The objective of this study was to investigate suitability of BALB/c mice as laboratory animal model and appropriateness of various routes of inoculation for the study of pathogenesis of paratuberculosis. Fifty-four mice aged 2-4 weeks were divided into 3 groups viz. oral, intraperitoneal and intravenous with 12 animals each and their respective control groups. The bovine isolate was grown in Middlebrook 7H9 medium with ADC and mycobactin J, harvested and final concentrations of 10⁷ cfu were suspended in pasteurised milk for oral (10 times) and 10⁶ and 10⁵ cfu in sterile saline for I/P and I/V inoculations (3 times), respectively. The infection was monitored for a period of 9 months by serological, immunological, bacteriological, molecular and sequential pathological methods at 3 time points (3, 6 and 9 months post infection, MPI).

Clinical symptoms such as depletion of perineal and mesenteric fat reserve, muscle wasting, emaciation and unthriftiness were observed more frequently in IP & IV groups than the oral group mice. Gross lesions suggestive of paratuberculosis infection were not observed in any group. Histologically, the small intestine particularly ileum showed broad, flat and fused villi with increased infiltration with lymphocytes and macrophages without formation of distinct granuloma in lamina propria from 3 to 9 MPI in all the 3 groups. Distinct granulomas were observed mostly in the lymphoid tissues. The mesenteric lymph nodes revealed multifocal granulomas at 9 MPI in the oral group, whereas animals from I/P and I/V groups had multiple granuloma at 6 and 9 MPI. The liver in the oral group showed only a few small aggregates of lymphoid cells, in the I/P group 1-2 focal granulomas, whereas in the I/V group, it had focal to multifocal granulomas from 3-9 MPI. The acid-fast bacilli were detected in the mesenteric lymph nodes of only one mouse from I/V group at 9 MPI. Serologically, there was rising titres of antibodies to MAP most prominently in the oral than in the I/P and I/V groups in the ELISA. None of the mice of three groups reacted positively to intradermal johnin skin test. Faecal excretion of the bacteria was not detected in culture. Only one mouse was found positive in tissue culture at 9 MPI from I/V group. One mouse each in I/P and I/V groups at 3 MPI in tissue PCR, and one mouse each in oral and I/V groups at 6 MPI were found to be positive in the faecal PCR. The results of this study suggested that BALB/c mouse was a suitable model and I/V route of inoculation was better than the other two routes for experimental reproduction of the disease.