakteriocynami i niektórymi czynnikami fizycznymi, takimi
jak wysokie ciśnienie lub impulsowe pole elektryczne daje większe szanse na zniszczenie nie tylko form
wegetatywnych bakterii saprofitycznych ale i bakterii chorobotwórczych, a także endospor. Działalność
bakteriocyn zależy od różnych czynników środowiskowych, w tym pH, temperatury i składu żywności.
Produkty spożywcze są uważane za złożone ekosystemy, w których interakcje między mikroorganizmami
mogą mieć ogromny wpływ na równowagę mikrobiologiczną i zdolność mikroorganizmów do namnażania
i wzrostu. Obecne odkrycia w molekularnej ekologii mikroorganizmów pozwalają na zgłębienie wiedzy na
temat wpływu bakteriocyn na ekosystemy żywności, a badania genomów bakteryjnych mogą przyczynić
się do odkrycia nowych źródeł bakteriocyn.
Słowa kluczowe: bakteriocyny, biokonserwacja, bakterie kwasu mlekowego
ADVANCES IN AGRICULTURAL SCIENCES
Adv. Agric. Sci. 2011, XIV (1–2), 35–44
Paulina Niedźwiedzka-Rystwej1, Agata Mękal1, Jarosław Poleszczuk2,
Wiesław Deptuła1
TLR RECEPTORS – SELECTED DATA
1
Departament of Microbiology and Immunology, University of Szczecin,
Felczaka 3c, 71-412 Szczecin, Poland, phone: 91 444 1605, fax. 91 444 1606,
email: [email protected]
2
Provincial Hospital in Kalisz, Department of Neurosurgery,
Poznańska 79, 62-800 Kalisz, Poland
Abstract. This paper presents the latest data regarding TLR receptors (Toll-like receptors) – markers
of natural immunity from PRR receptors (pathogen recognition receptors), the role of which is very
important to the anti-microbe immunity. The latest data indicate that the family of these receptors has
significantly grown owing to research with molecular biology methods, and presently TLR1-15 and TLR21,
22, 23 receptors are recorded. Furthermore, this paper describes the important role of such receptors
in epithelial cells in the digestive system, where they are also expressed, and therefore, they constitute
an important element of local immunity in the intestinal epithelium.
Key words: TLR, epithelial cells
INTRODUCTION
TLR receptors (Toll-like receptors) are markers of natural immunity, which – despite short
“history” in the literature, are quite well described (Beutler and Wagner 2002, Sabroe et al.
2003, Szczepański et al. 2004, Wagner 2004, Deptuła et al. 2006a, 2006b, Tokarz-Deptuła
et al. 2006, Śliwa et al. 2008). These are receptors from the PRR family (pathogen recognition
receptors) that recognise molecular pathogen patterns (PAMP) (Beutler and Wagner 2002,
Sabroe et al. 2003, Szczepański et al. 2004, Wagner 2004, Deptuła et al. 2006a, 2006b,
Tokarz-Deptuła et al. 2006, Śliwa et al. 2008). They are present on many cells, including immunity
system cells, namely lymphocytes, neutrophils, dendritic cells, mastocytes, monocytes, as well
as epithelial cells of the digestive and respiratory systems, endothelium of blood vessels, skin,
adipocytes, cardiomyocytes, fibroblasts and many other cells of organs in mammals (Beutler
and Wagner 2002, Sabroe et al. 2003, Szczepański et al. 2004, Wagner 2004, Deptuła et al.
2006a, 2006b, Tokarz-Deptuła et al. 2006, Śliwa et al. 2008). Owing to such location, they
have an unusual capacity of capturing and binding to microorganisms of bacterial, viral, as well
36
P. Niedźwiedzka-Rystwej, A. Mękal, J. Poleszczuk, W. Deptuła
as parasitic origin (Beutler and Wagner 2002, Sabroe et al. 2003, Szczepański et al. 2004,
Wagner 2004, Deptuła et al. 2006a, 2006b, Tokarz-Deptuła et al. 2006). Moreover, due to their
conservative structure and its location, they perform an important role of super-activators
in immune response of the vertebrates, including mammals, constituting the basis for defence
against microorganisms and parasites (Beutler and Wagner 2002, Sabroe et al. 2003,
Szczepański et al. 2004, Wagner 2004, Roach et al. 2005, Deptuła et al. 2006a, 2006b, Higgs
et al. 2006, Tokarz-Deptuła et al. 2006, Śliwa et al. 2008, Hwang et al. 2010).
TLR RECEPTORS
So far, in mammals, 13 TLR markers had been described, but the present studies (Roach
et al. 2005) indicate that there are more such receptors, as present records additionally
included receptor TLR14 and TLR15, as well as TLR21, 22 and 23 (Hwang et al. 2010).
Receptor TLR14 was described in frogs and fish, despite the fact that its function is not entirely
known (Roach et al. 2005, Hwang et al. 2010), and it was also recorded (Hwang et al. 2010)
that in Paralichthys olivaceus fish it takes part in bacterial infection with Edwardsiella tarda.
In turn, TLR15, which molecularly most differs from all other markers in the TLR family, was
described in chickens (Gallus gallus), including while infecting them with Salmonella enterica
(Higgs et al. 2006). In turn, receptors TLR21, 22 and 23 were described and registered
in selected species of fish, frogs and chickens (Roach et al. 2005).
It is worth noticing that the 13 TLR markets described at the time were grouped into five
sub-families: TLR2 (TLR1, TLR2, TLR6 or TLR1 and 2, as well as TLR2 and 6), TLR3, TLR4,
TLR5 and TLR9 (TLR7, TLR8, TLR9) (Beutler and Wagner 2002, Sabroe et al. 2003,
Szczepański et al. 2004, Wagner 2004, Deptuła et al. 2006a, 2006b, Tokarz-Deptuła et al.
2006, Śliwa et al. 2008). At present (Roach et al. 2005), on the basis of phylogenetic studies,
the described 18 TLR receptors (1–15 and 21–23) in vertebrates, have been grouped into six
sub-families: TLR1, TLR3, TLR4, TLR5, TLR7 and TLR11, whereas the sub-family of TLR1
receptor includes receptor TLR1 in all vertebrates, and TLR2 in mammals, fish and some birds,
as well as TLR6 and TLR10, which in the molecular aspect are very similar to TLR1 (Roach et
al. 2005). This sub-family also includes the newly described TLR14, identified in most detail
in frogs Xenopus and fish Tetraodeon and Fugu, as well as TLR15 described in chickens
(Roach et al. 2005). Another sub-family is formed by the group of TLR3 receptors, which
is very homogenous, and only comprises TLR3 marker in various species of mammals,
including humans and invertebrate animals. The situation is similar in the case of two further subfamilies, namely TLR4 and TLR5 receptors, which include, respectively, all TLR4 and TLR5
TLR receptors – selected data
37
receptors recorded in vertebrates (Roach et al. 2005). The next sub-family is formed by TLR7
receptor, which comprises TLR7, TLR8 and TLR9 receptors, identified in vertebrates, as well
as invertebrates. The last, sixth sub-family, is the sub-family of TLR11 receptor, which was
allocated with the previously recognised receptors TLR11, TLR12, TLR13, described in mammals
(mice, rats), as well as presently described receptor TLR21, which was characterized in fish
Takifugu rubipres, frog Xenopus, as well as in chickens, as well as newly described markers
TLR22 and TLR23 recorded in various species of fish (Roach et al. 2005).
TLR RECEPTORS AND EPITHELIAL CELLS OF THE INTESTINES
Among the difficult questions regarding the expression of TLR receptors, there are studies
regarding epithelial cells of the intestines, which are very important due to their function as
“gates” of some most frequent infections transferred via digestive system. Such studies are
rendered difficult due to the presence in the lamina propia of cells, directly under the intestinal
epithelium, of such elements as stromal cells, lymphocytes T, B, macrophyte and dendritic
cells (Abreu 2010). However, it has been evidenced that on epithelial cells of the smaller
intestine in humans, expression is recorded principally of TLR1, TLR2, TLR3, TLR4, TLR5
and TLR9 (Otte et al. 2004). Furthermore, it has been evidenced that on the surface
of intestinal epithelial cells in human colon, principally TLR2 and TLR4 markers are present,
yet the level of their expression is low (Cario and Podolsky 2000, Abreu et al. 2001, Otte et al.
2004). Moreover, in humans, on the surface of smaller intestine and colon cells, there is TLR3
receptor, while TLR5 is usually only expressed in the colon (Cario and Podolsky 2000).
In humans suffering from enteritis, increased levels of TLR4 has been recorded, or lower
increase in the levels of TLR2, TLR3, TLR5 and TLR9 (Cario and Podolsky 2000, Abreu 2010).
It must be noticed that the presence of TLR markers on the surface of intestinal epithelial cells
is limited in space, as in such cells there is apical surface, located from the side of intestinal
inside diameter, and basolateral surface from the side of lamina propria (Abreu 2010).
The studies have evidenced that for example expression of TLR2 and TLR4 receptors occurs
on the basolateral surface of enterocytes in the intestinal gland in foetus (Fusunyan et al.
2001). In turn, in mice, in the epithelium related to FAE (follicle associated epithelium), TLR2
receptors are located both on the apical, and basolateral surface (Chabot et al. 2006). In the
colon of a healthy human, the expression level of TLR2 and TLR4 is low, while high level
of TLR4 is recorded on apical surface in humans with Crohn disease, but not with ulcerous
colitis (Cario and Podolsky 2000). TLR5 expression in humans only refers to basolateral surface,
where, after the damage of epithelial barrier, as a result of flagellin stimulation, the receptor
38
P. Niedźwiedzka-Rystwej, A. Mękal, J. Poleszczuk, W. Deptuła
may contribute to production of cytokines and chemokines, such as IL-8 and CCL20 (Gewitz
et al. 2001, Rhee et al. 2005). In mice, the presence of TLR5 was evidenced on the apical
surface of epithelial cells of the ileum, where, under the influence of flagellin, chemokine KC
(keratinocyte chemoattractant) is produced, which is a homologue to IL-8 (Bambou et al.
2004). It was evidenced that the expression of TLR receptors on intestinal epithelial cells
is principally regulated by cytokines, which allows for their selective expression (Rehli et al.
2000, Suzuki et al. 2003, Mueller et al. 2006). And so, e.g. INF-γ and TNF induce transcription
of gene TLR4 (Suzuki et al. 2003), while IL-4 and IL-13 cause a decrease to its expression
(Mueller et al. 2006, Lotz et al. 2007). Furthermore, it is assumed that there is a correlation
between the functioning and expression of TLR markers and hormones secreted by
enteroendocrine cells, including somatostatin and cholecystokinin (Bogunovic et al. 2007,
Palazzo et al. 2007). In vitro studies evidenced that as a result of stimulation of enteroendocrine
cells with bacterial LPS, somatostatin is secreted, and on the surface of such cells TLR1, TLR2
and TLR4 appear (Bogunovic et al. 2007). In turn, as a result of flagellin impact on TLR5
and TLR9 receptors of enteroendocrine cells, cholecystokinin is released, which causes
contraction to cholecyst and narrowing of internal diameter of the smaller intestine (Palazzo
et al. 2007). Therefore, it is suggested that the expression of TLR receptors on enterocrine
cells contributes to the development of diarrhoea in response to the entering pathogens, which
may help to eliminate them from the body (Palazzo et al. 2007). It was evidenced that
the activation of TLR receptors related to epithelial cells of the intestines may also be inhibited
by various particles, referred to as negative regulators of TLR receptors, which is unfavourable
to the body (Burns et al. 2000, Zhang and Ghosh 2002, Dubuquoy et al. 2003, Melmed et al.
2003, Wald et al. 2003). Such negative regulators include TOLLIP protein (Toll-interacting
protein), which is an inhibitor to TLR2 and TLR4, which impacts on IRAK kinases
(IL-1R-associated kinases) (Burns et al. 2000, Zhang and Ghosh 2002). It was evidenced that
the protein, in vitro, is produced by intestinal epithelial cells (IEC), particularly as a result
of stimulation with LPS and lipoteichoic acid (Melmed et al. 2003). Intestinal epithelial cells
originating from patients suffering from enteritis did not evidence TOLLIP expression, which
probably contributes to the development of chronic inflammation (Steenholdt et al. 2009).
SIGGIR factor (single immunoglobulin IL-1R-related molecule), also referred to as TIR8, is also
a negative regulator for IL-1R, IL-33R, TLR4 i TLR9 (Wald et al. 2003). It was evidenced that
animals with shortage of the factor are more susceptible to enteritis (Garlanda et al. 2004, Xiao
et al. 2007). This group of factors also includes PPARγ (peroxisome proliferator activated
receptor-γ), which negatively regulates activation of NF-κB (nuclear factor κB) (Dubuquoy et al.
TLR receptors – selected data
39
2003). The expression of PPARγ is induced by intestinal receptor TLR4, owing to which it may
serve as a factor reducing enteritis (Dubuquoy et al. 2003). The studies evidenced that TLR
receptors are necessary for optimal proliferation of intestinal epithelial cells after their damage.
It was evidenced that in mice deprived of TLR4 receptor, as well as in mice revealing lack
of adaptor protein MyD88, necessary for transduction of the signal during activation of TLR
receptors, the intensity of cell proliferation due to damage of the epithelium by hialuronic acid
is significantly weakened (Zheng et al. 2009). It was also evidenced that as a result of damage
to the epithelium, owing to TLR4 marker, expression of cyclooxygenase 2 (COX2) is induced,
and then, the production takes place of prostaglandin E2 (PGE2) by IEC cell, as well as
induction of amphiregulin, which belongs to the family of epidermal growth factors (EGF) and
is a ligand for EGF receptor (EGFR) present on the surface of epithelial stem cells, which
induces their proliferation (Fukata et al. 2006, Fukata et al. 2009). Ligands of the EGFR also
include TGFα, EGF, β-cellulin, heparine-binding EGF (HB-EGF) and epiregulin (Giraud 2000).
Blocking of amphiregulin inhibits the phosphorylation of EGFR, which allows for drawing
conclusion that TLR4 may enhance proliferation of epithelial cells via induction of growth
factors (Fukata et al. 2007). The production of prostaglandin PGE2, and as a result proliferation
of epithelial cells, may also be induced by macrophytes and mesenchymal stromal cells (PSC),
also having TLR receptors (Brown et al. 2007, Fukata et al. 2009, Abreu 2010). It was evidenced
that in mice deprived of MyD88 protein or COX2, there is no expression of factors responsible
for induction of IEC cell proliferation. In turn, administration of synthetic derivative of PGE2
(dimetylo-PGE2) restores epithelial cell proliferation in such animals (Brown et al. 2007).
TLR receptors related to intestinal epithelium, other than regulation of epithelial cell proliferation,
also contribute to the production of anti-microbe peptides and lectins. It was evidenced that
correct recognition of ligands by TLR receptors, which are present at the surface of intestinal
epithelial cells (IEC), may lead to induction of the β-defensin expression – important antimicrobe proteins in humans and animals (Vora et al. 2004, Niedźwiedzka and Deptuła 2008).
The main source of anti-microbe peptides, including α-defensins, angiogenin 4 and REG3γ
protein (regenerating islet-derived protein 3γ), is formed by Paneth cells, present in intestinal
glands (Selsted and Ouellette 2005, Cash et al. 2006). The studies evidenced that the cells
may secrete α-defensins under the influence of various PAMP, such as LPS, lipoteichoic acid
or lipid A (Ayabe et al. 2000), while the lowered level of α-defensin expression was observed
in the small intestine in mice deprived of TLR9 (Lee et al. 2006). Furthermore, it was evidenced
that in mice, induced expression of REG3γ in Paneth cells may also occur as a result
of colonisation of the intestines by comensal bacteria, such as Bacteroides thetaiotaomicron
P. Niedźwiedzka-Rystwej, A. Mękal, J. Poleszczuk, W. Deptuła
40
(Cash et al. 2006). And so, in mice with diagnosed lack of adaptor protein MyD88, REG3γ was
not detectable, therefore, it is suggested that TLR receptors play an important role here (Brandl
et al. 2007, Vaishnava et al. 2008). It was evidenced that the production of REG3γ is regulated
principally by TLR2, which was confirmed by the studies indicating that mammals deprived
of the marker are much more prone to infections with Yersinia pseudotuberculosis (Dessein
et al. 2009). TLR receptors of intestinal epithelial cells may also contribute to immunoglobulin
production. It was evidenced that in humans, stimulation of these markers by PAMP leads to
expression of APRIL (a proliferation-inducing ligand) and TSLP (thymic stromal lymphopoietin),
which belong to cytokines inducing the switching in the lamina propia of immunoglobulin
classes from IgM and from IgA1 to IgA2 (He et al. 2007).
CONCLUSION
TLR receptors constitute a very important element of natural immunity, and the knowledge
on these markers continues to change. These facts not only refer to the number of such
receptors and their phylogenetic proximity, but also to the presence and role in particular
tissues and organs of the body. It was evidenced that intestinal epithelium constitutes
an important physiological barrier preventing against the penetration of microbes and enters
in favourable interactions with the bacterial flora of the intestines, and its role is also related
to TLR receptors, which recognise molecular patterns of pathogens (PAMP) of the intestinal
flora, which directly impacts on the appropriate functioning of the digestive system barrier, and
thus immunity of the entire body.
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in intestinal epithelial cells. J. Immunol. 173, 5398–5405.
Xiao H., Gulen M.F., Qin J., Yao J., Bulek K., Kish D., Altuntas C.Z., Wald D., Ma C., Zhou H.,
Tuohy V.K., Fairchild R.L., de la Motte C., Cua D., Vallance B.A., Li X. 2007. The Toll-interleukin-1
receptor member SIGIRR regulates colonic epithelial homeostasis, inflammation and tumorigenesis.
Immunity 26, 461–475.
Zhang G., Ghosh S. 2002. Negative regulation of Toll-like receptor-mediated signaling by Tollip. J.
Biol. Chem. 277, 7059–7065.
Zheng L., Riehl T., Stenson W.F. 2009. Regulation of colonic epithelial repair in mice by Toll-like
receptors and hyaluronic acid. Gastroenterology 6, 2041–2051.
RECEPTORY TLR – WYBRANE DANE
Streszczenie. Praca opisuje najnowsze doniesienia dotyczące receptorów Toll-podobnych (Toll-lie
receptors – TLR), które są bardzo istotnymi znacznikami odporności naturalnej, szczególnie w obronie
antyzarazkowej wobec bakterii, wirusów i pasożytów. Ostatnie dane wskazują na znaczący rozwój rodziny
tych receptorów, jako że w badaniach filogenetycznych zarejestrowano istnienie TLR1-15 oraz TLR21, 22,
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gdzie stanowią ważny element odporności lokalnej.
Słowa kluczowe: TLR, komórki nabłonkowe
ADVANCES IN AGRICULTURAL SCIENCES
Adv. Agric. Sci. 2011, XIV (1–2), 45–52
Paulina Niedźwiedzka-Rystwej, Wiesław Deptuła
HAEMATOLOGICAL AND BIOCHEMICAL FACTORS IN SELECTED SPECIES
OF REPTILES, RODENTS AND ANIMALS FROM ARTIODACTYLS ORDER
Department of Microbiology and Immunology, University of Szczecin,
Felczaka 3c, 71-412 Szczecin, Poland, phone: 91 444 1605, fax. 91 444 1606,
email: [email protected]
Abstract. Due to lack in the literature of data regarding haematological-biochemical factors in exotic
species of reptiles, rodents and animals from the Artiodactyls order, the number of which is increasing
in Poland, the paper presents the value of haematological parameters, such as erythrocytes, haematocrit,
haemoglobin, leucocytes, and biochemical factors, such as albumin, globulin, glucose, creatinine, bilirubin,
cholesterol, calcium, phosphorus, alanine aminotransferase, aspartic aminotransferase in selected
species of reptiles (python, iguana, crocodilus porosus and Nile crocodile, as well as olive ridley sea turtle),
rodents (house mouse – Mus musculus, hopping-mouse Notomys alexis, black rat – Rattus rattus, Central
rock-rat Zyzomys pedunculatus, Plains rat Pseudomys australis and Carpentarian Rock-rat Zyzomys
palatalis, Guinea pig – Cavia porcellus, golden hamster – Mesocricetus auratus, long-tailed chinchilla –
Chinchilla laginera and Mongolian jird – Meriones unguiculatus) and animals in the Artiodactyls order (axis
deer (chital), barking deer, bison, wild water buffalo, as well as llama and cattle).
Key words: haematological-biochemical factors, reptiles, rodents, Artiodactyls
The group of animals that can be met around people is significantly increasing, and although
it may seem many species are not fit for keeping in households, still these “untypical” animals
increasingly find their users. Such a situation is encouraged by the possibility of importing
of a broad range of species from abroad, including exotic countries. However, due to lack
of adjustment of such animals to the Polish environment, they often catch various diseases,
hence learning about and familiarisation with haematological and biochemical standards
for such animals is an important element in diagnosing many of their ailments and diseases, as
it allows for monitoring their health. Furthermore, even when “untypical animals” are not kept
in households, it happens that such exotic species are seen e.g. in zoos. It must be added that
even the most important worldwide studies regarding haematological and biochemical factors
in animals (Feldman et al. 2000), do not consider many species, hence the objective of this
study is to present the values of selected haematological and biochemical parameters in selected
46
P. Niedźwiedzka-Rystwej, W. Deptuła
reptiles, rodents, and Artiodactyls, which may be present in Poland, whereas the data in this
respect are not broadly available. Tables 1, 2, 3 present the value of haematological parameters
(erythrocytes, haematocrit, haemoglobin, leucocytes) and biochemical parameters (albumin,
globulin, glucose, creatinine, bilirubin, cholesterol, calcium, phosphorus, alanine aminotransferase,
aspartic aminotransferase) in selected species of reptiles (python, iguana, crocodilus porosus
and Nile crocodile, as well as olive ridley sea turtle), rodents (house mouse – Mus musculus,
hopping-mouse Notomys alexis, black rat – Rattus rattus, Central rock-rat Zyzomys pedunculatus,
Plains rat Pseudomys australis and Carpentarian rock-rat Zyzomys palatalis, Guinea pig – Cavia
porcellus, golden hamster – Mesocricetus auratus, long-tailed chinchilla – Chinchilla laginera
and Mongolian jird – Meriones unguiculatus), and Artiodactyls (axis deer (chital), barking deer,
bison, wild water buffalo, as well as llama and cattle).
When analysing the listed haematological values in reptiles (Table 1), with the following
representatives: python, iguana, crocodiles – saltwater (porosus) and Nile, and olive ridley sea
turtle, one must state that the number of erythrocytes in the species listed ranges from 0.6 x 1012/l
in saltwater crocodile to 5.8 x 1012/l in iguana. In turn, the value of haematocrit ranges between
14% in Nile crocodile to 52% in iguana. As regards haemoglobin concentration, the lowest
value amounting to 0.6 mmol/l was recorded for iguana, while the highest – 12.6 mmol/l in olive
ridley sea turtle. As regards the number of leucocytes in the reptiles analysed, it ranged
between 3.0 x 109/l in iguana and 26.2 x 109/l in Nile crocodile. In the area of biochemical
factors, the volume of albumin ranged from 6.0 g/l in olive ridley sea turtle to 28 g/l in iguana,
while the volume of globulin ranged from 16.5 g/l in Nile crocodile to 50.0 g/l in saltwater
crocodile. Glucose values ranged from 0.6 mmol/l in python to 12.1 mmol/l in saltwater
crocodile. Creatinine concentration amounted from 0 μmol/l in python to 97 μmol/l in olive ridley
sea turtle, hence it must be stated that this parameter recorded the greatest range. In turn,
the volume of bilirubin in the presented reptiles was rather similar, within the range from
1.7 μmol/l in olive ridley sea turtle to 17.1 μmol/l in iguana. As regards cholesterol, it was
observed that its concentration amounted to between 0 mmol/l in Nile crocodile, and 8.6 mmol/l
in iguana. As regards the quantities of calcium, both the lowest and the highest values were
determined in olive ridley sea turtle, amounting to from 0.8 to 13.0 mmol/l, while the levels
of phosphorus remained at the level of from 1.2 mmol/l in saltwater crocodile to 3.8 mmol/l
in olive ridley sea turtle. The volume of alanine aminotransferase (ALT) was the lowest in olive
ridley sea turtle and amounted to 2 U/l, while in iguana it was the highest, amounting to 67 U/l,
whereas the level of aspartic aminotransferase (AST) ranged significantly, and totalled from
4 U/l in iguana to 211 U/l in Nile crocodile.
Haematological and biochemical factors…
47
Table 1. Haematological-biochemical factors in selected species of reptiles
Species
of reptile
Parameters
12
Erythrocytes (10 /l)
Haematocrit (%)
Haemoglobin (mmol/l)
9
Leucocytes (10 /l)
Albumin (g/l)
Globulin (g/l)
Glucose (mmol/l)
Creatinine (μmol/l)
Bilirubin (μmol/l)
Cholesterol (mmol/l)
Ca (mmol/l)
P (mmol/l)
ALT (U/l)
AST (U/l)
Python
(Gabrisch
and Zwart
1995)
1–2.5
24–40
3.4–8.8
6–12
No data
No data
0.6–3.3
0–49
No data
No data
2.5–5.5
No data
No data
No data
Iguana (I.iguana)
(Gabrisch
and Zwart 1995)
1.0–5.8
25–52
0.6–1
3.0–15.0
10.0–28.0
No data
No data
8–80
6.8–17.1
2.7–8.6
No data
No data
5–67
4–90
Saltwater crocodile
(crocodilus
porosus) (Millan
et al. 1997)
0.6–1.3
17–41
4.7–12.2
6.4–25.7
14.0–23.0
27.0–50.0
4.5–12.1
20–51
No data
1.1–7.2
2.4–3.6
1.2–2.9
11–51
23–157
Nile crocodile
(Lovely et al.
2007)
0.4–1.0
14–22
4.7–9.5
3.8–26.2
11.1–19.4
16.5–42.6
1.8–4.8
17–56
No data
0–9.9
1.1–1.6
No data
15–63
14–211
Olive ridley sea
turtle (Santoro
and Meneses
2007)
No data
25–37
7.7–12.6
No data
6.0–9.0
No data
2.2–5.5
17–97
1.7–15.4
2.6–7.1
0.8–13.0
1.8–3.8
2–23
30–143
In parentheses after species, the literature item.
To conclude, it must be stated that regardless of zoological “proximity” of the presented
reptiles, the image of haematological-biochemical parameters is much differentiated among
them. Furthermore, the results obtained in the selected reptiles are most stable in the area
of haematological parameters as regards the number of erythrocytes, while the greatest range
of values was recorded for the number of leucocytes, whereas in the area of biochemical
indices, cholesterol concentration remains a rather stable parameter, whereas other parameters
such as volume of albumin, globulin, glucose, creatinine, bilirubin, and calcium and phosphorus
ions, as well as aminotransferase range significantly (Gabrisch and Zwart 1995, Millan et al.
1997, Santoro and Meneses 2007, Lovely et al. 2007).
In turn, when analysing the data regarding haematological factors in selected rodent
species (Table 2), namely house mouse (Mus musculus), hopping-mouse Notomys alexis,
black rat (Rattus rattus), central rock-rat Zyzomys pedunculatus, plains rat Pseudomys
australis and Carpentarian rock-rat Zyzomys palatalis, Guinea pig (Cavia porcellus), golden
hamster (Mesocricetus auratus), long-tailed chinchilla (Chinchilla laginera) and Mongolian jird
(Meriones unguiculatus) (Gabrisch and Zwart 1995, Feldman et al. 2000, Old et al. 2005, Old
et al. 2007, Ege et al. 2008), it must be stated that the number of erythrocytes in the listed
species ranges from 4.5 in Guinea pig to 12 x 1012/l in chinchilla. In turn, the percent
of haematocrit remains within the values from 27 in chinchilla to 55 in Guinea pig and golden
hamster. As regards haemoglobin concentration, it was recorded that the value ranges from
8 mmol/l in chinchilla to 17.9 in Mongolian jird. In turn, the number of leucocytes in the rodents
analysed ranged between 0.9 x 109/l in Zyzomys palatalis Carpentarian rock-rat and 14.42 x 109/l
in Guinea pig. In turn, as regards biochemical parameters of blood in the rodent species
analysed, it was determined that the albumin level ranged from 23 g/l in chinchilla to 51 g/l
P. Niedźwiedzka-Rystwej, W. Deptuła
48
in Australian Pseudomys australis species, whereas globulin level ranged from 9 g/l in chinchilla
to 42 g/l in hamster. In turn, glucose concentration amounted from 2.7 mmol/l in rat and Guinea
pig to 10.56 mmol/l in mice. Creatinine concentration ranged from 17.5 μmol/l in rat to 159.1 μmol/l
in Guinea pig. Bilirubin concentration remained within the limits of 1 μmol/l in Australian Notomys
alexis to 10.3 μmol/l in hamster. Also, large differences were recorded for cholesterol levels,
which ranged between 0.31 mmol/l in Guinea pig to 42 mmol/l in rat. As regards calcium
concentration, extreme values were observed in hamster, namely from 1.2 to 2.9 mmol/l,
whereas phosphorus levels remained at the level from 1 mmol/l in rat to 4.43 mmol/l
in Australian Zyzomys pedunculatus. Volume of alanine aminotransferase (ALT) amounted
to from 7 U/l in Australian mouse Notomys alexis to 86 in Zyzomys pedunculatus from
the same country, while the level of aspartic aminotransferase (AST) ranged from 45.7 U/l
in rat to 319 U/l in Australian Zyzomys pedunculatus.
Table 2. Haematological-biochemical factors in selected species of rodents
Notomys alexis - Spinifex hoppingmouse (Old et al. 2005)
Black rat (Rattus rattus) (Gabrisch
and Zwart 1995)
Zyzomys pedunculatus – Central
rock-rat (Old et al. 2005)
Pseudomys Australis – Plains rat
(Old et al. 2005)
Zyzomys palatalis - Carpentarian
Rock-rat
(Old et al. 2007)
Golden hamster (Mesocricetus
auratus) (Gabrisch and Zwart
1995)
Long-tailed chinchilla (Chinchilla
laginera) (Gabrisch and Zwart
1995)
Mongolian jird (Meriones
unguiculatus) (Feldmann et al.
2000)
7–10
7.7–8.3
No data
5.7–6.8
6.8–7.9
5.88–6.12
4.5–7
6–10
5–12
7.1–8.6
35–50
37–38
37.6–50.6
37–42
39–45
39–42
39–55
36–55
27–54
42–49
10–15
12.6–12.9
11–18
4–14
2.1–4.6
Albumin (g/l)
No data
Globulin (g/l)
Parameters
Erythrocytes
(1012/l)
Haematocrit
(%)
Haemoglobin
(mmol/l)
Leucocytes
(109/l)
Glucose
(mmol/l)
Creatinine
(μmol/l)
Bilirubin
(μmol/l)
Cholesterol
(mmol/l)
Guinea pig (Cavia porcellus)
(Gabrisch and Zwart 1995)
House mouse (Mus musculus)
(Gabrisch and Zwart 1995, Ege et
al. 2008)
Species
12.0–12.1 12.6–14.4 11.5–12.2 11.7–16.9
10–16
8–15.4 13.1–17.9
6.6–12.6
5.6–13.8
4.6–8.1
0.9–1.9
2.91–14.42
3–11
4–11.5
27–31
38–48
30–38
44–51
30–34
25.5–41.1
26–41
23–41
No data
No data
16–20
18–30
19–24
11–15
No data
18.9–24.7
27–42
9–22
No data
5.94–10.56
No data
2.8–7.5
No data
No data
No data
2.8–6.6
44.2
No data
17.5–70.8 No data
No data
31–47
3.1–9.2
1–2
No data
2–5
0–2
3.13
1.36–2.3
No data
88.4–159.1 80.4–87.5
1.63–1.93 0.31–1.67
0.64–3.5 1.04–2.59 No data
Ca (mmol/l)
No data
1.68–1.96
1.7–4.2
2.46–2.62 2.05–2.58 2.42–2.71
2.4–3.1
P (mmol/l)
No data
2.34–2.96
1–3.5
1.53–4.43 1.44–2.55 1.5–2.45
No data
ALT (U/l)
69.0–72.31
7–18
17.5–30.2
105.2–111.9 45.7–80.8 45.7–80.8 168–319
In parentheses after species, the literature item.
No data
4.3–10.3 3.42–6.84 No data
2.38–2.53 12.9–42
AST (U/l)
44.2
No data
0–1.59
No data
86
3.3–8.25 3.3–9.13
No data
1.2–2.9
No data
No data
1.09–2.64 1.29–2.58 No data
27–84
39–47
0–61
No data
10–35
No data
136–195
111–213
0–90
No data
96
No data
Haematological and biochemical factors…
49
To conclude on the values of haematological factors in the selected rodents, it must
be stated that the most stable parameter proved to be the number of erythrocytes, while
the greatest differences were recorded for the number of leucocytes. In turn, among biochemical
factors, which were less stable than haematological factors, calcium concentration proved
to be the most similar among all species, whereas the greatest differences were observed
for creatinine concentration. It must be added that the values recorded in this group of animals
in mice seem to be rather similar to the results obtained in other species.
In turn, when analysing the data in selected exotic species of the Artiodactyls order (Table 3),
namely axis deer (chital), Mongolian jird, bison, wild water buffalo, as well as llama (Gill 1992,
Hutchison et al. 1995, Feldmann et al. 2000, Anusz et al. 2007, Gupta et al. 2007), as well as
cattle (Deptuła et al. 1992, Deptuła et al. 1993, Feldmann et al. 2000), regarding the number
of erythrocytes, one may state that their number ranges from 2.1 in bison to 16.6 x 1012/l in llama.
In turn, the haematocrit percentage remains within the values from 10 in bison to 57 in wild
water buffalo. As regards haemoglobin concentration, it was recorded that the value ranges from
5.1 mmol/l in bison to 18.4 in chital. In turn, the number of leucocytes ranges from 1.3 x 109/l
in bison to 11.6 x 109/l in wild water buffalo. When analysing the data regarding biochemical
parameters in selected mammals in the Artiodactyls order, it must be noticed that the data are
rather limited and only selectively contain factors from the panel presented in Table 3. And so,
albumin level ranged from very low values of 2.77 g/l in llama to 42.5 g/l in Mongolian jird.
In turn, globulin level amounted to from 12.0 g/l in llama to 35.5 g/l in chital. Glucose
concentration in animals analysed has so far only been recorded in llama, and remained within
the range of 46–125 mmol/l. Creatinine concentration had so far only been measured in bison
and llama, with the levels of from 0.9 to 2.8 μmol/l. In turn, bilirubin level remained within
the limits of 0.1 μmol/l in llama and bison to 40 μmol/l in bison. Cholesterol level has not been
recorded for the animals under the present analysis. As regards calcium concentration, high
fluctuations were revealed, from 0.8 mmol/l in chital to 8.9 in llama. In reference to phosphorus
level, it was only assessed for llama, and amounted to from 4.1 to 7.0 mmol/l. The volume
of alanine aminotransferase (ALT) totalled from 16 U/l in chital to 429.6 U/l in bison, whereas
aspartic aminotransferase (AST) levels ranged from 6.1 U/l in bison to 208 U/l in llama.
To conclude on the values obtained in the area of haematological factors in the analysed
mammal species of the Artiodactyls order, it must be noticed that the most stable values have
so far been registered in the area of haemoglobin concentration, whereas leukocyte levels
were much differentiated. In turn, as regards biochemical factors, which were only rarely
analysed in the group of species discussed, it must be stated that the greatest continuity was
P. Niedźwiedzka-Rystwej, W. Deptuła
50
recorded for creatinine level, whereas the greatest differences referred to the volume of alanine
aminotransferase. Furthermore, it must be observed that the values obtained are usually
higher than the ones recorded in the popular Polish Artiodactyls, namely the cattle. Moreover,
it must be stated that the values are also highly varied among exotic mammals. It may
be concluded that the values of haematological-biochemical factors within Artiodactyls are
conditioned with their origin and varied environment in which they stay.
Table 3. Haematological and biochemical factors in the selected Artiodactyls
Parameters
12
Erythrocytes (10 /l)
Haematocrit (%)
Haemoglobin (mmol/l)
9
Leucocytes (10 /l)
Albumin (g/l)
Globulin (g/l)
Glucose (mmol/l)
Creatinine (μmol/l)
Bilirubin (μmol/l)
Cholesterol (mmol/l)
Ca (mmol/l)
P (mmol/l)
ALT (U/l)
AST (U/l)
Animal species (literature item in parentheses)
Class: Mammals
Order: Artiodactyls
axis deer
wild water
barking deer bison (Gill
llama
(chital)
buffalo
(Gupta et al. 1992, Anusz
(Hutchison
(Gupta et al.
(Feldman
2007)
et al. 2007)
et al. 1995)
2007)
et al. 2000)
No data
No data
2.1–11.1
8.1–9.3
10.1–16.6
48.5–52
50.5–54
10–50
51–57
20–26
14.3–18.4
14.9–16.6
5.1–17.7
20.7–22.9
10.3–16.0
1.9–2.6
2.1–3.7
1.3–9.1
9.1–11.6
No data
35.0–41.8
34.7–42.5
No data
No data
2.77–4.3
31.0–35.5
21.5–31.4
No data
No data
12.0–29.0
No data
No data
No data
No data
46–125
No data
No data
0.9–2.8
No data
1.5–2.4
0.3–4.1
2.2–7.6
0.1–40
No data
0–0.1
No data
No data
No data
No data
No data
0.8–1.2
0.9–1.1
No data
No data
8.1–8.9
No data
No data
No data
No data
4.1–7.0
16–20
38–46
48.8–429.6
No data
No data
22–38
56–60
6.1–69.7
No data
106–208
cattle (Deptuła
et al. 1992,
1993, Feldman
et al. 2000)
5.0–10.0
36–38
8.0–15.0
4.0–12.0
No data
34.0–49.0
62.1–70.8
No data
No data
No data
9.0–11.3
8.2–9.1
8.5–24.6
28.7–56.4
No data – no data available; in parentheses after species, the literature item.
It must also be added that the data presented in the area of immunological-biochemical
factors regarding selected species of reptiles, rodents and Artiodactyls highly vary. This results
in a situation where it is justified to treat such parameters as specific, and thus values
of haematological and biochemical factors should not be referred to among seemingly similar
species.
