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20171
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Subject: Animal Biotechnology × Author: DUTTA, MADHUSMITA × Type: Thesis × Thesis Type: M.V.Sc. ×
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    ThesisItemOpen Access
    MOLECULAR CHARACTERIZATION AND GENOTYPING OF STAPHYLOCOCCI ASSOCIATED WITH BOVINE MASTITIS
    (Assam Agricultural University, Khanapara,Guwahati, 2017-07) DUTTA, MADHUSMITA; Borah, P.
    Mastitis is an important disease of dairy cows and buffaloes causing huge economic losses in the form of reduced milk production. It is an inflammation of the mammary gland (udder) that causes physical and chemical changes in milk, and leads to pathological condition of the glandular tissue. It is generally associated with poor hygienic and husbandry practices. The present study was undertaken with a view to isolate and identify Staphylococcus aureus from both mastitic and apparently normal bovine milk samples. The study also included molecular typing of representative isolates and detection of important virulence-associated genes by PCR. A total of 204 milk samples comprising both clinically affected (14) and apparently normal (190) milk were used for this study. The apparently normal milk samples were subjected to California mastitis test, of which 85.79 % tested positive for sub-clinical mastitis. Bacteriological and biochemical examinations were performed to isolate and identify staphylococci associated with mastitis. A total of 60 (33.8%) out of 177 milk samples yielded Staphylococcus aureus, which were confirmed by polymerase chain reaction (PCR) amplification of conserved sequences of aroA gene. All the isolates (100 %) were found to possess three virulence-associated genes, namely surface protein A (spa), thermonuclease (nuc) and coagulase (coa) genes, while 58 (96.6%) of the isolates showed the presence of clumping factor A (clfA) gene. Antimicrobial susceptibility testing revealed that all the 60 isolates were resistant to Ampicillin and Cotrimoxazole, while the highest susceptibility (100%) was shown to Gentamicin, Kanamycin and Chloramphenicol followed by Streptomycin (80%). On the other hand, significantly lower susceptibility was shown to Ceftriaxone (13.33%), Tetracycline (8.33%) and Cefapime (1.67%). Out of the total 60 isolates, seven were subjected to PCR-RFLP of the coagulase (coa) gene. Polymorphism was shown by all the isolates (100%) with four different restriction patterns. Ten isolates were subjected to staphylococcal protein A (Spa) typing and PFGE. Spa typing revealed two different types, t165 and t1611. On the basis of phylogenetic analyses based on spa typing and PFGE, it was concluded that isolate number 9 of Spa type t165 is the ancestral strain, the clonal descendents of which are endemic in the study area causing subclinical bovine mastitis.