Molecular characterization of Pasteurella multocida isolates obtained from poultry, ruminant, cats and dogs using RAPD and REP-PCR analysis

Document Type: Original article

Authors

Department of Pathobiology, School of Veterinary Medicine, Shiraz University, Shiraz, Iran.

Abstract

In the present study, Randomly Amplified Polymorphic DNA‌ (RAPD) and Repetitive Extragenic Palindromic sequence-based Polymerase Chain Reaction (REP-PCR) were used to characterize 131 isolates of Pasteurella multocida, originating from different healthy and diseased animal species obtained from several geographical regions of Iran. The RAPD and REP-PCR generated amplified products in the range of 300 to 3400 bp and 200 to 2850 bp, respectively. Among all of the P. multocida isolates, cluster analysis revealed that 63 clusters and nine untypable isolates and 81 clusters and six untypable isolates were produced with RAPD and REP-PCR methods, respectively. The results indicated that the REP-PCR method showed a slightly higher level of discrimination power in differentiating of P. multocida isolates as compared with RAPD. The results showed that a considerable level of genetic diversity exists among P. multocida isolates even in the isolates with the same animal or geographical origins. There was no host- and region-specific pattern. In addition, the isolates obtained from the healthy and diseased animal did not reveal any correlation genotypic profiles, which could be supported by the hypothesis that P. multocida is a strictly opportunistic pathogen. In conclusion, because of a large amount of genetic heterogeneityin the P. multocida isolates, Pasteurellosis may be caused by different clones in the same herd or animal.

Keywords


1. Hunt ML, Adler B, Townsend KM. The molecular biology of Pasteurella multocida. Vet Microbiol 2000;72:3-25.

2. Ewers C, Lübke-Becker A, Bethe A, Kießling S, Filter M, Wieler LH. Virulence genotype of Pasteurella multocida strains isolated from different hosts with various disease status. Vet Microbiol 2006;114:304-317.

3. Ekundayo SO, Odugbo MO, Olabode AO, Okewole PA. Phenotypic variability among strains of Pasteurella multocida isolated from avian, bovine, caprine, leporine and ovine origin. Afr J Biotechnol 2008;7:1347-1350.

4. Donnio PY, Lerestif-Gautier AL, Avril JL. Characterization of Pasteurella spp. strains isolated from human infections. J Comp Pathol 2004;130:137-142.

5. Huber BS, Allred DV, Carmen JC, Frame DD, Whiting DG, Cryan JR, Olson TR, Jackson PJ, Hill K, Laker MT, Robison RA. Random amplified polymorphic DNA and amplified fragment length polymorphism analyses of Pasteurella multocida isolates from fatal fowl cholera infections. J Clin Microbiol 2002;40:2163-2168.

6. Carter GR. Studies on Pasteurella multocida. A hemagglutination test for the identification of serological types. Am J Vet Res 1955;16:481-484.

7. Arumugam ND, Ajam N, Blackall PJ, Asiah NM, Ramlan M, Maria J, Yuslan S, Thong KL. Capsular serotyping of Pasteurella multocida from various animal hosts-a comparison of phenotypic and genotypic methods. Trop Biomed 1996;28:55-63.

8. Harper M, Boyce JD, Adler B. Pasteurella multocida pathogenesis: 125 years after Pasteur. FEMS Microbiol Lett 2006;265:1-10.

9. Taylor JD, Fulton RW, Dabo SM, Lehenbauer TW, Confer AW. Comparison of genotypic and phenotypic characterization methods for Pasteurella multocida isolates from fatal cases of bovine respiratory disease. J Vet Diagn Invest 2010; 22:366-375.

10. Harper M, Boyce JD, Adler B. The key Surface Components of Pasteurella multocida: Capsule and Lipopolysaccharide. Curr Top Microbiol Immunol 2012; 361:39-51.