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Santoro M., Meneses A. 2007. Haematology and plasma chemistry of breeding olive ridley sea turtles
(Lepidochelys olivacea). Vet. Rec. 15, 318–319.
WSKAŹNIKI HEMATOLOGICZNE I BIOCHEMICZNE U WYBRANYCH
GATUNKÓW GADÓW, GRYZONI I ZWIERZĄT Z RZĘDU
PARZYSTOKOPYTNYCH
Streszczenie. Ze względu na brak w literaturze zestawień dotyczących wskaźników hematologicznobiochemicznych u egzotycznych gatunków gadów, gryzoni oraz zwierząt z rzędu parzystokopytnych,
których liczba z czasem zwiększa się w naszym kraju, praca prezentuje wartości parametrów hematologicznych, takich jak erytrocyty, hematokryt, hemoglobina, leukocyty oraz biochemicznych, takich jak
albumina, globulina, glukoza, kreatynina, bilirubina, cholesterol, wapń, fosfor, aminotransferaza alaninowa,
aminotransferaza asparaginianowa u wybranych gatunków gadów (pyton, jaszczurka iguana, krokodyl
różańcowy i nilowy oraz żółw oliwkowy), gryzoni (mysz domowa – Mus musculus, mysz Notomys alexis,
szczur śniady – Rattus rattus, szczur Zyzomys pedunculatus, szczur Pseudomys australis oraz Zyzomys
palatalis, świnka morska – Cavia porcellus, chomik syryjski – Mesocricetus auratus, szynszyla mała –
Chinchilla laginera i myszoskoczek mongolski – Meriones unguiculatus) oraz zwierząt z rzędu parzystokopytnych (jeleń axis (czytala), mundżak indyjski, żubr, bawół wodny oraz lama i bydło).
Słowa kluczowe: wskaźniki hematologiczno-biochemiczne, gady, gryzonie, zwierzęta parzystokopytne
ADVANCES IN AGRICULTURAL SCIENCES
Adv. Agric. Sci. 2011, XIV (1–2), 53–64
Antoni Jakubczak, Milena A. Stachelska
HEALTH BENEFITS RESULTING FROM PROBIOTIC BACTERIA CONSUMPTION
Food Technology and Nutrition Institute, The State College of Computer Science and Business
Administration in Łomża, Akademicka 14, 18-400 Łomża, Poland
Abstract. Probiotics are considered to be microbial food additives bringing a lot of benefits for humans’
health. They belong to a group of lactic acid-producing bacteria and are usually eaten together with
yoghurt, fermented milks and other fermented foods. They possess many beneficial features which
include the improvement of intestinal tract functioning, the enhancement of immune system, the synthesis
and improvement of bioavailability of nutrients, the decrease in lactose intolerance symptoms,
the reduction of allergy and certain cancer risks. It is hard to explain the mechanisms of exerting
of benefits for humans. However, it is known that they modify gut pH, have got an antagonizing activity
towards pathogens due to the production of antimicrobial compounds, which destroy pathogens.
Probiotics stimulate the immunomodulatory cells and produce lactase. There are many scientific
researches which examine the important role of probiotics being a part of a healthy diet not only for
humans but also for animals.
Key words: probiotics, biopreservation, lactic acid bacteria
INTRODUCTION
Nowadays, a huge interest in probiotic foods is observed among very demanding and health-conscious consumers. Many researchers have been carried out on living probiotic microorganisms to prove their strong impact on the functioning of immunological, digestive and respiratory systems. The results indicated that they significantly alleviate different infectious diseases
in children and elder people belonging to high-risk groups. Such characteristics of probiotic
microorganisms encourage food producers to supplement food products with the species
possessing such valuable features. It results in the appearance of a variety of probiotic foods
and drinks in the market. Such a growing interest in probiotic foods simultaneously induces
the development of methodology which enables to prove their efficacy and the safety for humans.
Two international organisations such as FAO and WHO started to work on the development
of guidelines for assessment of probiotics in food (FAO 2006).
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A. Jakubczak, M.A. Stachelska
The definition of probiotic bacteria is recently developed and used for the characterization
of alive bacteria which are proved to influence positively for health of humans and animals.
The first discovery of beneficial impact of probiotic bacteria was made by Eli Metchnikoff,
the Russian born Nobel Prize winner who worked at the Pasteur Institute at the beginning
of the last century. He stated that "The dependence of the intestinal microbes on the food
makes it possible to adopt measures to modify the flora in our bodies and to replace
the harmful microbes by useful microbes" (Metchnikoff 1907). His statement occurred to be
efficient and it is nowadays obvious that the children with have in their stools a relatively low
number of bacteria appearing in the form of peculiar, Y-shaped cells. On the other hand, such
morphological forms can be isolated in a high number from the intestines of healthy children.
It means that such bacteria might be given to the patients suffering from diarrhea to help
restore a healthy gut flora. Probiotic bacteria are considered to be live microorganisms which
when consumed in adequate numbers, attribute to bringing the health effect for the host.
Scientists have gained the relatively wide knowledge over the beneficial effects of probiotic
bacteria on human and animal health and essential progress has been made in the proper
selection and characterization of some probiotic bacteria proving their huge impact on health
and encouraging the consumption of food products containing their alive cells. Among probiotic
bacteria the members of the genera Lactobacillus and Bifidobacterium are worth being given
a special attention. They constitute typical inhabitants of humans and upper animals. They can
be found in their gastrointestinal tract. It was examined that the stomach contains a relatively
low number of bacteria (103 colony forming units per ml of gastric juice) whereas the bacterial
concentration increases throughout the gut achieving its final concentration in the colon
of 1012 bacteria/g. Bacteria start to colonize the gut at the moment of birth and continue their
colonisation throughout life (Mitsuoka 1992). Such bacteria are called the resident intestinal
microflora. They positively influence the well being of their host.
Their beneficial effect is strongly associated with their ability to maintain their presence
in the intestinal track and simultaneously to avoid colonization of some freshly ingested
microorganisms, including pathogens (van der Waaij et al. 1971, Vollaard and Clasener 1994).
It is unquestionable that dietary manipulation of gut microflora through the consumption
of milk probiotic products can increase the numbers of so called beneficial bacteria which might
lead to the improvement of the well being of their host. Such discoveries encourage the systematic
researches which might prove the highly positive influence of diets on the prevention of intestinal
putrefaction in prolonging life and maintaining the vitality of human body. It means that probiotic
bacteria are our future and its consumption with milk products can contribute to the improvement
of our functioning which also means that they should be investigated with the particular care.
Health benefits resulting from probiotic bacteria consumption
55
GUIDELINES FOR EVALUATION OF PROBIOTIC BACTERIA
In order to be applied in food products, probiotic microorganisms must be able to survive
while passing through the digestive tract and they should be able to proliferate in the intestines.
They must show resistance towards gastric juices and must be capable to grow in the environment of bile in digestive tract. They might be consumed with a food product which enables
them to survive while passing through the stomach and being exposured to bile. Probiotic
microorganisms are Gram positive bacteria and belong mainly to two genera, Lactobacillus
and Bifidobacterium (Holzapel et al. 1998, Klein et al. 1998). In order to be chosen to food
application for human they must possess beneficial effects for their host due to their growth
and activity in the human body (Collins et al. 1998, Morelli 2000). It is quite hard to estimate the
source of a microorganism. Infants are regarded to be born with having no probiotic bacteria
in their intestine. The origin of the intestinal microflora has not been fully explained yet. It is
necessary for them to maintain their effectiveness and it should be confirmed for each
potentially probiotic strain. There is also a necessity to carry out a numerous laboratory tests
in vitro to assesss the beneficial abilities of probiotics for human beings.
The easiest way to classify probiotic bacteria is to use the International Code of Nomenclature.
Probiotic properties are regarded to be strain related and the strain identification (genetic
typing) is recommended to be performed with the application of methodology such as pulse
field gel electrophoresis (PFGE). At first phenotypic tests should be carried out followed
by genetic identification by DNA/DNA hybridization, 16S RNA sequencing and other internationally
recognized methods. At last, the RDP (ribosomal data base project) should be done in order
to confirm their identity.
HEALTH BENEFITS OF PROBIOTIC BACTERIA
Consumption of probiotic bacteria with a variety of milk products can attribute to the health
benefits. There are some examinations which proved that probiotic strains in certain situations
do not possess any clinical effects (Andersson et al. 2001). It is commonly believed that the
beneficial influence of probiotic microorganisms for the host depends on their dosage
consumed with food and duration of consumption recommended by the manufacturer of each
individual strain or product which were calculated on the base of scientific investigation.
It is highly recommended that for each product there ought to be given the minimum daily
amount needed to contribute towards health benefits. Such amounts should be calculated after
carrying out a number of investigations both in vitro and in vivo.
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It has been found that consumption of probiotic bacteria can prevent diarrhea being a result
of contamination by some pathogenic bacteria and viruses. Infectious diarrhea is considered
to be a main world health problem and causes several million deaths per year. It is known that
the majority of deaths appear at children in developing countries. However, it is also assessed
that even 30% of the population in developed countries suffer from foodborne diarrhea
per year. The results of many investigations proved that probiotic bacteria can easily eliminate
the risk of diarrhea. Probiotic bacteria can be not only consumed in a form of food supplemented
with them, but also in a non-food form.
There were quite many researches carried out on with some of probiotic bacteria such as
Lactobacillus rhamnosus GG and Bifidobacterium lactis BB-12 in order to prevent (Saavedra
et al. 1994, Szajewska et al. 2001) and treat (Isolauri et al. 1991, Majamaa et al. 1995, Guarino
et al. 1997, Shornikova et al. 1997, Perdone et al. 1999, Guandalini et al. 2000) acute diarrhea
caused by rotaviruses in children. Apart from rotavirus, diarrhea may also be provoked by
many bacterial species which might even lead to death of humans. Many scientists indicated
that there are some probiotic strains which are able to inhibit the growth and adhesion
of a variety of enteropathogens (Coconnier et al. 1993, 1997, Bernet Camard et al. 1997,
Hudault et al. 1997, Gopal et al. 2001). Other examinations showed that such pathogens like
Salmonella can also be easily eliminated by some probiotic strains (Shu et al. 2000, Ogawa et
al. 2001). The biggest number of travelers’ diarrhea casus was caused by pathogens having
a bacterial nature and they were found to be eliminated by the application of some probiotic
bacteria (Hilton et al. 1997).
It has been proved that certain probiotic strains show antimicrobial activity towards infections
caused by Helicobacter pylori. H. pylori is a Gram negative pathogen which causes type B
gastritis, peptic ulcers and gastric cancer. It was indicated that lactic acid bacteria can prevent
the growth of this pathogen and simultaneously decrease urease enzyme activity which
is significant for this pathogen to survive in the acidic environment of the stomach (Midolo et al.
1995, Kabir et al. 1997, Aiba et al. 1998, Coconnier et al. 1998).
There were also some investigations carried out to check how probiotic bacteria are able
to prevent inflammatory diseases and bowel diseases such as pouchitis and Crohn’s disease,
as well as irritable bowel syndrome (Shanahan 2000). The potential role of probiotics in therapy
and prophylaxis may have a role to play in remediation (Gionchetti et al. 2000, Gupta et al.
2000). It can be stated that the intestinal microflora probably cause some inflammatory
conditions in the gut and some probiotics can presumably remediate such conditions by
the alteration in the composition of microflora.
Health benefits resulting from probiotic bacteria consumption
57
There is another benefit resulting from the consumption of probiotic bacteria. They were
proved to eliminate a risk of the onset of certain cancers. Some members of the gut microflora
are able to produce carcinogens such as nitrosamines. The application of lactobacilli
and bifidobacteria might modify the microflora composition in the guts thus preventing from
releasing nitrosamines responsible for cancer (Hosada et al. 1996). Furthermore, there is some
proof that cancer recurrences appearing for example in the urinary bladder can be decreased
by intestinal instillation of probiotics such as L. casei (Aso et al. 1995). Some observations in vitro
using L. rhamnosus GG and bifidobacteria as well as observations in vivo using L. rhamnosus
strains GG and LC-705 as well as Propionibacterium sp. indicated a significant decrease
in availability of carcinogenic aflatoxin in the lumen (El-Nezami et al. 2000, Oatley et al. 2000).
However, such observations are too insufficient to confirm the efficacy of probiotics in cancer
prevention. Many more researches should be carried out to state a strong preventive influence
of probiotics on cancer. Examinations should utilize internationally recognized markers
for cancer, or risk of cancer, and assess such markers and presence of carcinogenic lesions
or tumors over a suitably long period of time for prevention of primary cancer, and reduction
of the incidence of recurrences. What is more, probiotic bacteria support the functioning
of digestive system. They are able to alleviate constipation and its symptoms such as difficulty
in passing stool, excessive hardness of stool, slow transit through the bowel.
Some strains of probiotic bacteria actively participate in supporting the functioning of immune
systems. There are the two compartments which are important for the immune response. They
include the innate and adaptive immune systems. Macrophages, neutrophils, natural killer (NK)
cells and serum complement constitute the main components of the innate system and they
are the first line of defence against many microorganisms. The adaptive system (B and T cells)
is the additional line of defence. It should also be stated that cells of the innate system
modulate the beginning and subsequent direction of adaptive immune responses. Natural killer
cells such as gamma/delta T cells are responsible for regulating the development of allergic
airway disease. Many investigations have been carried out in vitro and in animals (Gill et al.
2000) which evidently indicate that probiotic strains can regulate immune parameters. It was
confirmed that dietary consumption of B. lactis HN019 and L. rhamnosus HN001 resulted
in measurable improvement of immune system functioning in the elderly (Arunachalam et al.
2000, Gill et al. 2001, Sheih et al. 2001). Such findings prove that probiotic regulation of immunity
system shows a great promise for further investigation. It is observed that probiotic microorganisms
can increase NK cell activity in the elderly (Gill et al. 2001) and nonspecific host defenses can
be regulated (Donnet-Hughes et al. 1999, Perdigon et al. 1999). There were some studies
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which identified some of the critical factors responsible for host’s defenses which include the
induction of mucus production or macrophage activation by lactobacilli signaling (Mack et al.
1999, Miettinen et al. 2000), stimulation of sIgA and neutrophils at the site of probiotic action
(for example the gut), and lack of release of inflammatory cytokines or stimulation of elevated
peripheral immunoglobulins (Kaila et al. 1992, Gardiner et al. 2001). It is also confirmed that
the stimulation of factors such as inflammatory cytokines might contribute to some health
benefits for the host.
There were also studies carried out to confirm the ability of probiotic bacteria to alleviate
the symptoms of allergy. In a double-blind, randomized, placebo-controlled trial, L. rhamnosus
GG was given to pregnant women for four weeks prior to delivery, then to newborns at high
risk of allergy for six months with the result that there was a substantial decrease in Elary
atopic disease (Kalliomaki et al. 2001). The results of such a research show a huge potential
of probiotic bacteria to regulate the immune response and prevent the appearance of allergic
diseases. Other investigations over infants allergic to cow’s milk showed that atopic dermatitis
was alleviated by consumption of probiotic strains L. rhamnosus GG and B. lactis BB-12
(Majamaa and Isolauri 1996, 1997, Isolauri et al. 2000). It is hard to explain the precise
mechanisms responsible for the prevention of appearance of allergy symptoms, not been
elucidated, but there are confirmed proofs that lactobacilli are able to reverse increased
intestinal permeability, enhance gut-specific IgA responses, promote gut barrier function
through restoration of normal microbes, and enhance transforming growth factor beta
and interleukin 10 production as well as cytokines that promote production of IgE (Kalliomaki
et al. 2001, Isolauri 2001).
Other studies indicated that probiotic bacteria prevent the appearance of cardiovascular
diseases. The consumption of probiotic lactobacilli and metabolic byproducts potentially
attribute to numerous benefits to the heart, through the prevention and therapy of different
ischemic heart syndromes (Oxman et al. 2001). They are also expected to decrease serum
cholesterol (De Roos and Katan 2000).
The next benefit taken from the consumption of probiotic bacteria products is connected
with urogenital tract disorders. With an exception of sexually transmitted diseases, it can be
said that very many infections of the vagina and bladder are provoked by microorganisms
which come from the bowel. There is a close relationship between presence of commensals,
especially lactobacilli in the vagina with health, and an absence of such bacteria in patients
suffering with urogenital infections. Disruption of balance in the normal vaginal flora composition
may be caused by wide-spectrum antibiotics, spermicides, hormones, dietary substances
Health benefits resulting from probiotic bacteria consumption
59
and factors which are not fully known. The results of some studies proved that probiotic bacteria
consumed with foods prezent some urogenital tract disorders. In order to confirm the efficacy
of probiotic bacteria activity, some criteria for their selection should be recommended (Reid
and Bruce 2001). They contain verification of safety, colonization ability in the vagina and
ability to reduce the pathogen count through competitive exclusion of adherence and inhibition
of patogen growth.
The beneficial role of probiotic bacteria can also be observed in the prevention of bacterial
vaginosis (BV). This is a disease of unknown etiology and is caused by the overgrowth
of different anaerobic bacterial species that lead to the elimination of lactobacilli, which should
constitute the dominating normal microflora of vagina. Women infected with BV may be at risk
of much more serious diseases such as endometriosis, pelvic inflammatory disease
and complications of pregnancy including pre-term labour (Hilton et al. 1995, Sieber and Dietz
1998, Reid et al. 2001). It was found that delivery of Lactobacillus acidophilus in yogurt led
to the prevention and therapy of candidal vaginitis (Hilton et al. 1992).
Another disease which can be avoided through the consumption of probiotic bacteria
is the urinary tract infection. There are calculations that several hundred million women suffer
from urinary tract infection (UTI) annually. Uropathogenic Escherichia coli coming from the bowel
is the most commonly met reason of this disease which refer to almost 85% of cases. Asymptomatic
bacteruria is very often fund in women, and may be followed by symptomatic UTI. The results
of some investigations prove that delivery of vaginal capsules of freeze dried Lactobacillus
strains GR-1 and B-54 once a week (Reid et al. 1995) and a capsule of Lactobacillus strains
GR-1 and RC-14 once a day (Reid et al. 2001b) might lead to the restoration of lactobacilli
mikroflora in the vagina which means the decrease in a risk of UTI recurrences.
CONCLUSIONS
There are many researchers which indicate the beneficial influence of some probiotic
bacteria on human health. It is known that some specific strains of probiotics are regarded as
safe for human use and can attribute to the inhancement of human health. Many diseases can
be avoided due to systematic consumption of some probiotic strains. Such diseases include
gastrointestinal infections, certain bowel disorders, allergy and urogenital infections. Probiotic
bacteria should be consumed by people who want to prevent themselves from certain diseases
and regulate host immunity. However, it should be stated that the regulatory status of probiotic
bacteria as a component of food is not estimated at an international level. The regulatory
procedures of supplementing food with probiotic bacteria are being developed and soon
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in more countries probiotic food products will be available thus contributing to the improvement
of human health. In order to confirm their effectiveness they must be identified by methods which
are internationally accepted molecular techniques and named on the base of the International
Code of Nomenclature. As there are too many doubts, much more researches should be
carried out to prove the beneficial influence of probiotic bacteria on people’s health. Many
attempts should be undertaken in order to make probiotic products more widely available
in particular to totally eliminate a risk of morbidity and mortality.
ACKNOWLEDGEMENTS
Presented work was supported by.
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Oatley J.T., Rarick M.D., Ji G.E., Linz J.E. 2000. Binding of aflatoxin B1 to bifidobacteria in vitro. J. Food
Prot. 63, 1133–1136.
Ogawa M., Shimizu K., Nomoto K., Takahashi M., Watanuki M., Tanaka R., Tanaka T., Hamabata T.,
Yamasaki S., Takeda Y. 2001. Protective effect of Lactobacillus casei strain Shirota on Shiga
toxin-producing Escherichia coli O157:H7 infection in infant rabbits. Infect. Immun. 69, 1101–1108.
Oxman T., Shapira M., Klein R., Avazov N., Rabinowitz B. 2001. Oral administration of Lactobacillus
induces cardioprotection. J. Altern. Complement. Med. 7(4), 345–354.
Perdigon G., Vintini E., Alvarez S., Medina M., Medici M. 1999. Study of the possible mechanisms involved
in the mucosal immune system activation by lactic acid bacteria. J. Dairy Sci. 82(6), 1108–1114.
Perdone C.A., Bernabeu A.O., Postaire E.R., Bouley C.F., Reinert P. 1999. The effect of supplementation
by Lactobacillus casei (strain DN-114 001) on accute diarrhea in children attending day care centers.
Int. J. Clin. Pract. 53, 179–184.
Reid G., Bruce A.W. 2001. Selection of Lactobacillus strains for urogenital probiotic applications. J. Infect.
Dis. 183, 77–80.
Reid G., Bruce A.W., Taylor M. 1995. Instillation of Lactobacillus and stimulation of indigenous organisms
to prevent recurrence of urinary tract infections. Microecol. Ther. 23, 32–45.
Reid G., Bruce A.W., Fraser N., Heinemann C., Owen J., Henning B. 2001. Oral probiotics can
resolve urogenital infections. FEMS Microbiol. Immunol. 30, 49–52.
Saavedra J.M., Bauman N.A., Oung I., Perman J.A., Yolken R.H. 1994. Feeding of Bifidobacterium
bifidum and Streptococcus thermophilus to infants in hospital for prevention of diarrhea and
shedding of rotavirus. Lancet. 344, 1046–1049.
Shanahan F. 2000. Probiotics and inflammatory bowel disease: Is there a scientific Rationale.
Inflamm. Bowel. Dis. 6, 107–115.
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subjects following dietary consumption of the lactic acid bacterium Lactobacillus rhamnosus
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KORZYŚCI ZDROWOTNE WYNIKAJĄCE Z KONSUMPCJI BAKTERII
PROBIOTYCZNYCH
Streszczenie. Probiotyki są uważane za dodatki do żywności przynoszące wiele korzyści dla zdrowia
ludzi. Należą one do grupy bakterii kwasu mlekowego i są zwykle spożywane wraz z jogurtem,
fermentowanymi napojami mlecznymi i innymi fermentowanymi produktami mleczarskimi. Posiadają wiele
korzystnych właściwości, które związane są z poprawą funkcjonowania przewodu pokarmowego,
wzmocnieniem systemu immunologicznego, syntezą i poprawą biodostępności składników odżywczych,
zmniejszeniem objawów nietolerancji laktozy, zmniejszeniem alergii i obniżeniem ryzyka niektórych
nowotworów. Trudno jest wyjaśnić mechanizmy odpowiadające za przynoszenie korzyści ludziom.
Jednak wiadomo, że probiotyki zmieniają kwasowość jelit, wykazują aktywność przeciwdrobnoustrojową
poprzez wytwarzanie związków niszczących patogeny. Probiotyki stymulują wytwarzanie laktazy. Wiele
badań naukowych zajmuje się badaniem właściwości probiotyków stanowiących istotną część właściwej
diety nie tylko ludzi, ale także zwierząt.
Słowa kluczowe: probiotyki, biokonserwacja, bakterie kwasu mlekowego
ADVANCES IN AGRICULTURAL SCIENCES
Adv. Agric. Sci. 2011, XIV (1–2), 65–70
Joanna Śliwa-Dominiak1, Arkadiusz Zupok2, Wiesław Deptuła1
VIRUSES OF ARCHAEA
1
Department of Microbiology and Immunology, University of Szczecin
Felczaka 3c, 71–412 Szczecin, Poland, e-mail: [email protected]
2
Student at the Chair of Microbiology and Immunology, Department of Natural Sciences,
University of Szczecin
Abstract. The paper presents the characteristics of bacteriophages infecting bacteria of Archaea domain.
The bacteria inhabit extreme environments with high salt content, acid content and high temperature.
So far, about 50 bacteriophages infecting the Archaea have been identified, which have capacities
allowing them to survive in extreme conditions. Among these bacterial viruses, most bacteriophages were
described infecting bacteria in the Euryarchaeota and Crenarchaeota phylum. Considering the ordination,
it was determined that bacteriophages infecting the Archaea principally belong to the Caudovirales order,
Myoviridae and Siphoviridae family, as well as Fusseloviridae, Lipothrixviridae, Guttaviridae and Rudiviriae
family.
Key words: bacteriophages, bacteria, environment, extreme conditions
Organisms belonging to the Archaea domain “live” in very extreme environments, as e.g.:
they may inhabit areas with high salt content (Niedźwiedzka & Deptuła 2007, Pagaling et al.
2007, Wolinowska 2008, Pietila et al. 2009), acidity (Niedźwiedzka & Deptuła 2007, Evans 2009,
Wolinowska 2008), as well as very high temperature (Prangishvili et al. 2006a, Niedźwiedzka
& Deptuła 2007, Wolinowska 2008, Evans 2009). As it turned out, life in such extreme conditions
is possible, but only few bacteria survive in such conditions (Wolinowska 2008). Hence, Archaea
include halophilous bacteria developing in heavily salted water reservoirs, e.g. Halobacterium
sp. (Halobacteriaceae) and acidophilous bacteria that develop in environments with pH lower
than 3, e.g. Acidianus sp. (Sulfolobaceae), as well as alkaliphilous bacteria, which develop
in environments with pH higher than 10 (Wolinowska 2008). The Archaea domain also includes
mathanogenic bacteria, e.g. Methanobacterium thermoautotrophicum (Methanobacteriaceae),
inhabiting anaerobic environments, the main respiratory product of which is methane; thermophilous bacteria, e.g. Pyrolobus fumarii (Pyrodictiaceae), occurring in warm environments,
where the temperature ranges from 45ºC to 60ºC, and psychrophilous bacteria, living in low
J. Śliwa-Dominiak, A. Zupok, W. Deptuła
66
temperatures not exceeding 20ºC (Wolinowska 2008). According to Bergey’s ordination
(Bergey et al. 2001), the Archaea domain is divided into the following phylum: Euryarchaeota
and Crenarchaeota (Table 1), although two further phylum are also provided: Nanoarchaeota
and Korarchaeota (Pagaling et al. 2007, Wolinowska 2008).
Table 1. The systematic of Archaea (1)
Domain
Phylum
Class
Methanobacteria
Order
Methanobacteriales
Methanococcales
Methanococci
Euryarchaeaota
Archaea
Methanomicrobiales
Methanosarcinales
Halobacteria
Halobacteriales
Thermoplasmata
Thermoplasmales
Thermococci
Archaeoglobi
Methanopyri
Thermococcales
Archaeoglobales
Methanopyrales
Thermoproteales
Crenarchaeota
Thermoprotei
Desulfurococcales
Sulfolobales
Family
Methanobacteriaceae
Methanothermaceae
Methanococcaceae
Methanocaldococcaceae
Methanomicrobiaceae
Methanocorpusculaceae
Methanospirillaceae
Methanosercinaceae
Methanoseataceae
Halobacteriaceae
Thermoplasmataceae
Picrophilaceae
Thermococcaceae
Archaeoglobaceae
Methanopyraceae
Thermoproteaceae
Thermofilaceae
Desulfurococcaceae
Pyridictiaceae
Sulfolobaceae
According to this ordination (Bergey et al. 2001), Euryarchaeota phylum is the most varied
in the aspect of existence of methanogenic, halophilous, thermophilous, and psychrophilous
bacterial species, and is divided into 7 classes (Table 1). Among the Methanobacteria and Methanococci classes, one can differentiate philogenetically the oldest organisms which in the course
of evolution developed genes in charge of production of substances supporting their metabolism
in difficult conditions (Bergey et al. 2001, Niedźwiedzka & Deptuła 2007). Other classes
in the Euryarchaeota phylum include: Halobacteria, Thermoplasmata, Thermococci, Archaeoglobi
Methanopyri (Table 1). The Crenarchaeota phylum represents just one class Thermoprotei
with three orders (Table 1), among which there are extreme thermophils and few psychrophiles
(Bergey et al. 2001, Wolinowska 2008). It must also be added that the aforementioned
Nanoarchaeota phylum only forms one species – Nanoarchaeum equitans, which is an absolute
symbiont that in order to live needs Ignicoccus hospitalis archeon belonging to the Crenarchaeota
(Wolinowska 2008). The fourth phylum of bacteria from the Archaea is Korarchaeota, which
is similar to the Nanoarchaeota and this phylum has not yet been considered in Bergey’s
Vruses of Archaea
67
ordination (Bergey et al. 2001). It is represented by sequences of nucleic acids obtained from
the analysis of the environment (Wolinowska 2008).
When characterising ordination groups of bacteria from the Archaea domain, it must
be stated that in recent years, apart from very sudden growth of information related to such
bacteria, also new species have been described (Wolinowska 2008). A very important discovery
among such information involved data regarding viruses present in the Archaea organisms.
What is most interesting, most of these bacteriophages were isolated from the environments
with the temperature of approx. 80ºC and acid pH < 3 (Rice et al. 2004). So far, about 50
bacteriophages infecting the Archaea have been identified (Pietila et al. 2009), which have
capacities allowing them to survive in extreme conditions (Zilling et al. 1996, Niedźwiedzka
& Deptuła 2007). Among many of them, there is Sulfolobus virus (Sulfolobaceae), the presence
of which was determined in the Kamtchatka Peninsula, Italy, Island and the USA, where DNA
was detected encoding so far unknown enzymes, the reactions of which are most efficiently
performed via the catalyst, namely high temperature (Niedźwiedzka & Deptuła 2007).
While describing bacteriophage infecting bacteria in the Archaea domain, it was determined
that among viruses belonging to Euryarchaeota phylum, approx. 20 viruses which infect such
bacteria were identified, and these are mainly viruses of the Caudovirales order. Among them,
the best characterised are the ones belonging to myoviruses ɸH and ɸCh1, infecting haloarchaea,
and belonging to siphoviruses ΨM1 and ΨM2, infecting Methanothermobacter (Prangishvili
et al. 2006). Among such viruses, only six were identified which are present in salty environments,
isolated from hyper-salty Australian lakes. These include virus infecting hyperalkalophilous archeon
Magadii natrialba (Halobacteriaceae), lytic virus HF1, and closely related to it virus HF2, infecting
Haloferax lucentense (Halobacteriaceae) and Halorubum coriense (Halobacteriaceae), as well
as cigar-shaped viruses HIS1 and HIS2 and lytical icosahedral virus SH1 infecting Haloarcula
hispanica (Halobacteriaceae) (Prangishvili 2006, Pagaling et al. 2007). Other bacteriophages
infecting bacteria belonging to the Archaea class include Pyrococcus abyssi virus (PAV1)
infecting hyperthermophilous Pyrococcus abyssi (Thermococcaceae), as well as bacteriophage
of unspecified name with genetic material dsDNA infecting Methanococcus voltae (Methanococcaceae) bacteria (Prangishvili et al. 2006).
Most Archaea bacteriophages were described among the bacteria from Crenarchaeota
phylum, because as many as 24 different bacteriophages, manifesting an unusual diversity
of shapes: from drops to bottles, and which were classified into 5 families of bacteriophages
(Table 2). The Fuselloviridae family, which includes Fusellovirus SSVI (Sulfobulus shibatae
virus) infecting Sulfolobus shibatae (Sulfolobaceae), are viruses belonging to the family with
68
J. Śliwa-Dominiak, A. Zupok, W. Deptuła
a form of cigar, and featuring the core containing a double-stranded, round DNA (dsDNA)
(Zilling et al. 1996, Pagaling 2007). The second family of bacteriophages of the Archaea
bacteria is Lipothrixviridae represented by Alphalipothrixviridae, Bethalipothrixviridae, Deltalipothrixviridae and Gammalipothrixviridae, while the main representatives of the family are viruses:
TTV1 (Thermoproteus virus 1), TTV2 (Thermoproteus virus 2) and TTV3 (Thermoproteus virus 3).
Genetic material of viruses from this family is linear dsDNA (www.ictvonline.org). The third
family is the Guttaviridae family, represented by new virus Sulfolobus – SNDV (Sulfolobulus
neozelandicus droplet-shaped virus), which in the cross-section reveals round, doublestranded dsDNA (www.ictvonline.org). The viruses morphologically resemble bacteriophage
T and lambda (Zilling et al. 1996). The fourth family is Rudiviridae, which includes virus SIRV1
(Sulfolobus Virus 1) (www.ictvonline.org). Many publications state that there is yet another
family (so far not considered in the ordination ICTV 2009), namely Bacilloviridae. The family
is represented by viruses TTV4 (Thermoproteus virus 4) and SIRV, which feature doublestranded, linear DNA (Zilling et al. 1996).
Very many studies on bacteriophages of the Archaea focused on their similarities
to bacteriophages of eubacteria (Prangishvili et al. 2006a). As it turned out, Euryarchaeota
viruses are morphologically similar to many bacteriophages, while viruses of hyperthermophilous
Crenarchaeota reveal unusual variety of morphology, by which they morphologically differ from
the known bacteriophages. It was determined that, according to such observations, Euryarchaeota
genome encodes many proteins similar to capsid proteins of bacteriophages of eubacteria.
Initial analysis of the genes of the Archaea bacteria from Crenarchaeota phylum did not reveal
any relations to bacteriophages, and also revealed very low number of genes encoding
bacteriophage proteins. Further studies on conservative domains of the Crenarchaeota genome
revealed that many genes had not been originally noticed. The presence of many proteins
homological to bacteria was detected, and even of Eucaryota, which allowed for determining
their probable function (Prangishvili et al. 2006a). In the course of further studies, it was
determined that viruses infecting bacteria from the Archaea domain are not evolutionarily
related to animal, plant viruses and bacteriophages. It was evidenced that their evolution
and creation of new species is related to independent growth in host genes or to a more
complex transfer of genes from other prokaryotes (Prangishvili et al. 2006a). The cycle
of infecting bacterial cells of the Archaea domain is still very little known (Prangishvili et al.
2006a). Many researchers focus on the analysis of lytic effect of viruses on the cells
of the Archea (Bize et al. 2009). At the example of SIRV2 virus (Islandicus archaeal sulfolobus
virus 2), the mechanism of releasing viruses from the Archaea cell was recognised (Bize et al.
Vruses of Archaea
69
2009). It was determined that it releases from the cell by switching on special cellular
structures in the Archaea (Bize et al. 2009). Its large size and pyramidal shape facilitate cutting
the Archaea cells across in several positions, including the rupture of layer S, while large
openings are formed in the membrane, through which virions enter inside the cell (Bize et al.
2009). The largest clusters of the Archaea viruses are observed in the cell cytoplasm, as well
as in the area of main chromosomes. Flow cytometry proved to be very useful for visualisation
of bacterial virus DNA. It was determined that these are lytical viruses causing degradation
of the Archaea cell (Bize et al. 2009), although according to Zilling et al. (Zilling et al. 1996),
there are also viruses that while infecting an Arachaea cell do not cause their lesion,
and among such mild viruses is TTV1 (Thermoproteus virus 1), which after replication allows
for cell survival.
To conclude, it must be determined that viruses infecting bacteria from the Archaea domain
are a very interesting object of study. Owing to their presence in extreme environments, they
contradict small biodiversity in the environments not inhabited by eukaryotic organisms
(Prangishvili et al. 2006). Owing to their properties, they can survive in unusually alkaline, acid
and hot environments. Increasingly, such properties are used in the chemical and biological
technology (Evans 2009). The future of nanotechnology involves the use of bacteriophages
of the Archaea e.g. the use of SIRV2 virus (Sulfolobus virus 2) owing to its resistance to high
temperatures and acid environment (Evans 2009).
REFERENCES
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of Systematic Bacteriology (ed.2). Wyd. Springer, New York.
Bize A., Karlsson E.A., Ekefjärd K., Quax T.E., Pina M., Prevost M.C., Forterre P., Tenaillon O.,
Bernander R., Prangishvili D. 2009. Unique virus release mechanism in the Archaea. Proc. Natl.
Acad. Sci. USA. 106, 11306–11311.
Evans D.J. 2009. Eksploitation of plant and archaeal viruses in bionanotechnology. Biochem. Soc.
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Pagaling E., Haigh R.D., Grant W.D., Cowan D.A., Jones B.E., Ma Y., Ventosa A., Heaphy S. 2007.
Sequence analysis of an archaeal virus isolated from a hypersaline lake in Inner Mongolia,China.
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Pietilä M.K., Roine E., Paulin L., Kalkkinen N., Bamford D.H. 2009. An ssDNA virus infecting Archaea:
a new lineage of viruses with a membrane envelope. Mol. Microbiol. 72, 307–319.
Prangishvili D., Forterre P., Garrett RA. 2006. Viruses of the Archaea: a unifying view. Nat. Rev.
Microbiol. 4, 837–48.
Prangishvili D., Garrett R.A., Koonin E.V. 2006a. Evolutionary genomics of archaeal viruses: unique
viral genomes in the third domain of life. Virus Res. 117, 52–67.
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Rice G., Tang L., Stedman K., Roberto F., Spuhler J., Gillitzer E., Johnson J.E., Douglas T.,
Young M. 2004. The structure of a thermophilic archaeal virus shows a double-stranded DNA viral
capsid type that spans all domains of life. PNAS. 20, 7716–7720.
Wolinowska R. 2008. Plazmidy Archaea. Post. Mikrobiol. 47, 457–463 [in Polish].
Zillig W., Prangishvilli D., Schleper C., Elferink M., Holz I., Albers S., Janekovic D., Götz D. 1996.
Viruses, plasmids and other genetic elements of thermophilic and hyperthermophilic Archaea.
FEMS Microbiol. Rev. 18, 225–236.
WIRUSY BAKTERII ARCHEA
Streszczenie. Praca przedstawia charakterystykę bakteriofagów infekujących bakterie z domeny Archea.
Bakterie te zasiedlają ekstremalne środowiska, o bardzo dużym zasoleniu, zakwaszeniu oraz wysokiej
temperaturze. Dotychczas zidentyfikowano około 50 bakteriofagów zakażających Archaea, które
posiadają zdolności pozwalające im przetrwać w ekstremalnych warunkach. Wśród tych wirusów
bakteryjnych najwięcej, opisano bakteriofagów infekujących bakterie należące do gromady Euryarchaeota,
oraz gromady Crenarchaeota. Biorąc pod uwagę systematykę, stwierdzono, że bakteriofagi infekujące
bakterie Archea należą przede wszystkim do rzędu Caudovirales, rodziny Myoviridae i Siphoviridae,
a także do rodziny Fusseloviridae, Lipothrix, Guttaviridae i Rudiviriae.
