Ovine Paratuberculosis: intra-herd incidence study and assessment of commercial (ID.VET, France) indirect milk ELISA test performance in dairy sheep

Ovine paratuberculosis is chronic disease affecting domestic and wild ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP). The infection causes significant economic losses worldwide and was associated to several human diseases as well. Until now, no confirmatory MAP screening technique revealing the disease stages in infected animals has been developed. This is mainly due to the lack of an efficient gold-standard method that can properly evaluate the performance of diagnostic assays. With this aim, from October 2019 to April 2020 an intra-herd cross-sectional and prospective incidence studies were carried out in two flocks with and without history of disease. To assess (i) the blood and milk MAP individual and bulk-milk seroprevalence by using an indirect milk ELISA (ID Screen® Paratuberculosis-Indirect Screening-ELISA test; ID.vet France); (ii) the incidence rate in a 6-month period; (iii) the faecal-excretors by qPCR IS900; and (iv) the performance and the accuracy of the ELISA test used, 200 paired serum, milk and faecal samples were collected. The 150 animals from positive flock were subdivided in three groups related to their age (young: 6 months, adult: <24 months and elder: than 24 months). Blood seroprevalence of 11.33% and 14% were detected at the start of the study (T0) and after 6 months (T6) on the same sheep respectively, recording an incidence rate of 3% and a significant increase in the mean serum antibody concentration in the Elders group (S/P%: p = 0.0005). An 8% (T0) and 10.67% (T6) milk seroprevalence, and an incidence rate of 7.1%, with a significant increase in the mean antibody concentration, were recorded in Elders group (S/P%: p<0.0001). Comparing blood and milk seroprevalences, no statistically significant differences were observed at T0 (p=0.3003) nor at T6 (p=0.5119). In the positive flock, 29 (19.33%) sheep was detected positive for MAP faecal excretion at T0: 3 animals (10.34%, n=29; mean Ct=22.23) as strongly and 26 (89.65%, n=29; average Ct=36.07) as weakly positive. The strongly faecal-excretory sheep (2% at T0 and 3.33% at T6; n=150) were in all three age groups and resulted positive to both, blood and milk ELISAs, except one sheep at T6. The bulk-milk analyses revealed that MAP positivity has a seasonal pattern, linked to the lactation stage. Considering the blood ELISA test and the qPCR (strong excretory) as gold-standard for MAP diagnosis, the used milk ELISA obtained an ‘Almost perfect agreement’ (k=82.6%) at T0 and a ‘Substantial agreement’ (k=70.0%) at T6, suggesting a new appropriate cut-off for milk in dairy sheep (ROC curve analysis: >21.38 S/P%; Se>80% e Sp>98%). This study suggests the possible use of milk matrix for MAP screening in dairy sheep, and a new appropriate cut-off value for ovine milk testing. Key words: Ovine paratuberculosis, milk ELISA performance, qPCR IS900, cut-off value.