Download NATIONAL VETERINARY RESEARCH INSTITUTE

NATIONAL VETERINARY
RESEARCH INSTITUTE
PARTYZANTOW 57
24-100 PULAWY, POLAND
tel. (48) 81 8863051
fax (48) 81 8862595
http://www.piwet.pulawy.pl
Pulawy
NATIONAL VETERINARY
RESEARCH INSTITUTE
established in 1945
as a scientific institution of the
Ministry of Agriculture
The major mission of the NVRI
is applied research in veterinary
medicine and provide services for
the public veterinary sector
Scheme of veterinary services in Poland
Ministry of Agriculture and Rural Development
National Veterinary Research Institute
National Reference Laboratories
Prime Minister’s Office
Veterinary Inspection
Chief Veterinary Officer
Regional Veterinary Diagnostic Laboratories
16
Branch laboratories
29
Private laboratories
60
Private practicioners
Voivodship Veterinary Inspectorate
Voivodship Veterinary Officer
16
District Veterinary Inspectorate
District Veterinary Officer
295
Border Veterinary Inspectorate
27
National Veterinary
Research Institute
Research staff
The Institute employs 350 persons
‰ 33 researchers with Sc.D. and Ph.D.
‰ 40 researchers with Ph.D
‰ 49 researchers with D.V.M. or M.Sc
‰ 102 technicians
Organization of the NVRI
‰ Laboratories representing basic disciplines
(bacteriology, virology, biochemistry, parasitology,
pathology,toxicology and pharmacology, food and
feedingstuffs hygiene
‰
Laboratories for the diseases of particular animal
species: equine, swine, poultry, fish
‰
Laboratories for diseases of special significance for
animals and husbandry (tuberculosis, foot and mounth
disease, mastitis) and zoonoses
The NVRI contributes the activity on
the following fields:
‰ Experimental research into animal diseases
‰ Reference activity dedicated to the regional
diagnostic laboratories and the Veterinary Inspection
‰ Active surveillance - implementation and execution of
the Multiannual Monitoring Programme
17.01.2005
Construction of the new
laboratories started
National reference laboratories (NRL)
A network of 14 laboratories within different
departments of NVRI appointed with the Act of
Ministry of Agriculture and Rural Development
of October 21, 2004
for the reference activities
The most important elements in the
functioning of the NRL are:
‰ Reference duties are fully integrated with
activity of NVRI departments undertaking
research and training
‰ Reference activity has its own budget
Activities of NRL (46)
Department/Laboratory
Reference activity
Microbiology
Brucellosis, antrax, listeriosis, tubercullosis
Virology
BSE, rabies, IBR/IPV, EAV, EVA
Swine Diseases
CSF, Aujeszky’s Dis., leptospirosis, TGE
Pathology
BSE, scrapie
Biochemistry
Bovine leukosis, MVV, CAEV
Cattle & Sheep Disease
Q fever, chlamydiosis, CBPP,
Poultry Diseases
HPAI, Newcastle Dis., Gumboro Dis., Marek Dis.,
mycoplasmosis,
Food and Mouth Disease
FMD, SVD
Fish Diseases
IHN, ISA, SV, VHS, IPN, BKD
Radiology & Toxicology
Dioxines, nitrosoamines
Pharmacology &Toxicology
Residues of pesticides, PCBs, toxic elements,
veterinary drugs, hormones, mycotoxins
Higiene of Food of Animal Origin
Food microbiology
Higiene of Animal Feedingstuff
PAP, medicated feedingstuff, microbiological agents
What are the duties of NRL?
‰ to collaborate with the regional laboratories in terms of
harmonization standards and diagnostic methods
‰ to carry out the proficiency tests
‰ to control the quality of diagnostic reagents used for the
tests reffered to the diagnosis of infectious diseases
‰ to collect and process epidemiological data related to animal
infectious diseases and official food control
‰ to perform laboratory examinations to confirm the results
done by authorized laboratories, if there are some doubts
‰ to train the employees of the authorised laboratories
NRL for Campylobacter
Located at Department of Hygiene of
Food of Animal Origin of NVRI
Campylobacter – main
activities (since 2004)
‰ Development of molecular identification
methods (PCR)
‰ Application of PCR and RFLP methods for
differentiation of the strains
‰ Isolation and identification of Campylobacter
from poultry
PCR methods
PCR test
m-PCR 1*
m-PCR 2**
PCR 3***
Primers
Sequence (5´→3´)
Target gene
Size of
PCR
amplicon
(bp)
MD16S1
MD16S2
ATCTAATGGCTTAACCATTAAAC
GGACGGTAACTAGTTTAGTAT T
16S rRNA
(C. coli and C. jejuni)
857
MDmapA1
MDmapA2
CTATTTTATTTTTGAGTGCTTGTG
GCTTTATTTGCCATTTGTTTTATTA
mapA
(C. jejuni)
589
COL3
MDCOL2
AATTGAAAATTGCTCCAACTATG
TGATTTTATTATTTGTAGCAGCG
ceuE
(C. coli)
462
HIP400F
HIP1134R
GAAGAGGGTTTGGGTGGT
AGCTAGCTTCGCATAATAACTTG
hipO
(C. jejuni)
735
CC18F
CC519R
GGTATGATTTCTACAAAGCGAGATA
AAAGACTATCGTCGCGTG
asp
(C. coli)
500
CL55
CL632
ATGCAAGTCGAACGATGAAGCGAC
CCACTCTAGATTACCAGT TTCCC
16S rRNA
(C. lari)
579
* Denis M., Soumet C., Rivoal K., Ermel G., Blivet D., Salvat G., Colin P.: Development of a m-PCR assay for simultaneous identification of
Campylobacter jejuni and C. coli. Lett. Appl. Microbiol. 1999, 29, 406-410.