Słowa kluczowe: bakteriofagi, bakterie, środowisko, ekstremalne warunki
ADVANCES IN AGRICULTURAL SCIENCES
Adv. Agric. Sci. 2011, XIV (1–2), 71–78
Joanna Śliwa-Dominiak, Wiesław Deptuła
THE CHARACTERISTICS OF SELECTED ENVIRONMENTAL BACTERIA
Department of Microbiology and Immunology, University of Szczecin,
Felczaka 3c, 71–412 Szczecin, Poland, e-mail: [email protected]
Abstract. The environment is the habitat of many microorganisms. Both water and soil are places
inhabited by them in large numbers and high diversity. This study presents the characteristics of selected,
interesting environmental bacteria, namely filamentous bacteria and actinomycetes. According to the
applicable ordination, filamentous bacteria belong to two phylum: Proteobacteria and Bacteroidetes.
These are Gram-negative microorganisms, typical of water environment, where they are present in large
numbers. They have the shape of longitudinal bacilli or rods, although sometimes they may be cylindrical.
They are always covered with a sheath, which may be incrusted with iron or manganese oxide. In the
water environment, they as regulators of many transformations, including participate in the self-cleaning
process of water reservoirs. Actinomycetes are bacteria which resemble fungi due to their “build”. These
are Gram-positive, aerobic in majority, acid-resistance, built of sporangia forming “mycelia”, from which
sporangiophores may extend, where sporangia with spores are formed to serve for spreading. Such
bacteria may also proliferate through fragmentation of filaments, or by generation of spores. Soil
environment is their characteristic place of habitat.
Key words: environment, water, soil, filamentous bacteria, actinomycetes
Water and soil are perfect habitats for microorganisms which occur there in large numbers
and high diversity (Śliwa-Dominiak et al. 2009). Water environment is a habitat for bacteria,
which may be divided into two main groups: autochthonous (local) microorganisms, namely
genera and species for whom water is the natural habitat for living and development;
and allochthonous (alien, acquired), namely genera and species for whom water is not a normal
environment, and which are transferred to waters from soil, air, plants, animals, human
and animal faeces, wastewater and other environments (Śliwa-Dominiak et al. 2009). Soil
is formed of solid mineral colloids to which microorganisms and organic colloids adhere, as present
in the form of humus compounds and soil dilutions created by water with diluted organic
and mineral compounds and gases (Schlegel 1996). Microorganisms constitute the quickestreacting component of soil biotic community, which is conditioned with the diversity of biochemical
functions proper to them and the unusually high physiological activity (Schlegel 1996). Soil is
principally accommodated by aerobic microorganisms, hence most bacteria are present in the
J. Śliwa-Dominiak, W. Deptuła
72
surface layer of soil, up to the depth of 30 cm, where air penetrates. In deeper layers, their
number quickly decreases (Schlegel 1996). Among the microorganisms rather generally present
in water and soil, there are ones referred to as filamentous bacteria and actinomycetes.
Filamentous bacteria are an interesting group of microorganisms, as they create a diversified
group of bacteria. Such microorganisms are present in large numbers in the water environment,
in the form of flocculent clusters. They are Gram-negative, with the shape of filament, usually
comprising cells of rod or cylindrical shape, covered with a sheath. A typical sheath is transparent,
although there are sheaths with varied colour, e.g.: yellow or brown, depending on whether
they contain sediments of iron or manganese oxide. Often, sheaths resemble ducts or tubes
usually, although not always, containing cells. In some bacteria, the sheath is so fine and strongly
connected with the cell that it is hard to detect under the microscope. Most filamentous bacteria
are capable of movement, which is done using cilia or flagella. Filamentous bacteria proliferate
in a way typical of bacteria, namely by division. Most of such bacteria are aerobic
and chemoorganotrophic microorganisms. The role of such bacteria in the water environment
frequently is the role of regulators of many transformations, such as biodegradation of organic
compounds and oxidation of mineral compounds. Furthermore, they participate in the selfcleaning process of water reservoirs.
Considering bacteriological taxonomy according to the first issue of Bergey’s manual
of systematic bacteriology ordination (Bergey et al. 1984), which divides bacteria into 33 section,
it must be stated that filamentous bacteria belong to section 22. The latest ordination (Bergey
et al. 2001) places the bacteria among two phylum: Proteobacteria and Bacteroidetes.
Notwithstanding such ordinations (Bergey et al. 1984, Bergey et al. 2001) filamentous bacteria
are represented by genera: Sphaerotilus, Leptothrix, Clonothrix, Haliscomenobacter, Crenothrix,
Lieskeella, Phragmidiothrix.
Sphaerotilus genus principally comprises simple rods, usually placed in single chains,
surrounded with a sheath of even width, which may be connected via special fixtures
to the walls in water reservoirs, to underwater plants, rocks or other surfaces. Sheaths are
usually thin, not incrusted with iron or manganese oxide. Optimal temperature for growth
of bacteria from this genus ranges from 20 to 30ºC. Such bacteria can be isolated from current
water or wastewater sludge. These are Gram-negative bacteria from the chemoorganotrophic
group. The bacteria feature aerobic metabolism and never participate in the fermentation
process. They can grown at the very low concentration of soluble oxygen (below 0.1 mg/1).
A typical species is Sphaerotilus natans, for which the living environment is fresh water highly
polluted with sewerage or industrial wastewater from various branches of agricultural industry.
The characteristics of selected environmental bacteria
73
Leptothrix genus principally includes simple rods in the form of chains surrounded with
a sheath, or in the form of freely floating single cells or diplococci, which move using a single
polar filament. Sheaths of such bacteria have a clear tendency to saturate or cover with iron
oxide or manganese oxide. Optimal temperature for growth of most strands of these
filamentous bacteria amounts to approx. 25ºC. Most frequently, they were isolated from current
water enriched with iron and manganese, not polluted with sewerage/wastewater. Bacteria
of Leptothrix genus are Gram-negative, belonging to chemoorganotrophs, with aerobic
metabolism, not fermentation-based. Growth of such bacteria may occur at low oxygen
concentration. A typical species of this bacterial genus is Leptothrix ochraceae, which probably
occurs in waters containing iron, yet poor with easily degrading organic matter.
Clonothrix genus includes bacteria the cells of which are cylindrical, and which occur,
similarly as the previous ones, in the form of filaments that may either be fixed to the medium,
or in the free state. They are surrounded by a more-or-less visible sheath incrusted with iron
oxide or manganese oxide, which compounds give them yellow-brown colour. Filaments
of such bacteria narrow to the end, and the ends may be single or seemingly bifurcated. Such
bacteria were not cultivated in laboratory conditions. These are Gram-negative bacteria,
probably belonging to chemoorganotrophs, with aerobic metabolism. Such bacteria do not
develop on artificial medium. Clonothrix fusa is a typical species, present in iron ponds.
Haliscomenobacer genus includes filamentous bacteria that usually have the shape of thin
rods forming chains, surrounded by narrow, hardly visible sheaths. The sheaths are not
incrusted with either iron oxide, or with manganese oxide. Chains formed by cells surrounded
with sheaths may create branches, which may damage sheaths that quickly regenerate.
As compared to the main filament, later branches are usually short. Cells outside sheaths are
rarely visible. Optimal temperature for growth of bacteria from this genus amounts to approx.
260C. They are most frequently isolated in large numbers from sewerage. Similarly as previous
genera of filamentous bacteria, they are Gram-negative and belong to aerobic chemoorganotrophs. For their development, thymine and vitamin B12 are necessary. Haliscomenobacter
hydrossis is a typical species of this bacterial genus, present in large numbers in waters
polluted with sewerage.
Crenothrix genus features cylindrical or disk-shaped cells, forming filaments strongly affixed
to the medium. Very thin sheaths surrounding filaments may be colourless, or may be incrusted
with iron oxide or manganese oxide. Such bacteria were not cultivated in laboratory conditions,
hence there is little information regarding their physiology and biochemistry. It is suspected
that, similarly as other filamentous bacteria, they are Gram-negative, aerobic chemoorganotrophs.
J. Śliwa-Dominiak, W. Deptuła
74
Sometimes, they reveal sliding motion. They are present in standing waters with low concentration
of organic matter, iron ions and traces of methane. Typical species include Crenothrix polyspor.
Lieskeella genus includes bacteria with rod-like cells, which create chains (filament chains).
Usually, such filament chains are wrapped around one filament, forming a double spiral
surrounded with a yellowish sheath incrusted with ferrous hydroxide. Such bacteria were not
cultivated in laboratory conditions. It is suspected that these are Gram-negative aerobic
chemoorganotrophs. Lieskeella bifida is a typical species isolated from the bottom of ponds
among marshes.
Phragmidiothrix genus includes bacteria of disk-shaped cells characterised with varying
size. The cells of such bacteria are laid as colourless, non-branched filaments affixed
to the medium and forming greyish-white tufts. Surrounded by very thin, delicate, jelly-like
and colourless sheaths that are not incrusted with iron or manganese compounds. Similarly
as the ones described above, they are not cultivated in pure cultures on microbiological media,
hence there is little information regarding this type of bacteria. It is assumed that these are
anaerobic microorganisms that tolerate hydrogen sulphide. Phragmaidiothrix multiseptata,
present in the north-Adriatic waters, is a typical species of this genus of filamentous bacteria.
Actinomycetes (Actinomycetales) is a large group of environmental microorganisms, initially
wrongly considered as filamentous fungi, due to some similarity. However, finding out
the details of their structure revealed their classification as prokaryotic organisms. This
is testified to by the structure of genetic apparatus, composition of cellular wall, lack of steroids
in the cytoplasmic membrane, similarity of cilia structure, as well as presence of chemoautotrophic
species, namely organisms using chemical energy obtained from oxidation of inorganic
compounds. Such bacteria are mainly present in soil, are Gram-positive, and their Latin name
derives from the first described species Actinomyces bovis, causing actinomycosis in cattle –
a disease of dense connective tissue (gums) and bone tissue (jaw) (Holt 1994).
While presenting the taxonomy of this bacterial group, it must be noticed that in the first
edition of Bergey’s ordination from the years 1984–1989 (Bergey et al. 1984), where bacteria
were divided into 33 sections, actinomycetes were classified among bacteria from sections 26–33,
forming a group of microorganisms characteristic of soil environment. According to the same
ordination of 1995 (Holt 1994), 35 taxonomic groups were identified, among which groups 16,
23–29 and partly 31–35 are bacteria present in the soil. In the present ordination (Bergey et al.
2001), actinomycetes principally belong to two phylum: Firmicutes and Actinobacteria.
Actinomycetes develop in the form of “mycelium" and produce substances preventing
the development of many harmful fungi and bacteria (Kotełko et al. 1977). They resemble
fungi, as they create a “mycelium”, the strands of which are, however, much thinner. Their
The characteristics of selected environmental bacteria
75
diameter does not exceed 1 μm (Kotełko et al. 1977). From the “mycelium”, there may grow
sporangiophores, where sporangia form with spores for microorganisms’ spreading (Kotełko
et al. 1977). In another case, the “mycelium” of those bacteria in old colonies may fall apart into
rod-like cells, and then spores are not created (Kotełko et al. 1977). In such bacteria, growth is
also recorded in the form of branched pseudo-mycelium, from which, owing to strand fusion,
yet not nucleoid fusion, heterokaryons may be created (Kotełko et al. 1977). Furthermore,
actinomycetes strands in pure cultures, sustained in the vegetative growth phase, may reveal
genetic changes, which probably result from nucleoid segregation in the homokaryotic strand
(Kotełko et al. 1977). It is worth adding that among prokaryotic organisms, heterokaryon
generation is an exception, while in actinomycetes this phenomenon is rather popular and well
known (Kotełko et al. 1977).
Actinomycetes grow well on simple media, such as agar, and are easily differentiated
by the manner of growth, formation of “mycelium”, which may be air-borne, substrate-based,
or by formation of sporangia and spores (Bergey et al. 1984, Schlegel 1996). Actinomycetes
similar to Nocardia may form both substrate-based and airborne “mycelium”, which in old
colonies falls apart into rod-like cells that do not produce spores (Bergey et al. 1984, Schlegel
1996). In turn, in actinomycetes of Streptomycetes genus, “mycelium” is always present,
strongly developed and featuring sporophores for spreading (Bergey et al. 1984, Schlegel
1996). Actinomycetes from Maduromyces genus form an internal “mycelium”, and a reproductive
mycelium (Bergey et al. 1984). In such bacteria, the internal mycelium is branched and features
no spores (Bergey et al. 1984). Such spores are present on the reproductive “mycelium”, which
is coloured from grey to brown, although it may also be yellow, red, blue, greenish or violet
(Bergey et al. 1984). Spore chains produced by actinomycetes of Maduromyces genus may be
simple or may form irregular coils, while their surface may be smooth or papillary (Bergey et al.
1984). Spores may also be formed on the internal “mycelium” (Bergey et al., 1984). This is so
in actinomycetes of Microbispora genus, where porophore growing on the internal “mycelium”
occurs on a broad base or a short sporophore (Bergey et al. 1984). Also in actinomycetes, one
may differentiate two types of spores, namely mobile and immobile spores (Bergey et al.
1984). Mobile spores are capable of movement owing to peritrichal flagella, and are present in
e.g. actinomycetes from Planomonospora or Spirillospora genera (Bergey et al. 1984). In turn,
immobile spores are present in actinomycetes e.g. from Microbispora or Streptosporangium
genus (Bergey et al. 1984). Actinomycete spores may also be of various shapes – from spherical
to elongated, and even claviform (Bergey et al. 1984). In actinomycetes from Planomonospora
genus, there are spores of cigar-shaped, cylindrical, mace-like shapes, while in actinomycetes
J. Śliwa-Dominiak, W. Deptuła
76
of Streptosporangium genus, they may be spherical, oval or rod-like (Bergey et al. 1984).
Actinomycete spores are usually not resistant to heat, yet resist drying (Schlegel 1996). The only
actinomycete forming spores resistant to high temperature is Thermoactinomycetes vulgaris
(Schlegel 1996). Actinomycetes also proliferate owing to fragmentation of strands, such as
Nocardia sp. or by production of spores, like Streptomyces (Kotełko et al. 1977). Proliferation
of various species and strands of actinomycetes is differentiated on the basis of colony shape,
colour, as well as size and smell, as well as on the basis of the type of sporophores produced
(Schlegel 1996).
A characteristic feature of actinomycetes is the odour of freshly ploughed soil in the spring,
which exactly comes from actinomycetes, and is caused by the substance referred to as
geosmin (Schlegel 1996).Optimal temperature for actinomycetes growth amounts to 20–30ºC,
while optimal pH – approx. 7 (Bergey et al. 1984). The exception is formed by actinomycetes
of Thermomonospora genus, which may grow in the temperature interval ranging from 40 to 48ºC
and environmental pH from 7 to 9 (Bergey et al. 1984). Many actinomycetes are capable
of degrading polysaccharides, as well as compounds that are hard to degrade, such as sterides,
cellulose, chitin, high fatty acids, or aromatic compounds (Schlegel 1996). One of the genera
of actinomycetes degrading cellulose includes Micromonospora actinomycetes that are popular
in the soil and in the rotting bottom sludge. Actinomycetes also show the capacity of producing
antibiotics, the example of which may be formed by actinomycetes of Streptomyces genus,
producing streptomycin and terramycin. Furthermore, e.g. Streptomyces actinomycetes
synthesise pesticides, insecticides, as well as antiviral compounds (Schlegel 1996). Such
bacteria are present in the soil, as well as in composts, manure, and on food products. Among
them, there are species that may enter into symbiosis with higher plants, as well as pathogens
causing diseases in humans and animals. Most of them is aerobic and acid-resistant,
and capable of degrading plant and animal debris, and capable of producing chemical
compounds, including antibiotics.
To conclude, it must be stated that the microorganisms described in this paper, namely
filamentous bacteria and actinomycetes, form very interesting objects, not visible to humans
with a “naked eye”. Such bacteria are typical of water and soil environment. They perform
many functions in the environment. Filamentous bacteria participate in the self-cleaning
processes of water reservoirs, while actinomycetes are characterised of performing various
biochemical processes, principally in the soil, e.g. degradation of polysaccharides, fatty acids,
or aromatic compounds, but also reveal the capacity of antibiotic production.
The characteristics of selected environmental bacteria
77
REFERENCES
Bergey D.H., Harrison F.C., Breed R.S., Hammer B.W., Huntoon F.M. 1984–1989. Bergey’s manual
of systematic bacteriology, ed.1. Springer, Nowy Jork.
Bergey D.H., Harrison F.C., Breed R.S., Hammer B.W., Huntoon F.M. 2001–2009. Bergey’s manual
of systematic bacteriology, ed.2. Springer, Nowy Jork.
Schlegel H.G. 1996. Mikrobiologia ogólna (wydanie drugie poprawione). PWN, Warszawa.
Holt J.G. 1994. Bergey’s manual of determinative bacteriology. Wyd. Williams & Willkins, Baltimore.
Kotełko K., Siedlaczek L., Lachowicz T. 1977. Biologia bakterii. PWN, Warszawa.
Śliwa-Dominiak J., Pawlikowska M., Deptuła W. 2009. Bakterie środowiska wodnego – wybrane dane.
Laboratorium 9, 56–59.
CHARAKTERYSTYKA WYBRANYCH BAKTERII ŚRODOWISKOWYCH
Streszczenie. Środowisko jest siedliskiem wielu drobnoustrojów. Zarówno woda, jak i gleba są miejscami
zasiedlanymi przez nie w dużej ilości i w bogatej różnorodności. Praca przedstawia charakterystykę
wybranych i zarazem ciekawych bakterii środowiskowych, to jest bakterii pochewkowych oraz promieniowców. Bakterie pochewkowe to zarazki należące według obowiązującej systematyki, w zasadzie
do dwóch gromad: Proteobacteria i Bacteroidetes. Są to mikroorganizmy Gram-ujemne, typowe dla
środowiska wodnego, w którym występują bardzo obficie. Mają kształt podłużnych laseczek lub pałeczek,
choć niekiedy mogą być cylindryczne. Otoczone są zawsze pochewką, która może być inkrustowana
tlenkiem żelaza lub manganu. W środowisku wodnym, pełnią one funkcje regulatorów wielu przemian,
w tym uczestniczą w procesie samooczyszczania zbiorników wodnych. Promieniowce to drobnoustroje,
które ze względu na swoją „budowę” przypominają grzyby. Są one Gram dodatnie, w większości tlenowce, kwasoodporne, zbudowane ze strzępek tworzących „grzybnie”, z których mogą wyrastać strzępki
zwane sporangioforami, na których tworzą się sporangia ze sporami, które służą do rozprzestrzeniania się
ich. Bakterie te mogą rozmnażać się także przez fragmentację nitek albo przez wytwarzanie zarodników.
Charakterystycznym miejscem ich występowania jest środowisko glebowe.
Słowa kluczowa: środowisko, woda, gleba, bakterie pochewkowe, promieniowce
ADVANCES IN AGRICULTURAL SCIENCES
Adv. Agric. Sci. 2011, XIV (1–2), 79–84
Małgorzata Pawlikowska, Wiesław Deptuła
RNA VIRUSES INFECTING FISH MARINE – SELECTED DATA
Department of Microbiology and Immunology, University of Szczecin
Felczaka 3c, 71-412 Szczecin, Poland, phone 91 444 1605, fax 91 444 1606
e-mail: [email protected]
Abstract. Viruses are structures present in all environments, including water environment. The environment is to be understood as the living environment or organisms in which they replicate, and are also
present in water alone, where viruses are capable of “surviving” for some time before they find a host. This
paper presents the characteristics of RNA viruses, both ssRNA with positive and negative polarity, as well
as dsRNA viruses, which infect sea fish, as they are slightly less recognised than DNA viruses.
Key words: fish, RNA, viruses
INTRODUCTION
Viruses present in marine waters are a source of various diseases in organisms living there,
for example they may impact on coral fading (Lohr et al. 2007), and are a cause of periodical
mass infections and deaths of both fish (Skall et al. 2005) and mammals (Jensen et al. 2002).
They infect cultures of salmons, shrimps, or oysters, causing significant economic losses
(Gomez et al. 2004, McLoughlin and Graham 2007), and for this reason constitute potential
risk factors for land-based animals and humans (Smith and Skilling 1980), although they
mainly control populations of marine organisms, creating their variety (Villarreal 2005).
The present study describes RNA viruses that infect sea fish. Among these viruses, there
are viruses with single stranded RNA (ssRNA), with positive and negative polarisation, and
double stranded RNA (dsRNA) (Table 1).
SSRNA VIRUSES WITH POSITIVE POLARITY
Among ssRNA viruses with positive polarity, capable of infecting fish, there are viruses
classified into Nodaviridae and Togaviridae families (Table 1).
M. Pawlikowska, W. Deptuła
80
Table 1. RNA viruses infected fishes
ssRNA(+)
Order
No order
Genetical
material
Family
Nodaviridae
ssRNA (-)
Mononegavirales
Togaviridae
No
order
dsRNA
No
order
Rhabdoviridae
Representative
BFNNV (Barfin Flounder
Nervous Necrosis Virus)
ACNNV (Atlantic cod
nervous necrosis virus)
DIEV (Dicentrarchus
labrax encephalitis virus)
JFNNV (Japanese
flounder nervous
necrosis virus)
LcEV (Lates calcarifer
encephalitis virus)
RGNNV (Redspotted
Grouper Nervous
Necrosis Virus)
SJNNV (Striped Jack
Nervous Necrosis Virus)
TPNNV (Tiger Puffer
Nervous Necrosis Virus)
SAV (Salmonidae
Alphavirus)
IHNV (Infectious
Heamtopoietic Necrosis
Virus)
HIRRV (Hirrame Virus)
SHRV (Snake Head
Rhabdovirus)
SVCV (Spring Viremia
Carp Virus)
EAV (Eel American
Virus)
VHSV (Viral Hemorrhagic
Septicemia Virus)
Host
Flounder (Pleuronectes sp.)
Atlantic cod (Gadus morhua)
Dicentrarchus labrax
(Dicentrarchus labrax)
Japanese flounder (Limanda
herzensteini)
Lates calcarifer (Lates calcarifer)
Redspotted grouper (Epinephelus
akaara)
Striped jack (Pseudocaranx
dentex)
Tiger puffer (Thamnacocnus
modestus)
Family Salmonidae
Atlantic cod, Pacific cods (Gadus
macrocephalus), Pacific herrings
(Clupea pallasii), Greenland halibut
(Reinhardtius hippoglossides),
Japanese flounder (Parachlithys
olivaceus), Amercian eel (Anguilla
rostrata)
Rabdovirus
Starry flounder (Platichtys
stellatus)
Orthomyxoviridae
ISAV (Infectious Salmon
Anaemia Virus)
Family Salmonicidae
Reovoridae
CSR (Chum Salmon
RNA virus)
Birnaviridae
genus Aquabirnavirus
Retroviridae
Retrovirus
Chum salmon (Oncyrhonchus
keta)
Atlantic salmon (Salmo salar), red
sea bream (Pagrus major),
American plaice (Hippoglossides
platessoides), Atlantic cods
(Gadus morhua)
Lemon shark (Negaprion
brevirostis), Puffer fish (Fugu
rubripes)
Winton et al. 1987, Herniou et al. 1998, Kitamura et al. 2000, Takano et al. 2001, Barker et al. 2002, Mjaaland
et al. 2002, Gagne et al. 2004, Gomez et al. 2004, Hoffmann et al. 2005, Nishizawa et al. 2005, Skall et al. 2005,
McLoughlin and Graham 2007, Nerland et al. 2007, Rise et al. 2008.
The Nodaviridae family. In this family, viruses infecting fish belong to the Betanodavirus
genus, and are a cause of viral nervous necrosis (VNN), also referred to as viral encephalopathy
and retinopathy (VER) (Thiery et al. 2004). The viruses have been divided into 8 groups
RNA viruses infecting fish marine – selected data
81
(clades), according to differences in the nucleotide sequence in T4 region of the capsid protein
(Gagne et al. 2004). These groups include the following viruses:
– Barfin Flounder Nervous Necrosis Virus (BFNNV),
– Atlantic cod nervous necrosis virus (ACNNV),
– Dicentrarchus labrax encephalitis virus (DIEV),
– Japanese Flounder Nervous Necrosis Virus (JFNNV),
– Lates calcarifer encephalitis virus (LcEV),
– Redspotted Grouper Nervous Necrosis Virus (RGNNV),
– Striped Jack Nervous Necrosis Virus (SJNNV),
– Tiger Puffer Nervous Necrosis Virus (TPNNV).
It must also be added that the genetic material of betanodaviruses was also identified
in round scad (Decapterus maruadsi), false kelpfish (Sebastiscus marmoratus), flounders
(Pleuronectes americanus), cultured Atlantic cod (Gadus morhua) and Atlantic halibut
(Hippoglossus hippoglossus) (Barker et al. 2002, Gomez et al. 2004, Nerland et al. 2007, Rise
et al. 2008). At present, it is assumed that diseases caused by betanodaviruses are recorded
in over 30 species of sea fish, included many cultured fish species (Cutrin et al. 2007).
The Togaviridae family. This family is represented by viruses from the Alphavirus genus
(Table 1), pathogenic to Salmonidae (McLoughlin and Graham 2007). It was evidenced that
sea lice, which are a reservoir of such viruses, are responsible for their transmission in salmonidae
(SAV) (McLoughlin and Graham 2007). Among alphaviruses in such fish, recognised viruses
include: SPDV (SAV1) – Salmonid Pancreas Disease Virus, SDV (SAV2) – rainbow trout
Sleeping Disease Virus, and NSAV (SAV3) – Norwegian Salmonid Alphavirus (McLoughlin
and Graham 2007).
SSRNA VIRUSES WITH NEGATIVE POLARITY
ssRNA viruses with negative polarity include the Rhabdoiviridae family belonging to the Mononegavirales order, and the Orthomyxoviridae family, not allocated to an order, which infect
marine organisms (Table 1).
The Rhabdoviridae family. Viruses belonging to this family are characterised with a broad
spectre of hosts; however, the ones present in the marine environment are only pathogenic
to fish, both wild and cultivated, and to marine mammals, and have been classified into
Novirhabdovirus and Vesiculovirus geni (Hoffmann et al. 2005). Viruses from the Novirhabdovirus
genus are pathogens for fish, and are represented by the infectious hematopoietic necrosis
virus (IHNV), Viral hemorrhagic septicemia virus (VHSV), Hirame rhabdovirus (HIRRV)
M. Pawlikowska, W. Deptuła
82
and Snakehead rhabdovirus (SHRV). In turn, representatives of the Vesiculovirus genus are
viruses also infecting fish, including Spring viraemia of carp virus (SVCV) or Eel American virus
(EAV) (Hoffmann et al. 2005). The VHSV virus is a major problem to rainbow trout cultures
in Europe, causing haemorrhages in many organs (Skall et al. 2005). For the first time, it was
isolated in Atlantic cod, and presently, it is also isolated in Pacific cods (Gadus macrocephalus),
Pacific herrings (Clupea pallasii), Greenland halibut (Reinhardtius hippoglossides), and Japanese
flounder (Parachlithys olivaceus) (Skall et al. 2005). The virus may also be transmitted by predating
from Pacific herrings onto other fish (Skall et al. 2005).
The Orthomyxoviridae family. This family includes Infectious Salmon Anaemia Virus
(ISAV), which infects both wild and cultivated salmonid fish, including Atlantic salmons, brown
trouts, trouts and herrings, resulting in pale gills, chronic anaemia, enlarged liver and spleen,
as well as necrosis of the liver (Mjaaland et al. 2002). The analysis of DNA sequence revealed
that European and Canadian strains of the ISAV virus differ, which points to significant
evolution, similarly as in the case of the influenza virus (Mjaaland et al. 2002).
DSRNA VIRUSES
Among the dsRNA viruses, only representatives of two families, Reoviridae and Birnaviridae
(Table 1), are capable of fish infections.
The Reoviridae family. The only representative of this family is the CSR virus from
the Aquareovirus genus, infecting the chum salmon (Oncorhynchus keta) (Winton et al. 1987).
The Birnaviridae family. Viruses from this family, belonging to the Aquabiranvirus genus,
are broadly spread and infect i.a. Atlantic salmon (Salmo salar), gilthead sea bream (Pagrus
major), American plaice (Hippoglossoides platessoides) and Atlantic cod (Gadus morhua)
(Kitamura et al. 2000, Takano et al. 2001), as well as flounders, American smelt (Osmerus
mordax), and Japanese amberjack (Seriola quinqueradiata) (Nishizawa et al. 2005). The best
recognised representative of the Aquabirnavirus genus is the infectious pancreatic necrosis
virus (IPNV) virus, which infects salmonid fish during their development in fresh water
(Romero-Bray et al. 2004). The IPN virus and serologically similar viruses present in many fish,
both living in fresh water and in the marine environment, allow to adopt a hypothesis that they
are the most widespread pathogens in the water fauna (Nishizawa et al. 2005).
RNA VIRUSES USING THE REVERSE TRANSCRIPTASE
This group of viruses includes viruses classified into the Retroviridae family (Table 1).
RNA viruses infecting fish marine – selected data
83
The Retroviridae family. In the case of viruses belonging to this family, the only genetic
material that could be isolated, as characteristic of this viral family, was of lemon shark (Negaprion
brevirostis) and tiger blowfish (Fugu rubripes) (Herniou et al. 1998). It is difficult to clearly
determine whether these fish species are typical hosts for these viruses, and the phylogenetic
analysis pointed to difference of the sequence obtained from the known sequences of retroviruses
present in animals (Herniou et al. 1998).
CONCLUSION
The data presented indicate that RNA viruses are very popular in the marine environment,
infecting many fish species. Many fish diseases, including among cultivated fish, caused
by RNA viruses, create an economic problem. This is a reason for the need to have a closer
look at viruses present in marine waters, including via research resulting not only in virus
detection, but also in determination of viral interactions with the hosts. Such research could
help to prevent many diseases, and to develop biological preparations, e.g. vaccines, to be applied
in cultures of infected fish.
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Thiery R., Cozien J., de Boisseson C., Kerbart-Boscher S., Nevarez L. 2004. Genomic classification
of new betanodavirus isolates by phylogenetic analysis of the coat protein gene suggests a low
host-fish species specificity. J. Gen. Virol. 85, 3079–3087.
Villarreal L.P. 2005. Viruses and the evolution of life. ASM Press, Washington.
Winton J.R., Lannan C.N., Fryer J.L., Hedrick R.P., Meyers T.R., Plumb J.A., Yamamoto T. 1987.
Morphological and biochemical properties of four members of a novel group of reoviruses isolated
from aquatic animals. Microbiol. Mol. Biol. Rev. 68, 353–364.
WIRUSY RNA INFEKUJĄCE RYBY MORSKIE – WYBRANE DANE
Streszczenie. Wirusy są strukturami, występującymi we wszystkich środowiskach, w tym także w środowisku wodnym. Środowisko to należy rozumieć jako środowisko życia organizmów, w których replikują,
jak też występują w samej wodzie, w której wirusy są w stanie „przetrwać” przez pewien czas, zanim np.
znajdą gospodarza. W niniejszej pracy przedstawiono charakterystykę wirusów RNA, zarówno ssRNA
o polarności dodatniej i ujemnej oraz wirusy dsRNA, infekujące ryby morskie, jako że są one nieco słabiej
poznane niż wirusy DNA.
Słowa kluczowe: ryby, RNA wirusy
ADVANCES IN AGRICULTURAL SCIENCES
Adv. Agric. Sci. 2011, XIV (1–2), 85–90
Małgorzata Pawlikowska, Wiesław Deptuła
RNA REGULATION OF BACTERIAL VIRULENCE – SELECTED DATA
Department of Microbiology and Immunology, University of Szczecin
Felczaka 3c, 71-412 Szczecin, Poland, phone: 91 444 1605, fax 91 444 1606
e-mail: [email protected]
Abstract. Pathogenic bacteria are a cause of many mortal diseases. For researchers, identification
and understanding of pathogenicity mechanism is a challenge. One of the paths for virulence occurrence
is regulated by RNA. This study presents the mechanism for regulation and expression of some virulence
factors by RNA, namely 5’ UTR fragments, riboswitches, and small non-coding RNA (sRNA).
Key words: RNA, 5’ UTR, riboswitches, sRNA
INTRODUCTION
During an infection, pathogenic bacteria must be capable of expressing their virulence
genes and of surviving in the host’s cells. The coordination of expression of genes in charge
of virulence factors and of environmental signals is the task of the so-called regulatory cascade.
Such regulation involves many proteins and mRNA which allows pathogens for metabolic
adaptation during an infection. Among the basic regulatory elements within mRNA, one can
differentiate 5’ UTR regulators, including RNA switches (riboswitches) and small non-coding
RNA (sRNA) (Winkler et Breaker 2005, Coppins et al. 2007).
5’ UTR REGULATORS
These RNA fragments are a part of untranslated region at the 5’ end of mRNA (5’ UTR),
used by bacteria, contrary to other organisms, to modify gene expression depending on the
temperature, pH, and the presence of metabolites (Winkler et Breaker 2005, Coppins et al.
2007, Gripenland et al. 2010). The 5’UTR region is located between the place of transcription
start and the start codon in mRNA. It is a region of varying length, containing several base
pairs. The transcription process may occur on the basis of various promoters, which allows
for formation of many potential 5’ UTR fragments and gives many post-transcription regulations
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(Loh et al. 2006). In pathogenic bacteria, they are used as factors modifying gene expression
on the basis of changes to temperature, pH, and the presence of metabolites (riboswitches).
The bacteria have a mechanism for temperature regulation operating like proteins,
a the DNA and RNA level, yet most frequently thermal sensors operate at the RNA level
and change the temperature directly (Hurme and Rhen 1998). This mechanism is important
to bacteria which need to change gene expression in response to the host’s temperature. This
is what happens in Listeria monocytogenes, where the role of thermal sensor is performed
by 116-nucleotide 5’ UTR fragment above the pfrA gene of mRNA, coding the sequence
forming additional structure in lower temperature, masking SD sequence (in charge of mRNA
attachment to the 30S ribosome unit), and thus inhibiting translation. An alternative additional
structure is formed at the temperature of 37ºC, which uncovers the SD region, and starts
translation and activation of transcription of the protein regulating listeriosin (PrfA) (Johansson
et al. 2002). PrfA is a necessary virulence factor of L. monocytogenes, which activates
virulence genes expression coding adhesive phagosomal escape factor (listeriolisin O),
and the factor modulating immunological response (Freitag et al. 2009). Also in Yersinia pestis,
the thermal sensor was found, which controls the LcrF virulence factor expression, which
occurs exclusively at the temperature of 37ºC and activates YopE expression, which blocks
phagocytosis (Fällman and Gustavsson 2005).
In the case of gene expression control with the change of pH, alx gene was found
in Escherichia coli, which codes the probable transporter involved in resistance to tellurium
(antibacterial factor), and its expression occurs in high alkalinity (Nechooshtan et al. 2009).
At pH 7.0, transcription of 5’UTR proceeds correctly, while in the case of pH change to alkaline,
formation of translation inactive structures occurs, and RNA polymerase pauses in two different
places of 5’UTR in order to stop the generation of inactive proteins (Nechooshtan et al. 2009).
RNA SWITCHES (RIBOSWITCHES)
Riboswitches are broadly spread among bacteria, archea and some eucarionta (funghi,
plants), which testifies to their significant biological importance (Nudler and Mironov 2004).
In some bacteria, riboswitches are multiple, e.g. in Bacillus subtilis they regulate 69 genes,
namely as much as 2% genome (Mandal et al. 2003). These are mRNA fragments in the 5’UTR
region. Their mechanism of action results from their attachment to particular ligands, which
may include aminoacids (e.g. lysine), nucleotides (e.g. guanin or adenin), and sugars (e.g.
glucosamino-6-phosphate), as they modify the biosynthesis expression and protein transport
for such ligands. The binding of a ligand to riboswitch causes the latter’s structural changes,
which results in a change of RNA polymerase capacity to continue the transcription process,
RNA regulation of bacterial virulence – selected data
87
or a change of mRNA translation capacity. Such binding occurs at the site referred to as RBS
(ribosome binding site), located at the UTR region, which is the place of mRNA binding
to the ribosome (Winkler et Breaker 2005, Coppins et al. 2007, Gripenland et al. 2010). One
class of riboswitches has been identified as controlling expression below the gene, switching
on genes important to DNA absorption, mobility and pathogenicity (Vibrio cholerae, Clostridium
difficile, Bacillus cereus), by binding the second messenger cyclic di-GMP (c-di-GMP)
(Sudarsan et al. 2008). An example of negative regulation by c-di-GMP is formed by riboswitch
situated in front of gbpA gene, which codes N-acetyl-glucosamine-binding protein A (GbpA),
important to Vibrio cholerae in colonisation of human intestines (Kirn et al. 2005).
SMALL NON-CODING RNA SPECIES
Small non-coding RNA species (sRNAs) occur in Bacteria, Arachea, and eukaryotes,
as elements regulating many biological processes (Storz et al. 2005). In bacteria, sRNAs
coordinate adaptation processes in response to environmental changes, integrate environmental
signals and control gene expression (Wassarmann 2002, Repoila et al. 2003, Gottesmann 2004).
sRNAs regulate gene expression both by evaporation to mRNA, impacting on its stability,
via translation and by binding to proteins and changing their function (Storz et al. 2005).
Participation of sRNAs in control and regulation of pathogenicity has been evidenced
in Staphylococcus aureus (Huntzinger et al. 2005, Novick 2006), Pseudomonas aeruginosa
(Heurlier et al. 2004), Vibrio cholerae (Miller et al. 2002) and Chlamydia trachomatis
(Grieshaber et al. 2006).
Virulence factors in Staphylococcus aureus principally include toxins, exoenzymes
and surface proteins coded by the agr system (Novick 2006). The system comprises two
divergent transcription units, RNAII and RNAIII. RNAII codes two main components (AgrA –
response regulator, AgrC – kinase sensor), AGRD propeptide and AgrB peptidase. In turn,
RNAIII is the first described regulatory sRNA, containing 514 nucleotides divided into
14 structures with double function: coding 26 aminoacids, δ-hemolysine (hld), and acting
as regulatory sRNA controlling virulence (Novick 2006). RNAIII is capable of binding to at least
three fragments (targets) of mRNA: hla mRNA coding α-hemolysine, spa mRNA coding protein
A, and rot mRNA coding transcription factor Rot (Huntzinger et al. 2005). This agr-dependent
expression of virulence factors (adhesine, haemolysin, protease, degradation enzymes)
is subject to various signals, including the number of cells with the growing cell number, also
the volume of RNAIII increases, which results in decrease in adhesine expression and
activation of haemolysin translation (Novick 2006). In Pseudomonas aeuroginosa, pathogenicity
is based on expression regulation of Type III secretion system, properties of the biofilm created
M. Pawlikowska, W. Deptuła
88
(adherence) and large volume of N-Acyl homoserine, lactone-regulated exotoxins, and secondary
metabolites (Heurlier et al. 2004). The main role is played by proteins RsmY, RsmZ and RsmA,
which inhibit the activity of CsrA protein responsible for carbon metabolism; furthermore, they
are in charge of biofilm formation, quorum sensing and type III secretion (Heurlier et al. 2004).
