Download Summary of general activities related to the disease

Activities in 2009
Paratuberculosis
María Laura Boschiroli
Agence Française de Sécurité Sanitaire des Aliments (AFSSA) /French Food Safety Agency
Laboratoire d'Etudes et de Recherches en Pathologie Animale & Zoonoses /
Animal Pathology and Zoonoses Research Laboratory
Unité Zoonoses Bactériennes / Bacterial Zoonoses Unit
23 avenue du Général de Gaulle, F-94706 Maisons-Alfort Cedex, FRANCE.
Tel: (+33[0]1) 49.77.13.00 – Fax: (+33[0]1) 49.77.13.44
E-mail: [email protected]
Summary of general activities related to the disease
1.
Test(s) in use/or available for the specified disease at your laboratory
Test
ELISA
PCR
For
Research
Diagnosis
Total
Antibody
0
12
12
PCR : 0
PCR : 0
0
/
1035
Bacterial DNA
RT PCR : 1035
MAP culture
MAP isolation
Avian tuberculin
control
Finished : 1344
Finished : 0
Ongoing : 542
Ongoing : 3
Living bacteria
1889
Living bacteria
86
0
86
Antigen
/
2
2
Agent identification
Isolation: samples are homogenised and afterwards decontaminated with 0.75% cetylpyridinium chloride. Eggbased media (Herrold) are used for inoculation, containing or not mycobactin and amphotericine B. Agent
identification is done on the basis of mycobactin dependence and on IS900 and ISMav1 amplification.
2.
Production and distribution of diagnostic reagents
Distribution of reference (3) or field strains (3) to national veterinary laboratories
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
Nil.
Annual reports of OIE Reference Laboratories and Collaborating Centres, 2009
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Paratuberculosis
4.
Preparation and supply of international reference standards for diagnostic tests or vaccines
Nil.
5.
Research and development of new procedures for diagnosis and control
Evaluation of different culture media for goat paratuberculosis:
We pursued a bacteriological study using different commercial and home made media (Herrold, Middlebrook 7H9
with a solid spongeous support, all with mycobactin) for paratuberculosis diagnosis on free or infected-considered
goat flocks. These flocks were chosen from a high intensive breeding region, and presenting different infectious
status. The study is still ongoing, but very promising results have been obtained with the liquid medium + solid
support since the bacillus can be identified earlier in these cultures compared with the solid slants.
Evaluation of new celluar antigens for early detection of caprine paratuberculosis:
In order to better characterise the intestinal immunological response in early phases of Map infection, an
experimental infection model in young goats has been set up (INRA Tours). Kids have been infected orally with a
Map strain isolated from a male adult with clinical signs of paratuberculosis. They have been followed up during 8
months in order to evaluate their infectious status and their induced immune response. Specific IFN-infection.
Animal have remained serologically negative, and, with the exception of only one animal, Map has never been
detected in faeces or in tissues neither by PCR nor by culture during the 8 months. On the contrary, histological
analysis showed the presence of inflammatory clusters in some infected animal after 4 months and in most animals
after 8 months, in association to Zhiel Neelsen staining and Map immunostaining in tissues. These results show
that in less than 8 months, experimental infection in goat kids can lead to a local and peripheral production of IFNMap in situ. This study
suggests that the existence of a tissular micro- environment leading to an early immunomodulation of presentingantigen cells which is and under-explored subject of Map infection. For better characterising immunomodulation,
we will further research on early markers of inflammatory responses and characterisation of cells infected by Map.
Molecular characterisation of M. avium subsp complex strains strains by MLVA:
Members of the Mycobacterium avium complex (MAC) are ubiquitous bacteria that can be found in water, food
and other environmental samples, and are considered opportunistic pathogens for numerous animal species,
mainly birds and pigs, as well as for human beings. We have recently demonstrated the usefulness of a PCR-based
Mycobacterial Interspersed Repetitive Units-Variable Number of Tandem Repeats (MIRU-VNTR) typing for
molecular characterization of M. avium subsp. paratuberculosis and M. avium strains exclusively isolated from
AIDS patients. Here we extended our analysis based on eight MIRU-VNTR markers to a strain collection
comprehensively comprising the other M. avium subspecies, including subsp. avium, subsp. hominissuis and
subsp. silvaticum, isolated from numerous animal species, HIV+ and HIV- humans and environmental sources. All
strains were fully typable with a discriminatory index of 0.885 almost equal to that obtained using IS1311
Restriction Fragment Length Polymorphism (RFLP) typing as a reference. In contrast to IS1311 RFLP, MIRUVNTR typing was able to further discriminate M. avium subsp. avium strains. MIRU-VNTR alleles strongly
associated or specific to M. avium subspecies were detected in several markers. Moreover, MIRU-VNTR-based
