Early-stage findings in an experimental calf model infected with Argentinean isolates of Mycobacterium avium subsp. paratuberculosis.

Mycobacterium avium subsp. paratuberculosis (MAP) is an important pathogen that causes granulomatous enteritis known as Johne's disease or paratuberculosis (PTB). In this study an experimental model of calves infected with Argentinean isolates of MAP for 180 days was used to provide more data of the early PTB stages. Calves were challenged by oral route with MAP strain IS900-RFLPA (MA; n = 3), MAP strain IS900-RFLPC (MC; n = 2) or mock infected (MI; n = 2), and response to infection was evaluated through peripheral cytokine expression, MAP tissue distribution and histopathological early-stage findings. Specific and varied levels of IFN-γ were only detected at 80 days post-infection in infected calves. These data indicate that specific IFN-γ is not a useful indicator for early detection of MAP infection in our calf model. At 110 days post-infection, TNF-α expression was higher than IL-10 in 4 of the 5 infected animals and a significant decrease of TNF-α expression was detected in infected vs. non-infected calves. All calves challenged were identified as infected by mesenteric lymph node tissue culture and real time IS900 PCR. In addition, for lymph nodes samples, the agreement between these techniques was almost perfect (κ = 0.86). Colonization of tissues and levels of tissue infection varied between individuals. Evidence of early MAP dissemination to extraintestinal tissues such as the liver was detected by culture in one animal (MAP strain IS900-RFLPA). In both groups microgranulomatous lesions were observed predominantly in the lymph nodes, with giant cells present only in the MA group. In summary, the findings described herein may indicate that local MAP strains induced specific immune responses with particularities that could suggest differences in their biological behavior. Further studies should be carried out in order to obtain an in-depth understanding of the influence of MAP strains in host-pathogen interactions and the outcome of disease.

Antibodies from healthy or paratuberculosis infected cows have different effects on Mycobacterium avium subspecies paratuberculosis invasion in a calf ileal loop model.

In this work, we used a calf ileal loop model to evaluate whether the preincubation of Mycobacterium avium subspecies paratuberculosis (MAP) with antibodies from healthy, MAP-positive or Lipoarabinomannan (LAM) immunized cows could affect the results of infection after 3.5 h. Bacterial load in tissue was assessed by Ziehl-Neelsen and by culture for each loop. MAP was detectable in all infected loops after 3.5 h.p.i.; although the presence of antibodies from MAP-positive cows significantly reduced bacterial load in loops as compared with antibodies from healthy donors (by Ziehl-Neelsen and culture, p-value < 0.003 and 0.0203, respectively). A possible direct effect of antibodies on MAP viability was shown to be not significant. Severity of histopathologic changes induced by MAP infection also varied according to the pretreatment: MAP induced less changes when inoculated in the presence of antibodies from MAP-positive cows as compared with antibodies from healthy donors. Overall, our results show that the presence of antibodies from MAP-positive cows reduced MAP invasion and consequent early histological changes in this ileal short-term loop model. These results may suggest a protective role of antibodies in the response against MAP at the portal of entry in cattle.

Early IgG2 in calves experimentally infected with Mycobacterium avium subsp. paratuberculosis.

The diagnosis of the early stages of paratuberculosis, caused by Mycobacterium avium subsp. paratuberculosis (Map), is a cumbersome task. In this study, an experimental Map-infection model of calves was used to improve the knowledge of early antibody response and to evaluate different in-house ELISAs in the detection of subclinical paratuberculosis. Calves were challenged with Map strain IS900-RFLPA (n = 3) or Map strain IS900-RFLPC (n = 2) (Argentinean isolated strains) or mock infected (n = 3), and their specific humoral response was evaluated. The diagnostic ELISA (IgG against Map protoplasmic antigen; PPA) could not detect the infection throughout the experimental period (180 days post-infection; dpi), whereas the IgG2/PPA-ELISA was able to identify infected calves at least once during the experiment. In addition, the use of crude Map extract detected most of the infections from 60 dpi onwards. Antibodies were also characterized by immunoblot: IgG2-reactivity to antigens of molecular weight lower than 50 kDa was detected in all infected calves. The experimental Map-infection model of calves used allows the study of the early humoral immune response in paratuberculosis. The evaluation of IgG2 specific to antigens lighter than 50 kDa emerges as an interesting alternative in calves naturally infected with paratuberculosis.