Puran ChandSanjiv Kumar2017-08-212017-08-212006http://krishikosh.egranth.ac.in/handle/1/5810029183Brucellosis is a highly infectious disease which is conventionally diagnosed using culture and isolation and serological methods. PCR assay has been shown to be a promising option for the diagnosis of brucellosis. However, a few studies have been reported with field samples for evaluating the assay as a diagnostic tool. In the present study, a genus-specific PCR assay has been assessed for the detection of Brucella melitensis DNA directly from various types of clinical samples collected from apparently healthy and recently parturated ewes. The assay is based on the gene encoding Brucella cell surface protein (BCSP-31), a 31 KDa protein in Brucella chromosome, giving a product of 223bp after PCR amplification. PCR assay was standardized using DNA extracted from pure culture of Brucella melitensis biovar 1. Detection limit of this PCR assay was found to be 167.6fg of DNA per reaction which corresponds to DNA extracted from 30 Brucella organisms. A total number of 40 ewes were examined of which 35 were belonged to a sheep farm known to have endemic brucellosis for more then two decades and 5 were from a brucellosis free sheep farm. The clinical samples including uterine discharge, milk and blood were collected from each animal at the same time. The samples of milk, uterine discharge and blood were processed for PCR amplification whereas milk and uterine discharge were subjected to culture and isolation for the recovery of Brucella melietnsis. Brucella melitensis was isolated from six animals (17.14%). All the isolates were identified to be Brucella melitensis biovar 1. Of these six Brucella melitensis, three (8.57%) were from uterine discharge and three (8.57%) from milk samples collected from different animals. The Brucella specific DNA was detected by PCR in 24 (68.57%) samples of uterine discharge, 11 (31.43%) samples of milk and 5 (14.28%) samples of blood. All these samples were from 35 animals belonging to the brucellosis endemic sheep farm. None of the samples from 5 ewes belonging to the brucellosis free farm gave positive result either in PCR or in culture and isolation. Taken together 25 ewes were detected positive by PCR. From these 25 (71.43%) ewes only one sample of uterine discharge was PCR negative but the milk and blood samples of this ewe were PCR positive. In addition, serological status of these animals was also determined by RBPT, mRBPT, SAT and ELISA, that gave positive results with three (8.57%), 11 (31.43%), 11 (31.43%) and 29 (82.86%) animals, respectively. All PCR positive animals were also positive in ELISA except one ewe. The number of ELISA positive animals was higher than that of PCR positive animals, but ELISA being an antibody detection assay it can not reveal the current presence of Brucella in the animal body. The presence of Brucella could only be established by demonstrating the presence of organism or its DNA in samples from ewes is very much likely to be detected by PCR as seen in the present study. The present study leads to the conclusion that PCR is a more sensitive technique as compare to the culture and isolation, and uterine discharge appears to be a better source than milk and blood for the same, if collected within 6-12 hours of parturition. Being simple, sensitive, fast and accurate diagnostic assay, PCR could be used as a routine laboratory test for the diagnosis of brucellosis in sheep.enThesis