Isolation, identification and molecular confirmation of Brucella melitensis from ovine and caprine flocks in Karnataka, India

Abstract

Sheep and goat brucellosis caused by Brucella melitensis, one of the most virulent Brucella species accounting for economic losses through abortion, stillbirths, reduction of milk yield and infertility. Disease has wide socioeconomic impact, in countries where, livestock sector is the major source of rural income. Early diagnosis is essential to minimise the spread of the disease besides public health importance. The present study reports the isolation, identification, biotyping and molecular confirmation of Brucella spp. in 18 different sheep and goat farms in Karnataka suspected to have brucellosis. A total of 550 serum samples, 25 aborted foetuses, uterine discharges and placental tissues were collected. The serum samples were subjected to Rose Bengal Plate Test (RBPT) and Competitive ELISA (c-ELISA). The clinical samples were processed for cultural isolation on Brucella Agar Media with selective antibiotic supplements. A total of 200 (36.36%) and 260 (47.27 %) serum samples were positive by RBPT and c-ELISA, respectively, further 195 (35.45 %) of them being positive by both the tests. Five Brucella isolates were recovered from 100 clinical samples. The isolates were characterized to their species by growing them on Brucella specific medium, biochemical reactions, CO2 requirement, H2S production, agglutination with A and M mono-specific antiserum, dye sensitivity to basic fuchsin and thionin. Further, molecular confirmation of the isolates was done by amplification of B. melitensis 16S (rRNA) sequence analysis by genus specific PCR and species specific IS711 repetitive DNA fragment by Brucella AMOS PCR. The present study envisages seroprevalence of at least 35.45 per cent and isolation rate of 25 per cent for B. melitensis warranting the need for institution of strict control measures.