Annual Reports

of OIE Reference Laboratories

and Collaborating Centres

Informes anuales de los

Laboratorios de Referencia

y Centros Colaboradores

de la OIE

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128 Annual reports of OIE Reference Laboratories and Collaborating Centres, 2006

Paratuberculosis Professor Ivo Pavlik

Veterinary Research Institute, Hudcova 70, 621 00 Brno, Czech Republic Tel.: (+420-5) 33.33.16.01, Fax: (+420-5) 33.33.12.29

pavlik@vri.cz, http://www.vri.cz

Summary of general activities related to the disease

1. Test(s) in use/or available for the specified disease at your laboratory

Test (Total) For Specificity Total

Complement fixation test (81) Antibody Paratuberculosis 81 wild ruminants

Immune diffusion test (275) Antibody Paratuberculosis 275 wild ruminants

Culture HPC-sedimentation (15 888)

M. avium subsp. paratuberculosis

Paratuberculosis 12 812 cattle faeces 376 cattle tissues 113 milk filters (cattle) 1 901 wild ruminant faeces 406 wild ruminant tissues 30 Crohn’s patient faeces 250 cow’s milk samples

Quantitative real-time PCR (42) M. avium subsp. paratuberculosis

F57 and IS900 1 42 milk samples

PCR (156) Isolates Quadruplex system for M. avium species2 and

M. avium complex members identification

144 Mycobactin J dependent: 107 cattle isolates 37 deer isolates 12 Mycobactin J. non-dependent

IS900 RFLP (246) Isolates Standardized method3 169 cattle origin 61 wild ruminants origin 5 goat 7 rabbit 2 sheep 2 environment

1 Specific fragments for paratuberculosis; home made technique, paper under preparation. 2 Home made technique for the identification of members of M. avium species and M. avium complex; paper

under preparation. 3 Pavlik, I., Horvathova, A., Dvorska, L., Bartl, J., Svastova, P., Du Maine, R., Rychlik, I.: Standardisation of

Restriction fragment length polymorphism for Mycobacterium avium subspecies paratuberculosis. Journal of Microbiological Methods, 1999, 38 (1-2), 155-167.

2. Production and distribution of diagnostic reagents

Mycobactin J for Mycobacterium avium subsp. paratuberculosis Mycobactin dependent strains isolation.

A total of 1 000 ml was used in our laboratory and a total of 485 ml were supplied to the laboratories in the Czech Republic: State Veterinary Diagnostic Institute, Prague (280 ml), Olomouc (175 ml) and Jihlava (30 ml).

A total of 170 ml were supplied to the EC countries: (50 ml) to The Netherlands, (20 ml) to Cyprus and (100 ml) to the Slovak Republic.

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Annual reports of OIE Reference Laboratories and Collaborating Centres, 2006 129

Activities specifically related to the mandate of OIE Reference Laboratories

3. International harmonisation and standardisation of methods for diagnostic testing or the production and testing of vaccines

None

4. Preparation and supply of international reference standards for diagnostic tests or vaccines

None

5. Research and development of new procedures for diagnosis and control

Conventional “quadruplex” PCR system for the identification of members of M. avium species (including M. avium subsp. paratuberculosis) and M. avium complex was routinely used and paper is under preparation.

Our laboratory developed the technique for isolation of the total ultra pure DNA (including DNA of M. avium subsp. paratuberculosis) from up to 50 ml of milk. Subsequently, the presence of M. avium subsp. paratuberculosis could be detected by two independent real time PCR systems with internal amplification control based on two specific independent loci: the multiple copy insertion sequence IS900 and a single copy fragment F57. The developed technique allows, on the assumption that 50 ml of the milk is analysed, to detect at least 3 M. avium subsp. paratuberculosis cells in 1 ml of the starting amount of milk using F57 fragment as a target; whereas, IS900 real time PCR system is more sensitive and is able to detect approximately 1 cell in 3 ml of the milk. Paper is under preparation.

6. Collection, analysis and dissemination of epizootiological data relevant to international disease control

RFLP analysis was carried out on the following M. avium subsp. paratuberculosis isolates from Argentina (23 cattle and 5 deer isolates), Austria (18 cattle isolates), Slovenia (9 cattle isolates), Scotland (2 deer isolates) and the Czech Republic (119 cattle, 54 deer, 5 goat 7 domestic rabbit, 2 sheep and 2 environmental isolates).

7. Provision of consultant expertise to OIE or to OIE Member Countries

Consultations and discussion about paratuberculosis control and/or certification program during the business trips in Europe (Slovakia, Austria, Germany, Spain, Ireland, Denmark, Belgium, The Netherlands, United Kingdom, Sweden, Norway, France, Greece and Cyprus) and in America (Argentina, Brazil, USA, Uruguay).