** Linton D., Lawson A. J., Owen R. J., Stanley J.: PCR detection, identification to species level, and fingerprinting of Campylobacter jejuni
and Campylobacter coli direct from diarrheic samples. J. Clin. Microbiol. 1997, 35, 2568-2572.
***Oyarzabal O. A., Wesley I. V., Barbaree J. M., Lauerman L. H., Conner D. E.: Specific detection of Campylobacter lari by PCR. Journal of
Microb. Methods, 29, 1997, 97-102.
Multiplex PCR results
M
1
2
3
4
5
6
7
8
9
10
M
500 bp →
Lanes: 1 – C. jejuni and C. coli, 2 – C. coli, 3 - C. jejuni, 4 – E. coli EDL 933, 5 – negative
control (H2O), 6 - C. jejuni and C. coli, 7 - C. coli, 8 – C. jejuni, 9 - E. coli EDL 933, 10 –
negative control (H2O), M - 100 bp DNA marker
Differentiation by molecular methods
Method
ERIC-PCR
flaA typing
RAPD-PCR
PFGE
Primer
name
Sequence (5´→3´)
ERIC1R
ERIC2
ATGTAAGCTCCTGGGATCAC
AAGTAAGTGACTGGGGTGAGCG
flaAF
flaAR
OPA-11
1254
Amplifictions conditions
94°C/4 min, 40 cycles: 25°C/45 s,
72°C/1 min, 94°C/45 s and 72°C/5
min
GGATTTCGTATTAACACAAATGGTGC
CTGTAGTAATCTTAAACATTTTG
94°C/5 min, 30 cycles:48°C/1 min,
72°C/2 min, 94°C/1 min and 72°C/5
min
CAATCGCCGT
CCGCAGCCAA
94°C/5 min, 40 cycles:37°C/1 min,
72°C/1 min, 94°C/1 min and
72°C/10 min
SmaI, SacII and both
ERIC-PCR results
M
3
6
50
21
60
W7 W11 W18 W20 W25 W35 W40 W41 W47 M
70
80
90
100
C. j. 3
C. j. 6
C. j. W20
C. j. W25
C. j. W35
C. j. W7
C. j. W11
C. j. W18
C. j. 21
C. j. W47
C. j. W40
C. j. W41
RFLP/PFGE (SmaI) results
3
10
6
20
21
30
W7 W11 W18
40
50
M
60
W20 W25 W35 W40 W41 W47
70
80
90
100
C. j. W40
C. j. W41
C. j. W25
C. j. W35
C. j. 21
C. j. W7
C. j. W11
C. j. W18
C. j. W20
C. j. 3
C. j. 6
C. j. W47
Proficiency tests
• Detection of Campylobacter in chicken
matrix, organized by FEPAS, UK,
November 2005
• Identification of Campylobacter, organized
by WHO Global Salm-Surv, External
Quality Assurance System, Denmark,
October 2006
Selected publications
•
K. Wieczorek, J. Osek: Multiplex PCR assays for simultaneous identification of
Campylobacter jejuni and C. coli. [in Polish]. Medycyna Weterynaryjna 2005, 61,
797
•
K. Wieczorek, J. Osek: Campylobacter as a cause of gastrointestinal infections.
A review. [in Polish]. Medycyna Weterynaryjna 2005, 61, 847
•
K. Wieczorek, J. Osek: The usefulness of some selected PRC techniques in
differentiating thermotolerant Campylobacter strains. [in Polish]. Zywnosc,
Nauka, Technologia, Jakosc 2005, 45, 132
•
K. Wieczorek, J. Osek: Prevalence of virulence markers in Campylobacter jejuni
and Campylobacter coli obtained from chicken carcasses. [in Polish]. Zoonozy,
2005, 92
•
K. Wieczorek, J. Osek: Identification of thermophilic Campylobacter jenuni and
Campylobacter coli by multiplex PCR. Hygiena Alimentorum XXVII, 2006, 253
•
K. Wieczorek, J. Osek: Differentiation of Campylobacter jejuni isolates by
random amplified polymorphic DNA method. [in Polish]. Medycyna
Weterynaryjna 2006, 62, 663
Contact
Prof. Jacek Osek, DVM, PhD
National Veterinary Research Institute
Department of Hygiene of Food of Animal Origin
Partyzantow 57
24-100 Pulawy, Poland
Phone: +48 81 8863051
Fax: +48 81 8862595
E.mail: [email protected]
www.piwet.pulawy.pl