Serological, cultural and molecular detection of Brucella infection in breeding bulls
Kanani, Amit (2007) Serological, cultural and molecular detection of Brucella infection in breeding bulls. PhD thesis, College of Veterinary Science and Animal Husbandry, Anand Agricultural University, Anand.
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Brucellosis is a widespread and economically important infectious disease of animals and humans caused by members of the genus Brucella. The disease is manifested by reproductive failure, which includes abortion, birth of unthrifty calves and retained placenta in female animals while lesions in male are largely confined to the genital organs including testicles, seminal vesicles and epididymes. The transmission of disease is by direct or indirect contact with infective excretors. Brucella contaminated semen presents a potential threat to the cattle industry as Brucella can spread through artificial insemination (AI). The correct and prompt diagnosis is important in controlling and eradicating the disease in animals. The present study was under taken to detect the seroprevalence of brucellosis and to detect the presence of Brucella organisms in semen of breeding bulls of AI Centres of Gujarat. The seroprevalence study was done by ELISA whereas RBPT and STAT were applied to detect their efficacy as compared to ELISA. To detect the presence of Brucella organisms in semen, cultural and PCR methods were used. Comparison among three genus specific primer pairs was made to detect their efficacy to detect Brucella DNA by PCR. A comparison was also made among serological, cultural and molecular methods to detect Brucella infection in bulls. A total of 194 serum samples from breeding bulls of different AI Centres of Gujarat were screened for presence of Brucella antibodies. Of these, 16 serum samples were found positive for Brucella antibodies by ELISA yielding an overall seroprevalence of 8.25%. The much higher seroprevalence (16.13%) was found in cattle than in buffalo bulls (0.99%). Sensitivity of RBPT and STAT was found to be of 50% and 62.5%, respectively, with considering ELISA as a gold standard test while specificity was found to be of 98.31% and 97.75%, respectively. Out of 101 semen samples of five AI Centres, Brucella could be recovered, using Brucella agar medium, from eight samples of three AI Centers. Out of these, six were from cattle bulls while two were from buffalo bulls. All the eight isolates were identified as Brucella organisms by cultural, morphological and biochemical characteristics and further confirmed by PCR using different genus specific primer pairs. In the process of standardization of methods for DNA extraction from semen QIAamp Mini Kit protocol was found most suitable among all the three methods tried. Of the 101 semen samples tested by three Brucella genus specifiec primer pairs, 19 were found positive by B4/B5 primer pair, 2 by JPF/JPR primer pair and 5 by F4/R2 primer pair. The B4/B5 primer pair found more suitable than other two as they resulted in highest positive numbers as well as gave all the samples positive that were positive by other two primer pairs. Real-time PCR assay based on intercalating dye SYBR Green I using B4/B5 primer pair was found suitable for quantifying the load of Brucella from the semen. On the basis of melt curve analysis, detection limits of real-time PCR assay was found up to 50 CFU/ml semen using 3 μl of template DNA. While load of Brucella organisms in semen of bulls of different AI Centres was ranged from 1.25×104 CFU/ml to 1.7×107 CFU/ml. On comparison of serological, cultural and molecular methods for detection of Brucella infection in 101 bulls, in serological methods 5.94%, 9.90% and 9.90% of bulls were found positive by RBPT, STAT and ELISA, respectively. Whereas, 7.92% of bulls were found culturally positive. Among the PCR assays 18.81%, 1.98% and 4.95% of bulls were found positive by B4/B5, JPF/JPR and F4/R2 primer pairs, respectively. The highest numbers of bulls were found positive by B4/B5 primer pair based PCR assay, whereas RBPT detected the least number of positive bulls. Among the three methods PCR assay resulted in highest number of positive bulls (19) followed by serological methods (11) and cultural isolation (8). The over all agreement between these methods was found 79.20%. Finally, the study revealed presence of Brucella antibody in serum and presence of Brucella organisms in the semen of breeding bulls of Gujarat. Simultaneously the seronegative bulls also revealed the presence of Brucella organisms and vice versa. Thus under Sexual Health Control Programme proper measures must be taken at State level for controlling Brucella infection. All breeding bulls must be tested periodically for detection of both Brucella antibody in serum and presence of Brucella organisms in semen. The bulls must be free from Brucella infection prior to use.
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