Browsing by Author "Mahavir Singh"
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ThesisItem Restricted Cloning, Expression and characterization of β-haemolysin of a local isolate of staphylococcus aureus(LUVAS, 2011) Mahavir Singh; Archana SharmaStaphylococcus aureus is a Gram-positive bacterium that causes several clinically important infections in man and animals, including mastitis in cattle and buffaloes. More than 30 different exotoxins are secreted by different strains of S. aureus and are involved in pathogenesis of various clinical conditions. The role of S. aureus beta-haemolysin has not yet been clearly understood. Recombinant techniques have allowed large scale and cost-effective production of biologicals for diverse applications. The present study was undertaken to clone, sequence, express and characterize beta-haemolysin of a local Indian strain of S. aureus. Pathogenic coagulase+ beta-haemolysin+ S. aureus was isolated from milk samples of 10 bovines suffering from clinical mastitis in Haryana. The bacteria were identified using various tests such as Gram’s staining, catalase production, mannitol fermentation, ‘hot-cold’ lysis on blood agar and titration of beta-haemolytic activity in culture supernatant, SA-hlb and SA-coa gene-specific multiplex PCR and antibiotic sensitivity pattern. PCR product of SA-hlb gene from one of the above isolates was cloned into pGEM-Teasy vector for nucleotide sequencing. The nucleotide sequence (n=891 nt) of SAhlb MV1 clone was deposited at GenBank NCBI website and assigned GenBank accession no. JN580071. BLASTN and BLASTP searches retrieved several orthologs with >95% homology to its nucleotide and deduced amino acid sequences. The protein sequence possessed ‘nSMase’ conserved domain and was a member of EEP superfamily. Most interestingly, its 3D structure had remarkable similarities to 3i41B (PDB) hlb from S. aureus strain RN4220 of USA origin and all the residues involved in catalytic, metal binding and phosphate binding sites were 100% conserved. PCR product of SA-hlb gene was also directionally cloned into SacI-XhoI sites of pQE-Tri System vector to allow expression of ~37 kDa recombinant SA-hlb.8xHis in JM107 transformants. The r-SA-hlb MV3 clone showing the highest level of expression as revealed by SDS-PAGE and verified by immunoblotting, was produced in large amounts and purified by Ni-chelate chromatography. The purified r-SA-hlb possessed hot-cold lytic activity against sheep and buffalo erythrocytes. The hot-cold activity was thermostable upto <90ºC for 30 min. and could be neutralized by antibodies in convalescent bovine sera. The purified r-SA-hlb also exhibited cytocidal effect on buffalo mononuclear cells, maximum being at 125 ng/ml concentration. It was useful as coated antigen at 2.5 μg/ml concentration for optimally measuring antibody levels in bovine sera by indirect ELISA. Various other applications of this purified recombinant S. aureus beta-haemolysin have been speculatedThesisItem Restricted Microbiological and immunological studies on buffalo subclinical mastitis(LUVAS, 2015) Mahavir Singh; Sharma, AnshuA total of 1356 quarter milk samples of 344 apparently healthy buffaloes from three organised dairy farms were screened for subclinical mastitis. Following IDF criteria, highest prevalence of subclinical mastitis was 6.67% in Government livestock farm followed by 5.83% in Hitech farm, 2.77% in LUVAS farm (April month) and lowest 1.43% in LUVAS farm (December month). Milk microbiome revealed that out of 114 staphylococci, 43.85% were S. aureus, and out of 61 streptococci, S. dysgalactiae (62.29%), S. agalactiae (27.86%) and S. uberis (9.83%) which were characterised phenotypically and confirmed by PCR assay using 16S rRNA specific primers. Among coagulase negative staphylococci (64), S. epidermidis was found most prevalent organisms followed by S. capitis, S. haemolyticus, S. hycius, S. chromogenes, S. simulans, S. saprophyticus. Antimicrobial sensitivity of isolates varied in different farms which depend on the use of antimicrobials and strains prevalent at that farm. In the present study, relative expression of proinflammatory cytokines revealed highest fold change in expression of TNFα followed by IL-1β, IL-6 and IFN-γ in milk of buffaloes with subclinical mastitis as compared to healthy ones. Lowest up regulation of IFN-γ was due to association of Gram positive bacteria. Significant increase in concentration of haptoglobin, milk amyloid A, lactate dehydrogenase and N-acetyl-β-D-glucosaminidase enzymes, pH, electrical conductivity, Na, Cl and significant decrease of Ca, Fe, Se, Zn, protein and fat values was observed in milk of subclinically mastitic buffaloes. All the parameters found better and reliable indicator of subclinical mastitis depending upon the availability of the reagents, kits and economy, can be used for screening of animals for subsequent bacteriological culture.ThesisItem Open Access Molecular Characterization Of Buffalo Pox Virus (BP4 Strain)(IVRI, Izatnagar, 1990) Mahavir Singh; P. P. Bhat