Characterization of Pasteurella multocida isolates by their outer memberane protein profiles, RAPD patterns and TOX A gene detection
Jain, Anshu (2004) Characterization of Pasteurella multocida isolates by their outer memberane protein profiles, RAPD patterns and TOX A gene detection. Masters thesis, College of Veterinary Science and Animal Husbandry, Anand Agricultural University, Anand.
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Pasteurella multocida is one of the most fascinating gram-negative bacteria and is a commensurable of the upper respiratory tract of many animal species. However, under predisposing circumstances the organism is the etiological agent of a wide range of economically important diseases. Thus to understand this microbial diversity, characterization of the isolates was done by their outer membrane protein profiles, RAPD patterns and tox A gene detection. For characterization of the isolates by their outer membrane protein (OMP) profiles, the cultures were grown in BHI broth and then subjected to sarkosyl method of OMP extraction. The electrophoretic protein profiles of the capsular type B showed the presence of 9 to 11 protein fractions. Based on band intensity, two polypeptides with approx. MW of 31.7 kDa and 34.9 kDa were considered to be the major OMPs. The OMP patterns of buffalo isolates revealed a total number of 9 bands with MW ranging from 21.1 to 89.2 kDa. While the H.S.Vaccine (P52) strain revealed a total number of 11 bands with protein fractions of 23.5 to 97.4 kDa. The OMP profiles from the field isolates of capsular type A revealed about 8 to 10 discernible protein bands. Based on band intensity, two polypeptides with MW of 31.7 kDa and 34.9 kDa were considered to be the major OMPs in all the isolates except one isolate of sheep. The rabbit isolates demonstrate a total number of 9 to 10 bands with molecular weights of 21 to 102 kDa. The protein fractions of sheep isolate revealed a total number of 8-9 bands with MW ranging from 21 to 135 kDa. The SDS-PAGE profiles of poultry isolates revealed a total of 9 bands with molecular weights of 18.6 to 88 kDa. The present research work was also carried out to study the effect of iron restriction on the OMP composition of P. multocida isolates. Among the capsular type B isolates, buffalo isolates expressed an additional protein of molecular weight 102 kDa while the vaccine strain (P52) did not express any additional protein as compared to iron sufficient medium. In capsular type A isolates, the rabbit isolates expressed additional protein band of molecular weight 77 kDa on a medium supplemented with iron chelator. Sheep isolates expressed additional protein band of 44 kDa in iron restricted medium and the poultry isolates, revealed protein bands of 44 kDa and 98 kDa when grown in iron restricted medium. To determine the genetic differences between the isolates, two random primers OPA- 11 and OPG-13 were used. The random primer OPA-11 generated nine different profiles. All the isolates belonging to different species viz. buffalo, rabbit, sheep and poultry isolates showed distinct RAPD profiles specific to their species of origin. All the buffalo isolates showed similar profile. While H.S. vaccine strain showed different profile from the buffalo isolate. The two rabbit isolates showed similar profile. The two sheep isolates showed two distinct RAPD profiles. The eleven poultry isolates showed four different RAPD profiles. The random primer OPG-13 showed only three RAPD profiles. All the capsular type A isolates of rabbit, sheep and poultry origin had identical RAPD pattern except one sheep isolate PS-1. Capsular type A exhibited all the bands present in capsular type B except the heaviest band of 1438 bp. PS-1 isolate expressed eight bands, all seven of capsular type B and an additional band of 773 bp. By Popgen and Network analysis it was found that 100% similarity exists among all the buffalo and among the rabbit isolates. While dissimilarity exists among the sheep as well as among the poultry isolate. The H.S. vaccine strain shows similarity to one of the poultry isolate. The tox A gene was detected in only 3 isolates (2 from sheep and one from poultry) of capsular type A while capsular type B isolates did not show any amplification product.
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