16s Rrna Based Metagenomics Analysis of Mastitis Milk in Bovine

Abstract

A total of 15 milk samples were included in the study primarily based on clinical signs of mastitis from HF and Sahiwal purebred animals. The samples were screened for SCC count immediately after reaching the laboratory. The pH of the samples ranged 5-9. The results of present study revealed higher concentration of chloride in all the samples except one and that does not have higher SCC count. The concentration of chloride is in concurrence of higher SCC concentration in samples. All the milk samples with increased SCC revealed higher chloride concentration. The results of WST revealed variable results as it is simply based on observations so any error in process may lead to false negative tests. The study revealed 15 isolates based on cultural, morphological, biochemical characterization. These included 5 coagulase negative Staphylococcus spp., and 8 E. coli and two yeasts. These were confirmed by the PCR based amplification with specific primers. Amplicons were subjected to Sanger sequencing and received data was analyzed. The BLAST based analysis confirmed the genus and species of all the isolates as Staphylococcus gallinarum, Staphylococcus succinus, Staphylococcus sciuri, Diutina rugosa and multiple strains of E. coli. The isolated yeasts were subjected to the amplification of 500bp amplicon from ITS primer. Sanger sequencing-based analysis confirmed these isolates as Diutina rugosa (previously known as Candida rugosa). It seems to be first report of isolation of Diutina rugosa from the clinical cases of subclinical mastitis in Indian cattle. The study for phylogeny linkage revealed Indian isolate in close homology with the isolate of Thailand and both share a same route followed by the isolates of China, Ghana and Mexico. The antibiotic sensitivity pattern revealed multidrug resistance in all the isolates of E. coli and Staphylococcus spp. The resistance was highest against Amoxyclave (100%), followed by Cephalexin (87.5%), Moxifloxacin (75%), Enrofloxacin (67.7%), Cloxacillin (66.7%), Ceftriaxone and Oxytetracycline (62.5%) and Gentamicin and Streptomycin (58.4%). The antibiotyping revealed 14 different antibiotypes. DNA of pooled samples was subjected to Sanger sequencing and data analysis. The refraction curve obtained from sequenced data clearly indicates the presence of large number of microbes in mastitic milk sample as compared to healthy sample. Similarly, the α-diversity suggests the more diversified microbial population in diseased milk samples. Data showed the taxonomical diversity one domain, two phylum, seven classes, 12 orders, 34 families, 81 genus and 197 species were missing from the milk sample of healthy animals. It signifies the diversification of udder environment in the cases of sub clinical mastitis. Moreover, in healthy udder archaea contributed 99.68% of total population and bacteria were only 0.31% as compared to 0.25% and 99.74% in mastitic udder. The presence of large number of bacterial populations in specific classes, families, genus and species are predominated by the bacteria absent from healthy uterus. Therefore, the present study recommends a.) use of discriminant analysis to discriminate microbial community of clinical and healthy mastitis and b.) regular use of antimicrobial susceptibility testing of mastitis.