Isolation and identification of Pasteurella muultocida of animal and avian origin by cultural, biochemical and molecular techniques
Javia, Bhaveshkumar B. (2004) Isolation and identification of Pasteurella muultocida of animal and avian origin by cultural, biochemical and molecular techniques. Masters thesis, College of Veterinary Sciecne and Animal Husbandry, Anand Agricultural University, Anand, Gujarat, INDIA.
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The present study was undertaken with a view of isolation and identification of P. multocida from suspected cases of pasteurellosis in different animals and birds. A total of 89 samples were collected from suspected cases, of which 13 (14.6%) P. multocida isolates(3 from buffalo, 2 from rabbit, 1 from sheep and 7 from poultry) were isolated using blood agar as primary culture medium. These isolates were studied for their biochemical behaviours, in vitro antibiotic sensitivity pattern, P. multocida species specific PCR (PM-PCR) and molecular characterization by PCR-SSCP and repetitive seuqnece based PCR. All the isolates produce non-haemolytic colonies on blood agar and failed to grow on MacConkey agar. On biochemical behaviors, all the 13 isolates (100%) were found positive for oxidase, catalase, indole production, nitrate reduction and fermentation of glucose, mannitol, sucrose and mannose. All the 13 isolates (100%) were negative for citrate utilization test and could not ferment maltose, arabinose, lactose, dulcitol, salicin and trehalose. All the isolates revealed similar biochemical behaviour. All the isolates (100%) were sensitive to enrofloxacin, flumequine, chloramphenicol and cephalexin, while twelve isolates (92.31%) were sensitive to pefloxacin, six isolated (46.15%) were sensitive to ciprofloxacin and four isolates (30.77%) were sensitive to amoxycillin. All the thirteen P. multocida isolates (100%) were found to be resistant to sulphadiazine. All the 13 field isolates along with one vaccine strain (P. multocida P32) and five other bacterial cultures were tested by PM-PCR, which revealed an amplified product of approximately 465 bp size for all the P. multocida isolates, while other bacterial culture did not. The PM-PCR product was subjected to SSCP analysis on 0.5X MDE gel with 10% glycerol. Total six different SSCP band patterns were obtained. These patterns contained a minimum one to maximum four ssDNA bands. Total numbers of bands across the patterns were found to be eight. In this study capability of rep-PCR [repetitive BOX elements, enterobacterial repetitive intergenc consensus (ERIC) and repetitive extragenic palindromic (REP) polymerase chain reaction (PCR)] was tested for molecular characterization of P. multocida isolates. Fingerprinting with BOX-, ERIC- and REP-PCR generated 14, 16 and 13 different bands with molecular weight ranging from 310-1160 bp, 55-900 bpand 44-500 bp, respectively. The number of bands amplified in different samples by BOX-, ERIC-, and REP-PCR were varied from 2 to 7, 6 to 11, and 1 to 10 with the band freqeucny ranging from 0.1429 to 0.7857, 0.0714 to 1.000 and 0.1429 to 0.9286 respectively. The bands of BOX-, ERIC-, and REP-PCR were 100%,75% and 100% polymorphic, respectively. Dendogram were constructed by POPGENE software using bands information from BOX-, ERIC-, and REP-PCR. Dendogram of BOX-PCR data showed 61% similarity among all buffalo isolates including vaccine strain and 91% similarity among both rabbit isolates. Poultry isolates were formed two groups with no similarity. Dendogram of ERIC-PCR data showed 100% similarity among all buffalo isolates including vaccine strain and between both the rabbit isolates. All the poultry isolates showed 92% similarity while sheep isolates showed 77% similarity with all poultry isolates. Dendogram of REP-PCR showed 100% similarity among all buffalo isolates including vaccine strain, between both the rabbit isolates and also among all the poultry isolates. Sheep isolate was branched out separately. REP-PCR found most efficient to group isolates from different host origin. Dendogram based on overall pooled data showed 89% similarity among all buffalo isolates including vaccine strain, 80% similarity between both rabbit isolates and 65% similarity among all poultry isolates. Sheep isolates showed 36% similarity to poultry isolates. Similar groups were also formed by NETWORK analysis. It is concluded that PM-PCR are useful for rapid and specific detection of P. multocida isolates. SSCP and rep-PCR are useful to identify and characterize P. multocida isolates which can not be classified ana characterized by conventional ways.
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