Evaluation of newly developed 'six recombinant secretary proteins based 'cocktail ELISA' and 'whole cell lysate' based 'indigenous ELISA' and tissue microscopy' with 'Gold standard' histo-pathology for the diagnosis of Johne's disease in slaughtered goats and buffaloes.

Three screening tests {(newly developed, six recombinant secretory proteins based 'cocktail ELISA', in-house robust 'indigenous ELISA' based on semi-purified protoplasmic antigens and tissue microscopy were evaluated with 'Gold standard', histo-pathology for the diagnosis of Johne's disease in goats and buffaloes. Serum and tissues {mesenteric lymph nodes and intestines) were driven from farmer's goats (n = 77) and buffaloes (n = 40) slaughtered for harvesting meat and farm goats (n = 77), died and necropsied. Twenty seven (35%) goats and 23 (57.5%) buffaloes were positive in all the four tests. Of 134 tissues screened by histo-pathology, 79.8% MLN and 76.8%, intestines, were positive for MAP infection. In tissue microscopy, 55.2 and 52.3%, goats and buffaloes were positive, respectively. Of 117 sera screened by i_ELISA, 58.4 and 70.0%, goats and buffaloes were positive, respectively. Whereas, c_ELISA detected 55.8 and 62.5%, goats and buffaloes, positives, respectively. Twelve tissues (70.5%) of goats necropsied were positive, both in tissue microscopy and histo-pathology. Most significant gross findings were serous atrophy of the fat and mild to moderate, diffuse thickening of terminal ileum, especially at ileo-caecal junction with or without transverse / longitudinal corrugations. In histo-pathology grade III and IV lesions were significantly low as compared to grade I and II. Of the four tests used for screening 268 samples, histo-pathology was most sensitive (78.3%), followed by i_ELISA (62.3%), c_ELISA (58.9%) and tissue microscopy (58.9%). Between two ELISA tests, c_ELISA using six recombinants secretory proteins, had higher specificity as compared to i_ELISA.

Trends and advances in the diagnosis and control of paratuberculosis in domestic livestock.

Paratuberculosis (pTB) is a chronic granulomatous enteritis caused by Mycobacterium avium subsp. paratuberculosis (MAP) in a wide variety of domestic and wild animals. Control of pTB is difficult due to the lack of sensitive, efficacious and cost-effective diagnostics and marker vaccines. Microscopy, culture, and PCR have been used for the screening of MAP infection in animals for quite a long time. Besides, giving variable sensitivity and specificity, these tests have not been considered ideal for large-scale screening of domestic livestock. Serological tests like ELISA easily detects anti-MAP antibodies. However, it cannot differentiate between the vaccinated and infected animals. Nanotechnology-based diagnostic tests are underway to improve the sensitivity and specificity. Newer generation diagnostic tests based on recombinant MAP secretory proteins would open new paradigm for the differentiation between infected and vaccinated animals and for early detection of the infection. Due to higher seroreactivity of secretory proteins vis-à-vis cellular proteins, the secretory proteins may be used as marker vaccine, which may aid in the control of pTB infection in animals. Secretory proteins can be potentially used to develop future diagnostics, surveillance and monitoring of the disease progression in animals and the marker vaccine for the control and eradication of pTB.

Comparative Evaluation of Different Test Combinations for Diagnosis of Mycobacterium avium Subspecies paratuberculosis Infecting Dairy Herds in India.

A total of 355 cows were sampled (serum, n = 315; faeces, n = 355; milk, n = 209) from dairy farms located in the Punjab state of India. Faeces and serum/milk samples were screened by acid fast staining and "indigenous ELISA," respectively. IS900 PCR was used to screen faeces and milk samples. Bio-load of MAP in dairy cows was 36.9, 15.6, 16.3, and 14.4%, using microscopy, serum ELISA, milk ELISA and milk PCR, respectively. Estimated kappa values between different test combinations: serum and milk ELISA, faecal microscopy and faecal PCR, milk ELISA and milk PCR, faecal PCR and serum ELISA were 0.325, 0.241, 0.682, and 0.677, respectively. Estimation of the relative sensitivity and specificity of different tests in the present study indicated that "serum ELISA" and "milk ELISA" were good screening tests, add "milk PCR" was "confirmatory test" for MAP infection. Combination of milk ELISA with milk PCR may be adopted as a model strategy for screening and diagnosis of JD in lactating/dairy cattle herds in Indian conditions.