Vibrio cholerae, as environmental and pathogenic bacterium, has the capacity of adapting to
various ecological niches, owing to multiple QSS system (quorum sensing system) and seven
sRNAs controlling virulence and biofilm formation (Miller et al. 2002). The main elements
regulating virulence include Qrr1-4, which act via LuxO and LuxU (kinases responsible
for luminescence), although virulence regulation is still under research (Miller et al. 2002). Also
in Chlamydia trachomatis, there is IhtA sRNA controlling the development cycle, which
is regulated by two histone-like proteins Hc1 and Hc2 (Grieshaber et al. 2006). Upon
the transformation of elementary body (EB) into reticular body (RB) inside the infected cell,
the volumes of IhtA sRNA increase and the Hc1 protein synthesis decreases. Reverse
situation takes place in the case of transformation of RB into EB (decrease in IhtA sRNA
volume, synthesis of Hc1 protein) (Grieshaber et al. 2006).
CONCLUSION
The presented data indicate that the RNA virulence factor regulation path is very popular.
Regulation of the expression of genes coding virulence factors depends on temperature or pH
changes. This is important for intracellular bacteria, such as Listeria monocytogenes or Yersinia
pestis, which are also present in external environment, while after entering human body
at the temperature of 37ºC start producing virulence factors (listeriosin O, LcrF). Another
regulator involves riboswitches, which after binding to a ligand impact on translation change.
This happens e.g. in Vibrio cholerae, Clostridium difficile, Bacillus cereus. Also, small noncoding RNA fragments (sRNA), by binding to mRNA, cause formation of toxins or enzymes
in Staphylococcus aureus, Pseudomonas aeruginosa and Vibrio cholerae, or control
the development cycle of Chlamydia trachomatis.
As evidenced by the examples presented, virulence regulation mechanisms are an interesting
element of pathogenic bacteria biology, as well as an interesting field for researchers who
commence learning about RNA’s role in virulence.
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Fällman M., Gustavsson A. 2005. Cellular mechanisms of bacterial internalization counteracted by Yersinia.
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pathogen. Nature Rev. Microbiol. 7, 623–628.
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of the histone-like protein Hc1 in Chlamydia trachomatis. Mol. Microbiol. 59, 541–550.
Gripenland J., Netteling S., Loh E., Tiensuu T., Toledo-Arana A., Johansson J. 2010. RNAs:
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Heurlier K., Williams F., Heeb S., Dormond C., Pessi G., Singer D., Camara M., Williams P., Haas D.
2004. Positive control of swarming, rhamnopolipid synthesis, and lipase production by the posttranscriptional RsmA/RsmZ system in Pseudomonas aeuroginosa PAO1. J. Bacteriol. 186, 2936–2945.
Huntzinger E., Boisset S., Saveanu C., Benito Y., Geissmann T., Namane A., Lina G., Etienne J.,
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to. Mol. Microbiol. 30, 1–6.
Johansson J., Mandin P., Renzoni A., Chiaruttini C., Springer M., Cossart P. 2002. An RNA
thermosensor controls expression of virulence genes in Listeria monocytogenes. Cell 110, 551–561.
Kirn T.J., Jude B.A., Taylor R.K. 2005. A colonization factor links Vibrio cholerae environmental
survival and human infection. Nature 438, 863–866.
Loh E., Gripenland J., Johansson J. 2006. Control of Listeria monocytogenes virulence by 5’-untranslated
RNA. Trends Microbiol. 14, 294–298.
Mandal M., Boese B., Barrick J.E., Winkler W.C., Breaker R.R. 2003. Riboswitches control fundamental
biochemical pathways in Bacillus subtilis and other bacteria. Cell 113, 577–586.
Miller M.B., Skorupski K., Lenz D.H., Taylor R.K., Bassler B.L. 2002. Parallel quorum sensing
systems converge to regulate virulence in Vibrio cholera. Cell 110, 303–314.
Nechooshtan G., Elgrably-Weiss M., Sheaffer A., Westhof E., Altuvia S. 2009. A pH-responsive
riboregulator. Genes Dev. 23, 2650–2662.
Novick R.P. 2006. Staphylococcal pathogenesis and pathogenicity factors: genetics and regulation.
In Gram positive pathogens; edn. 2 Edited by Fischetti A., Novick R.P, Feretti J., Portnoy D., Rood
J. ASM Press, 496–516.
Nudler E., Mironov A.S. 2004. The riboswitch control of bacterial metabolism. Trends Biochem. Sci.
29, 11–17.
Repoila F., Majdalani N., Gottesman S. 2003. Small non-coding RNAs, co-ordinators of adaptation
processes in Escherichia coli: the RpoS paradigm. Mol. Microbiol. 48, 855–861.
Storz G., Altuvia S., Wassarmann K.M. 2005. An abundance of RNA regulators. Annu. Rev. Biochem.
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Sudarsan N., Lee E. R., Weinberg Z., Moy R. H., Kim J. N., Link K. H., Breaker R. R. 2008.
Riboswitches in Eubacteria sense the second messenger cyclic Di-GMP. Science 321, 411–413.
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REGULOWANIE WIRULENCJI BAKTERII PRZEZ RNA – WYBRANE DANE
Streszczenie. Bakterie chorobotwórcze są przyczyną wielu śmiertelnych chorób. Wyzwaniem dla
naukowców jest poznanie i zrozumienie mechanizmów patogenności. Jedna z dróg powstawania
wirulencji regulowana jest przez RNA. W tej pracy przedstawiono mechanizm regulacji i ekspresji
niektórych czynników wirulencji przez RNA, czyli fragmenty 5’ UTR, ryboprzełączniki i małe niekodujące
RNA (sRNA).
Słowa kluczowe: RNA, 5’ UTR, ryboprzełączniki, sRNA
ADVANCES IN AGRICULTURAL SCIENCES
Adv. Agric. Sci. 2011, XIV (1–2), 91–103
Agata Mękal, Beata Tokarz-Deptuła, Alicja Trzeciak-Ryczek, Wiesław Deptuła
COMPLEMENT AND PROPERDIN, ELEMENT OF NON-SPECYFIC HUMORAL
IMMUNITY – IMPORTANT ELEMENT OF INNATE (NATURAL) IMMUNITY
Department of Microbiology and Immunology, University of Szczecin,
Felczaka 3c, 71-412 Szczecin, Poland, e-mail: [email protected]
Abstract. Complement system and properdin are important elements of non-specific immunity, defined
also as innate or natural immunity. Its main role is associated with effective recognition and elimination
of various pathogens. This paper describes the selected issues regarding the functioning of complement
system and properdin, namely activation pathways and regulating mechanisms. It explains their role
in innate immunity, as well as in adaptive immunity. This study gives examples of diseases associated
with the dysfunctions of these elements, and characterizes some mechanisms of pathogens that may
inhibit complement activity.
Key words: complement system, properdin, innate immunity
INTRODUCTION
Owing to the unique complexity and efficiency of the immune system in mammals,
it is possible to recognize non-self, potentially dangerous particles and self structures, which
ensures macroorganism’s protection against various microorganisms, including bacteria and
viruses. Complement system with properdin are one of the most important elements of nonspecific immunity, presently defined as natural or innate immunity, which significantly
contribute to effective recognition and elimination of pathogens (Hourcade 2006, Gołąb et al.
2007, Deptuła et al. 2008).
CHARACTERISTICS OF COMPLEMENT SYSTEM
Complement system comprises about 35 proteins with molecular weight of from 8 to 410 kDa,
present in the form of inactive precursors in serum and in various organs and tissues, including
liver, spleen, bone marrow, lungs, and adipose tissue (Kowalski 2000, Gołąb et al. 2007,
Chapel et al. 2009). Its function is principally related to opsonisation, chemotaxis, destroying
and killing microorganisms, elimination of immunological complexes, as well as initiation
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A. Mękal, B. Tokarz-Deptuła, A. Trzeciak-Ryczek, W. Deptuła
of local inflammation reactions Carroll 2004, Gołąb et al. 2007, Le Friec and Kemper 2009).
Initially, it was believed that complement, as a component of non-specific humoral immunity,
is an element of exclusively natural (innate) immunity, constituting the first line of the host’s defence
against permeating pathogens, yet later studies revealed it also participates in the regulation
of acquired (adaptive) immunity (Carroll 2004, Le Friec and Kemper 2009). Therefore, disorders
in activation of complement and its regulation mechanisms negatively affect the maintenance
of the macroorganism’s homeostasis, which may contribute to growth and development
of many different infections and diseases.
Complement activation in mammals may occur via three, and even five separate pathways:
classical, lectin pathway, and alternative, as well as fourth one as a result of direct decomposition
of complement components, and fifth one by initiation of alternative pathway, yet via properdin
(Hourcade 2006, Gołąb et al. 2007, Markiewski et al. 2007, Chapel et al. 2009, Le Friec
and Kemper 2009). Each of those pathways leads to accumulation of the key enzyme
of the complement cascade – C3 convertase, which is responsible for cutting C3 protein into
subunits C3a and C3b, which constitutes the main stage in the activation process (Gołąb et al.
2007, Le Friec and Kemper 2009). Conformation changes within one component result
in activation of proteolytic properties of another component, or in the acquisition of the capacity
to bind to the next component in the activation chain (Gołąb et al. 2007, Le Friec and Kemper
2009). Activation of classic pathway commences at the moment of binding C1q subunit of C1
protein to immunoglobulin present in the complex with antigen. C1 component of complement,
apart from C1q particle, also contains two subunits – C1r and C1s, which cause decomposition
of components C4 and C2, respectively, to C4a and C4b, as well as C2a and C2b (Xu et al.
2001, Gołąb et al. 2007, Le Friec and Kemper 2009). The resulting C4b2a complex with
proteolytic properties is referred to as C3 convertase. C3 convertase is the key protein complex
necessary for formation of C3b component, and then C4b2aC3b complex, referred to as C5
convertase (Pangburn and Rawal 2002, Gołąb et al. 2007, Le Friec and Kemper 2009). In turn,
C5 convertase decomposes C5 factor, as a result of which C5b is formed, which binds,
respectively, components C6, C7, C8 and C9. As a result of connecting these components,
membrane attack complex (MAC) is formed, which creates a channel in the membrane
of the target cell, leading to distortions to cell metabolism (Gołąb et al. 2007, Le Friec
and Kemper 2009). In turn, activation via lectin pathway, referred to as antibody-independent
pathway, occurs through mannose binding lectin (MBL) or a group of proteins referred to as ficolins,
which have the capacity of binding to carbohydrates present on the surface of various
pathogens, such as bacteria, viruses, fungi or protozoa (Gołąb et al. 2007, Thiel 2007, Le Friec
Complement and properdin, element of non-specyfic humoral immunity …
93
and Kemper 2009). MBL and ficolins then bind to serine proteases referred to as MASP (MBLassociated serine protease). The function of MASP proteases is similar to the function of C1,
therefore it is them that decompose C4, C2 and C3, as a result of which C3 convertase
(C4b2a) and C5 convertase (C4b2aC3b) are created (Gołąb et al. 2007, Le Friec and Kemper
2009, Thiel 2007). In turn, the alternative complement activation pathway involves the participation
of properdin, composed of factor B (beta-globulin), factor A (identical with fragment C3
of the complement), component D (convertase C3 of proactivator), component P (properdin)
and factors H and I (Deptuła et al. 2008). In the early phases of this pathway, factor B is bound
at the presence of Mg2+ ions with the form C3(H2O) of C3 protein, thus allowing factor D,
present principally in the active form, to decompose factor B into subunits Ba and Bb (Gołąb
et al. 2007, Le Friec and Kemper 2009). As a result of such changes, complex C3(H2O)Bb
is formed, referred to as initial form of C3 convertase. This form, in turn, transforms into active
C3bBb complex, referred to as final C3 convertase, which is affixed to the cell membrane,
and has the capacity of decomposing factor C3 into C3a and C3b (Gołąb et al. 2007, Le Friec
and Kemper 2009). C3bBb complex may also bind additional C3b fragments, thus becoming
convertase C5 of alternative pathway (C3bBb3b). The enzyme is located at the surface
of the target membrane and is stabilised by factor P, which protects it against regulator factors,
namely factor H and factor I (Gołąb et al. 2007, Deptuła et al. 2008, Le Friec and Kemper
2009). As mentioned earlier, also the fourth and fifth complement activation pathways have
been described (Kalowski et al. 1975, Hourcade 2006, Markiewski et al. 2007, Le Friec and
Kemper 2009). The fourth pathway comprises complement activation as a result of direct
decomposition of complement’s components under the influence of proteases participating
in the blood coagulation cascade, such as kalikrein, plasmin or thrombin (Markiewski et al.
2007). It was determined that thrombin may activate C5 component of the complement in mice
deprived of C3, where C5 convertase could not be formed. Furthermore, it was evidenced that
administration of thrombin and thromboplastin in rabbits induced complement activation
(Kalowski et al. 1975). It was recorded that the process could be, however, weakened
by thrombocytopenia, which suggests that thrombocytes are also involved in this phenomenon
(Kalowski et al. 1975). In turn, the fifth complement activation pathway involves direct initiation
of the alternative pathway by properdin P factor, yet not as in the alternative pathway by B factor,
which may recognize and bind to the surface of many pathogens, such as Neisseria
gonorrhoeae, or Escherichia coli strains, and even zymosan, component of yeast cell wall,
and then to C3b component (Hourcade 2006, Ferreira et al. 2010). C3bP complex binds
to factor B, and the created complex C3bBP is transformed under the influence of factor D into
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A. Mękal, B. Tokarz-Deptuła, A. Trzeciak-Ryczek, W. Deptuła
an active form of convertase C3 (C3bBbP). This convertase, in turn, decomposes C3, as
a result of which the generated C3b component binds to the pathogen surface via the second
place on properdin. The complex again cooperates with factors B and D, and then C3
is broken down to subunits, as a consequence of which convertase C5 and MAC are created
(Hourcade 2006). In relation to these facts, it is determined that among the proteins forming
part of complement system, properdin deserves special attention due to the fact that it is not
only a factor taking part in alternative complement activation pathway, but also it can be direct
initiator of complement activation (Hourcade 2006). Properdin is a protein the concentration
of which in human serum amounts to 4–25 μg/ml (Ferreira et al. 2010). It is present in the serum
in the form of cyclical polymers (dimers, trimers, tetramers), which are form as a result of bonding
monomers with weight of approx. 53 kDa (Hourcade 2006). Contrary to other complement
components, principally synthesised in the liver, properdin can be produced by monocytes,
T lymphocytes, including H-9, HuT78, Jurkat and T-All lymphoblastic T cells, mast cells,
granulocytes, HL-60, U-937, and Mono Mac 6 progenitor cells in bone marrow, and endothelial
cells (Ferreira et al. 2010). Its synthesis by the monocytary line cells is enhanced by phorbol
esters, bacterial LPS, IL-1β and TNFα, while its release from neutrophil multilamellar bodies
is stimulated among others by TNFα, C5a, IL-8 and LPS (Ferreira et al. 2010).
THE ROLE AND OPERATION OF COMPLEMENT SYSTEM
Complement system is principally associated with the natural immunity mechanisms,
yet already in Nussenzweig et al. 1971 proved that e.g. C3 component may be bound
on the surface of B cells, thus pointing to its role in the acquired immunity. Further studies
evidenced that complete C3 deficit results in weakening of the immune response related
to humoral immunity, while the classical pathway is an important mechanism involved
in successful capture of antigens and their keeping in lymphoid tissues, i.a. in spleenic lymph
nodes (Dunkelberger and Song 2010). Owing to expression of receptors CR – CR1 (CD35)
and CR2 (CD21) on the surface of B cells and folicular dendritic cells (FDC), complement
strengthens acquired immunity related to B lymphocytes (Carroll 2008, Dunkelberger and Song
2010). Receptor CR2 binds in the cell membrane to CD19 and CD81 particles, creating socalled co-receptor for B cells (CD21-CD19-CD81), while in the presence of antigens connected
to complement components, this fact allows for cross-binding to BCR receptor (Dunkelberger
and Song 2010, Le Friec and Kemper 2009). Such binding of CR2 and BCR leads to lowering
of the B-cell activation threshold and over 1000-fold increase of such cells’ response to
antigens (Dunkelberger and Song 2010). It is worth stressing that the expression of co-receptor
Complement and properdin, element of non-specyfic humoral immunity …
95
CD21-CD19-CD81 already takes place during B cell migration from bone marrow to peripheral
blood, which contributes to elimination of auto-reactive B cells and positive selection of B1 cells,
which are the main source of natural antibodies (Dunkelberger and Song 2010). Cross-binding
of co-receptor with BCR increases B-cell activity also in later stages of their differentiation,
principally towards memory B cells and naive B cells (Dunkelberger and Song 2010). The studies
evidenced that in mice deprived of CR1/2 and C3 component of complement, clear reduction
in IgM and IgG levels is observed, together with damage of IgM class switching to IgG, as well
as decrease in capacity of antigen capture in T cell – independent manner (Poydnzakova et al.
2003). Similar results were obtained during the analysis of antigen capture in T cell – dependent
manner, which referred both to bacteria (Streptococcus pneumoniae), and viruses (herpes virus,
West Nile virus) (Da Costa et al. 1999, Haas et al. 2002, Mehlhop and Diamond 2006).
At present, it is known that complement not only affects immune response related to B cells,
but also significantly contributes to regulation and functioning of T cells (Le Friec and Kemper
2009, Dunkelberger and Song 2010). It was evidenced that in mice with C3 deficit, infected
with influenza virus or lymphocytic choriomeningitis virus (LCMV), significant decrease
is observed in activity of CD4+ and CD8+ T cells (Kopf et al. 2002, Suresh et al. 2003).
On the basis of these studies, it was concluded that lack of C3 component leads to decrease
in the capacity of antigen opsonisation and their capture, as well as reduced activity of T cells.
In turn, in DAF (CD55) deficient mice, increased complement activity was observed, which
leads to enhancement of immune response dependent on T cells, particularly Th1 cells. Also,
increased secretion of IFNγ and IL-2 was observed, as well as inhibition of IL-10 secretion as
a result of stimulation with antigens (Heeger et al. 2005, Liu et al. 2005). DAF deficient mice
infected with LCMV also resulted in increase in activity of naive and CD8+ Th cells, yet this
required the presence of C3 or C5aR (Fang et al. 2007). Furthermore, it was evidenced that
DAF may function as a co-stimulating particle on the surface of human CD4+ T cells, inducing,
together with CD3, proliferation of such cells (Capasso et al. 2006). Apart from DAF,
co-stimulating particles during CD4+ T cell activation also include receptor CD46 (MCPmonocyte chemoattractant protein), which induces synthesis of, among others, IFNγ and IL-10.
As a result of simultaneous activation of CD46, C3b and TCR receptor in the presence of IL-2,
regulatory T (Treg) cells develop, which secrete IL-10 and granzyme B (Kemper et al. 2003).
It is suggested that Treg cells induced by complement system weaken the response of effector
T cells, which protects tissues against damage, as well as autoimmune diseases (O’Garra and
Vieira 2004). The studies evidence that in mice deprived of receptors C3aR and C5aR,
anafilatoxins C3a and C5a play the role of modulators in IL-12 production by APC cells, which
is the regulator in the development of Th1 and Th2 cells (Kohl et al. 2006). In the case of lack
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of C5aR in mice infected with type A influenza virus, decrease in the number of specific CD8+
T cells was also recorded (Kim et al. 2004). Complement activation related to creation
of channels in the cell membrane and lysis in pathogen cells, must be continuously regulated
(Le Friec and Kemper 2009). Main regulators of complement activation include RCA proteins,
among which one can differentiate membrane proteins and plasma proteins. Membrane proteins
are represented CR1 (complement receptor type1), CD55 (DAF – decay – accelerating factor),
CD46 (MCP – membrane cofactor protein), CD59 (protectin) and HRF (homologous restriction
factor), while plasma proteins include i.a. properdin factor I and factor H, factor C4bp,
and protein S (vitronectin) (Le Friec and Kemper 2009). The most “desired” regulation mechanism
is the prevention of further transformations of C3 and C4 components owing to the initiation
of their decomposition by factor I and co-factors, such as CD46 or CR1 (Le Friec and Kemper
2009). CD46 and CR1 move within the two-layer lipid membrane towards C3b and C4b,
binding with them, which facilitates their decomposition by properdin factor I. Similar
mechanism of action is also observed for factors H and C4bp, which are also co-factors
for factor I (Le Friec and Kemper 2009). If this mechanism is not effective enough,
then convertases C3 and C5 may be formed, as well as MAC complex on the host’s cells.
And so, the creation of C3/C5 convertase is prevented by so–called regulators, showing
decay-accelerating activity (DAA), and receptors CD55 and CR1, as well as properdin factors
H and C4bp (Le Friec and Kemper 2009). It is worth stressing that such proteins also have
capacity to decompose the already formed convertases. In turn, generation of MAC complex
is inhibited by CD59 and protein S (Le Friec and Kemper 2009). It was also evidenced that
some pathogens entering the macroorganism have the capacity of complement inhibition
almost at each phase of its activation, owing to developing various mechanisms, which allows
them to survive and proliferate in the host’s organism (Rooijakkers and van Strijp 2007,
Dunkelberger and Song 2010). For example, Staphylococcus aureus produces membrane
protein SpA (Staphylococcal protein A), which not only has the capacity of binding to Fc region
of immunoglobulins IgG, which leads to inhibition of the phagocytosis process, but also limits
the classical complement activation pathway by binding to C1q (Silverman et al. 2005).
A similar role is played by protein G and protein L (Rooijakkers and van Strijp 2007). St. aureus
can also produce Staphylococcal Complement Inhibitor (SCIN), which blocks all pathways
of its activation by effective inhibition of C3 convertases, as well as inhibits opsonisation
and reduces the effectiveness of phagocytosis (Rooijakkers et al. 2005). Bacteria may also
inhibit MAC formation or reduce its cytolytic properties, principally owing to the presence
of thick membrane wall in the case of Gram-negative bacteria (Dunkelberger and Song 2010).
Among MAC inhibitors, there is also surface protein with 80 kDa weight, present in Borrelia
Complement and properdin, element of non-specyfic humoral immunity …
97
burgdorferi, which is similar to human CD59, which also acts as inhibitor of this complex
(Pausa et al. 2003). Apart from this, pathogens may react with the host’s regulator proteins,
e.g. by binding properdin H factor, which causes an increase to degradation of C3b
component, and limitation to C3 convertase formation, and thus decrease in the complement’s
activity. This phenomenon was described in Neisseria (N.) meningitidis and N. gonorrhoeae
which, owing to binding properdin factor H, avoid its own destruction (Ngampasutadol et al.
2008). Another mechanism related to this process, involving pathogen inhibition of chemotaxis
and inflow of leucocytes, is related to receptors C5aR and FPR (formyl peptide receptor) that
take part in activation of such processes (Rooijakkers and van Strijp 2007). Moreover,
the process involves bacterial proteins that inhibit chemotaxis, which include CHIPS (chemotaxis
inhibitory protein of S.aureus) (de Haas et al. 2004). Some viruses also aim at inhibition
of complement system activity in order to increase their virulence, which was first described
at the example of measles virus (Karp et al. 1996). It was evidenced that as a result of influence
of this virus, CD46 receptor is destroyed, and production of IL-12 by APC cells is reduced,
which contributes to weakened functioning of the immune system (Karp et al. 1996). It was also
evidenced that CD46 receptor may also act as the role of receptor for bacteria, such as
N. meningitidis and N. gonorrhoeae (Lindahl et al. 2000). Moreover, many viruses from
the Picornaviridae family, e.g. echovirus and Coxsackie virus, have the capacity of binding to DAF
receptor, yet the places of their binding may differ, and additionally the participation of adhesive
particles ICAM-1 is required in this process (Shafren et al. 1997, Evans and Almond 1998).
There is also properdin, described over 50 years ago by Pillemer et al. (Pillemer et al. 1954)
as protein initiating complement alternative pathway, acting analogically to antibodies in the classical
pathway. Later studies revealed that properdin is also a positive regulatory factor and facilitates
activation of alternative pathway by 5-10-fold half-life elongation of the formed C3bBb
convertase, as well as owing to increased secretion of C3 convertase (Jeleyarova and Luty
1999). In turn, recent experiments evidenced that properdin can recognise and directly bind
to cell surface of various microorganisms, initiating the generally known complement activation
pathway via factor B (third pathway), as well as via factor P (fifth pathway) (Hourcade 2006,
Ferreira et al. 2010). Furthermore, properdin can also bind to host’s cells, such as apoptotic
or necrotic cells, which promotes their phagocytosis by macrophages (Hourcade 2006, Kemper
et al. 2008, Xu et al. 2008, Ferreira et al. 2010). In the case of apoptotic T-cells, properdin can
promote phagocytosis via two separate mechanisms (Hourcade 2006). One involves
its binding to apoptotic T-cells, and therefore complement activation in situ, which then allows
for contact with phagocytising cells. In turn, in the other case, after properdin binding to apoptotic
T-cells, there is a direct contact with phagocytising cells without prior complement activation
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A. Mękal, B. Tokarz-Deptuła, A. Trzeciak-Ryczek, W. Deptuła
(Hourcade 2006). It was also determined that some microorganisms, e.g. Streptococcus
pyogenes via exotixin B, can contribute to properdin degradation, and therefore to inhibition
of complement activation and of the phagocytosis process (Hourcade 2006). It must be added
that elimination of apoptotic cells is of significant importance in immunity processes, as it allows
for avoiding harmful inflammatory and autoimmunological responses, which occur as a result
of breaching the cell integration during apoptosis (Hourcade 2006). Therefore, if properdin
contributes to elimination of apoptotic T-cells through phagocytosis, one may suspect that
absence of this factor will result in increased number of such cells, and therefore development
of i.a. auto-immunological diseases, e.g. systemic lupus erythematosus (SLE) (Hourcade
2006). Apart from properdin, there are also two other proteins in charge of recognition
of apoptotic and necrotic cells, namely complement component C1 (complement-1) and MBL
(mannose binding lectin) protein, therefore one must point out that it is only the absence
of properdin and any of those "additional" factors (C1, MBL) will result in the development
of auto-immunological diseases (Hourcade 2006, Trouw et al. 2008). It was also evidenced
that persons with properdin deficit are more susceptible to meningococcal infections, as well as
to recurrent pneumonia and otitis media (Hourcade, 2006 Ferreira et al. 2010). At present,
it was evidenced that properdin means also extracellular pattern recognition molecules (PRM)
that recognise pathogen-associated molecular patterns (PAMP), such as two-strand viral RNA
(dsRNA) (e.g. rotaviruses), owing to which it can activate the immune system (Lesher et al.
2010, Zhang et al. 2010). Therefore, the factor can have a role analogical to pattern recognition
receptors (PRR), which include TLR and RLR receptors. It was determined that polyI:C acid –
pro-inflammatory factor present in viruses – activates alternative complement activation pathway
in a properdin-dependent manner in vitro and in vivo, while in the case of properdin deficit
in mice, inhibition of INF type I production and of NK cell activation was recorded, as well as
liver damage (Zhang et al. 2010). Furthermore, it was evidenced that as a result of methylation
of nucleosides of two-strand mammal RNA, namely. rRNA, tRNA, mRNA (e.g. 7-methylguanosine,
5-methylcytidine), properdin is not bound, hence alternative complement pathway is not
activated. Therefore, it is believed to be a key property that allows properdin to recognise viral RNA
and host’s RNA (Zhang et al. 2010). Zhang et al. suggest (Zhang et al. 2010) that extracellular
detection of viral dsRNA by properdin and activation of complement alternative pathway can
also enhance immunological response induced by TLR and RLR receptors. It was also evidenced
that stimulation with β-glucans forming part of fungi cell wall, i.a. Saccharomyces cerevisiae,
causes alternative complement pathway activation (Agarwal et al. 2010). It must be added that
it was also recorded that β-glucans can activate alternative complement pathway even
in the absence of properdin, contrary to zymosan that attacked this pathway exclusively
Complement and properdin, element of non-specyfic humoral immunity …
99
in the presence of properdin, while in the presence of properdin factor P caused 5-10-fold increase
in complement activation level, which proves that properdin does not directly bind to glucans,
but rather to C3 convertase (Agarwal et al. 2010).
COMPLEMENT SYSTEM AND THE SELECTED DISEASES
Complement system constitutes an important element of immune system, therefore dysfunctions
in its activation and regulation, principally related to deficits of various components, have
a negative impact on the maintenance of homeostasis, which may contribute to the development
of various diseases (Le Friec and Kemper 2009, Dunkelberger and Song 2010). And so,
in patients with C3 deficiency, lack of capacity to coat pathogens or immunological complexes
is observed, which results in the increased risk of various infections and diseases, including
auto-immunological diseases (Kowalski 2000, Chapel et al. 2009). In the case of complete
deficiencies in components of the classical pathway, increased susceptibility to auto-immunological
diseases is observed, while deficiencies to C3 component or properdin factors H and I, result
in increased risk of bacterial infections (Kowalski 2000). In the case of deficiencies of C3,
principally increased susceptibility is recorded to infections caused by Hemophilus influenzae,
Streptococcus pneumoniae and Streptococcus pyogenes (Kowalski 2000). Furthermore,
deficiency of components C3, C1, C2 or C4 promotes the occurrence of suppurative infections,
while deficiency of components C5-C9, MBL, as well as components of alternative pathway,
namely properdin factors B, D and P, which results in increased risk of infections caused
by Neisseria sp (Kowalski 2000, Sjoholm et al. 2006). Infections caused by Neisseria meningitidis
may also be a result of defects in the functioning of MAC, or deficiency of components
necessary to generate it (Dunkelberger and Song 2010). In turn, MBL deficiency in children
aged from 6 months to 2 years, therefore in the period between the loss of passively acquired
mother’s antibodies and the development of own immune system, causes infections with fever
(Dunkelberger and Song 2010). Furthermore, recurrent infections may be caused by mutations
or deficiencies in the area of properdin factors H, I and D, which cause the wear or deficiency
of C3 (Sjoholm et al. 2006). It is worth stressing that our improved knowledge on the role
of the complement system in the acquired immunity significantly contributes to better
understanding of relations between the complement and auto-immunisation (Le Friec and Kemper
2009). In rheumatic diseases, such as SLE (Systemic Lupus Erythematosus) or juvenile idiopathic
arthritis (JIA), there are genetically conditioned deficiencies of complement components, which
makes it difficult not only to eliminate immunological complexes, but also contributes to their
deposition in tissues, and thus to sustaining the inflammatory processes (Kowalski 2000).
The studies indicated that the deficit of components C1, C2, C4 or MBL is related to the deve-
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lopment of SLE, yet according to other authors, deficiency of components to the lectin pathway,
in particular MBL and C3, rather contributes to the development of circulatory system diseases
or arteriosclerosis (Le Friec and Kemper 2009). Deficiency of C1q or C2 components may lead
to the development of other auto-immunological diseases, e.g. the focal form of Lupus Erythematosus, glomerulonephritis, dermatomyosytis, or scleroderma (Kowalski 2000). In the case
of DAF receptor (CD55) deficiency in mice, increased morbitity with intestinitis diseases was
recorded (Le Friec and Kemper 2009). At present, it is also believed that C3 component
of the complement is one of the main mediators in damage to tissues occurring as a result
of reperfusion and implant rejection (Le Friec and Kemper 2009).
CONCLUSION
Proteins of the complement system is an evolutionally old and unusually complex element
of the immune system, which takes part in regulation of both innate and acquired immunity.
Among its proteins, one of the major components includes properdin which, as indicated
by the present study, can be the main initiator of the alternative complement activation pathway
via factor B and P, and also has the role of extracellular PRM particles that recognise PAMP
patterns. In turn, deficiencies of any of the complement components not only contribute
to the development of bacterial, or viral infections, but are also related to some autoimmunological diseases, such as SLE or glomerulonephritis. Furthermore, studies also indicated
correlation between the complement and damage to tissues, i.a. as a result of implant rejection.
These facts make the complement system interesting to researchers, both in the aspect
of its regulation and participation in immunological processes, and in the aspect of its reaction
with pathogens, which may significantly contribute to the development of not only effective
treatment of many diseases, but also to their prophylaxis.
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DOPEŁNIACZ I PROPERDYNA, ELEMENTY NIESWOISTEJ ODPORNOŚCI
HUMORALNEJ – WAŻNE SKŁADNIKI ODPORNOŚCI WRODZONEJ
(NATURALNEJ)
Streszczenie. Układ dopełniacza i properdyna są ważnymi elementami odporności nieswoistej,
określanej również jako odporność wrodzona lub naturalna, które odgrywają istotną rolę w efektywnym
rozpoznawaniu i usuwaniu różnych patogenów. Praca przedstawia wybrane zagadnienia dotyczące
funkcjonowania układu dopełniacza oraz properdyny, a mianowicie szlaki jego aktywacji oraz mechanizmy regulujące. Ponadto przybliża ich rolę w odporności wrodzonej, a także w odporności nabytej.
Przedstawiono również przykłady chorób związanych z zaburzeniami funkcjonowania tych elementów
i scharakteryzowano niektóre mechanizmy patogenów, które mogą hamować aktywność dopełniacza.
Słowa kluczowe: układ dopełniacza, properdyna, odporność wrodzona
II. ORIGINAL ARTICLES
ADVANCES IN AGRICULTURAL SCIENCES
Adv. Agric. Sci. 2011, XIV (1–2), 107–114
Paweł Nawrotek, Karol Fijałkowski, Danuta Czernomysy-Furowicz, Ewelina Michałek,
Alicja Solecka
IDENTIFICATION AND DIFFERENTIATION OF ZOONOTIC ESCHERICHIA COLI
STRAINS ISOLATED FROM HEALTHY SHEEP, BY MOLECULAR METHODS
Department of Immunology, Microbiology and Physiological Chemistry,
The West Pomeranian University of Technology, Szczecin,
Doktora Judyma 24, 71-466 Szczecin, Poland
Abstract. Specific pathogenic properties and zoonotic meaning of animal reservoir of Shiga toxinproducing E. coli strains (STEC), incline to constant epidemiologic research using fast, precise and reliable
methods. The aim of this study was molecular identification and analysis of genes that determine Shigalike toxins production. The research was based on 20 STEC strains isolated from digestive system
of healthy Polish Wrzosowka sheep: 10 adults and 10 lambs. Preliminary species identification of isolates
was preformed by PCR, detecting uspA gene – encoding the universal stress protein characteristic
for E. coli. However, for identification and differentiation of STEC strains with sltI, sltII or sltIIe genes PCR-RFLP was used. PCR confirmed species identity of all investigated E. coli strains. Forty per cent (8/20
E. coli strains) of all investigated isolates with virulence marker characteristic for STECs were slt positive.
Moreover, it was reported that 6/8 (75%) of all investigated STEC strains derived from adult sheep,
and only 2/8 (25%) from lambs. Additional differential analysis of slt genes by PCR-RFLP revealed
polymorphism of sltII genes encoding SLTII cytotoxin produced by strains pathogenic for human.
The present study proofs, that clinically healthy sheep can be an important environmental reservoir
of pathogenic STEC strains. However, the greatest percentage of those strains was observed in adult
sheep, pointing out their significance in transmission of infection to human. Molecular diagnostic enables
reliable and precise identification and analysis of potentially pathogenic zoonotic E. coli strains isolated
from asymptomatic infected animals.
Key words: Shiga toxin-producing E. coli strains, slt genes, molecular diagnostics, sheep
INTRODUCTION
Shiga toxin-producing Escherichia coli strains (STEC) are important group of enteric pathogens
of humans, mostly because of their zoonotic nature of infection (Brett et al. 2003, Nawrotek
2003, Bonyadian et al. 2010). Since 1982 they are connected in a developmental process
of serious human diseases, such as: haemorrhagic colitis (HC), haemolytic uraemic syndrome
(HUS) or thrombotic thrombocytopenic purpura (TTP) (Nawrotek 2003, Weiner 2008). Previous
studies have found over 400 serotypes of E. coli classified to that group of pathogens, among
them about 100 were isolated from human cases of diseases (Ramachandran et al. 2001,
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P. Nawrotek, K. Fijałkowski, D. Czernomysy-Furowicz, E. Michałek, A. Solecka
Weiner and Osek 2007, Bonyadian et al. 2010). It must be noted that predominant serotype
is E. coli O157 : H7 (Djordjevic et al. 2001). In animals, STEC rods (mostly representing
serogroups O138, O139 and O141), are the etiologic factor of edema disease of swine,
however other serotypes are responsible for calf diarrhoea (Nawrotek 2003).
STEC strains are phenotypically and genotypically different between each other, but ability
to produce Shiga cytotoxins is their common feature (Weiner and Osek 2007). Those toxins,
called also Shiga-like toxin (SLT), Shiga toxin (Stx) or verocytotoxin (VT), are mainly responsible
for acute course of infections (toxaemia). Inhibition of protein synthesis in e.g. in epithelial
cells of the small intestine and colon or in vascular endothelial cells of human capillary, and then its
destruction, upsetting ionic regulation and water balance and occurrence of bloody diarrhea
(Osek 2000, 2003) are the consequences of their cytotoxic effect. SLT toxins structurally
belong to holotoxins AB5 (complete toxins) and occur in many antigen varieties. Among them,
the most important are: SLTI and SLTII, produced by human pathogenic strains and SLTIIe
produced by E. coli which causes edema disease of swine (Scotland and Smith 1997). SLTI
cytotoxin are relatively homogenous group and are produced mostly by E. coli O26 : H11
serotype. In contrary, SLTII toxin consists of several different variants and it is synthesised
by dissimilar E. coli serotypes, mostly O157 : H7. Heterogenous, in terms of SLTIIe toxins
production, can be also animal STEC strains (Tschäpe et al. 1992, Scotland and Smith 1997).
