RAMANI PUSHPA, R.N (MAJOR)ANAND KUMAR, PSRINIVASA RAO, TTEJASWI, PENUGONDA2021-07-312021-07-312021-05https://krishikosh.egranth.ac.in/handle/1/5810171243THESESBovine mastitis is recognised as the most economically important disease affecting Dairy industry in India and all over the world. Most prevalent bacterial etiological agents identified in bovine mastitis are Staphylococcus aureus, Streptococcus species, E. coli and other Gram-negative organisms. The preferred treatment regime during the past decades for mastitis is antibiotic therapy. Streptococcus species are one of the most important causative agents of mastitis. Usually mastitis caused by Streptococci is of the subclinical type, so early detection is important. Among Streptococcus species S. agalactiae, S. dysgalactiae and S. uberis are predominant next to Staphylococcus species. The ability of Streptococcus to initiate growth in vivo and stably infect a host requires acquisition of virulence factors capable of neutralizing the mechanisms of host defence. Hence the present study is on antimicrobial susceptibility and detection of virulence factors of Streptococcus species associated with bovine mastitis. A total of 108 Bovine mastitis milk samples were examined and out of this 60 (55.5 %) samples were positive for Streptococcus species. The most prevalent isolate obtained was S. uberis 31 (51.7 %) followed by S. agalactiae 13 (21.7 %) and S. dysgalactiae 11 (18.3 %). All the isolates of Streptococcus spp. were identified based on morphology, cultural examination and further subjected to various biochemical tests viz., catalase test, oxidase test, ninhydrin test and sugar fermentation tests. Streptococcus selection broth was used for primary isolation followed by inoculation on to Edward’s medium with 5% sheep blood. The samples with Gram positive cocci in chains on Gram’s staining and cultures showing greyish, pinpointed colonies and/or esculin hydrolysis on edward’s medium were tentatively identified as Streptococcus species. All Streptococcus species isolates were further confirmed by PCR using genus and species specific primers. All the isolates of S. agalactiae were also subjected to CAMP test. Five Streptococcus isolates did not react to any of the specific primers used for S. uberis, S. agalactiae and S. dysgalactiae, hence categorised under Streptococcus other than these three species. Virulence factors of Streptococcus species were detected by using PCR. pauA gene was detected in eighteen (58.1 %) isolates and skc was detected in six (19.3 %) isolates of S. uberis. cfb gene (CAMP factor) was detected in twelve (92.3 %) isolates of S. agalactiae but phenotypically only five isolates were positive for CAMP test. In S. dysgalactiae mig gene was detected in 6 (54.5 %) isolates and skc gene was detected in one (9.1 %) isolate. The luxS gene responsible for biofilm formation was detected in eleven (35.4 %) isolates of S. uberis. xv On antibiotic sensitivity test the S. uberis isolates were resistant to ampicillin/ sulbactum followed by kanamycin, penicillin, ceftriaxone, tetracyclin, gentamicin and cotrimoxazole. Least resistance was observed for chloramphenicol, enrofloxacin and amoxicillin/ clavulanic acid. S. agalactiae isolates showed high resistance towards ampicillin/ sulbactum followed by kanamycin, penicillin G, enrofloxacin, chloramphenicol and gentamicin. Least resistance was found to ceftriaxone, amoxicillin/ clavulanic acid, tetracyclin and cotrimoxazole. Antibiogram of S. dysgalactiae revealed that the isolates were resistant to ampicillin/ sulbactum followed by kanamycin, enrofloxacin, penicillin G, chloramphenicol, gentamicin, ceftriaxone, tetracyclin and cotrimoxazole. These isolates showed least resistance towards Amoxicillin/ Clavulanic acid. Minimum inhibitory concentrations (MICs) of selected panel of antibiotics against S. uberis, S. agalactiae and S. dysgalactiae were studied by conducting modified resazurin dye microdilution assy. The MIC of S. uberis for penicillin-G is 15.62 μg/ml followed by 3.90 μg/ml for amoxicillin, 7.80 μg/ml for enrofloxacin, 78 μg/ml for gentamicin, 312.50 μg/ml for sulphamethoxazole + trimethoprim and 1250 μg/ml for sulphamethoxazole. For S. agalactiae the MICs for penicillin G, amoxicillin, enrofloxacin, gentamicin, sulphamethoxazole + trimethoprim and sulphamethoxazole were 15.62 μg/ml, 3.90 μg/ml, 31.25 μg/ml, 156 μg/ml, 625 μg/ml and 2500 μg/ml respectively. For S. dysgalactiae the MICs for penicillin G, amoxicillin, enrofloxacin, gentamicin, sulphamethoxazole + trimethoprim and sulphamethoxazole were 7.80 μg/ml, 0.98 μg/ml, 3.90 μg/ml, 39 μg/ml, 1250 μg/ml and 2500 μg/ml respectively.EnglishThesis