Sharma , N.S.Jain, Ankit2021-01-042021-01-042012https://krishikosh.egranth.ac.in/handle/1/5810158948Brucellosis is a re-emerging zoonosis. It is caused by members of genus Brucella. In the present study, 84 samples of vaginal mucus (35), foetal stomach contents (31), foetal membranes (11) and uterine discharges (7) collected from abortion/reproductive disorder in cattle (29) and buffaloes (55), nine (10.71%) were positive through isolation whereas, 12 (14.28%) were positive by PCR. Out of total 9 isolates, one isolates was typed as biotype 2, one as biotype 3 and seven isolates were typed as biotype 1. Prevalence of Brucella abortus in buffaloes and cattle was found 14.54% and 13.79% respectively. Foetal stomach content was the best sample for detection of Brucella organism. All the brucellosis positive animals were having history of abortion in 6-9th month of gestation. Antimicrobial sensitivity revealed that B. abortus was sensitive to doxicycline, oxytetracyclin, ampicillin, gentamicin, enrofloxacin, streptomycin, neomycin, cholarmphenicol, kanamycin and resistant to penicillin, cephalothin, cotrimoxazole and erythromycin. For rapid detection of Brucella from clinical samples, PCR was carried out. The amplicons of 905bp and 223bp were amplified using F4/R2 and B4/B5 primers respectively. New set of primers based on omp25 gene were designed. Amplified omp25 gene from a clinical isolate of B. abortus was cloned in to TA cloning vector. The cloned product was sequenced and the sequence data (Accession No. JQ839278.1) was analysed. For expression of omp25 gene, pPROExHT (b) prokaryotic expression vector (Invitrogen, USA) was used. His-tagged recombinant Omp25 protein was successfully expressed which showed a specific band of ~25kDa in SDS-PAGE analysis. Expressed recombinant Omp25 protein was successfully purified to homogeneity by Ni-NTA affinity chromatography under denaturing conditions and the purified recombinant protein was confirmed by Western blottingEnglishThesis