8. Provision of scientific and technical training to personnel from other OIE Member Countries

Kimberley Evelyn Sim, University of Aberdeen, Scotland, UK. Topic: Culture detection of Mycobacterium avium subsp. paratuberculosis. 1.9.2006-31.8.2007.

Catherine Murdoch, Aberdeen University, Scotland, UK. Topic: PCR detection of Mycobacterium avium subsp. paratuberculosis in milk. 1.9.2006-31.8.2007.

Nustes Lozano Manuel Juan, Tolima University, Ibagué, Colombia. Topic: Culture examination of Mycobacterium avium subsp. paratuberculosis. 1.8.2006-30.6.2007.

Dr. Fernando Paolicchi, Animal Health Group, INTA and Faculty of Agrarian Sciences, University of Mar del Plata, Argentina. Topic: Detection of Mycobacterium avium subsp. paratuberculosis in different matrices and typing of isolates by different molecular laboratory techniques. 17.5.-11.8.2006.

Dr. Johannes Lorenz Khol, University of Veterinary Medicine, Vienna, 2nd Medical Clinic for Ruminants and Swine, Austria. Topic: Detection of Mycobacterium avium subsp. paratuberculosis in different matrices and typing of isolates by different molecular laboratory techniques. 8.-26.5.2006 and 8.9.2006.

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130 Annual reports of OIE Reference Laboratories and Collaborating Centres, 2006

Dr. Louis Fischer, Geschäftsführer, Labovet GmbH, Labor für Veterinärdiagnostik und Hygiene, Vienna, Austria. Topic: Detection of Mycobacterium avium subsp. paratuberculosis in different matrices and typing of isolates by different molecular laboratory techniques. 8.9.2006.

Univ. Prof. Dr. Martin Wagner, Dipl. ECVPH, Coordinator Molecular Microbiology Group at the Institute for Milk Hygiene, & Technology and Food Science Coordinator Research Focus Food Safety and Risk Assessment, Department for Veterinary Public Health and Food Science, Vienna, Austria. Topic: Detection of Mycobacterium avium subsp. paratuberculosis in different matrices and typing of isolates by different molecular laboratory techniques. 8.9.2006.

Dr. Thomas Schlederer, Aureon Biosystems GmbH, Vienna, Austria. Topic: Detection of Mycobacterium avium subsp. paratuberculosis in different matrices and typing of isolates by different molecular laboratory techniques. 30.6.2006 and 8.9.2006.

9. Provision of diagnostic testing facilities to other OIE Member Countries

We have examined by culture cattle tissue samples and faeces from Austria, faeces from The Netherlands and Slovakia.

10. Organisation of international scientific meetings on behalf of OIE or other international bodies

None

11. Participation in international scientific collaborative studies

Our laboratory was involved in the following national and international projects dealing with the development of different the techniques (liquid culture, quantitative real time PCR etc.) for the detection of M. avium subsp. paratuberculosis in different matrices for the developing the certification program for paratuberculosis:

National grants

Healthy animals and safety food as a basis for the health of human population, part: Mycobacterial infections of animals. Institutional Research Plan, 15.1.2004-31.12.2008. Ministry of Agriculture, the Czech Republic, No.: MZE 0002716201.

European Union Grants:

Veterinary Network of Laboratories Researching into Improved Diagnosis and Epidemiology of Mycobacterial Diseases. Project Acronym: VENOMYC, No. SSPE-CT-2004-501903, EU, Brussels, 1.10.2003 – 30.9.2007 (Co-ordinator: Prof. Lucas Domínguez, Dpt. Patología Animal I, Sanidad Animal Facultad de Veterinaria U.C.M., Madrid, Spain).

Control and prevention of emerging and future pathogens at cellular and molecular level throughout the food chain. Integrated Project. Project Acronym: PathogenCombat. No. FOOD-CT-2005-007081. Framework VI Programme. EC Brussels, 1.4.2005-31.3.2009. Co-ordinator: Prof. Mogens Jakobsen, The Royal Veterinary and Agricultural University, Dept. of Food Science, Food Microbiology, Copenhagen, Denmark.

Development of improved tools for detection of paratuberculosis in livestock, M. avium subsp. paratuberculosis in food and for the assessment of the risk of human exposure. Acronym: ParaTBTools. No. FP6-2004-FOOD-3B-023106. STREP project, 2006-2009. Co-ordinator: Dr. Douwe Bakker, DLO Institute for Animal Science and Health, Lelystad, Netherlands.

Study on paratuberculosis in correlation with the food, the animal health and the environment. Research promotion foundation's framework programme for research and technological development 2003-2005. No. RPF'S FP 2003-2005; AEIFO 1104/02. Co-ordinator: Dr. Liapi Maria, Veterinary Officer at Cyprus Veterinary Services, Nicosia, Cyprus.

