Jain, V.K.Sindhu, Neelesh2017-08-192017-08-192007http://krishikosh.egranth.ac.in/handle/1/5810029124The present investigation was undertaken to standardize polymerase chain reaction (PCR) assay using ten different sets of primers for detection of sub clinical mastitis (SCM) directly from milk samples of apparently healthy animals. A total of 1230 milk samples including 367 samples from cow farm, 228 samples from buffalo farm and 635 samples brought in Veterinary College Central Laboratory, CCS Haryana Agricultural University, Hisar, were screened by PCR assay and other conventional tests (cultural examination, California mastitis test, Electrical conductivity test and Somatic cell count). Staphylococci isolates were characterized phenotypically and genotypically. Screening of cow farm using universal primer yielded 56.52 per cent animals and 33.24 per cent quarters to be positive for SCM whereas18.85 per cent quarters were found positive for Staphylococcus aureus by 16S-23S rRNA interspacer region primers. As many as 15.78 per cent animals and 5.70 per cent quarters were found positive by using universal primer while 15.38 per cent quarters were positive for Staphylococcus aureus by 16S-23S rRNA interspacer region primers from buffalo farm. A total of 15.90 per cent samples of College Central Laboratory were found positive for Staphylococcus aureus by 16S-23S rRNA interspacer region primers and majority of samples found negative by cultural examination and positive by PCR were from animals who received prior treatment with antibiotics. The Genus specific primers encoding gap gene and 16S-23S rRNA region could detect all Staphylococcus spp. Among species specific primers the primers encoding 16S-23S rRNA region were found to be 100 per cent sensitive followed by aroA gene (98.41 per cent), nuc gene (97.62 per cent) and coa gene (93.65 per cent). A total of 99.20 per cent of isolates were found positive by ubiquitous PCR assay. On the basis of results of different conventional diagnostic tests, CMT was found to be good animal side test for primary screening of large herd to identify subclinically infected animals within a very short period economically. Among phenotypic tests, thermostable nuclease test was most sensitive in comparison to latex and coagulase test. No false-positive reactions were detected by any of the tests applied on staphylococci other than Staphylococcus aureus. Genotypic characterization of staphylococcal isolates yielded similar results as were obtained by PCR of milk samples directly indicating PCR assay as an important tool and an alternative to phenotypic characterization. PCR was found to be most rapid, sensitive and specific assay when compared to bacteriological examination and other conventional tests and can be adapted for screening of large herd for detection of Staphylococcus aureus directly from milk.enThesis