results were consistent with a scenario of independent evolution of subsp. avium/silvaticum and subsp.
paratuberculosis from subsp. hominissuis, previously proposed based on multi-locus sequence analysis (MLSA).
MIRU-VNTR typing therefore appears to be a convenient typing method capable of distinguishing the three main
subspecies and strains of the complex and providing new epidemiological knowledge on MAC.
6.
Collection, analysis and dissemination of epizootiological data relevant to international disease
control
Nil
7.
Provision of consultant expertise to OIE or to OIE Members
Participation in the French Food Safety Agency (AFSSA) National Expert Committees for Animal Health:
Working group on Paratuberculosis in Ruminants.
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Annual reports of OIE Reference Laboratories and Collaborating Centres, 2009
Paratuberculosis
8.
Provision of scientific and technical training to personnel from other OIE Members
Nil
9.
Provision of diagnostic testing facilities to other OIE Members
Nil
10. Organisation of international scientific meetings on behalf of OIE or other international bodies
Nil
11. Participation in international scientific collaborative studies
STREP Projet on Paratuberculosis (6th PCRD): Together with the AFSSA-Niort, we take part in the “ParaTB
tools project”: 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. We participate of
Workpackage 3: Development of improved methods for the diagnosis of paratuberculosis in livestock and the
detection of in faeces, milk, blood and tissues. We also participate in Workpackage 5: Association between
ruminant immune responses and pathology in animals infected with M. avium subsp. paratuberculosis. In
particular, we take part in the goat experimental infection conducted by partner 10 (INRA-Tours). Faecal samples
(n=300) and organs (n=300) collected in kinetics have been analysed by PCR and by culture onto Herrold +
medium. Participation in ad hoc workpackage meetings: Fourth general meeting, León, Spain (1-3/07/2009).
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
Souriau A., Olivier M., Rossignol C., Valheim M., Laroucau K., Bertin C., Guilloteau L., 2009. "Etude de la
réponse immunitaire précoce vis-à-vis de Mycobactérium avium subsp paratuberculosis chez le jeune
caprin." Journées Scientifiques du département Santé Animale - INRA, Seignosse, France, 2009/05/25-28.
Thibault V. C., Grayon M., Heron I., Stevenson K., Willery E., Allix-Bégue C., Boschiroli M. L., Biet F., Supply
P., 2009. "Molecular typing approaches for epidemiological investigations of Paratuberculosis." Veterinary
Network of Laboratories Researching into Improved Diagnosis and Epidemiology of Mycobacterial Diseases
(VENoMYC) Final Meeting., Torino, Italy, 2009/06/17-20.
Radomski N., Thibault V. C., Karoui C., De Cruz K., Cochard T., Gutierrez M. C., Supply P., Biet F., Boschiroli
M. L., 2009. "Mycobacterium avium subspecies strains from human and animal origin." 30th Congress of the
European Society of Mycobacteriology, Porto, Portugal, 2009/07/5-8.
Thibault V. C., L. Y., Cochard T., Boschiroli M. L., Gutierrez M. C., Briet F., Lanotte P., 2009. "Identification
des espèces de mycobactéries au sein du complexe avium-intracellulaire par analyse de PCR en temps réel
de séquences d'insertions" IXèmes Journées de Mycobactériologie de Langue Française, Bandol, France,
2009/11/16.
Radomski N., Thibault V. C., Karoui C., De Cruz K., Cochard T., Gutierrez M. C., Supply P., Biet F., Boschiroli
M. L., 2009. "La phylogenie des sous-espèces du genre Mycobacterium avium par caractérisations
moléculaires de souches d'origines humaine et animale: comparaison des méthodes VNTR-MIRU et RFLPIS1311" IXèmes Journées de Mycobactériologie de Langue Française, Bandol, France, 2009/11/16.
Annual reports of OIE Reference Laboratories and Collaborating Centres, 2009
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Paratuberculosis

Scientific publications in peer-reviewed journals
Mercier P., Beaudeau F., Laroucau K., Bertin C., Boschiroli M. L., Baudry C., Seegers H., Malher X. 2009.
Comparative age-related responses to serological and faecal tests directed to Mycobacterium avium
paratuberculosis (Map) in French dairy goats. Small Ruminant Research (87):50-56.

Other communications
« Paratuberculose de Ruminants » (http://www.afssa.fr/Documents/SANT-Ra-Paratuberculose.pdf). On line since
March 2009.
13. Inscription of diagnostic kits on the OIE Register
i)
Did you participate in expert panels for the validation of candidate kits for inscription on the
OIE Register? If yes, for which kits?
Nil
ii)
Did you submit to the OIE candidate kits for inscription on the OIE Register? If yes, for
which kits?
Nil
_______________
This laboratory is accredited by the Laboratories Section of the French Committee for Accreditation (COFRAC) in compliance with the
ISO/IEC 17025 standard and COFRAC rules of application under the N° 1-1799 for test activities
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Annual reports of OIE Reference Laboratories and Collaborating Centres, 2009