SERODETECTION OF Brucella ANTIBODIES AND CHARACTERIZATION OF Brucella ISOLATES FROM REPRODUCTIVE DISORDERS OF CATTLE AND BUFFALOES IN AND AROUND ANAND
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Date
2016
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AAU, Anand
Abstract
Brucellosis is a zoonotic infection caused by gram negative, facultative intracellular bacteria Brucella that are pathogenic for a wide range of animals and human beings. The disease has a considerable impact on human and animal health, as well as socioeconomic impacts, especially in which rural income relies largely on livestock breeding and dairy products. Brucella organisms are coccobacilli or short rods, arranged singly and less frequently in pairs or small groups. The genus Brucella belong to the aproteobacteria group and consists of six recognized species: Brucella abortus, Brucella melitensis, Brucella suis. Brucella ovis. Brucella canis, Brucella neotomae and the strains recently discovered in marine mammals and in common vole (Microtus arvalis) and published under the respective species names oi Brucella ceti, Brucella pinnipedialis and Brucella microti. Diagnosis is usually based on serological tests and/or cultivation. Serological evidence suggested that brucellosis is highly endemic in many parts of India. The present study was undertaken to detect Brucella antibodies in serum, isolation of Brucella organisms from various reproductive disorders (abortion, R.O.P., endometritis, infertility and repeat breeder) and PCR based identification and characterization of Brucella isolates. For detection of Brucella antibodies in serum, two serological methods viz., i-ELISA and RBPT were carried out. For isolation of Brucella organisms Brucella agar medium (BAM) was used and for detection oi Brucella DNA by PCR three different genus specific primer pairs viz., B4/B5, JPF/JPR and F4/R2 were used. Species identification of various brucella isolates obtained from cattle and buffalo were carried out by AMOS PCR and Bruce-ladder PCR. During the study period, total 434 sera samples comprising of 361 from cattle and 73 from buffaloes were screened using i-ELISA, of which 51 (14.12%) and 22 (30.13%) were found positive, respectively. The overall seropositive sera among 434 sera samples was found to be 73 (16.80%). In present study, seroprevalence of Brucella antibodies from gynaecological disorder cases was studied from the 434 serum samples comprises of 361 from cattle and 73 buffaloes by RBPT. Forty eight sera samples were found to be positive by RBPT indicating seropositivity of 11.05%. Among the two serological tests employed for detection of Brucella antibody in serum of cattle and buffaloes, the highest positive results were obtained by ELISA (16.80%), followed by RBPT (11.05%). Prevalence of Brucella antibody in abortion and R.O.P. were studied using i- ELISA and RBPT, recorded 34.69% and 22.44% positivity in cattle and buffaloes, respectively whereas from reproductive disorders (endometritis, infertility and repeat breeder) 11.69% by i-ELISA and 7.25% by RBPT were found to be overall seropositive in cattle and buffaloes. Attempt for cultural isolation was done by inoculating 114 various samples like vaginal swabs, aborted material, milk and placenta. Of these, only 3 samples yielded Brucella organisms on Brucella agar medium. The isolates were identified by cultural, morphological, biochemical characteristics test and further confirmed by PCR using different genus and species specific primer pairs. Colony PCR was carried out using various genus specific primer pairs for the confirmation oi Brucella isolates for different gene. The desired product of 223 bp using B4/B5 (BCSP31 gene) primer pair was amplified in all the 3 isolate (including reference strain). However, desired product of 193 bp using JPF/JPR (omp2 gene) primer pairs was amplified except isolates C2 (cow). Whereas product of 905 bp using F4/R2 (16S rRNA gene) primer pairs was amplified in all the isolates. Furthermore for the species identification, multiplex PCR assays were employed that were AMOS PCR and Bruce-ladder PCR. AMOS PCR could amplify B. abortus specific primer, whereas Bruce-ladder also could able to identify all 3 isolates as B. abortus. All the isolates of Brucella abortus from cows (C1, C2 and C3) were found to be 100% sensitive to Streptomycin, Tetracycline, Amikacin, Erythromycin, Pefloxacin, Amoxyclav, Spectinomycin and Norfloxacin whereas all the three B. abortus isolates were found to be resistant to Ampicillin.
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VERTINARY MICROBIOLOGY, CHARACTERIZATION