ijvp

Indian Journal of Veterinary Pathology

0250-47580973-970X

Indian Association of Veterinary Pathologists

ijvp-29-1-thesis-abs-006

ABSTRACTS OF DISSERTATIONS OR ARTICLES

Molecular pathologyand dagnosis of paratuberculosis in sheep

Kumar

A. Anand

Dr¹Tripathi

B.N.

Dr¹

¹Degree/Year: PhD/2005 University: Indan Veterinary Research Institute, Izatnagar

162005

2916464

2005

IndianJournals.com

Paratuberculosis (Johne’ s disease), an economically important disease of the ruminants, is caused by M a. paratuberculosis. The pathogenesis of the infection during prolonged course is poorly understood and the diagnosis especially of subclinical infection is still considered a problem. The pathogenicity of caprine strain to sheep is not known, which may be important from the diagnosis and epidemiological points of view. The present study was carried out to elucidate cellular and molecular pathogenesis and to assess efficacies of a number of antemortem and post-mortem diagnostic methods in experimentally produced infection in sheep with caprine strain of M. a. paratuberculosis.

To achieve these objectives, a total of 31 crossbred lambs of either sex; aged 8–10 weeks were used for experimental study. They were divided into 3 groups. The infected group consisting of 15 lambs was orally administered with 4x 10 M a. paratuberculosis (caprine strain) at 10 occasions. The in-contact group comprising 8 lambs was kept along with the infected group of sheep. The uninfected control group of 8 animals received only placebo (PBS) treatment and was maintained separately. The identity of the culture was confirmed by cultural characteristics, mycobactin-J dependency and molecular methods demonstrating specific 1S900 and f57 genes of M.a. paratuberculosis. The duration of the experiment was 390 days and animals were serially euthanised at 60 days intervals. Several clinical, immunopathological and molecular parameters were studied over the time and also in sheep grouped according to the infection status.

The infected (n=15) and in-contact (n=8) sheep were further classified in various infection categories on the basis of histological lesions and positivity in 7 diagnostic tests. Amongst 15 infected sheep, 2 (13.3%) each were found to be in negative and suspected, 5 (33.3%) in mild and 3 (20%) each were in moderate and severe infection categories. Four (50%) sheep of the in-contact group had mild infection.

Clinically, 50 - 60%, sheep from the in-contact and infected groups were dull and showed various degree of wool shedding. The faecal shedding of the bacilli was observed in 8 (53.3%) infected and 4 (50%) in-contact sheep between 90 DPI and 390 DPI. The faecal culture detected 6 (40%) and 2 (25%) sheep from the infected and in-contact groups, respectively. 8 of 15 (53.3%) sheep in the infected group between 90 DPI and 180 DPI and 3 of 8 (37.5%) in the in- contact group between 120 DPI and 270 DPI were detected positive by agar-gel immunodiffusion (AGID) test. The humoral immune response was better evaluated by the absorbed ELISA and the significant rises in antibody titres were observed from 30 DPI onwards up to 330 DPI in the infected sheep. The in-contact sheep showed moderate increases in the antibody titres. Six of 15 (40%) infected sheep comprising 2 each from mild, moderate and severe infection categories were detected positive by blood PCR. The tissue PCR detected 9 (60%) sheep from the infected and 1 (1.5%) from the in-contact groups.

Gross lesions consisting of mild to severe thickening of the intestinal mucosa, enlarged mesenteric lymph nodes (MLN) and gelatinisation of fat started appearing after 90 DPI. The mild lesions in sheep were characterized by the presence of focal granulomas, mostly in the Peyer's patch area of the ileum and ileocaecal valve (ICV) and occasionally multinucleated giant cells without demonstration of acid-fast bacilli (AFB). The moderate lesions, observed in 3 sheep, consisted of multiple focal granulomas containing few to numerous AFB from proximal jejunum to the ICV, besides diffused infiltration of lymphoid cells and macrophages. The lesions in Peyer's patch area were more prominent. The severe lesions varying from large multifocal epithelioid granulomas to the formation of epithelioid cell sheets containing abundant AFB were observed in 3 sheep and were similar to the classical lesions of paratuberculosis. In the infected sheep, elevated levels of cytokine genes for IFN-a, TNF-a and IL-6 were observed in the intestine, lymph node and blood. The IL-2 gene expression was more in the intestine and lymph node. The expression IL-4 was more in the blood and lymph node.

Elevated numbers of CD2+, CD4+, CD8+, CD4+/CD8+ ratio and MHC II labelled cells were observed both in the intestine and lymph node. The elevated levels of cytokine expression and increased number of labelled lymphocyte subsets correlated well with the results of histopathology and immune responses.

The sensitivities of the clinical diagnostic methods were 80% for l/D johnin testing, over 73% for faecal smear and faecal PCR, 60% for AGID, over 53% for ELISA and faecal culture and 40% for blood PCR. Amongst postmortem diagnostic methods, highest sensitivity was obtained for immunoperoxidase (80%) followed by tissue PCR (and AFB demonstration in tissue (40%). The prevalence of paratuberculosis infection at unorganised farms was found to be 2.2% by faecal culture, 1.7% by AGID and 22.4% by ELISA.