MOLECULAR CHARACTERIZATION OF Pasteurella multocida ISOLATES OF BUFFALOES
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Date
2016-12
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SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA
Abstract
ABSTRACT:
Pasteurella multocida is a Gram negative pathogen responsible for
economically important diseases in ruminants especially in cattle and buffaloes. Young animals were more susceptible than adults and the carrier animals play an important role in spreading of Hemorrhagic septicaemia (HS). The present work was taken up for isolation and characterization of P. multocida from buffaloes.A total of 196 samples were collected from slaughtered animals, clinical suspected cases, field HS case and from field HS mortalities of buffaloes in Krishna
district, Andhrapradesh. Primarily the suspected samples were streaked directly on
Brain heart infusion (BHI) agar. The P. multocida suspected colonies were further
subjected to P. multocida species specific PCR (PM- PCR) and 23 (11.73%) were found positive. Out of 23 PM-PCR positive samples 16 were isolated as pure cultures of P. multocida by morphological tests, different cultural tests and biochemical tests. Furthermore the isolates were confirmed by conducting PM-PCR using a primer pair KMT1T7-KMT1SP6 and all the 16 isolates gave positive amplification of 460 bp product. The total isolation percentage 8.16% was observed. The antibiogram of P. multocida isolates revealed 100% sensitivity to
enrofloxacin, gentamicin, tetracycline, ampicillin, ceftriaxone and penicillin-G, 93.75% to chloramphenicol, ciprofloxacin, streptomycin and co-trimoxazole, followed by meropenem, aztreonam, sulphafurazole, nalidixic acid. The isolates found highly resistant to lincomycin, clindamycin, followed by sulphadiazine. The genotypic antibiotic resistance pattern of P. multocida resulted sul2 gene in 11 isolates, whereas 5 isolates had both sul1 and sul2 genes and none of the isolates harboured catA1, strA and strB genes. The capsular typing of P. multocida was conducted using Multiplex PCR, 14 isolates were classified as type A (87.5%) and two as type B (12.5%). The Cap A isolates were obtained from tonsils and Cap B isolates from whole blood and heat of field HS cases. Further the virulence genotyping of the isolates by PCR revealed that all Cap A isolates harboured (93.75%) hgbA, 100% hgbB, ompH, ptfA genes, whereas the Cap B isolates showed 100% prevalence of hgbA, hgbB, ompH, ptfA, pfhA and tbpA genes. The OMP profile of Cap A and Cap B isolates of P. multocida was analyzed on SDS-PAGE. The Cap A isolate yielded 6 polypeptide bands of approximate molecular weights of 19-76 kDa, whereas the Cap B isolate showed 6 polypeptide bands of 14.7- 80 kDa. Based on stain intensity, 38 kDa and 32.5 kDa were considered as major polypeptide bands in Cap A and Cap B isolates, respectively.
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