Genes responsible for determination of slt cytotoxins synthesis are present in many different
variants and are located on bacterial chromosome (sltIIe – chromosomal gen) or are transferred
by bacteriophages (sltI and sltII – phage genes), what is crucial during spreading the virulence
markers among E. coli strains (Rossmann et al. 1994, Scotland and Smith 1997, Bonyadian
et al. 2010).
Livestock – cows (mainly beef cattle), other ruminates and pigs are considered to be the main
reservoir of STECs, including both: healthy animals and those with diarrhoea symptoms (Nawrotek
2003, Weiner and Osek 2007). STECs were also isolated from horses, dogs, cats, deer and birds
(Blanco et al. 2003, Weiner and Osek 2007). The major source of human infection are raw
contaminated materials or unproper cooked, baked or processed food derived from animals
(mostly minced meat, milk and dairy products). Furthermore, there were reported cases of disease
after consumption of STEC contaminated vegetables and fruits, and also after direct animalhuman and human-animal contact (Ludwig et al. 2002, Blanco et al. 2003, Weiner 2008).
AIMS
Specific pathogenic properties and zoonotic significance of animal reservoir of Shiga toxinproducing E. coli strains, justify constant epidemiological monitoring using fast, precise and reliable
Identification and differentiation…
109
methods. The aim of this study was molecular identification and analysis of genes that determine
Shiga-like toxins production by STEC strains isolated from digestive system of healthy Poilsh
Wrzosowka sheep.
MATERIAL AND METHODS
The research was based on 20 E. coli strains isolated from digestive system (faecal swabs)
of healthy Poilsh Wrzosowka sheep: 10 adults (1–3 years old) and 10 lambs were used as
a material for this study. Two reference STEC strains: E. coli O157 : H7 – SLTII (National Institute
of Hygiene, Warsaw, Poland) and E. coli E68II/0141 – SLTIIe (National Veterinary Research
Institute, Pulawy, Poland), respectively sltII and sltIIe positive, were used as positive controls.
All isolates were kept frozen at -20ºC in Tryptone Soya Broth (TSB, Oxoid) with 10% glycerol.
For each experiment, 50 μL of bacteria suspension from the working stock was streaked
onto a Brain-Heart Infusion Agar (BHI agar, Oxoid) and grown for 18 h at 37ºC. A loop
of bacteria was removed from the plate and used to inoculate 1mL TSB broth and grown for
a further 18 h at 37ºC. After the incubation, bacterial DNA was isolated using commercially
available kit – Genomic Mini AX BACTERIA (A&A Biotechnology), according to manufacturer’s
instructions. Concentration and purity of isolated genomic DNA was evaluated fluorescently
using Quant-iT dsDNA BR assay kit (Invitrogen, Molecular Probes), according to manufacturer’s
instructions.
Preliminary species identification of isolates was preformed by PCR, detecting uspA gene
encoding universal stress protein characteristic for E. coli, as suggested by Oska (2003).
However, for identification and differentiation of STEC strains with sltI, sltII or sltIIe genes,
encoding Shiga-like toxins PCR-RFLP test was used, based upon previous studies (Nawrotek
2003). All PCR amplifications were carried out by AmpliTaq Gold PCR Master Mix (Applied
Biosystems), according to manufacturer’s instructions. Master Mix was prepared under
aseptic conditions (DNA/RNA UV-Cleaner, type UVC/T-AR, Biosan). All PCR reactions were
in duplicates, carried out in PeqStar 96 Universal Gradient thermocycler (Peqlab). Obtained
PCR amplicons were separated in a 2% (3% for RFLP products) agarose gel (Prona) with
addition of ethidium bromide 0.003% (v/v) (EtBr, Merck). Electrophoresis was carried out
in Sub-Cell GT Wide Mini with PowerPac Basic (Bio-Rad). The sizes of PCR-RFLP products
were compared to molecular mass marker – MassRuler Express DNA Ladder, 1000-100 bp
(Fermentas). The images of DNA bands were archived and then qualitative and quantitative
densitometric analysis was performed with IG/LHR InGenius LHR (Syngene Bio Imaging)
and GeneTools software (Syngene).
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P. Nawrotek, K. Fijałkowski, D. Czernomysy-Furowicz, E. Michałek, A. Solecka
RESULTS AND DISCUSSION
Preliminary species identification of isolates performed by means of PCR (uspA gene)
confirmed species identity of all investigated E. coli strains. However, each PCR amplification
for 8/20 E. coli strains isolated from healthy sheep revealed replicatable slt amplicons with
molecular weight of 227 bp, what made up 40% of all isolates with virulence marker characteristic
for STECs. Moreover, it was reported that 6/8 (75%) of all detected STEC strains derived from
adult sheep, and only 2/8 (25%) from lambs (Fig. 1). Additional differential analysis of slt genes
detected by PCR-RFLP revealed polymorphism of sltII genes (Fig. 2). Also of note is the
observation that detected STEC strains are genetically determined to SLTII cytotoxin synthesis,
which is also produced by human pathogenic strains and also present in asymptomatic
infected animals.
M H2O sltII 1
2
3
4
5
6
7
8
9
10
227 bp
E. coli strains
from adult sheep
M
1Y 2Y
3Y 4Y 5Y 6Y 7Y 8Y 9Y 10Y
E. coli strains
from lambs
227 bp
Fig. 1. Image of 2% agarose gel electrophoresis of slt amplicons from DNA of eight E. coli strains
isolated from adult sheep and lambs. Explanations: M – MassRuler Express DNA Ladder,
1000-100 bp; H2O – negative control; sltII – reference strain of E. coli O157 : H7 (sltII); 1–10
i 1Y–10Y – examined strains of E. coli; 3, 4, 5, 8, 9, 10, 6Y, 10Y – STEC positive strains
M
sltIIe
10
9
8
4
6Y
3
5
10Y
174 bp
103 bp
72 bp
52 bp
53 bp
Fig. 2. Image of 3% agarose gel electrophoresis of sltII (174 and 53 bp) and sltIIe (103, 72 and 52 bp)
amplicons after PCR-RFLP. Explanations: M – MassRuler Express DNA Ladder, 1000-100 bp;
sltIIe – reference strain of E. coli E68II/0141 (sltIIe); 10, 9, 8, 4, 6Y, 3, 5, 10Y – examined strains
of STEC
Identification and differentiation…
111
Although E. coli is human and animal symbiotic microorganisms, some strains can be clinically
or epidemiologically important during transmission of pathogens between human and animal
(Osek 2000, Dacko and Osek 2004). The natural reservoir of those microorganisms are mainly
healthy or with diarrhoea symptoms livestock, especially those raised for meat production
(Paton and Paton 1998, Twardoń et al. 2004). Since 1982 it is observed a constant increase
in number of single disease and cases of epidemic of bloody diarrhea, caused by Shiga toxinproducing E. coli strains (Nawrotek 2003, Weiner 2008). Cases of the diseases were mainly
noted after consumption of contaminated food of animals origin (Weiner and Osek 2007,
Weiner 2008).
The percentage of animals, including sheep, with STEC bacteria is vary. In Australia, it was
observed that 56–73% of sheep (Djordjevic et al. 2001, Blanco et al. 2003) have asymptomatic
STEC infection, what was higher than in our studies. Our results are consistent with reports
from Kudva et al. (1996), who reported similar percentage (43%) of asymptomatic carrier
animals of STEC strains in USA. Previous studies from other authors (Beutin et al. 1993,
Blanco et al. 2003) have also showed that this percentage is included between 55–95% of sheep.
Moreover, Blanco et al. (2003) observed that the percentage of lambs from which STEC
pathogens were isolated is definitely lower. Similar dependence was observed in our studies,
which showed at the same time domination of STEC in adult sheep. It could point out on potentially
greater coverage of those pathogens amongst that group of age, what in consequence causes
an increase the infection risk of human (e.g. in terms of food processing). HUS, HC and TTP
diseases are often reported after consumption of contaminated food of animals origin (Weiner
and Osek 2007, Weiner 2008).
STEC strains isolated from animals can vary regarding slt genes encoding Shiga-like toxins.
According to Blanco et al. (2003) only 1% of STECs isolated from sheep contained sltII gene
in their genome. However, most stains (82%) isolated from those animals belonged to human
pathogenic STEC serotypes, including 51% serotypes isolated previously from patients with
haemolytic uraemic syndrome. In further research those authors, using PCR, confirmed presence
of sltII gene in only 3% of strains isolated from sheeps, and the presence of sltI in 55%,
however 42% of those strains were characterised by the presence of both genes simultanesly.
In contrary, results obtained in our studies showed the greater percentage of STEC strains isolated
from healthy sheep only in the case of sltII gene. This is consistent with Ramachandran et al.
(2001) findings, what additionally point out the great involvement of strains containing both sltI
and sltII genes.
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P. Nawrotek, K. Fijałkowski, D. Czernomysy-Furowicz, E. Michałek, A. Solecka
In the USA 80 massive infections caused by STECs are reported every year. The number
of sporadic infections is there about 20 000 people. In this case, the major source of infection
is consumption of meat and milk (Weiner and Osek 2007). According to European Food Safety
Authority (EFSA), in 2004 number of human infections caused by Shiga toxin-producing E. coli
strains was at the 4th place, following Salmonella, Campylobacter and Yersinia. There was reported
81 confirmed diseases in Poland. About 1% of positive results (based on 41 929 food samples)
was found after analysing of food for STEC contaminations, in 18 EU countries and Norway.
The most of the positive results concerned raw beef and was noted in Italy (38.2%), Poland
(8.3%) and Spain (4%) (Weiner and Osek 2007). Those data point out the necessity of constant
monitoring of food for STECs contamination, and also animals, which are natural reservoir
of that microorganisms. Our findings are consistent with other authors reports (Paton and Paton
1998, Djordjevic et al. 2001, Blanco et al. 2003, Bonyadian et al. 2010), pointing out the involvement
of animals, including sheep, in epidemiologic chain of animal infections.
In conclusion, the present study proofs, that clinically healthy sheep can be an important
natural reservoir of pathogenic STEC strains, which is crucial in epidemiology of STEC infections.
However, the greater percentage of STEC strains observed in adult sheep can suggest their
involvement in transmission of infections in human.
Molecular diagnostic enables reliable and precise identification and analysis of the potential pathogenicity of zoonotic E. coli strains isolated from asymptomatic infected animals.
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IDENTYFIKACJA I RÓŻNICOWANIE ODZWIERZĘCYCH SZCZEPÓW
ESCHERICHIA COLI WYIZOLOWANYCH OD ZDROWYCH OWIEC,
Z UŻYCIEM METOD MOLEKULARNYCH
Streszczenie. Specyficzne właściwości chorobotwórcze oraz zoonotyczne znaczenie zwierzęcego
rezerwuaru shigatoksycznych szczepów E. coli (STEC), skłaniają do prowadzenia stałych badań epidemiologicznych, z użyciem szybkich, dokładnych i wiarygodnych metod. Celem podjętych badań była
diagnostyka oparta na metodach molekularnych, dotycząca identyfikacji oraz analizy genów determinujących wytwarzanie shigatoksyn 20 szczepów STEC, wyizolowanych z przewodu pokarmowego
zdrowych owiec rasy wrzosówka: 10 osobników dorosłych i 10 jagniąt. Wstępną identyfikację gatunkową
izolatów przeprowadzono z użyciem gatunkowo specyficznej reakcji PCR, ukierunkowanej na wykrycie
genu uspA kodującego uniwersalne białko stresowe charakterystyczne dla E. coli. Natomiast do identyfikacji i różnicowania szczepów STEC posiadających w swoim genomie geny sltI, sltII lub sltIIe kodujące
shigatoksyny, zastosowano test PCR-RFLP. W następstwie reakcji amplifikacji potwierdzono przynależność gatunkową wszystkich badanych szczepów E. coli oraz uzyskano, w przypadku ośmiu szczepów
wyizolowanych od zdrowych owiec, amplikony slt o masie molekularnej 227 pz, co stanowiło 40%
114
P. Nawrotek, K. Fijałkowski, D. Czernomysy-Furowicz, E. Michałek, A. Solecka
wszystkich badanych izolatów posiadających marker zjadliwości charakterystyczny dla szczepów STEC.
Jednocześnie zauważono, że aż sześć (75%) spośród wykrytych szczepów STEC, pochodziło od owiec
dorosłych, a tylko dwa (25%) od jagniąt. Dodatkowa analiza różnicowa wykrytych genów slt, przeprowadzona z użyciem testu PCR-RFLP, pozwoliła wykazać, że charakteryzują się one polimorfizmem
wskazującym na geny sltII kodujące cytotoksynę SLTII, wytwarzaną przez szczepy chorobotwórcze dla
człowieka. Uzyskane wyniki dowodzą, że zdrowe klinicznie owce mogą stanowić ważny środowiskowy
rezerwuar patogennych szczepów STEC. Natomiast większy odsetek tych szczepów zaobserwowany
w przypadku owiec dorosłych, może wskazywać na ich większe znaczenie w transmisji zakażenia na ludzi.
Diagnostyka molekularna umożliwia wiarygodną i precyzyjną identyfikację oraz analizę potencjalnej
chorobotwórczości odzwierzęcych pałeczek E. coli izolowanych od bezobjawowo zakażonych zwierząt.
Słowa kluczowe: shigatoksyczne szczepy E. coli, geny slt, diagnostyka molekularna, owce
ADVANCES IN AGRICULTURAL SCIENCES
Adv. Agric. Sci. 2011, XIV (1–2), 115–124
Beata Hukowska-Szematowicz1, Aleksandra Manelska2, Beata Tokarz-Deptuła1,
Wiesław Deptuła1
COMPARATIVE ANALYSIS OF THE GENE ENCODING VP60 CAPSID PROTEIN
IN VARIOUS STRAINS OF THE RHD (RABBIT HAEMORRHAGIC DISEASE)
VIRUS
1
Department of Microbiology and Immunology, University of Szczecin,
Felczaka 3c, 71-412 Szczecin, Poland, e-mail: [email protected]
2
Student of Biotechnology at the Department of Natural Sciences of the University of Szczecin
Abstract. The study presents a comparative analysis of the gene encoding structural protein VP60 in 32
strains of the RHD virus. The study evidenced 95.6% homology between these strains of the RHDV,
and on the basis of genetic interdependencies, in the homology tree, group I-VI was identified, while
in the phylogenetic tree – genetic groups G1-G6. The similarity analysis between the strains studied
revealed identity at the level of 92%, while the diversification observed, amounting to 8%, between
the analysed strains of the RHDV originating from various continents and identified in different years
proves their variability.
Key words: rabbit, VP60, RHD, genogroup
INTRODUCTION
The RHD virus is an infectious agent causing rabbit haemorrhagic disease, which belongs
to the Caliciviridae family and Lagovirus class (Annon 2009). The genome of the RHD virus
is a positively polarised single RNA strand terminated with poly-A tail (Meyers et al. 1991b),
comprising 7437 nucleotides contained in a sheathless capsid (Meyers et al. 1991a, Meyers et al.
1991b) structured as a regular icosehedron, with diameter of 28–40 nm. Single major polypeptide
of the RHD virus is the structural protein VP60 with the weight of 60kDa. Genetic studies
of the RHDV involved the analysis of nucleotide sequence of the entire RHDV genome identified
in 35 strains, and the entire gene encoding structural protein VP60 in 36 strains of the RHDV.
The first genetic studies of the gene encoding VP60 or its fragments involved determination
of homology between the analysed strains of the RHD virus (Chrobocińska 2007, quote
Hukowska-Szematowicz 2006, Matiz et al. 2006, Chrobocińska and Mizak 2007, McIntosh
et al. 2007, Farnos et al. 2007, Tian et al. 2007, Pawlikowska and Deptuła 2008, Pawlikowska
et al. 2008a, Pawlikowska et al. 2008b, Pawlikowska et al. 2008c, Działo et al. 2009, Pawlikowska
116
B. Hukowska-Szematowicz, A. Manelska, B. Tokarz-Deptuła, W. Deptuła
et al. 2009a, Pawlikowska et al. 2009b) and indicate that the homology of nucleotide sequence
of the gene in the analysed strains of the RHDV amounted to from 89.4–100% and was very
similar to homology recorded in the aminoacid sequence of VP60. In further studies,
phylogenetic interdependencies were analysed between the strains of the RHDV on the basis
of the sequence encoding VP60, expressed via phylogenetic trees constructed, where strains
were classified into genogroups (genetic groups) (Chrobocińska 2007, quote Hukowska-Szematowicz 2006, Forrester et al. 2006a, Forrester et al. 2006b, Matiz et al. 2006, Van de Bildt
et al. 2006, Farnos et al. 2007, Chrobocińska and Mizak 2007, McIntosh et al. 2007, Tian et al.
2007, Pawlikowska et al. 2008a, 2008b, 2008c, Działo et al. 2009, Niedźwiedzka-Rystwej et al.
2009, Pawlikowska et al. 2009a, Pawlikowska et al. 2009b, Alda et al. 2010) depending
on the year of strain identification or its geographic region. Such studies indicated that RHDV
strains form 2 (quote Hukowska-Szematowicz 2006, Farnos et al. 2007, Tian et al. 2007,
McIntosh et al. 2007, Działo et al. 2009, Niedźwiedzka-Rystwej et al. 2009, Pawlikowska et al.
2009a, Pawlikowska et al. 2009b), 3 (quote Hukowska-Szematowicz 2006, Matiz et al. 2006),
4 (Pawlikowska et al. 2008b), 6 (Chrobocińska 2007, quote Hukowska-Szematowicz 2006,
Van de Bildt et al. 2006, Chrobocińska and Mizak 2007) or 7 genetic groups (genogroups)
(Forrester et al. 2006b), and their classification into a particular group was more correlated
to the year of strain identification, while to a lesser extent to its geographical location.
AIM
The aim of the study was comparative analysis of the gene encoding VP60 capsid protein
in various strains of the RHD (rabbit haemorrhagic disease) virus. List of studied RHD virus
strains presented in Table 1. The sequences of 32 RHDV strains were taken from the Gene Bank.
MATERIAL AND METHODS
The material was formed by nucleotide sequences of the gene encoding VP60 protein
(of the length of 1740 nucleotides) 32 strains of the RHD virus (Table 1) collected from the Gene
Bank (Annon 2011). The comparative analysis of the nucleotide sequence in 32 strains
of the RHD virus was performed using DNAMAN 6.0 software. (Lynnon Biosoft, Canada). After
the alignment, namely set of matching sequences, homology and phylogenetic trees were
created. Homology trees allowed for determination to what extent the analysed 32 strains
of the RHDV are homological, while phylogenetic trees illustrate relationship and evolutionary
interdependencies between the strains. To assess the credibility of phylogenetic trees the
bootstrap method was used.
Comparative analysis of the gene encoding …
117
Table 1. List of studied RHD virus strains
No
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
Strain
WHN/China/0305
WHN/China/022005
WHN/China/012005
YL
JXCHA97
WXChina1984
TP
NJChina1985
00-08
95-10
03-24
95-05
00-13
00-Reu
99-05
HauteSaone France
05-01
Hagenow
Triptis
Eisenhuettenstadt
Meiningen
Hartmannsdorf
Frankfurt
Wriezen
Irleand25
Irleand19
Ast89
Mexico89
RHDV New Zealand
Rainham
RHF89 1989
KV0801 2008
RHDVa
RHDVa
RHDVa
RHDVa
RHDVa
RHDVa
RHDVa
RHDVa
RHDVa
RHDVa
RHDVa
RHDVa
RHDVa
Country of origin,
year of identification
China, 2005
China, 2005
China, 2005
China, no data
China, 1997
China, 1984
China, no data
China, 1985
France, 2000
France, 1995
France, 2003
France, 1995
France 2000
France, 2000
France, 1999
France, 1988
France, 2005
Germany, 1990
Germany, 1996
Germany, 1989
Germany, 1992
Germany, 1996
Germany, 1996
Germany, 1996
Ireland , no data
Ireland , no data
Spain, 1989
Mexiko, 1989
New Zealand, no data
UK, 1993
Korea, 1989
Korea, 2008
GenBank,
accesion number [23]
DQ069282
DQ069281
DQ069280
DQ530363
DQ205345
AF402614
AF453761
AY269825
AJ319594
AJ535094
AJ969628
AJ535092
AJ495856
AJ303106
AJ302016
U49726
AM085133
Y15441
Y15442
Y15440
Y15425
Y15425
Y15424
Y15427
AY928270
AY928269
Z24757
AF295785
AF231353
AJ006019
FJ212323
FJ212322
Explanation: RHDVa – antigenic variant of RHD virus.
RESULTS
As a result of comparison of the nucleotide sequence comprising 1740 nucleotides, it was
determined that the strains analysed reveal homology of the nucleotide sequence amounting
to 95.6%. Within this sequence, changes to the substitution type were recorded, namely
transversions and transitions. Most frequent mutations involved transversions, the number
of which amounted to 440, which constitutes 82.20% of all mutations. Transitions occurred
95 times (which constituted 17.80%). The homology tree generated (Fig. 1) pointed to 92%
homology between 32 strains of the RHD virus. The tree identifies VI groups. Group I
comprised 3 French strains 00-08, 00-13, 05-01 with homology of 97%. Group II included
strains: HauteSaone France, Mexico89, WXChina1984, RHDVNew Zealand, RHF891989,
AST89, and Eisenhuettenstadt with homology of 96%. In this group, the highest homology was
recorded between the pairs of strains Ast89 and Eisenhuettendtadt (99%). Group III included
strains 95-05, 95-10, Rainham, Frankfurt, Meiningen, Hagenow showing itentity at the level
of 96%. In this group, the highest homology of 99% was recorded for strains 95-10 and Rainham.
118
B. Hukowska-Szematowicz, A. Manelska, B. Tokarz-Deptuła, W. Deptuła
Fig. 1. Homology tree for 32 RHD strains obtained on the basis gene encoding VP60 capsid protein
Comparative analysis of the gene encoding …
119
Group IV comprises 3 strains: Wriezen, Ireland19 and Ireland25, which, similarly as in two previous
groups, reveal 96% of identity. Groups V and VI is formed by antigen variants 99-05, 03-24, 00-Reu,
Triptis, Hartmannsdorf, WHN/China/012005, WHN/China/022005, WHN/China/032005, TP,
NJChina1985, JXCHA97, YL and KV0801 2008. It must be noted that Group VI involves just
1 strain – Hartmannsdorf. Strains present in Group V arer characterised with 97% homology
between one another, while as compared to Hartmannsdorf strain, they show identity at the level
of 95%. The phylogenetic tree generated (Fig. 2) divided the strains analysed into 6 genetic
groups (G1-G6). Genetic group 1 (G1) included 3 strains: 00-08, Ast89, Eisenhuettenstadt, while
bootstrap factor amounted to 100. In this group, the greatest distance to other strains is observed
for 00-08 strain, which suggests it is the youngest in the phylogenetic aspect. Group 2 (G2) comprises 5 strains: WXChina1984, RHDVNew Zealand, HauteSaone France, Mexico89, RHF89 1989
with bootstrap factor amounting to 90. Group 3 (G3) is formed by strains: 00-13, 05-01, Ireland25,
Ireland19, Wreizen with bootstrap factor amounting to 100; while group 4 (G4) by strains: 95-10,
Rainham, Frankfurt, Meiningen, Hagenow, 99-05 with bootstrap factor amounting to 100.
Groups 5 and 6 (G5 and G6) include 13 strains being antigen variants-RHDVa: 00-Reu,
03-24, WHN/China/012005, 99-05, JXCHA97, TP, NJCHINA1985, YL, WHN/China/022005,
WHN/China/032005, KV0801 2008, Triptis, Hartmannsdorf – with bootstrap factor-100. It must
be noted that Group 6 is created individually by Hartmannsdorf strain. The greatest distance from
the remaining strains was recorded for KV0801 2008 strain, while the smallest – for 99-05 strain.
DISCUSSION
As a result of comparison of the nucleotide sequence comprising 95,6% nucleotides, it was
determined that the strains analysed reveal homology amounting to 95.6%. In homology tree
(Fig. 1) and phylogenetic tree (Fig. 2), the 32 analysed RHDV strains were classified into 6 group
(homology tree) and 6 genetic groups (G1-G6) (phylogenetic tree). In homology tree, Group I
was formed by 3 French RHDV strains from the years 2000–2005: 00-08, 00-13, and 05-01.
When analysing in detail the interdependencies in this group, it must be noticed that the French
strain 00-08 showed 90.5% homology to strain 05-01, namely 10% variation. Such a situation
results from the fact that between these two strains, there is 5-year time difference, hence
it can be suspected that such variation occurred. It can also be suspected that 05-01 strain
derives from 00-13 strain, which is indicated by the phylogenetic tree (observed on the basis
of branch length), and the bootstrap factor value of 100, which testifies to very high reliability
of the tree. It is also worth considering that strains 00-08 and 00-13 (both originating from 2000)
recorded 91.1% homology to each other, while at the phylogenetic tree were classified
into 2 different genogroups, which may mean that they evolved from 2 separate strains.
120
B. Hukowska-Szematowicz, A. Manelska, B. Tokarz-Deptuła, W. Deptuła
Fig. 2. Phylogenetic tree for 32 RHD strains obtained on the basis gene encoding VP60 capsid protein
Strain 00-08 is related to strains from 1989, yet is evolutionarily distant from them. In turn, strain
00-13 was classified into G3 group together with strain 05-01 (to which it showed 96.6%
homology), and with strains Ireland19, Ireland25 and Wriezen. A similar distribution of the strains
Comparative analysis of the gene encoding …
121
was obtained by Van de Bildt et al. (2006). Grouping of strains according to the year of identification was described by i.a. Nowotny et al., Le-Gall et al., Le Gall-Recule et al. (quote Hukowska-Szematowicz 2006), Niedźwiedzka-Rystwej et al. (2009), Pawlikowska et al. (2009a). In Group II,
homology tree included strains: HauteSaone France, Mexico89, WXChina1984, RHDV New
Zealand, RHF89 1989, AST89, and Eisenhuettenstadt, identified in the years 1984–1989
and with homology of 96%. In this group, the highest homology (99%) was recorded between
the strains AST89 and Eisenhuettendtadt. At the phylogenetic tree, Group 2 included just five
strains: WXChina1984, RHDV New Zealand, RHF89 1989, HauteSaone France, and Mexico89.
The remaining two strains, AST89 and Eisenhuettenstadt, were classified in Group 1. Distribution
of the RHDV strains present in Group II at the homology tree, while in two different groups
(1 and 2) at the phylogenetic tree, may suggest that these strains have 2 different ancestors.
Layout of the strains in the group, and high homology between them may be explained with
the same year of identification. Group III at the homology tree was formed by French (95-05,
95-10), English (Rainham), and German strains (Frankfurt, Meiningen, Hagenow), identified
in the years 1990–1995 and showing homology of 96%. Despite the fact that these 6 strains
at the homology tree constitute Group III, at the phylogenetic tree they were included in G4.
The smallest distance in this group is recorded for Rainham strain, which may point to its
earliest origin. Meiningen and Hagenow strains reveal identical distance, which may suggest
that their grouping depended on the place of their identification. Similar studies had been
previously performed by Moss et al. (guote Hukowska-Szematowicz 2006),who suggested
group formation on the basis of geographic region where the strains were identified. In turn,
2 French strains: 95-05 and 95-10, despite being identified in 1995, in the same geographic
region, recorded distance from one another. Strain grouping depending on the year of identification
and the geographic region was described by Pawlikowska et al. (2008a, 2008c) when analysing
European strains of the RHD virus. Group IV included 3 strains: German Wriezen (1996)
and two Irish – Ireland19 and Ireland25, which, similarly as strains from Group III showed 96%
homology. Irish strains record 98.6% homology to one another, while the German strain has 96.8%
homology to Ireland25 strain, while as compared to Ireland19–96.6% homology. Such a high
degree of homology can be explained with a similar year of identification of such strains, which may
indicate that Irish strains originate from the second half of the 1990s. At the phylogenetic tree,
these strains are placed in G3, together with French strains 00-13 and 05-01. Therefore,
one may suspect that both the strains: Wriezen, Ireland19 and Ireland25, as well as 00-13
and 05-01 had a common ancestor, although French strains were clearly distanced. A similar
distribution of strains was obtained in the studies by Forrester et al. (2006b), who also suggested
122
B. Hukowska-Szematowicz, A. Manelska, B. Tokarz-Deptuła, W. Deptuła
that the RHD virus was imported to Ireland from France or Germany. In own studies, Groups V
and VI included strains from the years 1985–2008, referred to as antigen variants: 99-05, 03-24,
00-Reu, Triptis, Hartmannsdorf, WHN/China/012005, WHN/China/022005, WHN/China/032005,
TP, NJ1985, JXCHA97, YL and KV08-01 2008. In own studies, Hartmannsdorf strain individually
created Group VI (both at the homology tree and at the phylogenetic tree), which can be explained
with recombination variability, which was described for this strain by Forrester et al. (2008).
A similar result in reference to Hartmannsdorf strain was obtained by Abrantes et al. (2008),
who analysed 43 strains of the RHD virus, including antigen variants. As a result of the analysis
performed, evidence of recombination was obtained, as its position was determined (part
of region C and region E). Similarly as in own studies, Hartsmanndorf strain recorded a varied
phylogenetic profile, depending on the analysed fragment of VP60 protein. Interdependencies
in Groups V and VI can be explained both with the year of identification and the geographic
origin of the RHDV strains. A clear evolutionary distance characterises Chinese strains
and Korean strain, identified in 2005 and 2008, respectively. Studies regarding RHDV strains
recognised as antigen variants were also carried out by Matiz et al. (2006), Chrobocińska (2007)
and Chrobocińska and Mizak (2007), Farnos et al. (2007), MacIntosh et al. (2007), and Tian et al.
(2007). The results of analyses described allowed for dividing the strains analysed into VI groups
(in the homology aspect) and 6 genetic groups (in the aspect of evolutionary interdependencies).
The strains analysed recorded homology of 92%, which corresponds to 8% difference, namely
points to low variability of the virus. Distribution of the strains in Groups G1-G4 depended
on the year of identification, while Groups G5 and G6 included antigen variants.
CONCLUSION
The comparative analysis of the gene encoding VP60 protein in 32 strains of the RHD virus
(Table 1) revealed their homology in 95.6%. The homology tree generated for these strains
divided them into 6 groups (I–VI) with homology of 92%, whereas phylogenetic tree divided
the strains analysed into 6 genetic groups (G1-G6). The highest homology, above 98%, was
recorded between the following pairs of strains: 00-Reu and 03-24, 99-05 and 03-24, 05-01
and 03-24, 05-01 and JXCHA97, 05-01 and KV0801 2008, 05-01 and NJChina1985, 05-01
and TP, 05-01 and Triptis, 05-01 and WHN/China/012005, 05-01 and WHN/China/022005, 05-01
and WHN/China032005, 05-01 and YL, Ast89 and Eisenhuettenstadt, 99-05 and NJChina1985,
99-05 and WHN/China/012005, 99-05 and YL, HauteSaone France and WXChina1984, Mexico89
and WXChina1984, NJChina1985 and WXChina1984, RHDV New Zealand and WXChina1984,
RHF89 1989 and WXChina1984. On the basis of the comparative analysis performed
Comparative analysis of the gene encoding …
123
on 32 strains of the RHD virus, one can state that the gene encoding structural protein VP60
is rather stable, and reveals 92% homology, which corresponds to 8% variation. This analysis
also indicated that the assessed 32 strains of the RHDV grouped depending on the year
of identification into four groups (G1-G4), while group five (G5) and six (G6) were formed
by antigen variants of RHDV.
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ANALIZA PORÓWNAWCZA GENU KODUJĄCEGO BIAŁKO KAPSYDU VP60
U RÓŻNYCH SZCZEPÓW WIRUSA RHD (RABBIT HAEMORRHAGIC DISEASE)
Streszczenie. W pracy przedstawiono analizę porównawczą genu kodującego strukturalne białko VP60
u 32 szczepów wirusa RHD. Badania wykazały 95,6% homologię pomiędzy tymi szczepami RHDV, a na
podstawie zależności genetycznych pomiędzy nimi wyodrębniono na drzewie homologii grupę I–VI,
natomiast na drzewie filogenetycznym grupy genetyczne G1–G6. Analiza podobieństwa pomiędzy badanymi szczepami wykazała identyczność na poziomie 92%, a stwierdzone zróżnicowanie, wynoszące 8%,
pomiędzy analizowanymi szczepami RHDV, pochodzącymi z różnych kontynentów i zidentyfikowanych
w różnych latach dowodzi ich zmienności.
Słowa kluczowe: królik, VP60, RHD, genogrupa
ADVANCES IN AGRICULTURAL SCIENCES
Adv. Agric. Sci. 2011, XIV (1–2), 125–130
Karol Fijałkowski, Anna Silecka, Danuta Czernomysy-Furowicz
SERUM PROTEIN FRACTIONS IN HEALTHY SOWS DURING PERINATAL PERIOD
Department of Immunology, Microbiology and Physiological Chemistry,
West Pomeranian University of Technology, Szczecin,
Doktora Judyma 24, 71-466 Szczecin, Poland
Abstract. Quantitative changes in swine serum protein fractions were determined during perinatal period.
Polish Landrace Swine were divided into 3 groups: the first concerned sows 2 weeks before parturition,
the second sows on the day of parturition and the third one sows 2 weeks after it. Total protein concentration was measured in all examined sera. Afterwards, electrophoresis and densitometric analysis were
performed. Significant differences in protein fraction concentration were observed between the groups.
The most considerable contrast in protein concentration was shown in albumin, α and γ globulin fractions
between groups one (24.93 g/l, 2.94 g/l, 28.90 g/l) and two (31.45 g/l, 1.23 g/l, 21.59 g/l).
Key words: total protein concentration, electrophoresis, serum fractions, proteinogram
INTRODUCTION
Protein analysis is performed in serum or plasma and may be divided into 3 stages. The first,
concerns total protein concentration, the second, the analysis of particular protein fractions,
while the third, the concentration of selected protein (Dembińska-Kieć and Drożdż 2002).
The interpretation of serum protein composition includes evaluation of concentration changes
in particular protein during morbidity process and their function in organism. The determination
of swine plasma protein is utilised as a marker of stud health (Janowski et al. 1997), while
concentration of selected proteins, as CRP and Hp, is particularly useful in demonstration
of inflammation changes accompanying asymptomatic infections. Moreover, serum protein
measurement allows estimating the advancement level of pathological processes in viral
and bacterial infections as well as the general health status. Recently, the determination
of serum acute phase proteins is more and more often employed in veterinary diagnostics
as one of animal health criteria (Kostro et al. 2003).
The aim of this study was to determine quantitative changes of particular protein fractions
in serum of sows in perinatal period.
126
K. Fijałkowski, A. Silecka, D. Czernomysy-Furowicz
MATERIAL AND METHODS
Sera for this study were obtained from 12 sows (Polish Landrace Swine). All animals
originated from one farm. Blood was collected 3 times: 2 weeks before parturition, on the day
of parturition and 2 weeks after it. The animals did not show any symptoms of poor condition or
inflammation and parturitions were physiological.
Total protein concentration was determined by means of Biuret method (with wave length
540 nm) on 96 flat-bottomed microplates (Nunc) using microplate reader Elx800 (Bio-Tek
Instruments INC.). The solution of Bovine Serum Albumin (Sigma) was employed to established
calibration curve. The electrophoresis was performed on agarose gel (HYDRAGEL HR7, SEBIA)
during 40 minutes with tension 80V. DS-3 (Cormay) was used for densitometric reading.
The calculations were performed with computer program GraphPad Prism, while statistical
analysis was based on t-student test.
RESULTS AND DISCUSSION
The immunological research concerning animals during pregnancy often shows characteristic
features of acute phase reaction. First of all, it is manifested by moderate or significant increase
of some serum proteins (positive acute phase proteins) and decrease of albumin (negative
acute phase protein). The decrease of albumin and increase of α and β globulin fractions was
observed in this study. Enumerated elements are typical for pregnancy period in sows as well other
breeding animal females (Hitzig 1983, Halliwell 1989). The results of total protein and protein
fraction concentration are shown in Table 1.
Table 1. Statistical analysis of examined parameters in sows n = 12 (g/l)
Parameters
TP
A/G
Albumin
α1
α2
β1
β2
γ
2 weeks before
parturition
The day of parturition
2 weeks after
parturition
77.83 ± 1.27
(76.36 – 82.90)
0.47 ± 0.05
(0.40 – 0.55)
24.93 ± 2.09
(21.96 – 28.30)
294 ± 0.76
(2.03 – 4.16)
8.56 ± 0.80
(7.38 – 9.74)
5.00 ± 0.60
(4.36 – 5.73)
7.48 ± 1.75
(5.59 – 11.09)
28.90 ± 3.30
(23.7 – 34.33)
76.31 ± 2.77
(72.76 – 80.62)
0.71 ± 0.12
(0.58 – 0.89)
31.45 ± 2,41
(28.23 – 34.84)
1.23 ± 0.27
(0.91 – 1.58)
8.52 ± 1.69
(6.54 – 10.99)
4.88 ± 0.74
(4.30 – 6.17)
8.70 ± 1.62
(6.90 – 11.29)
21.59 ± 3.40
(16.97 – 28.59)
77.93 ± 1.69
(75.13 – 80.17)
0.63 ± 2.10
(0,56 – 0.73)
30.07 ± 1.75
(27.72 – 31.95)
3.19 ± 0.16
(2.99 – 3.4)
9.20 ± 1.01
(8.11 – 11.19)
4.52 ±0.36
(4.26 – 5.16)
9.12 ± 0.25
(8.67 – 9.42)
21.83 ± 3.15
(16.29 – 25.07)
Statistically significant
differences between
mean values
ns
**1 – 2,3
**1,3 – 2
ns
ns
ns
**1 – 2,3
1, 2, 3 – sows 2 weeks before parturition, on the day of parturition, 2 weeks before parturition, respectively,
**
– significant differences P  0.05 and P  0.01, ns – insignificant differences, ± – standard deviation, ( - ) – utter
values.
Serum protein fractions in healthy sows during perinatal period
127
Total protein concentration. Total protein concentration was similar in all three sow
groups. The obtained values were consistent with reference values established by Eder (1987),
Winnicka (2008) and many other authors (Bogadzińska 1994, Krzymowski 1998). The value
range for pregnant sows in this study (76.36–82.90) was similar to the one obtained by Green
et al. (1982): 71.4–78.78 g/l.
