Bovine mastitis is an important disease that afflicts dairy industry through decrease in milk quality and quantity. Various species of bacteria such as E. coli, S. aureus, S. agalacticae, P. aeruginosa and K. pneumoniae are involved in causing mastitis and affecting milk production consequently. In the present investigation, all E. coli isolates were subjected to PCR amplification targeting 23S rRNA gene using species-specific primers and amplicon size matched with those reported by Riffon et al. (2001). Prevalence of E. coli in mastitis cases generally suggests poor udder hygiene and the results obtained in this study are in agreement with those obtained by ,Bradely et al., 2002, Khaled et al., 2010., Sayed, 2014, who have also reported higher incidence of E.coli. The biochemical characterization of isolates and fermentation of sugars are comparable with studies of Kruthi (2006) and Pachaury (2011).

All 12 isolates of S. aureus complied with standard biochemical and sugar fermentation tests, and results obtained were in concurrence with that of Upadhyay (2009), who carried out fermentation studies with S. aureus obtained from milk of mastitic cattle and goat. There have been reports of amplification of other genomic fragments viz.,23S rRNA for genotypic confirmation of S. aureus strains isolated from bovine mastitis (Straub et al.,1999; Stephan et al., 2001; Salasia et al., 2004; Sanjiv et al., 2008; Bhanderi et al., 2009; Momtaz et al., 2010 and Upadhyay et al., 2010), that generated species specific amplicon of 1250 bp. Khaled et al. (2010) have also used 23S rRNA gene based system, albeit generated species-specific amplicon of 1318 bp using a different set of primers.

Streptococcus spp. isolates were biochemically confirmed and subjected to PCR amplification, targeting 23S rRNA spacer region, by using S. agalactiae, S. dysgalactiae and S. uberis species-specific primers that generated 587 bp, 403 bp and 95 bp amplicons. 23S rRNA spacer region based studies were conducted by Riffon et al. (2001) for genotypic confirmation of Streptococcus agalactiae organisms isolated from bovine mastitic milk and they observed 587 bp PCR product.  16S-23S rRNA spacer region based studies were conducted by Forsman et al. (1997) for genotypic confirmation of Streptococcus spp. isolated from bovine mastitic milk and they observed 280 bp, 270 bp, and 330 bp PCR product for S. agalactiae, S. dysgalactiae and S. uberis respectively. Phuektes et al. (2001) also used 16S-23S rRNA spacer region for differentiating S. agalactiae, S. dysgalactiae and S. uberis based on amplicon size. There have been reports (Khaled et al., 2010), where amplification of other genomic sites viz., 16S rRNA gene  for differentiating S. agalactiae (405 bp) and S. dysgalactiae (281 bp) by amplicon size. 16S rRNA, 23S rRNA and 16S-23S rRNA spacer region based PCR have been developed by Hassan et al. (2001) for S. uberis and they observed 445 bp, 451 bp, and 330 bp respectively for 16S rRNA , 23S rRNA and 16S-23S rRNA spacer region.

In the present study, all 8 isolates which had been biochemically identified as P. aeruginosa were subjected to genotyping using species-specific primers reported earlier (Spilker et al., 2004). All 8 isolates subjected to PCR using species specific primers produced the product of 956 bp as reported by Spilker et al. (2004). All the isolates could be amplified using the said PCR indicates higher sensitivity and specificity of this test.

In the present investigation, all 5 isolates, which had been biochemically identified as K. pneumoniae were subjected to PCR amplification targeting 16S–23S rDNA internal transcribed spacer (ITS) region based specific primers (Liu et al., 2008) and our results are completely in agreement with that of Liu et al., 2008.

In conclusion, high percentage of positive samples and relatively high occurrence of environmental microorganisms indicates urgent need to emphisize udder hygiene.