Lakshmi Kavitha, K(MAJOR)Ramani Pushpa, R.N.Srinivasa Rao, TJAGADESH, J2019-07-162019-07-162019-04http://krishikosh.egranth.ac.in/handle/1/5810114852THESESThe Gram-negative bacterium, Pasteurella multocida, is an important worldwide primary and opportunistic pathogen as well as the common commensal of many wild and domesticated animals. The present work was taken up to characterize and observe genetic diversity of bovine, ovine and porcine isolates of P. multocida. A total of 283 samples were collected from apparently healthy slaughtered animals (276) and clinical cases (7). Primarily the suspected samples were streaked directly on Brain heart infusion (BHI) agar. Further when all the suspected samples subjected to PMPCR, 86 (30.3%) were found positive. Out of 86 PM-PCR positive samples, 31 were identified (bovine 8, ovine 11 and porcine 12) as pure cultures of P. multocida by morphological tests, different cultural and biochemical tests. Further the 31 isolates were finally confirmed by employing PM-PCR using a primer set KMT1T6-KMT1SP6 with 460 bp amplicon and all the isolates were found positive in PM-PCR. The total isolation percentage of 10.9% was observed. The capsular typing was done by Uniplex PCR targeting Cap A and Cap D capsule specific gene primers, yielded 20 Cap A isolates, 8 Cap D isolates and 3 untypable isolates. The virulence gene profile of isolates showed high prevalence of hgbA gene (77.41%) among the three iron binding proteins, followed by hgbB gene in 41.9% of isolates, among these lowest prevalence was observed in bovine isolates. Gene tbpA has lowest prevalence (19.35%) among the three iron binding proteins. Moreover, the tbpA gene was highly associated with ovine isolates. The higher prevalence of adhesion related gene such as ptfA (76.66%) was observed. This gene was detected 100% in porcine isolates. Dermonecrotxin gene was noticed in high percentage in ovine isolates (27.27%), while only one isolate of bovine harbored this gene. The pfhA gene revealed clear association to Cap A strains (50%), and none of the other capsule types of P. multocida harbored this gene. The dermonecrotoxin toxA was found in 25% of Cap D strains, followed by 10% of Cap A. Moreover, P. multocida strains of capsule type A, and D showed high prevalence of the ompH, ptfA and hgbA compared to cap-negative strains. Molecular typing techniques (REP-PCR, ERIC-PCR and (GTG)5-PCR) differentiated all 49 strains (isolates from this study and previous isolates) into different profiles. All the isolates were found genetically distinct from standard P. multocida strain P52 (Vaccine strain of India). Genetic diversity among the isolates of P. multocida and their genetic dissimilarity with P52 vaccine strain showed an indication about vaccine strategy. This study provided a clear evidence of presence of more than one isolate type in host species and also provided indication of high genetic variation among field isolates of P. multocida and may be the reason of vaccine failure and outbreaks. Furthermore the isolation of different capsular types from single host species warrens the need for the use of polyvalent vaccine against Pasteurella infections in their respective hosts.ennullThesis