MOLECULAR CHARACTERIZATION OF Pasteurella multocida AND Mannheimia haemolytica FROM SHEEP PNEUMONIA
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Date
2019-01
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SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517502. (A.P.) INDIA
Abstract
Pasteurella multocida and Mannheimia haemolytica are responsible for major economic problems in ruminant production worldwide. These organisms cause respiratory diseases and shipping fever under stress in sheep. The objective of the present study is molecular characterization of Pasteurella multocida and Mannheimia haemolytica from sheep pneumonia.
A total of 195 lung samples were collected and were streaked on brain heart infusion agar. The suspected colonies were subjected to P. multocida species specific PCR (PM- PCR) and M. haemolytica species specific PCR using PHSSA and Rpt2 genes by multiplex PCR and found 12 (6.15%) and 15 (7.69%) positives, respectively. Out of 12 PM-PCR positive samples, 7 pure cultures of P. multocida were obtained. Further the isolates were confirmed by conducting PM-PCR and all gave positive amplification of 460 bp product. The total isolation percentage 3.59% was observed. Among the 15 PHSSA and Rpt2 positive samples of M. haemolytica 10 were isolated as pure cultures. Later the cultures were confirmed by PCR. The isolation percentage of M. haemolytica observed was 5.12%. P. multocida capsule biosynthesis gene showed Cap A and Cap D with 28.57% and 57.14%, respectively. One isolate was untypable. The distribution of virulence genes showed pfhA (14.28%), tbpA (71.41%), ompH (57.14%), ptfA (0%), hgbA (57.14%), hgbB (0%) and toxA (57.14%). It was observed Cap D association with toxA gene and Cap A with pfhA gene. The virulence genotyping of M. haemolytica was conducted using lktA gene in which 30% of the isolates possesed this gene. The isolates of P. multocida revealed antibiotic sensitivity to gentamicin, cotrimoxazole, ceftriaxone, ampicillin, sulfamethoxazole, streptomycin, amoxicillin-clavulanic acid, nalidixic acid (100%), followed by chloramphenicol and colistin (71.42%), amoxicillin (57.1%), enrofloxacin (4.85%), clindamycin (28.57%). However the isolates were resistant or intermediately resistant to tetracycline (100%). The distribution of antibiotic resistance genes were catA1 (100%), strA (57.14%), strB (71.42%) and tetB (42.85%). The results of sensitive antibiogram pattern of M. haemolytica revealed gentamicin, cotrimoxazole, ceftriaxone (100%), ampicillin, sulfamethoxazole (90%), chloramphenicol, nalidixic acid (80%), amoxicillin-clavulanic acid (60%), tetracycline (40%), amoxicillin (3%), colistin, enrofloxacin (1%). The distribution of antibiotic resistance genes showed tetB (40%) and absence of other genes studied. The gross pathology of the infected lungs showed cranioventral consolidation and pleural adhesions. The histopathological changes observed in the affected tissues were infiltration of neutrophils and mononuclear cells into the bronchial lumen, alveoli and bronchopneumonia was evident. The research work paved way for development of conventional vaccines using different capsular types. It also paved way for future development of rapid diagnostic kits and probable recombinant vaccines using the properties of virulence genes.
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