Mastitis is one of the most commonly occurring diseases of dairy animals. It is the most important cause of economic losses to the dairy industry in India and throughout the world. In the present study prevalence of microorganisms isolated from mastitic milk and their antibiotic resistance was studied. A total of sixty nine milk samples from mastitic animals (clinical and subclinical) were tested using sodium lauryl sulphate test and those positive were selected and transported to the laboratory for isolation and identification of the causative agent. Out of these samples fifty samples yielded bacterial growth when tested on selective/non selective medium. Of these fifty samples, seven samples had single bacterial growth whereas rest of the 43 samples had mixed growth. Among the bacterial isolates it was found that the highest prevalence was of Streptococcus agalactiae and Staphylococcus aureus followed by E. coli and Klebsiella pneumoniae. Antibiotic sensitivity test revealed that Streptococcus agalactiae revealed the highest sensitivity to ofloxacin, ciprofloxacin, gentamicin and resistance to amoxicillin and doxycycline; Staphylococcus aureus revealed the highest sensitivity to ciprofloxacin, doxycycline, azithromycin and sparfloxacin and resistance to amoxycillin and gentamicin.Similarly, E. coli revealed the highest sensitivity to azithromycin and chloramphenicol and resistance to amoxicillin and teicoplanin, and Klebsiella pneumoniae revealed highest sensitivity to azithromycin, gentamicin and resistance to amoxicillin, teicoplanin and erythromycin. Thus, from the present study it could be concluded that Streptococcus agalactiae, Staphylococcus aureus, E. coli and Klebsiella pneumoniae were the most frequently isolated organisms from mastitic animals and azithromycin and the third generation fluoroquinolones (ciprofloxacin, sparfloxacin and ofloxacin) were the most sensitive drugs.

In the present study, a de novo multiplex polymerase chain reaction using genus specific primers for four organisms' viz., E. coli, Klebsiella pneumoniae, Staphylococcus aureus and Streptococcus agalactiae were designed to identify and differentiate all the four organisms in a single reaction. Further, DNA was extracted from mastitic milk using three different methods and was subjected to multiplex polymerase chain reaction to compare and identify the best method out of these three methods based on the amplification results of multiplex polymerase chain reaction. It was observed from the study that multiplex PCR was able to amplify three organisms (Streptococcus agalactiae, Klebsiella pneumoniae and E. coli) successfully whereas was unable to amplify Staphylococcus aureus in the multiplex PCR. Mutiplex PCR when evaluated on the field samples using mastitic milk revealed SDS Triton DNA extraction method to be superior to Power Food Microbial DNA isolation Kit and Milk Bacterial DNA isolation kit method in isolating the bacterial DNA from the mastitic milk as indicated by amplification in multiplex PCR reaction.