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Annual reports of OIE Reference Laboratories and Collaborating Centres, 2006 131

Development of improved tools for detection of paratuberculosis in livestock, M. avium subsp. paratuberculosis in food and for the assessment of the risk of human exposure. Acronym: ParaTBTools. No. FP6-2004-FOOD-3B-023106. STREP project, 2006-2009. Co-ordinator: Dr. Douwe Bakker, DLO Institute for Animal Science and Health, Lelystad, Netherlands.

12. Publication and dissemination of information relevant to the work of OIE (including list of scientific publications, internet publishing activities, presentations at international conferences)

Presentations at international conferences and meetings

Beran V., Trcka I., Kopecna M., Moravkova M., Kralik P. & Pavlik I.: Different culture techniques for Mycobacterium avium subsp. paratuberculosis detection. In: Proceedings of workshop of VENOMYC project, 26th – 29th April, Jena, Germany, 2006, 15-16.

Pavlik I., Beran V., Trcka I. & Rozsypalova Z.: Samples of biological material from cattle for laboratory detection of causal agent of paratuberculosis: design and results of OIE inter-laboratory ring test. In: Proceedings of workshop of VENOMYC project, 26th – 29th April, Jena, Germany, 2006, 44-46.

Kohler H., Burket B., Pavlik I., Seelhorst T. & Martin G.: Evaluation of serological tests for the diagnostics of paratuberculosis – calculation of predictive values and sample size in monitoring programmes. In: Proceedings of workshop of VENOMYC project, 26th – 29th April, Jena, Germany, 2006, 28-29.

Kralik P., Tesinska I., Bartos M &, Pavlik I.: Quantification of Mycobacterium avium subsp. paratuberculosis (MAP) in milk. PathogenCombat Consortium Meeting, Ghent, Belgium, 14th – 17th March 2006.

Pavlik I., Trcka I., Koubek P., Heroldova M., Kopecna M., Lamka J., Cvetnic Z., Moser I., Hotzel H., Ocepek M., Mrlik V. & Pate M.: Mycobacterial infections in domestic and wild carnivores originating from Central Europe. In: Proceedings of workshop of VENOMYC project “Wildlife Reservoirs of Mycobacterial Infections, Dublin, Ireland, 13th – 16th September 2006, 13-14.

Pavlik I. (invited lecture): The experience with the control of paratuberculosis in the Czech Republic during the years 1988 to 2004. In: Biotechnology 2006, Ceske Budejovice, Czech Republic, February 15th to 16th, 2006, Rehout, V. (ed.), Scientific Pedagogical Publishing, ISBN 8085645-53-X, 839-844.

Pavlik I. (invited lecture): Bovine and avian tuberculosis, paratuberculosis and conditionally pathogenic mycobacteria in cattle, pigs and other animals in the Czech Republic: prevalence, diagnostics, sources of infection, control and prevention. In: Proceedings of XVI Reunión Científico Técnica de la AAVLD (Asociación Argentina de Veterinarios de Laboratorios de Diagnóstico), Simposio Internacional de Micobacterias, 7th December 2006, Mar del Plata, Argentina, 178-181.

Pavlik I. (invited lecture): Bovine tuberculosis, paratuberculosis, avian tuberculosis and mycobacterioses in the Czech Republic during the last 15 years. Innsbruck, Austria, 7th June, 2006.

Pavlik I. (invited lecture): Tuberculosis in wild animals: current situation and possibility for control. Section: Advances in Veterinary Medicine on 143. Annual Convention of American Veterinary Medical Association, Honolulu, Hawaii, USA, 15th – 19th July 2006.

Pavlik I. (invited lecture): Bovine and avian tuberculosis, paratuberculosis and conditionally pathogenic mycobacteria in cattle, pigs and other animals in the Czech Republic: prevalence, diagnostics, sources of infection, control and prevention. In: Proceedings of XVI Reunión Científico Técnica de las AAVLD (Asociación Argentina de Veterinarios de Laboratorios de Diagnóstico), Simposio Internacional de Micobacterias, 7th December 2006, Mar del Plata, Argentina, 178-181.

Pavlik I. (invited lecture): Paratuberculosis in domestic and wild animals. Division Laboratorios Veterinarios dr. Miguel C. Rubino, Montevideo, Uruquay, 11th December 2006.

Scientific publications in peer-reviewed journals

Bartos M., Hlozek P., Svastova P., Dvorska L., Bull T., Matlova L., Parmova I., Kuhn I., Stubbs J., Moravkova M., Kintr J., Beran V., Melicharek I., Ocepek M. & Pavlik, I.: Identification of members of Mycobacterium avium species by Accu-Probes, serotyping, and single IS900, IS901, IS1245 and IS901-flanking region PCR with internal standards. Journal of Microbiological Methods, 2006, 64 (3), 333-345.

