Genetic diversity of Mycobacterium avium sp. paratuberculosis by mycobacterial interspersed repetitive Unit-Variable number tandem repeat and multi-locus short-sequence repeat one-sentence summary: Genetic diversity of Mycobacterium avium sp. paratuberculosis isolates.

Background: Paratuberculosis is an enteric disease caused by Mycobacterium avium sp. paratuberculosis (MAP) that affects mainly ruminant producing losses to the livestock industry. Many molecular epidemiological methods have been used to discriminate MAP isolates. Method: The aim of this study was to describe the genetic diversity of the Argentinean MAP isolates using a combination of two molecular systems, the mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) ("automated and "non-automated") and the multi-locus short-sequence repeat (MLSSR) system. Results: Thirty-two isolates were identified as MAP of C type by IS900 polymerase chain reaction (PCA) and IS1311 PCA-restriction enzyme analysis. The main patterns found by both MIRU-VNTR systems were INMV1 (54.5%), INMV2 (24.2%) and INMV11 (9.1%). The INMV5, INMV8 and INMV16 were represented with one isolate each (3.0%). Only 4 MIRU-VNTR loci were polymorphic. Conclusion: Those isolates sharing the same INMV patterns were analyzed by MLSSR, being locus 2 the most polymorphic one showing isolates with 9, 10, 11, and more than 11 "G" repeats. Besides, the global discriminatory power among isolates could be increased using both techniques. Based on these results, a short version of the "automated" MIRU-VNTR could be used as a screening tool to group isolates genetically related and subsequently perform the SSR using locus 2 on those isolates sharing the same INMV pattern.

Development and Validation of a Novel ELISA for the Specific Detection of Antibodies against Mycobacterium avium Subspecies paratuberculosis Based on a Chimeric Polyprotein.

Bovine paratuberculosis (PTB) is caused by Mycobacterium avium subsp. paratuberculosis (MAP). The optimization of detection tests specific for MAP is crucial to improve PTB control. In this work, we aimed to develop and validate a diagnostic tool based on an ELISA to specifically detect anti-MAP antibodies from bovine serum samples. For that purpose, we designed a recombinant polyprotein containing four specific antigens from MAP and optimized the ELISA. The validation consisted of the assessment of 10 sera from PTB-infected and healthy bovines with different OD values. The diagnostic performance of the polyprotein-ELISA was evaluated by testing 130 bovine serum samples (47 healthy, 48 MAP-infected, and 35 M. bovis-infected bovines). The ELISA using the polyprotein yielded an area under the ROC curve (AUC) of 0.9912 (95% CI, 0.9758-1.007; P < 0.0001). Moreover, for this ELISA, the cut-off selected from the ROC curve based on the point with a sensitivity of 95.56% (95% CI, 0.8485-0.9946) and specificity of 97.92 (95% CI, 0.8893-0.9995) was 0.3328. Similar results were obtained with an ELISA using the commercial Paratuberculosis Protoplasmatic Antigen (PPA). However, the ELISA with the polyprotein antigen showed a better performance against sera from animals infected with Mycobacterium bovis compared to the ELISA with PPA: lower cross-reactivity (2.85% versus 25.71%). These results demonstrate a very low cross-reactivity of the polyprotein with antibodies present in serum samples from animals infected with M. bovis. The designed polyprotein and the validated ELISA could be very useful for the specific identification of MAP-infected animals in herds.