Introduction : Mycobacterium avium ssp. paratuberculosis (Map) est l’agent étiologique de la paratuberculose, qui affecte les ruminants et est suspectée d’être transmissible à l’homme en lien avec la maladie de Crohn, une inflammation chronique de l’intestin. Le contrôle de cette maladie à forte prévalence, nécessite de mieux identifier et connaître son agent. Aujourd’hui, le typage des souches repose sur la RFLP-IS900 qui est peu discriminante et peu sensible. Objectif : Dans cette optique, mon projet de thèse a eu pour objectif de développer une méthode de typage moléculaire novatrice utilisant les minisatellites afin d’évaluer le degré de diversité génétique de la sous-espèce paratuberculosis. Ce travail nécessite la construction d’une collection de souches et le développement d’une méthode de typage hautement discriminante. Résultats : Nos résultats obtenus avec huit marqueurs MIRU-VNTR (Mycobacterial Interspersed Repetitive Unit-Variable Number Tandem Repeat) montrent que cette méthode est rapide, adaptée à Map et plus discriminante de la RFLP-IS900. Par ailleurs, ces mêmes marqueurs nous ont permis de typer une collection de souches de M. avium ssp. avium et M. avium ssp. hominissuis. Le typage MIRU-VNTR a également permis de différencier la souche vaccinale Map 316F, en fonction de son origine, laboratoire Weybridge (Royaume-Uni) et laboratoire Mérial (France). Dans le but d’affiner la discrimination de ces souches, nous nous sommes intéressés aux marqueurs microsatellites SSR (Short Sequence Repeat) analysés par séquençage. Onze loci SSR ont été analysés et évalués sur une collection de 127 souches de Map préalablement typées par MIRU-VNTR et RFLP- IS900. Les résultats suggèrent que la stratégie la plus adaptée pour la discrimination des souches consiste à utiliser dans un premier temps le typage MIRU-VNTR ; le typage SSR et la RFLP-IS900 peuvent permettre une discrimination complémentaire. Conclusion : Ce travail de thèse nous a permis de développer une méthode de typage rapide, fiable et discriminante pour l’étude de la sous-espèce paratuberculosis, mais également du complexe MAC. / Background: Mycobacterium avium subsp. paratuberculosis (Map) is the etiological agent of paratuberculosis, affecting wide range of domestic ruminants and suspected to be involved in Crohn’s disease in human. The control of this disease, highly prevalent, requires better identification methods and better knowledge of the etiological agent. Currently, the gold standard method to type Map strains is the IS900-RFLP. But this technique is not very sensitive and not very discriminating. Objective: In this context, the project of my thesis aimed to develop an innovative method of molecular typing using the minisatellites in order to evaluate the degree of diversity of Map. This work requires the construction of a collection of strains and the development of a new molecular typing method. Results: Our results obtained with eight MIRU-VNTR markers (Mycobacterial Interspersed Repetitive Unit-Variable Number Tandem Repeat) show that MIRU-VNTR typing is a fast typing method, adapted to Map and more discriminative than IS900-RFLP. In addition, these 8 markers could be applied to type a collection of Mycobacterium avium subsp. avium and Mycobacterium avium subsp. hominissuis. MIRU-VNTR typing differentiated the vaccine strain 316F according to the laboratory of origin Weybridge (UK) or Mérial (France). In order to improve the discrimination of the strains, we used the microsatellites markers SSR (Short Sequence Repeat) using sequencing analysis of the Map genome. Eleven SSR were analysed and evaluated on a collection of 127 Map strains previously typed by MIRU-VNTR and IS900-RFLP typing. Our results suggested that the most adapted strategy to discriminate the Map strains consisted in using MIRU-VNTR typing, and that SSR and RFLP-IS900 typing to better discriminate the clustered isolates. Conclusion: The work of my thesis enabled to develop fast, reliable and robust typing method for the study of Map and also MAC subspecies.

The role of Mycobacterium avium subsp paratuberculosis (MAP) in the etiology and pathogenesis of inflammatory bowel disease (IBD) including Crohn's Disease (CD), has been investigated. The fastidious characteristics and cross reactivity of MAP with other members in Mycobacteria have produced significant challenges in their detection and identification. In this two year pilot study, an array of three PCR molecular assays based on the detection of sequences from the16S rRNA, IS1245, and IS900 genes, belonging to members of the MAC, have been developed and optimized into a common protocol to be used as a rapid and accurate diagnostic tool regarding M. avium complex (MAC) infection. The PCR protocol time was reduced by half, and the sensitivity and specificity of the molecular assays has been significantly improved barring the need for southern hybridization. This improved methodology was employed for the molecular typing of MAC in 100 resected, full-thickness tissue samples removed from IBD patients. The tissue samples were homogenized, decontaminated, and inoculated into two mycobacterial culture media systems. A total of 328 Bactec and Mycobacteria Growth Indicator Tube (MIGT) cultures were evaluated for positive MAC growth. Harvested cells were then subjected to genomic DNA extraction and subsequent PCR typing. The I6 S rRNA-based PCR resulted in detection of 26/28 (93%) MAC in Bactec cultures. Specifically, 25/28 (89%) of positive MAC indicated the presence of IS1245 specific to M. avium subsp avium (MAV), and 6/28 (21%) produced results consistent with the presence of IS900 following nested PCR. Moreover, 20/100 (20%) of MGIT cultures were positive for MAP. Sequence analysis was performed on amplified regions of the IS900 element from seven isolates. A nucleotide alignment revealed that 2/7 isolates demonstrated 100% homology to Bovine MAP and 5/7 isolates showed 96-99% homology to sequenced Bovine MAP published in GenBank. The detection of at least two Bovine derived MAP in IBD tissue will have great impact on the epidemiology and reclassification of IBD. The significant homology of the other five isolates to Bovine derived MAP suggests a diversity in the geographical distribution of MAP regarding Johne's disease and CD. Ultimately, the etiology, diagnosis, and the treatment of IBD as well as control and prevention measures may be enhanced with better tools for investigating emerging infectious diseases.