Protein fraction concentration
α1-globulin concentration. The decrease of α1-globulin fraction on the day of parturition
was observed in comparison to both other group. Mean values in sows 2 weeks before and
after parturition were similar. Green et al. (1982) observed in their research values about 1 g/l
lower, in the range between 0.8 g/l and 1.4 g/l.
α2-globulin concentration. Mean concentration of α2-globulin fraction 2 weeks before
parturition was 8.56 g/l which is comparable with reference values established by Eder (1987).
The results obtained in animals on the day of parturition and 2 week after it were also
physiological according to Green et al. (1982) and Eder (1987). All the results concerning
α fraction (after summing up α1 and α2 fraction) 2 weeks before, on the day and 2 weeks after
parturition, were located within the reference values proposed by Winnicka (2008).
β1-globulin concentration. The insignificant decrease of β1-globulin fraction was observed,
starting from 2 weeks before parturition, through the day of parturition, ending on 2 weeks after it.
It may be assumed that higher values of β1-globulin fraction in pregnant animals are due
to physiological increase of transferrin (Hitzig 1983, Ciesielski, 1997, Szutowicz 1997, Dembińska-Kieś and Drożdż 2002).
β2-globulin concentration. Mean values of β2-globulin concentration in sows on the day
of parturition and 2 weeks after it were within reference values (Eder 1987). As in the case
of α fraction, totalled β fraction results (in all examined groups) were within reference values
established by Winnicka (2008).
γ-globulin concentration. The values for γ-globulin obtained in this study were lower than
the ones established by Green et al. (1982). The difference may be connected with dissimilar
advancement of pregnancy. Bogadzińska et al. (1994) underline high diversity of quantitative
plasma content connected with age, physiological state, susceptibility on stress or even the time
of day. γ-globulin concentration in serum of sows on the day and 2 weeks after parturition were
coherent with reference values proposed by Winnicka (2008) and only slightly went beyond
references established by Eder (1987).
Protein fractions in sows 2 weeks before parturition. All proteinograms show the increase
of acute phase proteins, which was predominantly observed in α-globulin and β-globulin
concentration. On the other hand, albumin fraction was in decrease (Fig.1A).
128
K. Fijałkowski, A. Silecka, D. Czernomysy-Furowicz
A
B
C
Fig. 1. Proteinograms of serum: sows two weeks before parturition (A), on the day of parturition (B),
two weeks after parturition (C)
Albumin decrease is concerned physiological during the pregnancy in majority of pregnant
females (Hitzig 1983). The concentration of this fraction was comparable in sows on the day
of parturition and 2 weeks after it. Obtained results were within the reference values established
by Eder (1987), Bogdzińska et al. (1994) and Winnicka (2008). Whereas Krakowski (1998)
and Lechowski (1998) proclaim higher albumin concentration.
The increase of γ-globulins was observed in majority of examined animals, which indicates
the higher concentration of immunoglobulins.
Protein fractions in sows on the day of parturition. The decrease of γ-globulins was
observed in this group. The concentration of this fraction was significantly lower comparing
to values obtained in sows before parturition (Fig. 1B.).
That situation may be due to immunoglobulin transfer to mammary gland and colostrum
respectively. The decrease of serum immunoglobulins on the day of parturition may be
a consequence of some immunoglobulin passage (nearly before the birth), from maternal
blood to colostrum due to selective transportation. The immunoglobulins that pass from blood
to colostrum are mostly IgG1 (80% in swine colostrum), but also IgA and IgM. The latter ones
may be produced in mammary gland (Halliwell 1989, Minakowski and Weidner 1998, Gasiński
1999).
γ-globulin fraction might be also influenced by the increase of CRP – the most important
acute phase protein in swine. According to Lechowski et al. (1998), the increase of CRP may
be induced by different kinds of stress factors, for instance relocation to paritorio or parturition
itself.
Protein fractions in sows 2 weeks after parturition. Proteinograms of sows 2 weeks after
parturition resembled proteinograms of healthy, matured and not pregnant females (Fig. 1C.).
γ-globulin concentration in 2 week time after parturition reverts to the state observed in healthy,
matured animals as a consequence of colostrum production termination. The immunoglobulin
concentration is definitely lower in milk than in colostrum (Halliwell 1989, Gasiński 1999, Lipiński
Serum protein fractions in healthy sows during perinatal period
129
1999, Burek and Grela 2001, 2002, Rekiel 2002). The place of immunoglobulin production also
changes; in contrast to colostrum immunoglobulins, the majority of antibodies presented in milk
originate not from serum, but from mammary gland (Halliwell 1989).
CONCLUSION
The results show that serum is characterised by high content variability connected with age,
physiological state and stress factor – parturition between others. Even though proteinograms
of perinatal period concerns physiological state of examined animals, they manifest many features
characteristic for pathological changes. The animal monitoring by means of electrophoretic
fractioning, which leads to determination of mutual rate of particular protein share, allows
estimating immunological condition and, indirectly, welfare of examined animals. Moreover,
it provides undertaking of adequate immunoprophylactic and zoohygienic procedures.
REFERENCES
Bogdzińska M., Araszkiewicz J., Kapelański W., Gabrych I., Masewicz M . 1994. Evaluation
of the relationship between growth and development, and total protein concentration and its fractions
in blood plasma of Polish Landrace and Duroc boars. ATR im. Jana i Jędrzeja Śniadeckich w Bydgoszczy, Zesz. Nauk. 189, Zootech. 26, 47–56 [in Polish].
Burek R., Grela E. 2001. [Milking capacity of sows]. Trzoda Chlewna 12, 49–51 [in Polish].
Burek R., Grela E. 2002. Colostrum in piglets’ rearing. Trzoda Chlewna 4, 49–50 [in Polish].
Ciesielski D. 1997. Plasma proteins and their role in immunological response. Biochemia kliniczna
i analityczna, (red.) S. Angielski Warszawa, Państwowy Zakład Wydawnictw Lekarskich [in Polish].
Dembińska-Kieć A., Drożdż R. 2002. Plasma proteins, laboratory diagnostics with clinical biochemistry
elements. Red. A. Dembińska-Kieć, J.W. Naskalski, Wrocław, Urban & Partner [in Polish].
Eder H. 1987. Blood and Lymph, Veterinary Physiology Handbook, Scheunert A, Trautmann A, Berlin
und Hamburg, Verlag Paul Parey [in German].
Gasiński M. 1999. Appropriatep sow preparation for reproduction – the breeding success guaranty.
Trzoda Chlewna 11, 49–51 [in Polish].
Green S.A., Jenkins S.J., Clark P.A. 1982. A comparison of chemical and electrophoretic methods of serum
protein determinations in clinically normal domestic animals of various ages. Cornell Vet. 7, 416–426.
Halliwell R.E.W. 1989. Veterinary clinical immunology. Philadelphia/London/Toronto/Montreal/Sydney/Tokyo,
W.B. Saunders Company.
Hitzig W.H. 1983. Plasma proteins, Warszawa, Wyd. PZWL [in Polish].
Janowski H., Szweda W., Janowski T.E. 1997. Detailed pathology and therapy of swine diseases.
Wyd. ART Olsztyn [in Polish].
Kostro K., Luft-Deptuła D., Gliński Z., Miazga A. 2003 Role of acute phase proteins in animal
pathology. Życie Weter. 78 (1), 19–25 [in Polish].
Krakowski L., Krzyżanowski J., Wrona Z. 1998. Selected parameters changes of nonspecific immunity
in piglets during neonatal period. Med. Weter. 54 (11), 750–752 [in Polish].
Lechowski R., Sawosz E., Kluciński W., Chachułowa J., Siwicki A.K. 1998 Different stress factors
influence on acute phase proteins, gamma globulins, total protein and lysozyme activity in swine
serum. Med. Weter. 54 (9), 619–621 [in Polish].
130
K. Fijałkowski, A. Silecka, D. Czernomysy-Furowicz
Lipiński K. 1999. Swine milk as a source of nutrients [Trzoda Chlewna]. 5, 27–28 [in Polish].
Minakowski W., Weidner S. 1998. Vertebrate biochemistry. Warszawa, PWN [in Polish].
Rekiel A. 2002. Mechanisms of natural immunity acquisition in piglets [Trzoda Chlewna]. 12, 38–41
[in Polish].
Szutowicz A. 1997. Plasma proteins [Biochemia kliniczna]. (Ed.) S. Angielski, Sopot, Perseusz [in Polish].
Winnicka A. 2008. Reference values of elementary laboratory tests in veterinary. Warszawa, Wydaw.
SGGW [in Polish].
ANALIZA FRAKCJI BIAŁKOWYCH SUROWICY ZDROWYCH MACIOR
W OKRESIE OKOŁOPORODOWYM
Streszczenie. Określono ilościowe zmiany frakcji białek surowicy u macior w okresie okołoporodowym.
Przebadano 3 grupy macior rasy polskiej białej zwisłouchej. Pierwszą stanowiły maciory 2 tygodnie przed
porodem, drugą maciory w dniu porodu, trzecią maciory 2 tygodnie po porodzie. W surowicy każdej
maciory oznaczano poziom białka całkowitego. Następnie wykonywano rozdział elektroforetyczny surowicy i dokonywano jego analizy densytometrycznej. Zaobserwowano znaczne różnice w stężeniach frakcji
białkowych pomiędzy trzema grupami badanych zwierząt. Największy kontrast w stężeniu białek stwierdzony został we frakcjach albuminowej, α-globulinowej i γ-globulinowej, między surowicą macior 2 tygodnie przed porodem a w dniu porodu. Średnie stężenie frakcji albuminowej, α-globulinowej i γ-globulinowej
w surowicy macior 2 tygodnie przed porodem wynosiło odpowiednio: 24,93 g/l, 2,94 g/l, 28,90 g/l,
u macior w dniu porodu odpowiednio 31,45 g/l, 1,23 g/l, 21,59 g/l.
Słowa kluczowe: białko całkowite, elektroforeza, frakcje surowicy, proteinogram
ADVANCES IN AGRICULTURAL SCIENCES
Adv. Agric. Sci. 2011, XIV (1–2), 131–134
Anna Silecka, Karol Fijałkowski, Danuta Czernomysy-Furowicz
SERUM PROTEIN FRACTIONS IN PIGLETS – COMPARISON OF TWO TYPES
OF AGAROSE CARRIERS
Department of Immunology, Microbiology and Physiological Chemistry,
West Pomeranian University of Technology, Szczecin,
Doktora Judyma 24, 71-466 Szczecin, Poland
Abstract. The electrophoresis of 18 sera taken from clinically healthy piglets was performed by means
of two types of agarose gel (Protein Gel 100 and Hydragel HR). Six fractions were obtained with
Protein Gel 100. The mean concentration for each fraction was as follows: albumin – 29.07 g/L,
α1 – 2.82 g/L, α2 – 7.09 g/L, β1 – 4.06 g/L, β2 – 6.29 g/L, γ – 9.54 g/l. The employment of Hydragel HR
revealed also six fractions: albumin – 25.07 g/l, α1 – 1.03 g/l, α2 – 10.69 g/l, 1 – 4.80 g/l, 2 – 6.56 g/l,
γ – 10.61 g/l. Albumin / globulin ratio equals 0.98 g/l and 0.74 g/l, for Protein Gel 100 and Hydragel HR
agarose gel respectively.
Key words: albumin, electrophoresis, globulin, protein fraction, total protein
INTRODUCTION
Agarose electrophoresis is one of the most popular methods for protein fractioning. It reveals
homogenic albumin and heterogenic globulin fractions. The evaluation of changes in protein
quantitative ratio as well as in particular fraction concentration is of great diagnostic importance
(Bigoszewski et al. 2001). For instance, it provides with information about organ or even whole
system malfunctioning. Total protein concentration is also a significant indicator of animal
physiological state. Therefore, serum protein analysis can be applied in prophylaxis and therapy,
which leads to limitation of economic loss (Kostro et al. 2003).
Proteinograms have not been so widely used in veterinary as they are in medicine due to
lack of standardisation. Six protein fractions are recognised in human serum (α1, α2, β1, β2, γ),
(Tomaszewski 1993), while in the case of animals its number differs. This variation is not only
observed between species, but also within it. Five to seven fractions can be seen in swine
(Scheunert and Trautmann 1987), six to seven in canine, five to ten in equine serum (Coffman
1969, Osbaldiston 1972, Kristensen et al. 1977, Halliwell 1989). This controversy emerges from
different physical and chemical properties of agarose gels as well as different electrophoretic
parameters.
132
A. Silecka, K. Fijałkowski, D. Czernomysy-Furowicz
The aim of this study was to establish prteinogram of piglets on to types of agarose carriers.
MATERIAL AND METHODS
Blood samples obtained form 18 piglets was used in this research. All 2 week old animals
originated from one place and underwent the same nutritional conditions.
Total protein concentration and protein fraction electrophoretic mobility and quantity were
determined in the sera. Total protein concentration was measured by means of Biuret method
on Elx800 microplate reader (Bio-Tek Instruments INC., Universal Microplate reader), using 96
format flat-bottomed microplate (bioMérieux, 123 x 81 mm). BSA (Bovine Serum Albumin,
Sigma) solution served for preparation of calibration curve. Electrophoresis was performed
on two carrier types: Protein Gel 100, (Cormay) – 20 minutes, voltage 100V and Hydragel HR
(Sebia) – 40 minutes, voltage 80V. DS-3 (Cormay) was employed for densitometric reading.
All statistical analyses were conducted with Statistica 7.1 software.
RESULTS AND DISCUSSION
Six protein fractions (albumin, α1, α2, β1, β2, γ) were obtained on both carriers. β2, γ showed
no differences, while albumin and α1 concentration was significantly higher on Protein Gel 100
(Table 1). On the other hand, higher values of α2 and β1 was observed in the case of Hydragel
HR. Taking into consideration, the fact, that research was performed at the same time, with
the same condition and using the same serum samples, the revealed differences must be
connected with type and sensitivity of employed carriers.
Table 1. Statistical analysis of examined parameters in piglets n = 18 (g/l)
Parameters
Protein Gel 100
Albumin
α1
α2
β1
β2
γ
Statistically significant
differences between
mean values
58.77 ± 6.23
(52.01 – 67.81)
TP
A/G
Hydragel HR
0.98 ± 0.08
(0.61 – 0.85)
29.07 ± 4.17
(22.39 – 36.40)
2.82 ± 0.39
(2.20 – 3.53)
7.09 ± 0.82
(5.39 – 8.37)
4.06 ± 0.65
(2.70 – 5.62)
6.29 ± 1.03
(4.10 – 8.23)
9.54 ± 2.82
(6.79 – 15.66)
0.74 ± 0.08
(0.61 – 0.85)
25.07 ± 2.79
(21.55 – 29.19)
1.03 ± 0.18
(0.76 – 1.28)
10.69 ± 1.28
(9.68 – 13.09)
4.80 ± 0.76
(3.97 – 6.10)
6.56 ± 1.66
(3.29 – 8.15)
10.61 ± 1,99
(7.90 – 14.42)
**
*
*
**
ns
ns
*,** – significant differences P  0.05 and P  0.01, ns – insignificant differences, ± – standard deviation, (-) – utter
values.
Serum protein fractions in piglets – comparison of two types of agarose carriers
133
Fig. 1. Protein Gel 100
Fig. 2. Hydragel HR
Serum protein electrophoretic analysis on agarose gel is an elementary diagnostic test
performed in order to observe changes between particular protein fraction concentrations.
Moreover, it may be treated as an introduction for further analysis of individual acute phase
proteins.
Summarizing, application of Hydragel HR and Protein gel 100 led to distinguishing
of the same protein fraction number. Therefore, determination of reference values for Protein
Gel 100, is particularly important, as this carrier is more economical and more frequent in use.
REFERENCES
Bigoszewski M., Rychlik A., Depta A. 2001. Acute phase proteins in domestic animals. Med. Weter.
57(3), 151–154.
Coffman J.R. 1969. Clinical application of serum protein electrophoresis in the horse. In: Proceedings,
th
14 Ann Meeting Am Assoc Equine Practitioners, Philadalphia, 265–279.
134
A. Silecka, K. Fijałkowski, D. Czernomysy-Furowicz
Green S.A., Jenkins S.J., Clark P.A. 1982. A comparison of chemical and electrophoretic methods
of serum protein determinations in clinically normal domestic animals of various ages. Cornell Vet.
7, 416–426.
Scheunert A., Trautmann A. 1987. Blood and Lymph, Veterinary – Physiology Handbook, Berlin
und Hamburg, Verlag Paul Parey.
Halliwell R.E.W. 1989. Veterinary Clinical Immunology, Philadelphia/London/Toronto/Montreal/Sydney/Tokyo,
W.B. Saunders Company 162–165.
Kostro K., Luft-Deptuła D., Gliński Z., Miazga A. 2003. Role of acute phase proteins in animal pathology.
Życie Weter. 78 (1), 19–25.
Osbaldiston G.W. 1972. Serum protein fraction in domestic animals. Br. Vet. J. 128, 386–393.
Tomaszewski J.J. 1993. Laboratory diagnostics, PZWL, 44–45 [in Polish].
ANALIZA OBRAZU ELEKTROFORETYCZNEGO SUROWICY KLINICZNIE
ZDROWYCH PROSIĄT Z ZASTOSOWANIEM DWÓCH TYPÓW NOŚNIKA
AGAROZOWEGO
Streszczenie. Rozdział elektroforetyczny 18 surowic pochodzących od klinicznie zdrowych prosiąt
przeprowadzono na dwóch typach żeli agarozowych: Protein Gel 100 i Hydragel HR. Po elektroforezie
i odczytach densytometrycznych żelów Protein Gel 100 wyodrębnionych zostało 6 frakcji białek. Średnie
stężenie uzyskanych frakcji wynosiło: frakcja albuminowa – 29,07 g/l, frakcja α1 – 2,82 g/l, frakcja
α2 – 7,09 g/l, frakcja β1 – 4,06 g/l, frakcja β2 – 6,29 g/l, frakcja γ – 9,54 g/l. Podobnie na żelu Hydragel HR
również wyróżnionych zostało 6 frakcji białkowych. Średnie stężenie tych frakcji wynosiło: frakcja
albuminowa – 25,07 g/l, frakcja α1 – 1,03 g/l, frakcja α2 – 10,69 g/l, frakcja β1 – 4,80 g/l, frakcja β2 – 6,56 g/l,
frakcja γ – 10,61 g/l. Stosunek albumin do globulin wynosił 0,98 g/l (Protein Gel 100) i 0,74 g/l
(Hydragel HR).
Słowa kluczowe: albumina, białko całkowite, elektroforeza, frakcja surowicy, globuliny, proteinogram
ADVANCES IN AGRICULTURAL SCIENCES
Adv. Agric. Sci. 2011, XIV (1–2), 135–146
Beata Tokarz-Deptuła1, Bartłomiej Pejsak2, Wiesław Deptuła1
WHITE AND RED BLOOD CELL INDICES IN HEALTHY RABBITS
1
Department of Microbiology and Immunology, University of Szczecin,
Felczaka 3c, 71-412 Szczecin, Poland, e-mail: [email protected]
2
PhD Student at the Department of Microbiology and Immunology, University of Szczecin,
Felczaka 3c, 71-412 Szczecin
Abstract. This paper illustrates selected haematological parameters such as haemoglobin concentration,
number of erythrocytes, thrombocytes, total number of leukocytes and nember of lymphocytes,
neutrophils, eosinophils, basophils and monocytes in healthy rabbits – domestic mixes. These data were
achieved in own investigations that were performed in 1989–1990 and then were compared with results
described by another authors. The group of rabbits under investigation was numerous, therefore results
presented in this paper can serve as reference parameters of haematological factors in rabbits living in our
region.
Key words: rabbit, haematological factors
INTRODUCTION
Home rabbit, being a utility animal, originates from wild rabbit (Oryctolagus cuniculus).
Its systematic allocation is the class: mammals (Mammalia); subclass: theria (Theria), order:
lagomorphs (Lagomorpha); suborder: gnawing animals (Duplicidentata); family: leporids
(Leporidae) (quote Deptuła 2003). Apart from utility value, it is also an important animal
in laboratory and scientific experiments, in vivo and in vitro, constituting a convenient animal
research model, in studies of various infections and diseases in humans, e.g. infection with HIV
virus, or studies on implants and histocompatibility, and in household animals, e.g. viral
infections (Fox and Laird 1970, Śmigielska 1977, Ezema et al. 1984, Gamboa and Miller 1984,
Szubertowska and Gromysz-Kałkowska 1986, Wolford et al. 1986, Anderson and Henck 1994,
Jain 1994, Reagan et al. 1995, Tokarz-Deptuła 1998, Wells et al. 1999, Aleman et al. 2000,
Pearce 2000, Rohilla et al. 2000, Kim et al. 2002, Deptuła et al. 2003, Tokarz-Deptuła and Deptuła
2005, Yang et al. 2005, Hukowska-Szematowicz 2006, Niedźwiedzka 2008, Jeklova et al.
2009, Tokarz-Deptuła 2009). However, its use in such research requires specific knowledge,
136
B. Tokarz-Deptuła, B. Pejsak, W. Deptuła
principally in the area of broadly understood animal adaptation (Wolford et al. 1986, Jain 1994,
Rohilla et al. 2000, Deptuła et al. 2003, Jeklova et al. 2009). Among major parameters, which
form the basis for such various experiments involving such animals, there are haematological
indices which, as suggested by many authors, can depend on the age, sex, race or technology
of breeding, feeding, physiological condition, and bio-climatic conditions (Fox and Laird 1970,
Ezema et al. 1984, Gamboa and Miller 1984, Wolford et al. 1986, Parkanyi and Rafay 1989,
Anderson and Henck 1994, Jain 1994, Tokarz-Deptuła 1998, Wells et al. 1999, Aleman et al.
2000, Pearce 2000, Rohilla et al. 2000, Kim et al. 2002, Deptuła et al. 2003, Tokarz-Deptuła
and Deptuła 2005, Yang et al. 2005, Hukowska-Szematowicz 2006, Niedźwiedzka 2008,
Jeklova et al. 2009, Tokarz-Deptuła 2009). Studies by various authors (Chomicz 1967, Fox and
Laird 1970, Kabata et al. 1991) also indicate that the gender of rabbits affects the white blood
cell image, but principally lymphocytes, which obtain lower values in females than in males.
Some studies (Królak 1998, Kim et al. 2002) evidence that with the age of rabbits, the obtained
values of haematological indices increase. There are, however, also studies (Gromysz-Kałkowska
et al. 1981, Bortolotti et al. 1989) where the above is not confirmed. There are also studies
(Zaorska 1973, Kabata et al 1991) indicating that the haematological blood image of rabbits
at the same age (1–2 years) and similar weight (3–5 kg) is rather stable. Furthermore, it was
evidenced (Zaorska 1973, Parkanyi and Rafay 1989) that in rabbits aged 2 months, 1 year and
2 years, nutritional conditions do not affect haematological indices. It was also recorded
(Zaorska 1973, Kabata et al 1991) that rabbits of white New Zealand race have a rather lower
number of leucocytes and lymphocytes as compared to mixed-breed ones, while the number
of monocytes is three times lower in mixed breed as compared to rabbits of white New Zealand
race (Zaorska 1973, Kabata et al 1991). It was also determined (Bortolotti et al. 1989) that
pregnancy in female rabbits results in lowered number of lymphocytes and increase in neutrophils
and erythrocytes.
PURPOSE OF THE STUDY
The present study is aimed at presenting the haematological image (% haematocrit, haemoglobin concentration, erythrocyte number, total leucocyte number, and the number of lymphocytes,
neutrophils, eosinophils, basophils, and monocytes) in healthy rabbits, obtained in 10-year long
own observations, and at comparing them to analogical haematological indices obtained
by different authors (Table 3, 4).
White and red blood cell indices in healthy rabbits
137
MATERIAL AND METHODS
This study uses the results of own studies in the area of haematological studies on 200 rabbits
examined in the years 1989–1999. The studies involved mixed-race rabbits weighing from
2.0 to 4.2 kg, and aged 3–6 months, of both sexes. These were conventional animals from
licensed farm, remaining under continuous veterinary-zootechnical supervision (Anon 1987).
Animals used in the experiment remained at the vivarium of the Department of Microbiology
at the Faculty of Natural Sciences, University of Szczecin, where they were placed in cages
with parameters predefined for rabbits (Anon 2006), and the room featured conditions conformant
to the recommended national standards (lighting, temperature and ventilation) (Anon 2006).
The animals were subject to a 14-day adaptation period and were fed with full-portion LSK
rabbit feed in the quantity of 150–200 g/day and had unlimited access to water. Blood was
drawn from the edge vein of rabbit ear, and haematological indices were identified, with routine
methods used in laboratory diagnostics, which involved: percent (%) of haematocrit, haemoglobin
concentration, number of erythrocytes, total number of leucocytes, and the number of lymphocytes,
neutrophils, eosinophils, basophils and monocytes. The results of the studies (Table 1) were
confirmed to results obtained in rabbits by various authors (Table 3). Furthermore, in Tables 2
and 4, haematological results were presented as minimum and maximum values from Tables
with own studies results (Table 1) and results of other authors (Table 3).
DISCUSSION OF THE RESULTS
The image of changes to haematological indices presented in the present own studies in the
period from 1989 to1999 (Table 1, 2) indicates that the values regarding% of haematocrit
amounted to from 31 to 51%, haemoglobin concentration – from 3.6 to 12.3 mmol/l, number
of erythrocytes – from 2.4 to 6.1 x 1012/l, leucocytes – from 3.3 to 10.0 x 109/l, lymphocytes –
from 55 to 88.0%, neutrophils – from 4.0 to 40.0%, eosinophils – from 0.0 to 3.8%, basophils –
from 0.0 to 7.0%, and monocytes – from 0.0 to 9.0%. The results referring to % haematocrit,
haemoglobin concentration and% of lymphocytes (Table 1, 2) obtained in own studies are very
similar. In turn, the results regarding the number of erythrocytes, leucocytes and % of neutrophils
slightly differ (Table 1, 2), whereas results in the area of percent of eosinophils, basophils
and monocytes indicate clear disparity (Table 1, 2). It must be added that in the case of such
studies, the material on which the studies were performed was very “even”, and the experiment
was carried out on a large sample of animals in practically uniform standard conditions
for rabbits. Therefore, it seems, the obtained values can be adopted as reference values
for domestic mixed-breed rabbits aged 3–6 months, and weighing 2.0–4.0 kg. In the studies carried
138
B. Tokarz-Deptuła, B. Pejsak, W. Deptuła
out by our team, and performed at a later date (Niedźwiedzka-Rystwej and Deptua 2010),
also performed on domestic mixed-breed rabbits of similar weight (3.2–4.2 kg), assessing such
results with differentiation of rabbit sexes, it was evidenced that analogical haematological
indices in these animals remain at a similar level (Table 1, 2). The analysis of the results
obtained in own studies (Table 1, 2) also indicates that the recorded fluctuations in haematological
parameters are similar to results presented in studies by various authors (Pinna-Pintor
and Grassini 1957, Didishelm et al. 1959, Barański et al. 1962, Chomicz 1967, Lewandowska
1967, Michalska 1967, Siński and Krzysztofowicz 1967, Chowaniec and Markiewicz 1970, Fox
and Laird 1970, Laird et al. 1970, Kotche and Gottschalk 1972, Zaorska 1973, Dąbrowski
1975, Schlam et al. 1975, Śmigielska 1977, Gromysz-Kałkowska et al. 1981, Lineburg 1986,
Szubertowska and Gromysz-Kałkowska 1986, Annon 1987, Rafay and Parkanyi 1987, Knorr
and Wenzel 1988, Niyo et al. 1988, Bortolotti et al. 1989, Gibasiewicz 1989, Hewitt et al. 1989,
Katkiewicz 1989, Parkanyi and Rafay 1989, Toth and Kruger 1989, Vyhnalek and Klusakowa
1989, Deptuła et al. 1990, Müller et al. 1990, Pawelski 1990, Górecka 1991, Kabata et al.
1991, Moiser and Hesketh 1992, Plassiert et al. 1992, Słaby 1992, Morton and Abbot 1993,
Polak 1993, Dacasto et al. 1994, Doubek and Svoboda 1994, Hilyer 1994, Mojzyk 1994,
Poźniak 1995, Reagan et al. 1995, Zalewska 1995, Brylińska and Kwiatkowska 1996, Polanin
1996, Iwasieczko 1997, Królak 1998, Okerman 1999, Sadło 1999, Sitarska et al. 2000). It must
be added that in the studies of these indices, it was noticed that in the case of haematocrit
percentage, it achieves the values from 26.3 to 55.0%, while haemoglobin concentration –
from 5.6 to 15.2 mmol/l. Similarly, the number of erythrocytes ranged from 2.7 to 11.2 x 1012/l,
leucocytes from 3.0 to 14.1 x 109/l, lymphocytes from 7.4 to 90.0%, neutrophils from 0 to 50.0%,
eosinophils from 0 to 30.0%, while the values of basophils and monocytes ranged 0–23.0%.
It seems that the rather greater differentiation in the area of these haematological indices than
in the values from own studies is probably due to physiological condition of the animals
or the research methodology. Notwithstanding these facts, it must be stated that the comparison
of the values of haematological indices obtained in own studies (Table 1, 2) to the results
of studies carried out in this respect by various authors (Table 3, 4) still points to clear similarity
of the results obtained, in particular in the area of haematocrit percentage, haemoglobin
concentration, number of erythrocytes and total number of leucocytes, while certain discrepancies
refer to the percent of lymphocytes, neutrophils, eosinophils, basophils and monocytes.
Regardless of the above facts, it must be assumed that the results of studies obtained
on such a large number of animals, observing uniform properties of animals and conditions
during the experiment indicate that they can be adopted as reference values for our region.
Table 1. Minimum and maximum values of haematological indices in rabbits obtained in own studies
Parameter
measured
Haematocrit
%
Haemoglobin
mmol/l
Erythrocytes
12
10 /l
Leucocytes
9
10 /l
Lymphocytes
%
Neutrophils
%
Eosinophils
%
Basophils
%
Monocytes
%
Literature item
Polak K.
Polanin A.
1993
1996
Deptuła W.
1990
Górecka D.
1991
Iwasieczko
S. 1997
Królamk M.
1998
Mojzyk C.
1994
Poźniak R.
1995
Sadło J.
1999
Słaby K.
1992
Zalewska K.
1995
35–41
–
–
46–51
–
–
–
31–36
–
–
–
3.6–9.2
6.5–8.9
5.0–7.5
5.3–8.1
6.9–12.3
5.2–7.9
5.7–8.5
–
5.2–7.9
7.0–9.5
6.5–7.6
3.7–4.6
3.8–4.4
–
4.8–5.5
2.4–3.2
3.1–3.6
3.7–5.5
3.4–3.8
4.9–6.1
3.5–4.0
3.4–3.8
4.3–10
3.3–7.7
6.0–8.1
5.2–6.9
5.7–9.3
5.9–7.6
5.3–8.2
4.9–7.0
3.8–7.4
3.5–6.0
5.1–6.8
59–80.6
68–80
55–79
70–88
74–87
74–78
63.3–77.7
73.5–79.2
63.4–77.8
67–82
71–82
15.2–33.4
17–28
19–40
4–9
11–12
20–24
19.3–28.3
18.4–23.6
14.2–27.0
15.0–29.5
22–28
0–0.2
0.0–2.0
0.0–0.4
2–3
0.3–3.7
0.0–0.7
0.3–1.0
0.5–1.7
1.2–3.7
0.0–1.5
1.1–3.8
0.7–2.4
0.0–3.0
0.0–2.0
2–7
0.1–1.5
0.0–0.5
0.6–1.3
0.5–1.2
0.4–2.1
1.5–3.5
0.2–0.5
5
1–3
0.0–3.0
6–9
0.0–1.3
0.0–0.7
0.6–1.0
0.4–1.0
1.7–3.7
0.0–1.5
0.1–0.2
Table 2. Minimum and maximum values of haematological indices in rabbits obtained in own studies presented in Table 1
Haematocrit
%
31.0–51.0
Haemoglobin
mmol/l
3.6–12.3
Erythrocytes
12
10 /l
2.4–6.1
Leucocytes
9
10 /l
3.3–10.0
Parameter measured
Lymphocytes
%
55.0–88.0
Neutrophils
%
4.0–43.0
Eosinophils
%
0.0–3.8
Basophils
%
0.0–7.0
Monocytes
%
0.0–9.0
Table 3. Minimum and maximum values of haematological indices in rabbits obtained by various authors
Didishelm P.
1959
Gibasiewicz W.
1989
Gromysz-Kałkowska K.
1981
35
36–55
43.6
–
–
38
12.1
7.4
12
9.6
14.0
–
–
10–16
15.2
8.4–12.4
–
12.8
6
5.7
4–6
5.7
4.6
6.0
–
–
6–10.1
6.7
2.7–6.3
11.2
6.0
7.6
8.1
3.8–12
7.8
7.6
8.5
11.5
–
3–11
12.4
6.3–14.1
5.0
9.2
12
11.9
4–6
3.2–12
Hewitt C.D.
1989
Dąbrowski Z.
1975
–
Fox R.R.
1970
Dacasto M.
1994
40.7
36
Doubek J.
1994
Chowaniec W.
1970
34.9
–
Brylińska J.
1996
40
–
Bortolotti A.
1989
–
Barański S.
1962
Haematocrit
%
Haemoglobin
mmol/l
Erythrocytes
12
10 /l
Leucocytes
9
10 /l
Lymphocytes
%
Neutrophils
%
Eosinophils
%
Basophils
%
Monocytes
%
Anon
1987
Parameter
measured
Chomicz L.
1967
Literature item
55
–
54
–
55
50–80
–
64
–
–
8–50
35.9
–
–
–
30
10–42
–
30
–
–
–
1–3
2.8
–
–
–
1
0–5
–
1
–
–
2.5
–
1–3
–
–
–
–
–
0–1
–
–
–
2
–
1–4
–
–
–
–
14
0–0.3
3
–
–
20–90
59
68
20–90
8–50
35
32
1–3
1
1–3
1–4
2
–
Table 3a. Minimum and maximum values of haematological indices in rabbits obtained by various authors
12.9±1.1
12.4
13.5
12.7
6.3
5.5–7.5
6.2
6.2
5.7±0.4
4–6
6.4
6.1
–
6–12
6.2
8.1
6.4±1.6
3.8–12.0
12.9
6.9
Niyo K.A.
1988
13.4
Muller D.
1990
12.7
10–16
38
Morton D.
1993
39–41
Moiser K.J.
1992
Laird C.
1970
–
41
Mi8chalska Z.
1967
Kotche W.
1972
40.8±2.4
33–48
Lineburg A.
1986
Knorr F.
1972
–
Kabata J.
1991
Katkiewicz M.
1989
Haematocrit
%
Haemoglobin
mmol/l
Erythrocytes
12
10 /l
Leucocytes
9
10 /l
Lymphocytes
%
Neutrophils
%
Eosinophils
%
Basophils
%
Monocytes
%
Hilyer E.V.
1994
Parameter
measured
Lewandowska I.
1967
Literature item
–
26.3
–
41
–
–
7.7
8–15
13
–
3.6–6
–
4.5–7.0
5.7
–
5.5
6.4–11.8
–
9.0
6.4
4.6
30
30–85
53
63
–
20–90
–
42
–
–
–
–
64
67
20–52
31
32
–
8–50
–
–
–
–
–
–
29.9
28
0–5
0.8
1.3
–
1–3
–
–
–
–
–
–
1.4
–
2–7
4.1
2.4
–
0.5–30
–
–
–
–
–
–
3.5
–
1–12
6.3
2
–
1–4
–
–
–
–
–
–
1
–
Table 3b. Minimum and maximum values of haematological indices in rabbits obtained by various authors
–
–
7.2–0.1
–
13
11.2
–
–
–
11.6
4.5–7.4
–
5.8
5.4
–
5.7
–
5.5
–
3.1–9.9
–
6.5
7.1
9.4
7.4
4.1–11.2
7.7
22–95
63.1
56.4
53.3
71
66.7
14–83
–
29.8
29.9
–
23
–
0–8
2.9
1.4
–
23
0–8
0.2
3.5
–
0.5–16
3.9
1
–
–
33.5
–
–
10.6
4.9–5.0
4.9
–
–
6.5
5.6–7.7
7.5
9.5
6.2
–
58.3
–
42
7.4
7.4
50
–
32.6
–
52
20
19.5
1.2
1
–
0.1
–
–
–
2.8
0–4
2
4
–
3.1
–
–
–
1.8
0–6
2.3
1.3
–
2.5
–
6
–
2
Siński E.
1967
Zaorska T.
1973
–
Toth L.A.
1989
–
Vyhnalek J.
1989
Schlam O.W.
1975
–
Śmigielska J.
1977
Reagan W.J.
1995
41
Szubertowska E.
1986
Rafay J.
1987
–
Sitarska E.
2000
Plassiert G.
1992
30–44
Pawelski S.
1990
Pinna-Pintor P.
1957
Haematocrit
%
Haemoglobin
mmol/l
Erythrocytes
12
10 /l
Leucocytes
9
10 /l
Lymphocytes
%
Neutrophils
%
Eosinophils
%
Basophils
%
Monocytes
%
Parkanyi V.
1989
Parameter
measured
Okerman L.
1999
Literature item
39
30–53
34
36.1–38.2
5.6–11.8
11.5
10.3–10.7
4–9
5.2
10
–
63.9
44
0–30
30.4
–
0–2
23
–
23
–
9.1
45
Table 4. Minimum and maximum values of haematological indices in rabbits obtained in studies presented by various authors in Table 3
Haematocrit
%
26.3–53.0
Haemoglobin
mmol/l
5.6–15.2
Erythrocytes
12
10 /l
2.7–11.2
Leucocytes
9
10 /l
3.0–14.1
Parameter measured
Lymphocytes
%
7.4–90.0
Neutrophils
%
0–50.0
Eosinophils
%
0–30.0
Basophils
%
0–23.0
Monocytes
%
0–23.0
White and red blood cell indices in healthy rabbits
143
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WSKAŹNIKI BIAŁO I CZERWONOKRWINKOWE U KRÓLIKÓW ZDROWYCH
Streszczenie. Praca przedstawia wybrane wskaźniki hematologiczne (stężenie hemoglobiny, liczbę
erytrocytów, trombocytów, ogólna liczbę leukocytów oraz ilość limfocytów, neutrofilów, eozynofilów,
bazofilów i monocytów) u królików zdrowych mieszańców krajowych, które uzyskano w badaniach własnych realizowanych w latach 1989–1990, a które odniesiono do badań obrazu biało- i czerwonokrwinkowego przeprowadzonych przez innych autorów. Zaprezentowane wyniki na tak licznej grupie zwierząt,
dają możliwość wypracowania danych referencyjnych w zakresie wskaźników hematologicznych u królików dla naszego regionu.
