The study was conducted at Bhola districts, Bangladesh from May to August, 2019. A total of 70 milking buffaloes were used in this study from 2 different buffalo farms. A structured questionnaire was developed and all data were directly collected from the farm owners by farm visits. Efforts have been made to avoid obvious mechanical errors while recording the data.
California Mastitis Test A standard protocol and aseptic measurements were taken to collect milk samples from the farms. The milk was collected from all four quarters separately and tested on-farm by California Mastitis Test (CMT) kit (California Mastitis Test®, Portland) according to manufactures instructions. Briefly, a plastic paddle with four receptacles was used for this purpose. After cleaning the teats using antiseptic, 2 ml of foremilk was stripped from 4 teats of each buffalo cow separately into the respective cup of the paddle. An equal amount of CMT reagent was added to milk in each cup of the plastic paddle. Then the reagent and milk were mixed in the cups of the plastic paddle by a swirling motion. The result was recorded immediately according to manufacture instruction by visual examination. No coagulation or gel formation of milk was regarded as negative for SCM and coagulation or gel formation of milk was regarded as positive for SCM. The positive milk sample was preserved at -200C for further molecular study and send to the Department of Microbiology and Veterinary Public Health, Chattagram Veterinary and Animal Sciences University, Khulshi, Chattogram-4225 for further analysis.
Bacteriological Analyses of Milk Samples For the isolation and identification of bacteria, four common bacterial species were targeted in this experiment. CMT positive samples were used in these bacterial isolation experiments. The 20µl of milk sample was streaked on 5% bovine blood agar plate and incubated up to 72 h at 37°C and the plate was examined every 24 h interval for optimum growth of mastitic bacteria. Bacterial species were primarily identified based on colony morphology, presences or absences of hemolysis, gram staining, catalase and coagulase test. Subsequently, suspected Staphylococcal colonies were sub-cultured on mannitol salt agar (Oxoid Ltd., UK) and incubated for 24 h at 37°C. Characteristic yellowish colony on mannitol salt agar, gram-positive grape-like cluster cocci with catalase-positive isolates were identified as Staphylococcus spp. For, E. coli species the presumptive colony was inoculated onto MacConkey agar (Oxoid Ltd., UK) medium and incubated at 37°C for 24 h. Large pink colonies yielded on the MacConkey agar were further subculture onto Eosin methylene blue (EMB) agar (Oxoid Ltd., UK), and after recommended incubation, only distinctive metallic green sheen colonies were confirmed as E. coli. For Streptococcus spp. all presumptive colonies were subcultured onto blood agar and characteristic dewdrop like a colony with ring-shaped hemolysis and, catalase-negative, gram-positive chain former cocci were identified as Streptococcus spp. Bacillus spp. were confirmed based on colony morphology (irregular, large, raised gray color colony) with characteristic hemolysis pattern and large gram-positive bacilli with positive catalase test. All bacterial isolates were then preserved at -80°C using 50% glycerol until further examination.
Molecular Confirmation of Staphylococcus aureus and E. coli Two common bacterial primers were used in this experiment due to the excessive cost of this primer namely, S. aureus and E. coli. Bacterial genomic DNA was extracted by using the boiling lysis method described by Millar et al. (2000). Finally, Staphylococcus isolates and E. coli isolates were confirmed by the PCR amplification by the following primers. The PCR amplification conditions were initial denaturation for 2 min at 95°C, followed by 30 cycles for the 30s at 95°C, 35s at 56°C, and 60 s 72°C; and final extension at 72°C for 2 min. Finally, amplified PCR products were visualized in a UV chamber after completing gel electrophoresis on 1% agarose.
Antimicrobial Susceptibility Test All bacterial isolates were selected for culture sensitivity testing against 12 different antimicrobials compounds using disc diffusion methods according to the Clinical and Laboratory Standards Institute (CLSI) guideline (CLSI. 2010). The following antibiotic discs (Oxoid Ltd., UK) were used, namely: enrofloxacin (10 μg), ciprofloxacin (5 μg), gentamicin (30 μg), tetracycline (30 μg), erythromycin (15 μg), amoxicillin (10 μg), trimethoprim-sulfamethoxazole (1.25 + 23.75 μg), cefoxitin (10 μg), ceftriaxone (10 μg), vancomycin (5mg), penicillin (10 IU) and streptomycin (100 μg). For each isolate, the zone of inhibition around each disc was measured and interpreted as susceptible (S), intermediate (I) and resistant (R) according to CLSI referred value of veterinary pathogens (CLSI. 2010).
Statistical Analysis Descriptive statistical analysis was performed to assess the prevalence of reproductive diseases and disorders, risk factors of mastitis by SPSS 17 statistical software.