Investigation of genetic polymorphisms in Streptococcus and Staphylococcus agents isolated from bovine milk samples

2. ABSTRACT The aims of the present study were to isolate and identify Staphylococcus aureus (5. aureus) and Streptococcus agalactiae (S. agalactiae) from California Mastitis Test (CMT)-positive milk samples of dairy cow populations located in the east and south east of Turkey, and then to characterize the coagulase (coa) positive isolates for S. aureus and CAMP-factor (cfb) positive isolates for S. agalactiae by means of Polymerase Chain Reaction based-Restriction Fragment Length Polymorphism (RFLP) analysis and Random Amplified Polymorphic DNA (RAPD) assay. In the bacteriological culture of milk samples (n = 700) that were positive in CMT for S.aureus, growth was obtained from 367 samples. Among these, species-specific PCR identified 200 (28.6%) isolates as S. aureus. The PCR amplification of the coa gene showed that 161 of the S. aureus isolates were coa positive. In coa PCR, while most of the isolates (n = 135, 83.9%) were determined to produce a single band with the molecular sizes ranging from 500 to 1400 bp, a small number of isolates (n = 26, 16.1%) yielded two amplification products. Coa-RFLP analysis by restriction enzymes of Alul and Hin61 revealed 23 and 22 band patterns, respectively. RAPD assay based upon Primer 786 and OPS 11 produced 32 and 30 different profiles, respectively. In addition, milk samples collected from 820 dairy cows at five different regions were examined for the presence of S. agalactiae. In the bacteriological culture of milk samples (n =820) that were positive in CMT, growth was obtained from 199 samples. Among these, both species-specific PCR and cfb-PCRidentified 85 (10.4%) isolates as S. agalactiae. After digestion of the amplified cfb-PCR products with the restriction enzymes AM and Tsp5091, all isolates appeared to have identical restriction profiles. RAPD assay based upon OPB 1 8 produced 18 different profiles. In conclusion, it was determined that a high degree of heterogeneity existed in the S. aureus and S. agalactiae strains isolated from dairy cow populations located in the east and south east of Turkey. RAPD assay and coa- RFLP analysis for S. aureus and RAPD assay for S. agalactiae can be used as good alternatives to conventional methods in typing S. aureus and S. agalactiae isolates of bovine origin. It is suggested that the findings of this study will help develop effective control and treatment strategies (e.g. development of new vaccines by using predominant strains, selection of effective antibiotics) against Staphylococcal and Streptococcal mastitis and therefore reduce the incidence of the disease. Keywords: Dairy cattle, Mastitis, S. aureus, S. agalactiae, PCR, RFLP, RAPD. 4