Mhase, P. P.Rohokale, J. S.2017-06-212017-06-212015http://krishikosh.egranth.ac.in/handle/1/5810022300Mastitis is multi-etiological disease of dairy animals characterized by physical, chemical and usually bacteriological changes in milk and pathological changes in glandular tissues of udder. Mastitis has evolved as a disease of highest economic significance particularly to the dairy industry worldwide. Financial losses arise from the costs incurred on treatment, culling, decreased milk production, sometimes permanent loss of udder, decreased milk value and even death of animal. For monitoring udder health, it is necessary to implicate the reliable and affordable diagnostics method and constant need to improvise them. In the present study easy, cheap and rapid MALDI-TOF MS technique was applied for detection of pathogens associated with mastitis keeping PCR results as reference particularly for major pathogens. During present investigation a total of 235 dairy animals from unorganized and organized farms located in Pune and Satara region were examined using CMT to study prevalence of mastitis. All 136 CMT positive samples were further processed for bacterial culture in enrichment medium which generated 127 culturally positive samples from which 203 pure bacterial colonies were isolated culturally as single or as mixed infections. The major pathogens were identified upon growing them on selective media like EMB and MacConkey’s agar for E. coli and on MSA agar for S. aureus, respectively. They were confirmed by biochemical catalase, oxidase and IMViC tests. Accordingly, total 80 (62.9 %) isolates were detected as S. aureus and 53 ( 41.7 %) as E. coli. All the isolates were processed for antibiotic sensitivity testing and overall rate of sensitivity indicated, highest rate of sensitivity in 159 isolates (78.3 %) for gentamicin followed by enrofloxacin 155 (76.3 %), ceftriaxone 122 (60 %), ciprofloxacin 97 (47.7 %), chloramphenicol 73 (35.9 %), streptomycin 44 (21.6 %), oxytetracycline 25 (12.3 %) and penicillin 9 (4.4 %) and overall rate of resistance pattern of all isolates indicated highest rate of isolates i.e. 192 (94.5 %) being resistant to Penicillin followed by oxytetracycline 149 (73.4 %), streptomycin 132 (65 %), chloramphenicol 88 (43.4 %), ciprofloxacin 78 (38.4 %), ceftriaxone 52 (25.6 %), gentamycin 36(17.7 %) and enrofloxacin 34(16.7 %). Culturally detected E. coli and S. aureus were subjected to molecular detection with PCR technique so as to use the results as reference for evaluating MALDI TOF MS. Using femA gene PCR, 80 isolates identified culturally were specifically (100 %) confirmed as S. aureus which generated 132bp size product upon electrophoresis. Total 53 E. coli isolates when treated with 16SrRNA PCR were also confirmed specifically (100 %) which yielded 232 bp size bands on electrophoresis of their PCR end product. Randomly selected isolates of E. coli (n = 25) and S. aureus (n = 25) were further processed with MALDI-TOF MS technique; keeping PCR result as reference. Along with these the other gram positive (n = 32) and gram negative (n=38) bacterial isolates were also processed which were generated during bacterial culture of milk sample for their identification. MALDI-TOF MS technique was found highly accurate and rapid in detection of major bacterial pathogens like E. coli and S. aureus with highest specificity (100 %) other unidentified bacterial isolates associated with mastitis from freshly cultured media were identified confirmed on genus and probable species level with MALDI TOF MS as Salmonella anatum 4 (5.7%), Streptococcus dysgalactiae, Enterobacter cloacae, S. xylosus, Proteus mirabilis, Clostridium difficile, Enterococcus faecium, all were 3 in no. ( 4.2 %), S. hominis, Klebsiella oxytoca, Stenotrophomonas spp., S. schleiferi, Klebsiella oxytoca, Pseudomonas flurescens, S. hominis, S. epidermidis, S. cohnii, Proteus vulgaris, Streptococcus faecalis, Clostridium beijerinckii all were found 2.85 % ( 2 in no.) Lactobacillus agilis, Aeromonas encheleia, Lactobacillus aviarius, Camphylobacter jejuni (all were 1.42 %). Some of the different species were also detected during present studies which were Cuprividus hector, Azoarcus communis, Arthrobacter polychromogen. While some organisms remained as unidentified as peak score observed was not optimum. From the present findings it may be concluded that, MALDI-TOF MS is a robust assay in case of detection of disorders presenting polymicrobial etiologies like in case of mastitis with high confidence and shortest possible time. MALDITOF MS is a fast and truthful technique which has capability to replace conventional identification of multiple bacterial strains usually associated with udder environment and milk of bovine mastitis.ennullThesis