Słowa kluczowe: królik, wskaźniki hematologiczne
ADVANCES IN AGRICULTURAL SCIENCES
Adv. Agric. Sci. 2011, XIV (1–2), 147–154
Jolanta Karakulska, Anita Stępień, Karol Fijałkowski, Danuta Czernomysy-Furowicz
THE EFFECT OF PENICILLIN AND AMOXICILLIN/CLAVULANIC ACID
ON THE INDUCTION OF L FORM OF STAPHYLOCOCCI ISOLATED
FROM MASTITIC MILK OF COWS
Department of Immunology, Microbiology and Physiological Chemistry
West Pomeranian University of Technology, Szczecin
Doktora Judyma 24, 71-466 Szczecin, Poland, e-mail: [email protected]
Abstract. Many antibiotics used to treat mastitis affect the bacterial cell wall, and can induce the L form
of various microorganisms. L forms allow bacteria to resist antibiotic treatment and survive in the mammary
gland. The research was based on 17 Staphylococcus spp. strains, isolated from milk of cows with
subclinical mastitis from a single herd. The bacteria were classified into Staphylococcus genus and were
identified to particular staphylococcal species on the basis of gap gene presence and analysis of the gap
gene polymorphism by RFLP with AluI restriction enzyme. Based on the analysis of gap gene
polymorphism S. aureus (29.4%), S. chromogenes (23.5%), S. haemolyticus (17.6%), S. capitis (5.9%),
S. epidermidis (5.9%), S. equorum (5.9%) and Staphylococcus spp. (11.8%) were identified. The susceptibility of staphylococci to penicillin G, oxacillin, ampicillin, amoxicillin/clavulanic acid, cephalothin, cefoxitin,
cefuroxime, cefoperazone and ceftiofur was analyzed. Among 17 analyzed staphylococci, 23.5% strains
were resistant and 23.5% strains were sensitive to all β-lactam antibiotics. In the test with cefoxitin, seven
(41.2%) methicillin-resistant strains were identified. Staphylococcal strains were stimulated to develop
L forms with penicillin (Penicillinium Procainicum L 1200000 IU) and amoxicillin/clavulanic acid (Augmentin,
457 mg). 12 strains (70.6%) developed their L forms under influence of penicillin and amoxicillin/clavulanic
acid. Among staphylococci which were able to produce L forms, 83.3% strains stimulated with penicillin
and 91.7% strains stimulated with amoxicillin/clavulanic acid developed their L forms after 10 minutes
of incubation with the antibiotic. In turn, after 24 hours of incubation there were 100% and 91.7% strains,
respectively.
Key words: Staphylococcus, L form, penicillin, amoxicillin/clavulanic acid, mastitis
INTRODUCTION
For the first time, the L forms were described in Streptobacillus moniliformis by Klieneberger
in 1935 (Klieneberger 1935). The L forms are also called the cell wall-defective bacteria (CWD),
(Owens 1987). Staphylococcal L form colonies have different morphology from the parent
strains, and form colonies that look like fried eggs (Owens 1988). These are live bacterial cells,
but they are devoid of the cell wall, as a result of antibiotic or other substance activity. They can
occur in vivo. As an example, the L forms in the mammary gland of cows avoid antibiotics,
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J. Karakulska, A. Stępień, K. Fijałkowski, D. Czernomysy-Furowicz
for which the target is a cell wall and may cause a relapse of the disease after therapy.
In enrichment cultures of L forms can also appear their intermediate forms, which are the effect
of partial damage of the cell wall. The intermediate forms do not have morphology typical
for L forms (Shimokawa and Nakayama 1997, Stoitsova et al. 2000). It has been shown that
antibiotics acting on the cell wall generate L forms of staphylococci (Molander et al. 1964).
Many antibiotics used to treat mastitis affect the bacterial cell wall, and can induce the L form
of various microorganisms. L forms allow bacteria to resist antibiotic treatment and survive
in the mammary gland. Therefore, when choosing an antibiotic for the treatment of mastitis,
especially its acute form caused by S. aureus, the possibility of induction of L forms by this
antibiotic should be considered. The increase of pH in the mammary gland and levels
of electrolytes and proteins in serum during inflammation, favor the formation of L forms in vivo.
In addition, low level of complement and decreased phagocyte activity in the mammary gland
are factors that enable the survival of L forms during infection (Owens 1987).
The aim of this study was to analyze the sensitivity of staphylococcal strains, isolated from
mastitic milk, to β-lactam antibiotics, and to determine the influence of penicillin and amoxicillin/
/clavulanic acid on the induction of L forms in vitro.
MATERIAL AND METHODS
The research was based on 17 Staphylococcus spp. strains, isolated from milk of cows with
subclinical mastitis from a single herd. As control strains S. aureus ATCC 43300 (American
Type Culture Collection), S. aureus ATCC 25923 (American Type Culture Collection)
and S. epidermidis PCM 2651 (Polish Collection of Microorganisms) were used.
Isolation and identification of strains. Milk samples in the volume of 0.1 ml were plated
on the supplemented Baird-Parker Agar (Oxoid) and incubated for 24–48 hours at 37ºC. Initial
identification of isolates was based on the colony morphology, Gram staining, oxidase (Oxidase
Reagent, bioMérieux) and catalase (H2O2) activity and the ability to ferment glucose in anaerobic
conditions (OF Basal Medium, Oxoid). The bacteria were classified into Staphylococcus genus
and were identified to particular staphylococcal species on the basis of gap gene presence
and analysis of the gap gene polymorphism by RFLP method. The presence of the gap gene
was determined with primers and reaction conditions described previously by Yugueros et al.
(2000). PCR products of gap gene were digested with AluI restriction enzyme (Fermentas),
according to the manufacturer’s procedure. PCR and RFLP products were separated
by electrophoresis and were analyzed using GeneTools software (Syngene). Staphylococcal
DNA was isolated using Genomic Mini kit (A&A Biotechnology, Gdynia). The PCR was
performed in the Mastercycler Personal thermocycler (Eppendorf).
The effect of penicillin and amoxicillin/clavulanic acid…
149
Disc diffusion susceptibility test. The susceptibility to nine β-lactam antibiotics: penicillin
G (P 10U), oxacillin (OX 1 μg), ampicillin (AMP 10 μg), amoxicillin/clavulanic acid (AMC 30 μg),
cephalothin (KF 30 μg), cefoxitin (FOX 30 μg), cefuroxime (CXM 30 μg), cefoperazone (CFP
75 μg) and ceftiofur (EFT 30 μg) was analyzed. The test was carried out by the disc diffusion
method on the Mueller-Hinton agar (Oxoid), as recommended by Hryniewicz et al. (2005)
and according to CLSI (Clinical and Laboratory Standards Institute, 2007).
Induction of L forms. Staphylococcal strains were stimulated to develop L forms with
penicillin and amoxicillin/clavulanic acid, according to the method described by Owens
(1988) and Wróblewska et al. (2006), with own modifications. A suspension of each strain
(1.5 x 108 CFU/ml) in Tryptic Soy Broth medium (Oxoid), initially heated to 35ºC, was incubated
for 2 hours at 35ºC, in order to allow full development of the bacteria cell wall. After
the incubation, 10 ml of this suspension was added to 990 ml pre-warmed to 35ºC Brain Heart
Infusion medium (Oxoid) supplemented with 5% NaCl, 5% sucrose, 0.5% yeast extract (Difco),
10% horse serum with 100 U/ml penicillin G and 100 ug/ml of amoxicillin/clavulanic acid.
The sources of antibiotics were two commercially available drugs, respectively: Penicillinium
Procainicum L 1200000 IU (powder for suspension, Polfa Tarchomin S.A.) and Augmentin
(400 mg amoxicillin and 57 mg clavulanic acid, powder for suspension, GlaxoSmithKline). After
10 minutes and 24 hours of incubation at 35ºC, 15 ml of bacterial suspension was streaked
onto enrichment Brain Heart Infusion Agar (with 5% NaCl, 5% sucrose, 0.5% yeast extract)
and incubated for 48 h at 35ºC. Observations were carried out for eight days.
RESULTS
The presence of gap gene in the genomes of all the 17 analyzed strains confirmed their
belonging to the Staphylococcus genus. Based on the analysis of gap gene polymorphism
S. aureus (n = 5, 29.4%), S. chromogenes (n = 4, 23.5%), S. haemolyticus (n = 3, 17.6%), S. capitis
(n = 1, 5.9%), S. epidermidis (n = 1, 5.9%), S. equorum (n = 1, 5.9%) and Staphylococcus spp.
(n = 2, 11.8%) were identified. All strains were characterized by the typical for staphylococci
culture properties, they were catalase-positive, oxidase-negative and fermented glucose, under
aerobic and anaerobic conditions. The results indicate intra- and interspecific variation
in susceptibility of isolated staphylococci to the nine β-lactam antibiotics (Table 1). Among
17 investigated strains, 23.5% (2 strains of S. chromogenes, S. epidermidis and one strain
of Staphylococcus spp.) were resistant, and 23.5% (2 strains of S. aureus, one strain
of S. haemolyticus, and one strain of Staphylococcus spp.) were sensitive to all antibiotics.
On the basis of the results obtained in the test with cefoxitin, seven (41.2%) methicillin-resistant
strains were identified: one of the five S. aureus, three of four S. chromogenes, one S. epidermidis,
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J. Karakulska, A. Stępień, K. Fijałkowski, D. Czernomysy-Furowicz
one S. equorum, and one of two Staphylococcus spp. The sensitivity to the oxacillin was noted
in three strains, and the resistant to the oxacillin was found in four strains among all investigated
methicillin-resistant staphylococci. S. aureus ATCC 43300 and S. epidermidis PCM 2651 were
also methicillin-resistant.
Table 1. Disc diffusion susceptibility test
Strain
No.
S. aureus
8
S. aureus
13
S. aureus
14
S. aureus
15
S. aureus
16
S. chromogenes
1
S. chromogenes
2
S. chromogenes
4
S. chromogenes
7
S. haemolyticus
5
S. haemolyticus
6
S. haemolyticus
9
S. capitis
17
S. epidermidis
3
S. equorum
10
S. spp.
11
S. spp.
12
S. aureus ATCC 25923
S. aureus ATCC 43300
S. epidermidis PCM 2651
P
10 U
S
S
S
S
R
R
R
R
R
S
S
R
R
R
R
R
S
S
R
R
OX
1 μg
S
S
S
S
R
R
R
S
S
S
S
S
I
R
S
R
S
S
R
R
AMP
10 μg
R
S
S
R
R
R
R
R
R
S
R
S
R
R
R
R
S
S
R
R
AMC
30 μg
S
S
S
S
R
R
R
S
S
S
S
S
S
R
S
R
S
S
R
S
KF
30 μg
S
S
S
S
S
R
R
R
S
S
R
S
S
R
S
R
S
S
S
S
FOX
30 μg
R
S
S
S
S
R
R
R
S
S
S
S
S
R
R
R
S
S
R
R
CXM
30 μg
R
S
S
S
S
R
R
R
S
S
R
S
S
R
R
R
S
S
S
S
CFP
75 μg
R
S
S
S
S
R
R
R
S
S
R
S
S
R
R
R
S
S
I
S
EFT
30 μg
R
S
S
S
S
R
R
R
S
S
S
S
S
R
R
R
S
S
R
S
S – sensitivity, R – resistance, I – intermediate sensitivity, P – penicillin, OX – oxacillin, AMP – ampicillin,
AMC – amoxicillin/clavulanic acid, KF – cephalothin, FOX – cefoxitin, CXM – cefuroxime, CFP – cefoperazone,
EFT – ceftiofur.
The results obtained in the test of induction of L forms of staphylococci with penicillin
and amoxicillin/clavulanic acid are shown in Table 2. The reference bacterial strains and 70.6%
among all examined staphylococcal strains isolated from mastitic milk, developed their L forms
under influence of penicillin and amoxicillin/clavulanic acid. Other strains (29.4%) did not
produce L forms under the influence of those two antibiotics, irrespectively of incubation time
with an antibiotic. L form colonies were larger (sometimes significantly) than their parent forms.
Those colonies had a conical profile and thick, pigmented ("fried egg") or not pigmented core
in the center of the colony, visibly distinguished from the rest of the structure of the colony
(Fig. 1). According to Gram staining method, the L forms were Gram-negative, and were rarely
arranged in typical grape-like clusters or packets. Among the 12 strains with the ability
to produce L forms, 83.3% strains stimulated with penicillin and 91.7% strains stimulated with
amoxicillin/clavulanic acid developed their L forms after 10 minutes of incubation with
the antibiotic. In turn, after 24 hours of incubation there were 100% and 91.7% strains, respectively.
All strains that developed L forms, produced also their intermediate forms. The morphology
of colony of intermediate forms differed from both initial (“parent”) forms and L forms. They
The effect of penicillin and amoxicillin/clavulanic acid…
151
formed colonies much larger than the "parent forms", but a core in the center of the colony was
not observed. Additionally, in 4 strains (No 5, 10, 13, 16) which did not create L forms, intermediate
forms were observed.
Table 2. Induction of L form test
Penicillin G
Strain
No.
S. aureus
S. aureus
S. aureus
S. aureus
S. aureus
S. chromogenes
S. chromogenes
S. chromogenes
S. chromogenes
S. haemolyticus
S. haemolyticus
S. haemolyticus
S. capitis
S. epidermidis
S. equorum
S. spp.
S. spp.
S. aureus ATCC 25923
S. aureus ATCC 43300
S. epidermidis PCM 2651
8
13
14
15
16
1
2
4
7
5
6
9
17
3
10
11
12
Amoxicillin/clavulanic acid
Time of incubation with antibiotic
10 min.
+
–
–
+
–
+
–
+
+
–
+
+
+
+
–
–
+
+
+
+
24 h
+
–
–
+
–
+
+
+
+
–
+
+
+
+
–
+
+
+
+
+
10 min.
+
–
–
–
–
+
+
+
+
–
+
+
+
+
–
+
+
+
–
+
24 h
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+ presence of L form, – absence of L form.
Fig. 1. L forms of staphylococci on Brain Heart Infusion Agar
DISCUSSION
β-lactam antibiotics are frequently used in mastitis therapy. In our study, seventeen investigated
staphylococcal strains, isolated from mastitic milk, showed different sensitivity to the β-lactams
antibiotics. Forty one percent of methicillin-resistant strains, belonging to S. chromogenes
(n = 3), S. aureus (n = 1), S. epidermidis (n = 1), S. equorum (n = 1) and S. spp. (n = 1) were
identified. In vitro resistance of staphylococci to methicillin also implies resistance in vivo to all
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J. Karakulska, A. Stępień, K. Fijałkowski, D. Czernomysy-Furowicz
β-lactam antibiotics, and preclude their use in therapy (Hryniewicz et al. 2005). Among methicillinresistant strains, 57.1% showed in vitro resistance to all investigated β-lactam antibiotics.
Owens and Nickerson (1989), demonstrated that L form are involved in bovine mastitis treatment
failures, and emphasized the importance of identification these forms when staphylococcal strains
are isolated from mastitic milk. In this work, only 5 (29.4%) of 17 staphylococcal strains did not
show ability to produce L forms under the influence of penicillin or amoxicillin/clavulanic acid.
However, in the case of four of these strains intermediate forms were identified, as described
by Owens and Nickerson (1989), Jakubczak et al. (2002) and Wróblewska et al. (2006).
In addition, intermediate forms were observed in cultures of all strains which produced
the L forms. The different strains showed varying susceptibility to develop L forms, depending
on the antibiotic, which corresponds with the results obtained by Jakubczak et al. (2002).
All identified L forms of staphylococci were Gram-negative and did not show the typical,
staphylococcal arrangement of cells. The results of our findings regarding cell morphology
of the L forms of staphylococci and their reaction to the Gram stain are consistent with other
authors (Fuller et al. 2005).
Owens (1988) and Jakubczak et al. (2002), examining the induction of L forms of S. aureus
strains isolated from mastitis by penicillin and amoxicillin/clavulanic acid, observed more
L forms after 10 minutes of incubation with antibiotics, than after 24 hours. However, Wróblewska
et al. (2006) found more L forms after 24 hours incubation of S. epidermidis strains with
penicillin. In our study, as a result of stimulation with penicillin, more strains produced L form
after 24 hours than after 10 minutes. However, under the influence of amoxicillin/clavulanic
acid the same number of strains developed L form after 10 minutes and after 24 hours
of incubation. Owens (1988) demonstrated that not all investigated strains of Staphylococcus
aureus isolated from milk of cows with mastitis produce L forms. Furthermore, he observed that
the ability to develop L form depend on neither the sensitivity or resistance of microorganisms
to the β-lactams, nor the mechanism of bacterial resistance to drugs. In the experience all
β-lactam antibiotics (including amoxicillin/clavulanic acid and penicillin) induced the creation
of L forms. Additionally, Wróblewska et al. (2006) noted that the penicillin-resistant Staphylococcus
epidermidis strains also produce the L form after stimulation with this antibiotic. Our results
correspond with those obtained by Owens (1988) and Wróblewska et al. (2006). We found,
that eight out of ten strains resistant to penicillin and four out of five strains resistant
to amoxicillin/clavulanic acid, were able to produce L forms.
In this study, all the observed colonies of intermediate forms and L forms were larger
and noticeably different from the initial colony (parent forms). Similar observations, regarding
the morphology of the L form colonies, were noted by Wróblewska et al. (2006). On the other
The effect of penicillin and amoxicillin/clavulanic acid…
153
hand, Jakubczak et al. (2002) observed the L form colonies of irregular shape ("fried eggs")
but much smaller than the colonies of the parent forms. They also observed the formation
of intermediate forms, which colonies were also smaller than the colonies of initial forms but did
not have the characteristic, centrally located core, typical for L forms. Comparable results
concerning the morphology of the L forms were obtained by Fuller et al. (2005). They described
the L form colonies as smaller in comparison with the colonies of strains before the antibiotic
induction, with a characteristic conical profile, resulting from the presence of the core
in the middle of the colony.
Although staphylococcal L forms are isolated for a long time (also from mastitis), until now
they have not been completely characterized (Owens 1988, Jakubczak et al. 2002). They are
considered as a kind of spore form, protecting the bacteria during the antibiotic therapy
and allowing the resumption of pathogenic activity after treatment. A large percentage
of sensitive to the induction of L forms staphylococci recorded in the literature justifies
the implementation of these tests for routine diagnostics and monitoring staphylococcal
infections, including mastitis. Results of standard drug susceptibility tests, supplemented with
the results of the induction of L forms test can provide a valuable suggestions regarding
antibiotic choice for the treatment of particular cases of staphylococcal diseases.
REFERENCES
CLSI. 2007. Clinical and Laboratory Standards Institute. Seventeenth Informational Supplement. CLSI
document M 100-S17. Clinical and Laboratory Standards Institute, 940 West Valley Road, Suite
1400, Wayne, Pennsylvania 1898 USA.
Fuller E., Elmer C., Nattress F., Ellis R., Horne G., Cook P., Fawcett T. 2005. β-lactam resistance
in Staphylococcus aureus cells that do not require a cell wall for integrity. Antimicrob. Agents
Chemother. 49, 5075–5080.
Hryniewicz W., Sulikowska A., Szczypa K., Gniadkowski M., Skoczyńska A. 2005. Rekomendacje
doboru testów do oznaczania wrażliwości bakterii na antybiotyki i chemioterapeutyki [Recommendations
for susceptibility testing to antimicrobial agents of selected bacterial species]. Post. Mikrobiol. 44,
175–192 [in Polish].
Jakubczak A., Sachanowicz J., Kleczkowski M., Bukowski K. 2002. Wpływ wybranych antybiotyków
na wytwarzanie form L szczepów Staphylococcus aureus [Influence of selected antibiotics in inducing
of L-forms of Staphylococcus aureus]. Med. Weter. 58, 807–809 [in Polish].
Klieneberger E. 1935. The natural occurrence of pleuropneumonia-like organism in apparent symbiosis
with Streptobacillus moniliformis and other bacteria. J. Pathol. Bacteriol. 40, 93–105.
Molander C.W., Kagan B.M., Weinberger H.J., Heimlich E.M., Busser R.J. 1964. Induction by antibiotics
and comparative sensitivity of L-phase variants of Staphylococcus aureus. J. Bacteriol. 88, 591–594.
Owens W.E. 1987. Isolation of Staphylococcus aureus L forms from experimentally induced bovine
mastitis. J. Clin. Microbiol. 25, 1956–1961.
Owens W.E. 1988. Evaluation of various antibiotics for induction of L forms from Staphylococcus
aureus strains isolated from bovine mastitis. J. Clin. Microbiol. 26, 2187–2190.
Owens W.E., Nickerson S.C. 1989. Morphologic study of Staphylococcus aureus L-form, reverting,
and intermediate colonies in situ. J. Clin. Microbiol. 27, 1382–1386.
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Shimokawa O., Nakayama H. 1997. Inactivation of penicillin-induced staphylococcal L-forms by human
serum high density lipoprotein. FEMS Microbiol. Lett. 156, 113–117.
Stoitsova S., Michailova L., Markova N., Dimova I., Jordanova M., Dilova K. 2000. Cell-wall-deficient
forms of Staphylococcus aureus as lung pathogens: an ultrastructural study. Folia Microbiol.
(Praha) 45, 359–363.
Wróblewska J., Janicka G., Gospodarek E., Szymankiewicz M. 2006. L-forms of Staphylococcus
epidermidis induced by penicillin. Pol. J. Microbiol. 55, 243–244.
Yugueros J., Temprano A., Berzal B., Sánchez M., Hernanz C., Luengo J.M., Naharro G. 2000.
Glyceraldehyde-3-phosphate dehydrogenase-encoding gene as a useful taxonomic tool for
Staphylococcus spp. J. Clin. Microbiol. 38, 4351–4355.
WPŁYW PENICYLINY I AMOKSYCYLINY Z KWASEM KLAWULANOWYM
NA INDUKCJĘ FORM L GRONKOWCÓW WYIZOLOWANYCH Z MLEKA
MASTITOWEGO KRÓW
Streszczenie. Wiele antybiotyków stosowanych w leczeniu mastitis oddziałuje na ścianę komórkową
bakterii i jest zdolnych do wzbudzania form L u różnych mikroorganizmów. Formy L umożliwiają
drobnoustrojom stawianie oporu terapii antybiotykowej i przetrwanie w gruczole mlekowym. Materiałem
do badań było 17 szczepów Staphylococcus spp., wyizolowanych z prób mleka pobranego od krów
z podkliniczną postacią mastitis, pochodzących z jednego stada. Ocenę przynależności szczepów
do rodzaju Staphylococcus oraz identyfikację gatunkową przeprowadzano odpowiednio na podstawie
obecności genu gap oraz analizy polimorfizmu genu gap techniką RFLP, przy użyciu enzymu restrykcyjnego AluI. Na podstawie analizy polimorfizmu genu gap zidentyfikowano szczepy S. aureus (n = 5,
29,4%), S. chromogenes (n = 4, 23,5%), S. haemolyticus (n = 3, 17,6%), S. capitis (n = 1, 5,9%),
S. epidermidis (n = 1, 5,9%), S. equorum (n = 1, 5,9%) i Staphylococcus spp. (n = 2, 11,8%). Analizowano
wrażliwość szczepów na 9 antybiotyków β-laktamowych: penicylinę G, oksacylinę, ampicylinę,
amoksycylinę z kwasem klawulanowym, cefalotynę, cefoksytynę, cefuroksym, cefoperazon i ceftiofur.
Spośród 17 analizowanych gronkowców, 23,5% szczepów było opornych i 23,5% było wrażliwych na
wszystkie antybiotyki β-laktamowe. Przy użyciu testu z cefoksytyną, zidentyfikowano siedem (41,2%)
szczepów metycylinoopornych. Gronkowce stymulowano do tworzenia form L penicyliną (Penicillinium
Procainicum L 1200000 j.m.) oraz amoksycyliną z kwasem klawulanowym (Augmentin 457 mg).
12 szczepów (70,6%) tworzyło formy L, zarówno pod wpływem penicyliny, jak i amoksycyliny z kwasem
klawulanowym. Wśród gronkowców zdolnych do tworzenia form L, 83,3% szczepów stymulowanych
penicyliną oraz 91,7% szczepów stymulowanych amoksycyliną z kwasem klawulanowym tworzyło formy
L po 10 minutach inkubacji z antybiotykiem. Z kolei, po 24 godzinach inkubacji gronkowców z antybiotykami, formy L tworzyło odpowiednio 100 i 91,7% szczepów.
Słowa kluczowe: Staphylococcus, formy L, penicylina, amoksycylina/kwas klawulanowy, mastitis
ADVANCES IN AGRICULTURAL SCIENCES
Adv. Agric. Sci. 2011, XIV (1–2), 155–164
Karol Fijałkowski, Danuta Czernomysy-Furowicz, Paweł Nawrotek
INFLUENCE OF STAPHYLOCOCCUS AUREUS EXOSECRETIONS ISOLATED
FROM BOVINE MASTITIS ON LEUKOCYTE MORPHOLOGY IN VITRO
Department of Immunology, Microbiology and Physiological Chemistry,
West Pomeranian University of Technology, Szczecin,
Doktora Judyma 24, 71-466 Szczecin, Poland
Abstract. The aim of the study was the determination of the influence of S. aureus exosecretions isolated
from bovine mastitis on leukocyte morphology in vitro. Three types of in vitro cell cultures were grown:
culture contained polymorphonuclear cells (PMN cultures), mononuclear cells (MNC cultures) and both
PMN and MNC cells (MIX cultures). Thirty S. aureus strains were isolated from milk samples collected
from cows with clinical mastitis. Supernatants, which were used to treat cells, were prepared from 18 h
bacterial cultures. All the investigated supernatants were classified on the basis of their properties
in the study into 3 groups: SA-SN (superantigen-like supernatants), LT-SN (leukotoxic-like supernatants)
and 0-SN (other supernatants). In general, supernatants demonstrated various effects on neutrophil
and lymphocyte morphology, and the effect was dependent on the cell culture type (presence of certain
leukocyte type). All superantigen-like supernatants caused statistically significant increase of the percentage of transforming lymphocytes. Supernatants containing leukotoxins-like factors showed strong
cytotoxic effect on neutrophils. However supernatants containing superantigenic factors caused
accelerated death of those cells (neutrophils). This proves that both leukotoxic-like and superantigen-like
factors produced by S. aureus strains are involved in destroying the most important type of immune
response of bovine mammary gland.
Key words: superantigen, leukotoxin, S. aureus, leukocyte morphology, proliferation
INTRODUCTION
Among all species of Staphylococcus genus, Staphylococcus aureus is considered to be
the most virulent one (Iwatsuki et al. 2006). S. aureus is characterized by a very high virulence,
as it is able to produce a large array of virulence factors. Those factors are: cell wall components,
surface proteins, enzymes and toxins (Foster 2005). Toxins produced by S. aureus, according
to their activity can be divided into two main groups. The first group consists of toxins that are
cytolytic towards leukocytes (leukotoxins) and are able to kill phagocytes directly – building into
the cell membrane they form pores causing death of leukocytes (Shuberth et al. 2001). This
type of toxins can also cause massive release of inflammatory mediators. Superantigenic
toxins are virulence factors that belong to second group of exotoxins. Superantigens cause
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K. Fijałkowski, D. Czernomysy-Furowicz, P. Nawrotek
abnormal, excessive and non-specific activation of the immune system, that in consequences
leads to immunosupression. Superantigens can also selectively accelerate the death of bovine
and human neutrophils in vitro, but only in the presence of blood mononuclear cells. Both,
superantigens and leukotoxins are exotoxins, play a crucial role in the initiation and exacerbation
of S. aureus mastitis in cattle (Loeffler et al. 1988, Riollet 2001, Chang et al. 2005). Until now,
very few data are available regarding ability of producing these both virulence factors at the
same time and the principles of their involvement in inflammation process (Fitzgerald et al.
2000, Schuberth et al. 2001). Based on the previous studies, it can be assumed that the effects
of action of those two groups of exotoxin are mostly depend on the type of cells present
in cultures. The aim of the study was the determination of the influence of S. aureus exosecretions
isolated from bovine mastitis on leukocyte morphology in vitro.
MATERIAL AND METHODS
Bacterial strains. Prior to this study, a collection of thirty S. aureus and two S. intermedius
(comparative control) strains isolated from milk of cows with clinical mastitis in one herd from
Western Pomerania (Poland) were used for preparing supernatants. The isolates were identified
as S. aureus on the basis of their biochemical properties (api STAPH, bioMerieux, France)
and by PCR using primers for the S. aureus specific nuc gene (gene encoding nuclease),
as described by Brakstad et al. (1992).
All the strains were tested for the production of superantigen-like and leukotoxin-like factors
using indirect methods, by evaluation of the influence of prepared supernatants on morphology
of leukocytes in vitro.
Preparation of supernatants from bacterial cultures. All isolates of S. aureus were
plated out on blood agar plates (Columbia agar base with 5% sheep blood, Grasso, Poland)
and cultured for 24 h at 37ºC. After the incubation, one colony-forming unit of each isolate was
transferred into 9 ml of BHI broth (Brain Heart Infusion, Oxoid, UK) and incubated for 18 h at
37ºC. After centrifugation at 5000 x g for 15 min, supernatants were filtered using antibacterial
low protein binding filters (Millex GP, Millipore, Ireland), aliquoted and frozen at -20ºC until
further use. Additionally, supernatants which killed neutrophils at a statistically significant level
after 12h incubation were heat inactivated by incubating in a water bath at 60ºC for 30 minutes
(Schuberth et al. 2001).
Isolation of leukocytes. Leukocytes were isolated from peripheral blood of three years old
Holstein-Friesian (HF) cows, free of intramammary infection, as previously described (Schuberth
et al. 2001, Mehrzad et al. 2002). The cows were clinically healthy and were at the same stage
in lactation cycle.
Influence of Staphylococcus aureus exosecretions isolated…
157
Blood samples were aseptically collected by jugular venipuncture into vacutainer tubes with
EDTA as a coagulant (Greiner Bio-One, UK). Blood was layered on the Histo-paque gradient
(Sigma-Aldrich, Germany) and centrifuged at 400 x g for 30 minutes. Mononuclear cells
(MNCs) were harvested from the interface and washed three times with PBS (400 x g, 200 x g,
200 x g, 18ºC) and cells were adjusted to 1 x 107 cells/ml of culture medium containing RPMNI
1640 supplemented with 10% FBS (Sigma-Aldrich). Based on morphologic examination, more
than 94% of the cells in the final preparation were lymphocytes, and more than 97% of cells
were viable as indicated by trypan blue staining.
Polymorphonuclear leukocytes were harvested from the pellet below the Histo-paque
gradient after the hypotonic lysis of pelleted erythrocytes. The process was performed
by adding an equal volume of double-distilled water (hypotonic conditions) and then, by adding
2x concentrated PBS (Sigma-Aldrich) to regain the isotonicity of the solution. The remaining
cells were washed twice with PBS (200 x g, 200 x g, 18ºC), resuspended in RPMI supplemented
with 10% FBS were added, to give a final concentration of 1 x 107 cells per ml. Greater than
90% of the cells in the final preparation were neutrophils based on morphologic examination,
and more than 97% were viable (trypan blue staining).
In vitro treatments of leukocytes. For the assay, 1000 µl of leukocyte suspension in complete
culture medium (RPMI 1640 without phenol red, fetal bovine serum (5%), sodium pyruvate
(1 mM), penicillin (100 U/ml), streptomycin (100 μg/ml), amphotericin B (2.5 μg/ml), L-glutamine
2.05 mM, Sigma-Aldrich) were incubated in flat-bottom 24-well microtiter plates (Becton
Dickinson and Company, USA), in the incubator (temperature 37ºC, 5% CO2, RS Biotech) with
200 µl of each supernatant. Culture contained polymorphonuclear cells (PMN cultures),
mononuclear cells (MNC cultures) and both PMN and MNC cells (MIX cultures). The final
concentration of leukocytes were: 2 x 106 MNC or PMN cells in one millilitre of MNC or PMN
cultures respectively, and in MIX cultures – 1 x 106 MNC cells/ml and 1 x 106 PMN cells/ml.
As a control, 200 µl of BHI broth instead of supernatant was added and as a positive control
(presence of superantigen-like factors in the supernatants) purified staphylococcal enterotoxin
A (SEA) was used. SEA was purchased from Sigma and was used at the concentration
of 10 ng/ml (Mullarky et al. 2001).
Evaluation of leukocytes morphology. In order to prepare microscopic preparations,
leukocyte culture were mixed gently for 10 minutes. Content of each well from plate was
transferred to aseptic 1.5 ml tube. The cell suspension was centrifuged (250 x g, 10 min, 18ºC),
supernatant was discarded and the pellet was resuspended in 50 μl PBS. The cell suspension
was transferred on microscopic slides, previously covered with glycerol and gelatine mixture
(50% glycerol, 8% gelatin, 0.1% phenol). Prepared smears were preserved in 70% ethanol
and stained in Wright’s stain (0.4%, Sigma-Aldrich). All microscopic preparations were evaluated
158
K. Fijałkowski, D. Czernomysy-Furowicz, P. Nawrotek
and quantitated by light microscopy under 100x objective (Axioskop 2, Carl Zeiss with Power
Shot G6 camera, Canon and Zoom Browser Ex software.
All obtained results are presented as the number of transforming (lymphocytes) or damaged
(lymphocytes and granulocytes) cells per 100 cell of certain leukocyte type. Evaluation
of lymphocyte transformation and proliferation was investigated according to criteria described
previously by Kupryś et al. (1998), including: cell size and changes in cell morphology –
cytoplasmic increased, eccentric position of the nucleus and changes in the chromatin structure.
Neutrophils were classified to damaged cells according to Genestier et al. (2005) description,
as characteristic for leukotoxic effect, such as: cell swelling, loss of membrane integrity, release
of intracellular contents, lack of genetic material content or fragmentation of the nucleus,
and cytoplasmic vacuolation. Lymphocyte were classified into the group of damaged cells
if any irregular changes in lymphocyte pattern were observed.
Microscopic preparations were made in 96 hours MNC cultures, 12 hours PMN cultures
and 12 and 96 hours MIX cultures. In the microscopic preparations prepared from MNC cultures
morphological changes of lymphocytes were assessed. In the microscopic preparations
prepared from PMN cultures – changes of neutrophils, and in preparations from MIX cultures
both, changes of lymphocytes (only 96 h cultures) and changes of neutrophils (only 12 h cultures)
were evaluated.
STATISTICAL ANALYSIS
Data are presented as median with minimum and maximum values and interquartile range
(IRQ). The statistical significance of the differences between treatments and controls were
analysed by Student’s t test. All statistical analyses were conducted with Statistica 7.1
software. All experiments were carried out in triplicate.
RESULTS AND DISCUSSION
In general, supernatants demonstrated various effects on neutrophil and lymphocyte
morphology, and the effect was dependent on the cell culture type (presence of certain
leukocyte type). All the investigated supernatants were classified on the basis of their
properties in the study into 3 groups: SA-SN, LT-SN and 0-SN. Group LT-SN (leukotoxic-like
factors) consisted of supernatants that considerably increased number of damaged cells after
12 h of PMN and MIX cultures, and which lost this property after heat treatment. The third
group of supernatants – group SA-SN (superantigen-like factors) – contained supernatants that
caused an increase in number of transforming lymphocytes in 96h MNC and MIX cultures
(similarly to SEA) and which had no effect on neutrophils in PMN cultures. Additionally,
Influence of Staphylococcus aureus exosecretions isolated…
159
supernatants prepared from S. intermedius cultures were classified into S.I.-SN group.
The experiments were designed and results were interpreted according to the group classification.
In all 96 h MNC and MIX cultures incubated with supernatants from SA-SN group and in the
positive control, statistically significant increase of transforming lymphocytes percentage was
observed (compared to the negative control), (Fig. 1). The percentage of transforming lymphocytes
in 96 h MNC cultures incubated with SA-SN as well as in the positive control (SEA) was 24%.
The percentage of transforming lymphocyte in the negative control was 7%. In MIX cultures
incubated with SA-SN, the average percentage of transforming lymphocytes after 96 h, was
22% and in contrast to MNC cultures, was not statistically significant in comparison
to the percentage of transforming lymphocytes in the negative control (10% of transforming
cells) as well as in the positive control (27% of transforming lymphocytes). SA-SN did not
caused significant increase of damaged neutrophils in PMN cultures. However, the percentage
of damaged neutrophils in MIX cultures incubated with this group of supernatant, was
statistically significant higher than in the negative control (Fig. 2).
B
N
0
SA
-S
N
N
S.
I.S
LT
-S
N
0SN
0
10
S.
I.S
10
20
0SN
20
30
LT
-S
N
Transforming lymphocyte
(%)
30
SA
-S
N
Transforming lymphocyte
(%)
A
Fig. 1. Influence of the group of supernatants on the percentage of transforming lymphocyte in MNC (A)
and MIX (B) cultures. The results represented the median with minimum and maximum values
and interquartile range (IRQ)
A
B
100
Demaged neutrophils
(%)
80
60
40
20
60
40
20
N
S.
I.S
0SN
LT
-S
N
N
S.
I.S
0SN
LT
-S
N
0
SA
-S
N
0
80
SA
-S
N
Demaged neutrophils
(%)
100
Fig. 2. Influence of the group of supernatants on the percentage of damaged neutrophils in PMN (A)
and MIX (B) cultures. Explanations as in Figure 1
K. Fijałkowski, D. Czernomysy-Furowicz, P. Nawrotek
160
Supernatants from LT-SN group did not induce activation and proliferation of lymphocytes
in MNC and MIX cultures compared to the negative control. However, six supernatants from
that group caused statistically significant increase of damaged lymphocytes in MNC cultures,
and nine supernatants – in MIX cultures (Fig. 3). In 12h MIX and PMN cultures all supernatants
from LT-SN caused statistically significant increase of damaged neutrophils compared
to the negative control. All of these supernatants lost this activity after they were heat
inactivated by incubating in a water bath at 60ºC for 30 min, according to Schuberth et al.
