Application of a peptide-mediated magnetic separation-phage assay for detection of viable Mycobacterium avium subsp. paratuberculosis to bovine bulk tank milk and feces samples.

Naturally contaminated bovine bulk tank milk (n = 44) and feces (n = 39) were tested for the presence of viable Mycobacterium avium subsp. paratuberculosis by a novel peptide-mediated magnetic separation-phage (PMS-phage) assay. Counts of viable M. avium subsp. paratuberculosis cells ranging from 1 to 110 PFU/50 ml of milk and 6 to 41,111 PFU/g of feces were indicated by the PMS-phage assay.

Gamma interferon responses to proteome-determined specific recombinant proteins in cattle experimentally- and naturally-infected with paratuberculosis.

Johne's disease (JD), is a fatal enteritis of animals caused by infection with Mycobacterium avium subspecies paratuberculosis (Map). Diagnosis of subclinical JD is problematic as test sensitivity is limited. Th1 responses to Map are activated early, thus detection of a cell-mediated response, indicated by measuring interferon gamma (IFN-γ) stimulated by mycobacterial antigens, may give the first indication of sub-clinical infection. Crude extracts of Map (PPDJ) have been used to detect the cell-mediated response in infected cattle. More specific, quantifiable antigens may improve test specificity and reproducibility. Map-specific proteins, MAP_3651c and MAP_0268c, raised a cell-mediated immune response in sub-clinically infected sheep. Results presented in this manuscript demonstrate these proteins elicit a cell-mediated response in experimental and natural infections of cattle. Individual ranked IFN-γ responses of experimentally infected calves to PPDJ showed a high, statistically significant association with ranked responses of recombinant Map antigens. Responses of infected animals were higher than the control group. Threshold values determined using data from an experimental infection were applied to naturally infected animals. Some animals exhibited responses above these threshold values. Responses to MAP_3651c on a farm categorised as high-risk for JD showed strong evidence (P<0.001) that responses were significantly different to lower-risk farms. The IGRA test may prove to be an additional tool for the diagnosis of JD, and inclusion of specific antigens a refinement however, understanding and interpretation of IGRA results remain challenging and further investigation will be required to determine whether the IGRA test can detect exposure and hence predict clinical JD.

A New Experimental Infection Model in Ferrets Based on Aerosolised Mycobacterium bovis.

There is significant interest in developing vaccines to control bovine tuberculosis, especially in wildlife species where this disease continues to persist in reservoir species such as the European Badger (Meles meles). However, gaining access to populations of badgers (protected under UK law) is problematic and not always possible. In this study, a new infection model has been developed in ferrets (Mustela furo), a species which is closely related to the badger. Groups of ferrets were infected using a Madison infection chamber and were examined postmortem for the presence of tuberculous lesions and to provide tissue samples for confirmation of Mycobacterium bovis by culture. An infectious dose was defined, that establishes infection within the lungs and associated lymph nodes with subsequent spread to the mesentery lymph nodes. This model, which emphasises respiratory tract infection, will be used to evaluate vaccines for the control of bovine tuberculosis in wildlife species.