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132 Annual reports of OIE Reference Laboratories and Collaborating Centres, 2006

Trcka I., Lamka J., Suchy R., Kopecna M., Beran V., Moravkova M., Horvathova A., Bartos M., Parmova I. & Pavlik I.: Mycobacterial infections in European wild boars (Sus scrofa) in the Czech Republic during the years 2002 to 2005. Veterinarni Medicina, 2006, 51 (5), 320-332. http://www.vri.cz/docs/vetmed/51-5-320.pdf

O'Brien R., Mackintosh C.G., Bakker D., Kopecna M., Pavlik I., Griffin J.F.T.: Immunological and molecular characterization of susceptibility in relationship to bacterial strain differences in Mycobacterium avium subsp paratuberculosis infection in the red deer (Cervus elaphus). Infection and Immunity, 2006, 74 (6), 3530-3537.

Kopecna M., Ondrus S., Literak I., Klimes J., Horvathova A., Moravkova M., Bartos M., Trcka I. & Pavlik I.: Detection of Mycobacterium avium subsp. paratuberculosis in two brown bears in the Central European Carpathians. Journal of Wildlife Diseases, 2006, 42 (3), 691-695.

Hosek J., Svastova P., Moravkova M., Pavlik I. & Bartos M.: Methods of mycobacterial DNA isolation from different biological material: a review. Veterinarni Medicina, 2006, 51 (5), 180-192. http://www.vri.cz/docs/vetmed/51-5-180.pdf

Hasonova L. & Pavlik I.: Economic impact of paratuberculosis in dairy cattle herds: a review. Veterinarni Medicina, 2006, 51 (5), 193-211. http://www.vri.cz/docs/vetmed/51-5-193.pdf

Beran V., Havelkova M., Kaustova J., Dvorska L., Pavlik I.: Cell wall deficient forms of mycobacteria: a review. Veterinarni Medicina, 2006, 51 (7), 365-389. http://www.vri.cz/docs/vetmed/51-7-365.pdf

Other communications

Hasonova L., Trcka I., Pavlik I.: Paratuberculosis in dairy cattle (in Czech). Nas Chov, 2006, (6), 35-39.

Pavlik I.: Mycobacterial infections in animals and humans (in Czech). Veterinarstvi, 56 (11), 713-714.

Other publications related to mycobacteriology

Pavlik I.: The experience of new European Union Member States concerning the control of bovine tuberculosis. Veterinary Microbiology, 2006, 112, 221-230.

Pate M., Svara T., Gombac M., Paller T., Zolnir-Dovc M., Emersic I., Prodinger W.M., Bartos M., Zdovc I., Krt B., Pavlik I., Cvetnic Z., Pogacnik M., Ocepek M.: Outbreak of tuberculosis caused by Mycobacterium caprae in a zoological garden. Journal of Veterinary Medicine series B, 2006, 53, 387-392.

Shitaye J.E., Getahun B., Alemayehu T., Skoric M., Treml F., Fictum P., Vrbas V., Pavlik I.: A prevalence study of bovine tuberculosis by using abattoir meat inspection and tuberculin skin testing data, histopathological and IS6110 PCR examination of tissues with tuberculous lesions in cattle in Ethiopia. Veterinarni Medicina, 2006, 51 (11), 512-522. http://www.vri.cz/docs/vetmed/51-11-512.pdf

Cvetnic Z., Spicic S., Katalinic-Jankovic V., Marjanovic S., Obrovac M., Benic M., Mitak M., Pavlik I.: Mycobacterium caprae infection in cattle and pigs on one family farm in Croatia: a case report. Veterinarni Medicina, 2006, 51 (11), 523-531. http://www.vri.cz/docs/vetmed/51-11-523.pdf

Pavlik I.: Control and eradication of bovine tuberculosis in central Europe. In: Mycobacterium bovis Infection in Animals and Humans, Second Edition, Thoen, C.O., Steele, J.H., Gilsdor, M.J. (eds.), Blackwell Publishing, 2006, ISBN-13: 978-0-8138-0919-9, pp 136-148, 329 pp.

Pavlik I.: Bovine tuberculosis in Russia and the former states of the Soviet Union. Mycobacterium bovis Infection in Animals and Humans, Second Edition, Thoen, C.O., Steele, J.H., Gilsdor, M.J. (eds.), Blackwell Publishing, 2006, ISBN-13: 978-0-8138-0919-9, pp 173-198, 329 pp.

Pavlik I. & Trcka I.: Bovine tuberculosis in animals in the Czech Republic and other Central European countries between 1990 and 2004 (in Czech). Veterinarstvi, 2006, 56, 294-298.

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