(2001). Neither percentage of transforming lymphocytes nor damaged leukocytes in MIX
and MNC cultures increased after incubation with supernatants from 0-SN and S.I.-SN,
in comparison to the negative control.
A
B
50
Demaged lymphocyte
(%)
15
10
5
30
20
10
N
S.
I.S
0SN
LT
-S
N
N
S.
I.S
0SN
LT
-S
N
0
SA
-S
N
0
40
SA
-S
N
Demaged lymphocyte
(%)
20
Fig. 3. Influence of the group of supernatants on the percentage of damaged lymphocyte in MNC (A)
and MIX (B) cultures. Explanations as in Figure 1
Classification of supernatants to SA-SN group was based on their ability to evoke blastogenesis
and lymphocyte proliferation. This criteria of classification of virulence factors produced
by S. aureus strains is consistent with previous reports from Schuberth et al. (2001). Furthermore,
obtained results were compared to the positive control containing pure staphylococcal
enterotoxin A. As suggested by Mullarky et al. (2001) SEA and SEC, are the most frequently
produced enterotoxins by S. aureus strains isolated from cows with mastitis. Induction
of lymphocyte proliferation in the presence of supernatants containing superantigen-like factors
was observed in both MNC and MIX cultures. The greatest percentage of proliferating
lymphocytes in those cultures was noted after 96 hours of incubation. It is widely known that
ability to activate the lymphocytes is common feature of all superantigens, however until now
the research in bovine leukocyte culture were conducted only with SEA, SEB, SEC1, SEC2,
SEE, TSST-1 (Schmaltz et al. 1995, Yokomizo et al. 1995, Schuberth et al. 1996, 1998,
Ferens et al. 1998, Hendricks et al. 2000).
Influence of Staphylococcus aureus exosecretions isolated…
161
Besides MNC cells activation, four of the five supernatants with superantigenic-like properties
showed cytotoxic activity against neutrophils in 12 h MIX cultures, in contrast such activity was
not detected in pure PMN cultures. This findings were consistent with previous reports from
Schuberth et al. (2001), who observed that damage of neutrophils caused by superantigenic
factors can be a result of indirect action of regulatory proteins (e.g. cytokines) released
by superantigen-activated MNC cells, explaining, why cytotoxic effect at the presence of SA-SN
supernatants was not observed in PMN cultures. Schuberth et al. (2000) reported, that different
superantigenic factors (SEA, SEB, SEE), showing distinct affinity to MHC II and Vβ chains
on TCR receptors, could indirectly cause accelerated death of neutrophils in in vitro cultures
of leukocytes. Moreover, the authors suggest that this effect of accelerated death of neutrophils
could be, with the high probability, characteristic for all known superantigens and other
supeantigen-like putative factors. As it is also suggested by Shuberth et al. (2001), cytotoxic
effect against PMNs observed in superantigens presence is dependent on the superantigenic
factor concentration. Determination of accelerated death of neutrophils caused by superantigeniclike factors in MIX cultures incubated with four of the five supernatants, can thus provide
a higher concentration of these proteins in the four supernatants. Summarizing, the analysis
of obtained results and on the findings from other authors, it can be concluded, that a common
feature of leukotoxins and superantigens is their negative effects (directly or indirectly cytotoxic)
on bovine neutrophils.
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Hendricks A., Leibold W., Kaever V., Schuberth H.J. 2000. Prostaglandin E2 is variably induced by
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Iwatsuki K., Yamasaki O., Morizane S., Oono T. 2006. Staphylococcal cutaneous infections:
Invasion, evasion and aggression. J. Dermat. Sci. 42, 203–214.
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Kupryś I., Józefowicz G., Kuna P. 1998. The effect of heparyn on immunoglobulin E (IgE) synthesis
by peripheral blood mononuclear cells (MNCs) stimulated with interleukin – 4 (IL-4) or lipopolisaccharide
(LPS). Alergia Astma Immunol. 3 (1), 51–55 [in Polish].
Loeffler D.A., Creasy M.T., Norcross N.L., Paape M.J. 1988. Enzyme-linked immunosorbent assay
for detection of leukocidin toxin from Staphylococcus aureus in bovine milk samples. J. Clin. Microbiol.
26, 1331–1334.
Mehrzad J., Duchateau L., Burvenich C. 2009. Phagocytic and bactericidal activity of blood and
milk-resident neutrophils against Staphylococcus aureus in primiparous and multiparous cows
during early lactation, Veter. Microbiol. 134, 106–112.
Mullarky I.K., Su C., Frieze N., Park Y.H., Sordillo L.M. 2001. Staphylococcus aureus agr genotypes
with enterotoxin production capabilities can resist neutrophil bactericidal activity. Infect. Immun.
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Riollet C., Rainard P., Poutrel B. 2001. Cell subpopulations and cytokine expression in cow milk
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Schuberth H.J., Hendricks A., Leibold W. 1998. There is no regulatory role for induced nitric oxide
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Schuberth H.J., Kreueger C., Zerbe H., Bleckmann E., Leibold W. 2001. Characterization of leukocytotoxic and superantigen-like factors produced by Staphylococcus aureus isolates from milk
of cows with mastitis. Veter. Microbiol. 82, 187–199.
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Yokomizo Y., Mori Y., Shimoji Y., Shimizu S., Sentsui H., Kodama M., Igarashi H. 1995. Proliferative
response and cytokine production of bovine peripheral blood mononuclear cells induced
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Sci. 57, 299–305.
OCENA WPŁYWU EGZOGENNYCH CZYNNIKÓW WIRULENCJI
WYTWARZANYCH PRZEZ SZCZEPY S. AUREUS IZOLOWANE OD KRÓW
Z OBJAWAMI MASTITIS NA MORFOLOGIĘ LEUKOCYTÓW W HODOWLACH
IN VITRO
Streszczenie. Celem badań była ocena wpływu egzogennych czynników wirulencji wytwarzanych przez
szczepy S. aureus izolowane od krów z objawami mastitis na morfologię leukocytów w hodowlach in vitro.
Zakładano trzy rodzaje hodowli leukocytów: hodowle zawierające komórki polimorfonuklearne (hodowle
PMN), hodowle zawierające komórki mononuklearne (hodowle MNC) oraz hodowle zawierające komórki
PMN i MNC (hodowle MIX). Ocenie poddawano supernatanty przygotowane z hodowli 30 szczepów
S. aureus. Szczepy te wyizolowane zostały z mleka krów rasy polskiej holsztyno-fryzyjskiej z klinicznymi
objawami mastitis. Na podstawie uzyskanych wyników supernatanty zakwalifikowano do trzech różnych
grup: SA-SN (supernatanty o właściwościach superantygenowych), LT-SN (supernatanty o właściwościach leukotoksycznych) i 0-SN (pozostałe supernatanty). Na podstawie uzyskanych wyników stwierdzono
odmienny, zależny od rodzaju hodowli wpływ badanych supernatantów na morfologię neutrofilów
i limfocytów w hodowlach in vitro. Supernatanty o właściwościach superantygenowych powodowały
Influence of Staphylococcus aureus exosecretions isolated…
163
statystycznie istotne zwiększenie odsetka limfocytów transformujących. Supernatanty w których wykryto
obecność czynników o charakterze leukotoksycznym, wykazywały silny, cytotoksyczny wpływ na
neutrofile, natomiast supernatanty zawierające superantygeny powodowały przyśpieszoną śmierć tych
komórek. Otrzymane wyniki dowodzą, że zarówno czynniki leukotoksyczne jak i supernatygenowe wytwarzane przez szczepy S. aureus uczestniczą w niszczeniu neutrofilów – najistotniejszych komórek odpornościowych gruczołu mlekowego krów.
Słowa kluczowe: superantygen, leukotoksyna, S. aureus, morfologia leukocytów, proliferacja
ADVANCES IN AGRICULTURAL SCIENCES
Adv. Agric. Sci. 2011, XIV (1–2), 165–172
Milena A. Stachelska, Antoni Jakubczak
MICROBIOLOGICAL ACTIVITY OF SALTS OF COUMARIC AND CINNAMIC
ACIDS AGAINST ESCHERICHIA COLI O157:H7 AND STAPHYLOCOCCUS
AUREUS IN VITRO
Food Technology and Nutrition Institute, The State College of Computer Science and Business
Administration in Łomża, Akademicka 14, 18-400 Łomża, Poland
Abstract. A huge demand for healthy and microbiological safe food is observed among consumers. They
expect manufacturers to produce convenient food which is ready to eat after heating up. Such products
prepared in sterile conditions can be easily contaminated with food pathogens. Contamination may
be caused through the contact with production staff and contaminated storage surfaces. There is a risk
of appearing pathogenic microflora in food which is resistant towards the adverse environmental factors.
Such microorganisms may involve Escherichia coli O157:H7 and Staphylococcus aureus. The research
was carried out to assess the microbiological activity of salts of coumaric and cinnamic acids towards
E. coli O157:H7 and S. aureus in vitro and to indicate the inhibition zones on medium plates. Such
substances naturally occur in plant tissues and constitute an alternative for artificial preservatives.
Key words: salts of phenolic acids, antimicrobial activity, Escherichia coli O157:H7, Staphylococcus
aureus
INTRODUCTION
Phenolic acids are widely spread in plant tissues. This group includes coumaric and cinnamic
acids. They are proved to be natural preservatives used in foods. Search for new preservatives
is still valid, because the preservatives used so far apart from having the desired effect
on elimination of bacteria, yeasts and moulds from food, leads also to the appearance of side
effects, including food intolerances and allergic reactions. Phenolic acids appear in fruit
and vegetables as well as in coffee and tea (Halliwell and Gutteridge 1984, Harborne
and Williams 1984, Güner et al. 2000, Gülçin et al. 2003). They exhibit antimicrobial activity.
Their lack of toxicity on the human body provides grounds for the application of these
compounds as new preservatives in food (Acar et al. 2010).
This study evaluates the antibacterial activity of salts of coumaric and cinnamic acids towards
E. coli O157:H7 and S. aureus in vitro. Many studies indicated that phenolic acids have antioxidant
effects (Zheng et al. 2007) and might have cardio protective effect (Goyal et al. 2010). What is
166
M.A. Stachelska, A. Jakubczak
more, they also possess the anti-tumoural properties both in vitro and in vivo (Escribano et al.
1999). They constitute biologically active compounds isolated from plant species and are used
for the elimination of pathogenic micro-organisms (Essawi and Srour 2000). They are proved
to show the antimicrobial potential against foodborne pathogens and may be used to successfully
eliminate them from food products (Slinkard and Singleton 1977, Simic 1988, Yen et al. 1993,
Park et al. 1997, Reische et al. 1998, Tanaka et al. 1998, Yanga et al. 2002, Vahidi et al. 2002,
Tepe et al. 2005).
A huge variety of different plants containing phenolic acids are used in traditional medicine
today due to their antimicrobial activity (Oke et al. 2009, Zampini et al. 2009, Mboss et al. 2010).
Different antibiotics have found their application in treatment of many infectious diseases for
a long time (Löliger 1991, Komali et al. 1999, Lozano et al. 1999). The use of antioxidants
in foods. In free radicals and food additives. London: Taylor and Francis.
However, it is indicated that pathogen bacteria show the antimicrobial resistance towards
drugs causing difficulties with the treatment of human infections (Moller et al. 1999, Nørbæk
and Kondo 2002). Such phenomenon encourages scientists to look for new antimicrobial
substances coming from different plant sources which constitute the new generation of natural
antimicrobial chemotherapeutic agents (Ali-Shtayeh et al. 1998, Duh et al. 1999, Ahmad and
Beg 2001). Such plant extracts rich with phenolic acids fulfill the role of natural agents in food
preservation (Baratta et al. 1998). They are used to inhibit foodborne bacteria and extend
the shelf life of processed food (Dapkevicius et al. 1998, Burits and Bucar 2000, Bhaskarwar
et al. 2008).
There are different naturally occurring extracts including essential oils, herbs and spices
which show antimicrobial activity and inhibit as well as eliminate the growth of food spoilage
and pathogens (Oussalah et al. 2006). The aim of the studies shown in this paper is to assess
the antimicrobial activity of selected salts of phenolic acids on the growth of E. coli O157:H7
and S. aureus in vitro.
MATERIAL AND METHODS
Pure substance. The material consisted of selected salts of phenolic acids such as a potassium salt of o-coumaric acid, a sodium salt of o-coumaric acid, a lithium salt of o-coumaric
acid, a potassium salt of m-coumaric acid, a sodium salt of m-coumaric acid, a lithium salt
of m-coumaric acid, a potassium salt of p-coumaric acid, a sodium salt of p-coumaric acid,
a lithium salt of p-coumaric acid as well as a potassium salt of cinnamic acid, a sodium salt
Microbiological activity of salts of coumaric and cinnamic acids…
167
of cinnamic acid and a lithium salt of cinnamic acid. The 5% solutions of each substances were
prepared to check their antimicrobial activity towards E. coli O157:H7 and S. aureus in vitro.
Microbial strain. The strains used for microbiological analysis were obtained from ATCC
collections (the American Type Culture Collection, USA) and they were bought in Promochem,
Łomianki, Poland. The strains included E. coli O157:H7 ATCC 8739 and S. aureus ATCC 3220.
Maintenance and preparation of cultures. Cultures of E. coli O157:H7 and S. aureus
ATCC 3220. Were isolated from bovine faeces and maintained on tryptone soy broth agar
(TSBA) slants at 4ºC (bioMérieux, Warszawa, Poland).
Preparation of liquid bacterial culture in tryptic soy broth. A 16-h-old culture inoculated
in tryptone soy broth (bioMérieux, Warszawa, Poland) at temperature 37ºC was taken for further
experiment. The optical density of this culture after inoculation was determined at 625 nm
(Ultraspec III, Pharmacia, Sweden). The incubation was stopped when the optical density
achieved a value in the range of 0.8–1.0. The culture suspensions were diluted to an absorbance of 0.1 and used as such for the antimicrobial tests.
Placing the culture dilution on a plate with medium. The medium used for further
experiment was sorbitol MacConkey agar (bioMérieux, Warszawa, Poland) for E. coli O157:H7
inoculation and Columbia agar with sheep blood (bioMérieux, Warszawa, Poland) for S. aureus
inoculation. They were poured into each sterilised Petri dish (Ø 10 cm).
A 1-ml of a 16-h culture diluted to achieve an absorbance of 0.1 was placed onto
the surface of pre-dried sorbitol MacConkey agar Petri dishes (Ø 10 cm) and Columbia agar
with sheep blood Petri dishes (Ø 10 cm), then allowed to remain in contact for 1 min. The Petri
dishes were allowed to dry for 20 min at room temperature.
Agar-well diffusion method. The antimicrobial activity of the samples was assayed by the
Agar-well diffusion method (Perez et al. 1990).
Seven equidistant holes were made in the two different medium Petri dishes using sterile
cork borers (Ø 7 mm). A 0.05 ml of six different salts was added to each hole and one hole was
filled with 0.05 ml of tryptone soy broth as a control sample using a pipettor (Eppensdorf).
The Petri dishes were incubated at 37ºC for 24 h.
Measuring the inhibition zone diameter. At the end of the incubation period, inhibition
zones which appeared on the medium Petri dishes were calculated in millimeters. The experiment
was repeated ten times and the inhibition zones were compared with those of reference discs.
Reference discs used for control were as following: nystatin (100 U), ketoconazole (50 μg),
tetracycline (30 μg), ampicillin (10 μg), penicillin (10 U), oxacillin (1 μg), tetracycline (30 μg) and
gentamycin (10 μg). Then the mean values were presented with an accuracy of 0.1 mm.
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M.A. Stachelska, A. Jakubczak
RESULTS AND DISCUSSION
In the present investigation, the antimicrobial effects of twelve salts of phenolic acids
possessing similar chemical structures were tested against a gram-negative bacteria E. coli
O157:H7 and a gram-positive coccus S. aureus. The salts proved to possess the inhibitory
activity towards the investigated bacteria and yeast. The antimicrobial properties of the phenolic
salts and their potential application in pathogen elimination were quantitatively estimated
by the measurement of inhibition zone and zone diameter. The antimicrobial activities of salts
of phenolic acids against E. coli O157:H7 and S. aureus are presented in Fig. 1.
Fig. 1. Antibacterial properties of salts of phenolic acids against Staphylococcus aureus and E. coli
O157:H7. 1 – a potassium salt of o-coumaric acid, 2 – a sodium salt of o-coumaric acid, 3 – a lithium
salt of o-coumaric acid, 4 – a potassium salt of m-coumaric acid, 5 – a sodium salt of m-coumaric
acid, 6 – a lithium salt of m-coumaric acid, 7 – a potassium salt of p-coumaric acid, 8 – a sodium
salt of p-coumaric acid, 9 – a lithium salt of p-coumaric acid, 10 – a potassium salt of cinnamic
acid, 11 – a sodium salt of cinnamic acid, 12 – a lithium salt of cinnamic acid.
It can be said that the salts of phenolic acids show a relatively narrow antibacterial spectrum
against gram-negative bacteria and quite a wide spectrum against the gram-positive bacteria.
It was observed that a lithium salt of o-coumaric acid and a potassium salt of p-coumaric acid
were indicated to have the most inhibitory activity towards S. aureus. The inhibition zone
diameter for a lithium salt of o-coumaric acid towards S. aureus was measured as 22 mm,
whereas for a potassium salt of p-coumaric acid towards S. aureus was measured as 20 mm.
However, these substances did not possess such an influential effect on the growth
inhibition of E. coli O157:H7. It was best effected by a sodium salt of p-coumaric acid.
The inhibition zone diameter of a sodium salt of p-coumaric acid towards E. coli O157:H7 was
measured as 12 mm.
Microbiological activity of salts of coumaric and cinnamic acids…
169
Quite influential against S. aureus were also a lithium salt of p-coumaric acid as well as
a potassium salt of m-coumaric acid. They gave similar results. The inhibition zone diameters
amounted to 17 mm.
It can also be said that salts of coumaric acids are more effective in comparison to the salts
of cinnamic acids towards S. aureus. Both salts of coumaric and cinnamic acids show similar
not so significant effect on the growth inhibition of E. coli O157:H7 apart from a sodium salt
of cinnamic acid which possess a strong antibacterial activity giving a wide inhibition zone and
causing decolouration of sorbitol MacConkey agar Petri dish. The inhibition zone diameter
amounted to 44 mm.
The weakest effect against S. aureus posess the salts of cinnamic acid, where the most
effective is a sodium salt of cinnamic acid with an inhibition zone measured as 14 mm, while
a lithium salt of cinnamic acid with an inhibition zone measured as 13 mm and a potassium salt
of cinnamic acid with an inhibition zone measured as 11 mm.
As far as E. coli O157:H7 is concerned, it is relatively weakly affected by as a potassium salt
of o-coumaric acid, a sodium salt of o-coumaric acid and a lithium salt of o-coumaric acid
which give the following inhibition zones: 7 mm, 9 mm and 9 mm.
CONCLUSIONS
It can be summarized that the salts of phenolic acids express different antimicrobial activity
against the tested foodborne pathogens. Each pathogen has its own characteristic features
and natural phenolic substances should be chosen individually for effective elimination
of contamination in food. Many further experiments should be carried out in order to develop
the most successful methods of elimination of pathogens from food products.
ACKNOWLEDGEMENTS
This work was supported by grant no. N N312 427639.
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AKTYWNOŚĆ MIKROBIOLOGICZNA SOLI KWASU KUMAROWEGO
I CYNAMONOWEGO W STOSUNKU DO ESCHERICHIA COLI O157:H7
I STAPHYLOCOCCUS AUREUS IN VITRO
Streszczenie. Wśród konsumentów obserwuje się ogromny popyt na zdrową i bezpieczną pod względem
mikrobiologicznym żywność. Od producentów żywności oczekują oni żywności wygodnej, która jest
gotowa do spożycia bezpośrednio po podgrzaniu. Takie produkty mogą z łatwością ulec zanieczyszczeniu
bakteriami chorobotwórczymi. Zanieczyszczenia mogą być wywołane poprzez kontakt z pracownikami
produkcji i skażonymi powierzchniami magazynowymi. Istnieje ryzyko pojawienia mikroflory chorobotwórczej w żywności, która jest odporna na niekorzystne czynniki środowiskowe. Do takich mikroorganizmów
zalicza się Escherichia coli O157:H7 i Staphylococcus aureus. Badania in vitro przeprowadzone zostały
w celu oceny aktywności mikrobiologicznej soli kwasu kumarowego i cynamonowego w stosunku do
E. coli O157:H7 i S. aureus poprzez określenie stref hamowania wzrostu. Substancje te naturalnie
występują w tkankach roślinnych i stanowią alternatywę dla konserwantów chemicznych.
Słowa kluczowe: sole kwasów fenolowych, aktywność przeciwdrobnoustrojowa, Escherichia coli
O157:H7, Staphylococcus aureus
ADVANCES IN AGRICULTURAL SCIENCES
Adv. Agric. Sci. 2011, XIV (1–2), 173–180
Karol Fijałkowski, Paweł Nawrotek, Danuta Czernomysy-Furowicz
USEFULNESS OF NEUTRAL RED UPTAKE METHOD FOR INVESTIGATION
OF THE BOVINE LEUKOCYTE VIABILITY AND LYMPHOCYTE PROLIFERATION
Department of Immunology, Microbiology and Physiological Chemistry,
West Pomeranian University of Technology, Szczecin,
Doktora Judyma 24, 71-466 Szczecin, Poland
Abstract. The application of the neutral red uptake (NRU) assay, as a non-radioactive, simple and cheap
assay which can be used in multiple leukocyte in vitro study was evaluated. In the research,
the colorimetric NRU assay was compared with MTT reduction test in bovine mononuclear and polymorphonuclear cell cultures. The possibility to use the colorimetric NRU assay for measuring proliferation
induced by phytohemagglutinin (PHA) and number of live leukocyte was also studied. In general,
the sensitivity of the NRU assay was similar to MTT reduction assay. In a range from 10 000–20 000 000
cells/well a linear correlation between the optical signal (OD at 540 nm) and the cell number was found,
in both MTT reduction and NRU assays. A very good and statistically significant correlation between MTT
and NRU was also obtained. Summarizing, the neutral red uptake assay similarly to MTT reduction assay
is a valid test for evaluation of lymphocyte proliferation and number of bovine leukocyte. Due to very high
correlation, it can be used as a second, comparative assay in leukocyte study in in vitro cultures,
especially when MTT reduction test is performed.
Key words: neutral red uptake, MTT reduction, lymphocyte proliferation, leukocyte number
INTRODUCTION
The lymphocyte proliferation and leukocyte viability as well as their general number are
usually measured by the [3H] thymidine incorporation and MTT reduction assays. In order
to find a suitable non-radioactive and also alternative to the MTT reduction assay, the trials
of evaluation usefulness of another assays are taken (Rai-el-Balhaa et al. 1985, Iwata and Inoue
1993). Here the proliferative potential and the total number of live cells were measured, using
neutral red uptake assay (NRU) which then was compared with MTT reduction test. The MTT
test is based on the reduction of water-soluble yellow dye MTT to purple, water insoluble
formazan crystals. This reaction is carried out by mitochondrial dehydrogenase (mitochondrial
redox activity) and occurs only in living cells. Increase in the number of living cells or their
metabolic activity, causes increases in the amount of formazan formed, and thus increase
in absorbance of the sample (proliferation as a function of mitochondrial activity of living cells
(Mossman 1983, Bounous et al. 1992). A main component of NRU assay is a dye, neutral red.
174
K. Fijałkowski, P. Nawrotek, D. Czernomysy-Furowicz
Living cells (including leukocyte) absorb the dye by the active transport and store it in the lysosomes, whereas dead cells do not incorporate neutral red. Changes in number or physiological
state of cells exhibit a change in the amount of absorbed dye. The incorporation of neutral red
into lysosomes is an energy dependent process. Moreover, the loss or impairment of absorption
of neutral red may be due to loss of cell membrane integrity (Pipe et al. 1995, Stoika et al. 2002).
The aim of the study was the evaluation of the usefulness of neutral red uptake assay
for the determination of the total number of live leukocytes and proliferative potential
of lymphocytes. The NRU assay was compared with the conventional MTT reduction assay
which is commonly used for this study.
MATERIAL AND METHODS
Isolation of leukocytes. Leukocytes were isolated from peripheral blood of three years old
Holstein-Friesian (HF) cows, free of intramammary infection, as previously described
(Schuberth et al. 2001). The cows were clinically healthy. Blood samples were aseptically
collected by jugular venipuncture into vacutainer tubes with EDTA as a coagulant (Greiner
Bio-One, UK). Blood was layered on Histo-paque gradient (Sigma-Aldrich, Germany) gradient
and centrifuged at 400 x g for 30 minutes.
Mononuclear cells (MNCs) were harvested from the interface and washed three times with
PBS (400 x g, 200 x g, 200 x g, 18ºC). Mononuclear cells were adjusted to 2 x 107 cells/ml of RPMI
1640 without phenol red, Sigma-Aldrich). Based on morphologic examination, more than 94%
of the cells in the final preparation were lymphocytes, and more than 97% of cells were viable
as indicated by trypan blue staining.
Polymorphonuclear leukocytes were harvested from the pellet below the Histo-paque
gradient after the hypotonic lysis of pelleted erythrocytes. The process was performed
by adding an equal volume of double-distilled water (hypotonic conditions) and then, after
30 seconds, by adding 2x concentrated PBS (Sigma-Aldrich) to regain the isotonicity
of the solution. The remaining cells were washed twice with PBS (200 x g, 200 x g, 18ºC),
resuspended in RPMI without phenol red, and antibiotics supplemented with 10% FBS (SigmaAldrich) were added, to give a final concentration of 2 x 107 cells per ml. Greater than 90%
of the cells in the final preparation were neutrophils based on morphologic examination,
and more than 97% were viable (trypan blue staining).
Isolated cells were diluted to an appropriate density in the culture medium on 24-well plates
(Becton Dickinson and Company, USA) in the total volume 1 ml. Individual wells contained the
PMN and MNC cell cultures. The final cell density in individual wells were 1 x 104 do 2 x 107 cell/ml.
In the prepared suspensions MTT reduction and NRU tests were performed.
MTT reduction test. The MTT viability and activity assay was done as previously described
by Zolnai et al. 1998 (originally described by Mossman 1983), with slight modifications. 20 µl
Usefulness of neutral red uptake method…
175
of MTT solution (5 mg/ml in RPMI 1640 without phenol red, Sigma-Aldrich) was added
to the wells, and plates were incubated another 4 hours under culture conditions. At the end
of the incubation, the plates were centrifuged at 2000 x g for 10 min and the culture supernatant
was discarded. In the next step, 200 µl of DMSO (dimethyl sulfoxide, Sigma-Aldrich) was
added to each well, and the plates were vigorously shaken. The amount of MTT formazan
formed during the incubation was measured with an ELISA reader (ELx800 Universal Microplate
Reader – Biotek Instruments, USA) at a wavelength of 540 nm and reference wavelength
of 630 nm. The results were expressed as the percent of control values.
Neutral red uptake assay. Absorption of neutral red dye by leukocytes was measured as
described by Pipe et al. (1995) and Stoika et al. (2002), with slight modifications. 10 µl of NR
solution (3.3 mg/ml DPBS, Sigma-Aldrich) was added to the wells and plates were incubated
another 4 hours under culture conditions. At the end of the incubation period, plates were
centrifuged at 400 x g for 10 min and medium was carefully removed. PMNs were quickly
washed with PBS. Plates were centrifuged again (400 x g, 10 min) and the wash solution was
carefully removed. Incorporated neutral red was then solubilized in a solution of 1% acetic acid
in 50% ethanol. The cultures were shaken on a gyrating shaker for 15 min to enable
solubilization of all NR in neutrophils. The amount of neutral red taken up during the incubation
was measured with an ELISA reader (ELx800 Universal Microplate Reader) at a wavelength
of 540 nm. The results were expressed as the percentage of control values.
Lymphocyte proliferation test. To determine the usefulness of the NRU assay for the determination of lymphocyte proliferation the following experiment was performed: 1000 µl of 2 x 106
leukocytes/ml in complete culture medium (RPMI 1640 without phenol red, fetal bovine serum
(5%), sodium pyruvate (1 mM), penicillin (100 U/ml), streptomycin (100 μg/ml), amphotericin B
(2.5 μg/ml), L-glutamine 2.05 mM, Sigma-Aldrich) were incubated for 24, 48, 72 and 96 hours
in flat-bottom wells of 24-well microtiter plates (Becton Dickinson and Company, USA) with PHA
(LF-7, Biomed, Poland) as non-specific mitogen at the final concentration 250 μg/ml. Cultures were
incubated (Galaxy S, RS Biotech, UK), in typical cell culture conditions (37ºC, 5% CO2, humid atmosphere). After incubation period MTT reduction and NRU assays were performed as stated before.
All the percentage values were calculated by the same formula: Percent of control
(%) = (OD sample - OD background) / (OD control - OD background) x 100, where OD is optical
density.
Statistical analysis. Data are presented as the means ± SEM. The statistical significance
of the differences between treatments and controls were analyzed by Student’s t test.
The relationship between results obtained in MTT and NRU assays was analyzed by Pearson’s
correlation coefficient. All statistical analyses were conducted with Statistica 7.1 software.
All experiments were carried out in triplicate.
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K. Fijałkowski, P. Nawrotek, D. Czernomysy-Furowicz
RESULTS AND DISCUSSION
MTT reduction test and the incorporation of neutral red assay are the methods that are
known for many years and to this day, are the most popular techniques in the determination
of cytotoxicity, cell activity and cell proliferation (Borenfreund and Puerner 1985, Rai-el-Balhaa
et al. 1985, Gerlier and Thomasset 1986, Borenfreund et al. 1988, Weichert et al. 1991, Chung et al.
1993, Maravelias et al. 1999, Yang et al. 2009). Both the tests are based on the activity
of the various mechanisms of cellular metabolism. MTT reduction depends on the redox activity
of mitochondria. (Gerlier and Thomasset 1986), and absorption of neutral red on the integrity
of the cell membrane and active transport to lysosomes (Babich et al. 1988, Babich
and Borenfreund 1990). Usefulness of the MTT assay to determine the cytotoxicity of different
types of cells including the blood leukocytes, has been confirmed in numerous studies
performed around the world by several researchers (Vega et al. 1987, Borenfreund et al. 1988,
Weichert et al. 1991). There are numerous data, demonstrating the possibilities of applying
the MTT test to determination of the activity and proliferation of lymphocytes isolated from
different animal species, including cows (Rai-el-Balhaa et al. 1985, Weichert et al. 1991, Iwata
and Inoue 1993, Zolnai et al. 1998, Siwicki et al. 2003). High correlation of MTT test with
the the test of a very high sensitivity, based on the [3H] thymidine incorporation into DNA was
confirmed (Randall and HayGlass 1995, Wagner et al. 1999). Nowadays, [3H] thymidine
incorporation and MTT assays are most often used to measure the proliferation and number
of live cells including leukocyte(Rai-el-Balhaa et al. 1985, Iwata and Inoue 1993). However both
techniques have a number of disadvantages. [3H] thymidyne assay needs an expensive
equipment and produce radioactive waste (Wagner et al. 1999). MTT reduction test does not
work properly with leukocytes isolated from some animal species (Bounous et al. 1992).
Another disadvantage of the MTT assay is the production of the formazan crystals which have
to be solubilized prior to the evaluation of the absorbance. The formazan crystals could not
be solubilized completely. The precipitation of the serum proteins of the culture medium
by the solvent was also observed (Sladowski et al. 1993, Wagner et al. 1999). Additionally,
its sensitivity when lymphocyte proliferation is measured often remains unclear (Gerlier
and Thomasset 1986, Chen et al. 1990, Bounous et al. 1992).
Here we describe the establishment of the NRU assay, as a non-radioactive, simple and cheap
assay which can be used in multiple leukocyte in vitro study. In the research, the NRU assay
was compared with MTT reduction assay in bovine mononuclear cell and polymorphonuclear
cell cultures. The possibility to use the colorimetric NRU assay for measuring proliferation
induced by phytohemagglutinin (PHA) and live leukocyte number was also studied. So far,
the incorporation of neutral red test was used mainly in research related to the determination
Usefulness of neutral red uptake method…
177
of cytotoxicity, cell viability and to identify and detect the activated state of the cells. However,
there are studies showing that neutral red may be absorbed and accumulated in lysosomes
of leukocytes, including lymphocytes, monocytes and neutrophils. It is known that neutral red
as a vital stain is accumulated mainly in the lysosomes of monocytes and neutrophils, but
it also can be engulfed by lymphocytes (Winkler 1974, Sipka et al. 2000). The more activated
the cells are, the greater is the uptake of neutral red (Antal et al. 1995).
In our study a range from 10 000–20 000 000 cells/well a linear correlation between the optical
signal (OD at 540 nm) and the cell number was found in both MTT reduction and NRU assays
(Fig. 1). The results of the stimulation assays by PHA (LF-7) with respect to the incubation
period NRU and MTT assays are present in Fig. 2. Comparing the results of both assays,
the data clearly demonstrate a high correspondence concerning the decrease and increase
of lymphocyte stimulation. After the stimulation a very good and statistically significant correlation
between MTT and NRU was also obtained. Applying statistical analysis we found that
the correlation coefficients for MNC culture was 0.941 and 0.913 for PMN (Table 1).
A
B
0,26
0,32
0,30
Y = 0,04694x - 0,0577
0,28
R2 =
0,24
Y = 0,04332x - 0,0897
R2 = 0,9856
0,991
0,22
OD (λ = 540nm - 630nm)
OD (λ = 540nm - 630nm)
0,26
0,24
0,22
0,20
0,18
0,16
0,14
0,12
0,20
0,18
0,16
0,14
0,12
0,10
0,10
0,08
0,08
0,06
2,5
3,0
3,5
4,0
4,5
5,0
5,5
6,0
Cell density (log)
6,5
7,0
0,06
3,5
7,5
4,0
4,5
5,0
C
7,0
7,5
95% Confidence interval
Y = 0,21911x - 0,8342
0,6
R2 = 0,9732
R2 = 0,9664
0,5
OD (λ=540nm)
0,5
OD (λ = 540nm)
6,5
0,7
Y = 0,20762x - 0,7577
0,4
0,3
0,4
0,3
0,2
0,2
0,1
0,1
0,0
3,8
6,0
D
0,7
0,6
5,5
Cell density (log)
95% confidence interval
4,0
4,2
4,4
4,6
4,8
5,0
5,2
Cell density (log)
5,4
5,6
5,8
6,0
6,2
6,4
95% confidence interval
0,0
3,8
4,0
4,2
4,4
4,6
4,8
5,0
5,2
Cell density (log)
5,4
5,6
5,8
6,0
6,2
6,4
95% confidence interval
Fig. 1. Effect of leukocyte cell number on absorbance at 540 nm measured: A) MNC – MTT assay,
B) PMN – MTT assay, C) MNC – NRU assay, D) PMN – NRU assay
K. Fijałkowski, P. Nawrotek, D. Czernomysy-Furowicz
178
A
*
*
120
140
*
Cell viability
(% of control)
140
Cell viability
(% of control)
B
*
100
120
*
100
80
80
0
24
48
72
Time (h)
96
0
24
48
72
96
Time (h)
* – statistically significant differences between supernatants versus negative (100%) control (P 0.05 Student’s t test).
Fig. 2. Influence of PHA mitogen (LF-7, 250 μg/ml) on the leukocyte viability in MNC cultures MTT assay
(A), NRU assay (B);
Table 1. Pearson’s correlation coefficient between results obtained in MTT and NRU assay after
stimulation by PHA (LF-7)
Leukocyte type
Correlation coefficient
MNC
0.9413*
PMN
0.9132*
* correlation statistically significant (p ≤ 0.05).
Summarizing, the NRU assay similarly to MTT reduction assay is easy to handle, a large
number of probes can be assayed in a relatively short time and no radioactivity is necessary.
For the measurement of the colored product a common ELISA reader can be used.
The neutral red uptake assay similarly to MTT reduction assay is a valid test for evaluation
of lymphocyte proliferation and number of bovine leukocyte. Due to very high correlation, it can
be pronounced that NRU assay can be used as a second, comparative assay in leukocyte
study in in vitro cultures, especially when MTT reduction test is performed.
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K. Fijałkowski, P. Nawrotek, D. Czernomysy-Furowicz
OCENA MOŻLIWOŚCI ZASTOSOWANIA TESTU WYCHWYTU I GROMADZENIA
CZERWIENI OBOJĘTNEJ W BADANIACH ŻYWOTNOŚCI I PROLIFERACJI
LEUKOCYTÓW BYDLĘCYCH IN VITRO
Streszczenie. Celem pracy była ocena możliwości wykorzystania nieradioaktywnego testu pochłaniania
i gromadzenia czerwieni obojętnej (NRU), w badaniach bydlęcych leukocytów in vitro. Badaniom poddano
możliwość wykorzystania testu NRU do pomiarów proliferacji limfocytów, indukowanej niespecyficznym
mitogenem – fitohemaglutyniną oraz pośredniego oznaczania liczby żywych leukocytów. Badania prowadzono z użyciem świeżo izolowanych leukocytów bydlęcych. Wyniki uzyskane w teście NRU porównywano z wynikami uzyskanymi po przeprowadzeniu testu redukcji soli tetrazolowej – testu MTT. Na podstawie otrzymanych wyników stwierdzono, że czułość testu NRU w hodowlach bydlęcych leukocytów jest
podobna i porównywalna do testu redukcji MTT. Zarówno dla testu MTT jak i NRU, w zakresie od 10 000
– 20 000 000 komórek/mililitr stwierdzono liniową i wysoce skorelowaną zależność, między absorbancją
(długości fali 540 nm), a liczbą komórek. Przeprowadzone badania pozwoliły na określenie przydatności
zastosowanego testu NRU, który zgodnie z uzyskanymi wynikami może być wykorzystany do oceny
proliferacji limfocytów jak i pośredniego oznaczania liczby żywych leukocytów w hodowlach in vitro.
Wysoka korelacja pomiędzy testami, dowodzi również, że test NRU może być wykorzystany jako kolejny,
alternatywny test w badaniach bydlęcych leukocytów in vitro.
Słowa kluczowe: pochłanianie i gromadzenie czerwieni obojętnej, redukcja MTT, proliferacja limfocytów,
liczba leukocytów