11. Matsuki T, Watanabe K, Tanaka R. Genus- and species-specific PCR primers for the detection and identification of bifidobacteria. In: Tannock GW (ed.). Probiotics and Prebiotics. Where Are We Going? Wymondham: Caister Academic Press, 2002, 85-105.

12. Ventura M, van Sinderen D, Fitzgerald GF, Zink R. Insights into the taxonomy, genetics and physiology of bifidobacteria. Anton Leeuw 2004;86,205–223.

13. Dziva F, Muhairwa AP, Bisgaard M, Christensen, H. Diagnostic and typing options for investigating diseases associated with Pasteurella multocida. Vet Microbiol 2008;128:1-22.

14. Dziva F, Christensen H, van Leengoed LAMG, Mohan K, Olsen JE. Differentiation of Pasteurella multocida isolates from cases of atrophic rhinitis in pigs from Zimbabwe by RAPD and ribotyping. Vet Microbiol 2004;102:117-122.

15. Gilson E, Clément JM, Brutlag D, Hofnung MA. A family of dispersed repetitive extragenic palindromic DNA sequences in E. coli. EMBO J 1984;3:1417-1421.

16. Olive DM, Bean P. Principles and applications of methods for DNA-based typing of microbial organisms. J Clin Microbiol 1999;37:1661-1669.

17. Blackall PJ, Miflin JK. Identification and typing of Pasteurella multocida: a review. Avian Pathol 2000;29:271-287.

18. da Silva RB, Valicente FH. Molecular characterization of Bacillus thuringiensis using REP-PCR. SpringerPlus 2013;2:641.

19. Taylor JD, Doyle DJ, Blackall PJ, Confer AW. Use of REP-PCR and 16s rRNA gene sequencing for comparison of Mannheimia haemolytica isolates obtained from fatal cases of bovine respiratory disease in the USA and australia. Aust Vet J 2014; 92:15-23.

20. Shivachandra SB, Kumar AA, Chaudhuri P. Molecular characterization of avian strains of Pasteurella multocida serogroup-A:1 based on amplification of repetitive regions by PCR. Comp Immunol Microbiol Infect Dis 2008;31:47-62.

21. Chen HI, Hulten K, Clarridge JE. Taxonomic subgroups of P. multocida correlate with clinical presentation. J Clin Microbiol 2002;40:3438–3441.

22. Saxena MK, Singh VP, Kumar AA, et al. REP-PCR analysis of Pasteurella multocida isolates from wild and domestic animals in India. Vet Res Commun 2006; 30:851-861.

23. Townsend KM, Frost AJ, Lee CW, Papadimitriou JM, Dawkins HJ. Development of PCR assays for species- and type-specific identification of Pasteurella multocida isolates. J Clin Microbiol 1998;36:1096-1100.

24. Dziva F, Christensen H, Olsen JE, Mohan, K. Random amplification of polymorphic DNA and phenotypic typing of Zimbabwean isolates of Pasteurella multocida. Vet Microbiol 2001;82:361-372.

25. Ozbey G, Kilic A, Ertas HB, Muz A. Random amplified polymorphic DNA (RAPD) analysis of Pasteurella multocida and Manheimia haemolytica strains isolated from cattle, sheep and goats. Vet Med–Czech 2004;49:65-69.

26. Versalovic J, Koeuth T, Lupski JR. Distribution of repetitive DNA sequences in eubacteria and application of fingerprinting of bacterial genomes. Nucleic Acids Res 1991;19:6823-6831.

27. Lee KE, Jeoung HY, Lee JY, Lee MH, Choi HW, Chang KS. Phenotypic characterization and Random Amplified Polymorphic DNA (RAPD) analysis of Pasteurella multocida isolated from Korean pigs. J Vet Med Sci2011;74:567-573.

28. Gunawardana GA, Townsend KM, Frost AJ. Molecular characterization of avian Pasteurella multocida isolates from Australia and Vietnam by REP-PCR and PFGE. