Raw milk samples of 503 individual buffaloes were collected from dairy farms located in Agra and Mathura cities in North India. Multiple tests (Indirect Fluorescent Agglutination test (i_FAT), IS900 PCR, Microscopy, Indigenous ELISA kit (i_ELISA), Dot-ELISA (d_ELISA) and Latex agglutination test (LAT)) based bio-load and bio-type profile of Mycobacterium avium subspecies paratuberculosis (MAP) was studied. Cumulatively average bio-load was 61.2 percent using three antigen and three antibody based. In i_FAT, IS900 PCR and microscopy, 43.5, 13.3 and 40.9 percent milk were positive for MAP, respectively. Whereas, 32.8, 49.3 and 44.1 percent milk samples were positive in i_ELISA, d_ELISA and LAT, respectively. Bio-typing of representative milk samples using IS900 PCR positive raw milk (67), 13.4 percent were infected with 'Indian Bison Type' biotype using IS1311 PCR_REA. Study concluded that 'Indian Bison type' was the predominant bio-type infecting lactating buffaloes of this region. Raw milk was highly convenient sample in buffaloes and 'milk samples' were first time screened without initial processing of milk samples. Detection limits of each tests was improved. Results of five tests (d_ELISA, LAT, i_ELISA, microscopy, i_FAT were comparable, except IS900 PCR. High bio-load of MAP in milk of buffaloes was major health hazard for human health. High bio-load of MAP was alarming and calls for initiation of Johne's disease control programs in the country.

Fascioliasis causes huge economic losses in livestock industry by reducing the growth, fertility rate, meat and milk yield. In the present study, somatic antigens isolated from Fasciola gigantica were assessed for the early detection of infection by developing antibody detection enzyme immunoassay. The somatic antigens extracted from adult F. gigantica worms was separated on SDS-PAGE and immunogenicity was determined by Western blot (WB). The results showed polypeptides bands between 15 to 95 kDa, and most prominent bands were 15, 28, 36, 38, 55, 72 and 95 kDa polypeptides. The WB revealed a single polypeptide of size between 36 to 55 kDa as antigenic. The sensitivity and specificity of ELISA test established for 36 to 55 kDa somatic antigens was 95.45 percent (95 percent CI: 77.16 percent to 99.88 percent) and 87.1 percent (95 percent CI: 70.17 percent to 96.37 percent), respectively. Kappa value revealed that the strength of agreement is considered to be very 'good'. In house established ELISA was implemented in the field and 12.1 percent (31/256) large ruminants were found positive, while 4.68 percent (12/256) with postmortem examination. The fasciolosis did not show significant (P GT 0.05) association with host type, breed and age groups, while significant (P LT 0.01) association with host sexes. The result confirmed that in-house established ELISA test had good value for serodiagnosis of fasciolosis in cattle and buffaloes for large scale epidemiological studies.