Isolation of Brucella and its quantification in semen by real time PCR

Polymerase chain reaction (PCR) assay was employed for the detection of Brucella DNA in semen of cattle and buffalo breeding bulls. A genus specific primer pair which amplified a 223 bp fragment of gene encoding a 31-kDa Brucella abortus antigen was used in assay. Cultural isolation of Brucella organism from semen was also performed. PCR resulted in more numbers of positive samples as compared to cultural isolation since it detected Brucella DNA in 19 out of 101 semen samples, while Brucella organism was isolated from only 8 semen samples of same bulls. The PCR may be used as supplementary test for detection of Brucella infection in bulls. Real time quantification revealed the load of Brucella organisms in semen ranging from 1.25×104 CFU/ml to 1.7×107 CFU/ml.