Serological, cultural and molecular detection of Brucella infection in bovine including quantification in milk by real time PCR
Patel, Tanmay (2007) Serological, cultural and molecular detection of Brucella infection in bovine including quantification in milk by real time PCR. Masters 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 transmission of the disease is by direct or indirect contact with infective excretors. Brucella contaminated milk presents a potential threat to the new born calves and human beings as it can spread through ingestion and causes infectious abortion and undulant fever. The correct and prompt diagnosis is important in controlling and eradicating the disease in animals. The present study was under taken to detect the Brucella antibodies in serum as well as in milk and for detection of Brucella organisms in bovine milk. The ELISA was used for detection of Brucella antibodies in serum in conjunction with RBPT and STAT to detect their efficacy as compared to ELISA, whereas, ELISA and MRT were employed for detection of antibodies in milk. To detect the presence of Brucella organisms in milk, 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. The real-time was used for quantification of Brucella in milk. A comparison was also made among antibody detection, cultural and molecular methods to detect Brucella infection in bovines. A total of 231 bovine serum samples were screened for presence of Brucella antibodies. Of these, 67 (29.00%) serum samples were found positive for Brucella antibodies by ELISA. The much higher seropositivity (38.29%) was found in cattle than in buffaloes (26.63%). Sensitivity of RBPT and STAT were found to be of 25.37% and 61.19%, respectively, with considering ELISA as a gold standard test while specificity was found to be of 99.39% and 98.78%, respectively. Out of 53 bovine milk samples screened 15 (28.30%) and 08 (15.09%) were found positive for Brucella antibodies by ELISA and MRT, respectively. Considering ELISA as a gold standard test, the sensitivity and specificity of MRT were found to be 40.00% and 94.73%, respectively. In comparison of serum antibody and milk antibody detection tests on total 53 serum and milk samples from common individual animals, the ELISA detected antibodies in more number of serum samples (37.73%) than that of milk (28.30%). Considering serum ELISA as a gold standard test, the sensitivity of RBPT, STAT, MRT and milk-ELISA were found to be of 40.00%, 70.00%, 30.00% and 55.00%, respectively, while specificity was found to be of 100.00%, 100.00%, 93.93% and 87.87%, respectively. The overall agreement of 77.34% for RBPT, 88.67% for STAT, 69.81% for MRT and 75.47% for milk-ELISA were found with the serum ELISA. Brucella could be recovered only from four milk samples (2 each from cows and buffaloes) of the 53 cultivated on Brucella agar medium. The isolates were identified as Brucella organisms by cultural, morphological and biochemical characteristricts and further confirmed by PCR using different genus specific primer pairs. For DNA extraction from milk the method described by Romero and Lopez-Goni (1996) was found most suitable. Of the 53 milk samples tested by three Brucella genus specific primer pairs, 9 were found positive by B4/B5 primer pair, 1 by JPF/JPR primer pair and 2 by F4/R2 primer pair. The B4/B5 primer pair was found more suitable than other two as the same 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 milk. On the basis of melt curve analysis, a detection limit of real-time PCR assay was found 50 fg DNA or 10 CFU/ml of milk (considering 5 fg is equal to one Brucella cell) using 5 μl of template DNA. While, load of Brucella organisms in milk of bovines were ranged from 1.128 x 104 CFU/ml to 172.800 x 104 CFU/ml of milk. On comparison of serum antibody detection, milk antibody detection, cultural and molecular methods for detection of Brucella infection in 53 bovines, in serum antibody detection tests RBPT, STAT and ELISA detected 15.09%, 26.41% and 37.73% of positive bovines in serum, respectively. Whereas, MRT and ELISA detected 15.09% and 28.30% of positive bovines in milk, respectively. While, 7.54% of bovines were found culturally positive. Among the PCR assays 16.98%, 1.88% and 3.77% of bovines were found positive by B4/B5, JPF/JPR and F4/R2 primer pairs, respectively. The highest numbers of bovines were found positive by serum ELISA whereas, cultural method detected the least number of positive bovines. Among the four, tests detected Brucella antibodies in serum were resulted in highest number of positive bovines (20, 37.73%) followed by tests detected Brucella antibodies in milk (17, 32.07%), PCR assays (09, 16.98%) and cultural isolation (04, 7.54%). The over all agreement between these methods was found 58.49%. Finally, the study revealed presence of Brucella antibody in serum as well as in milk and presence of Brucella organisms in the bovine milk. Simultaneously the seronegative bovine also revealed the presence of Brucella organisms and vice versa. Thus under Health Control Programme to eradicate the brucellosis from animals as well as for public health point of view proper measures must be taken at State level for controlling brucellosis. Therefore all animals must be tested periodically for detection of both Brucella antibody and presence of organisms.
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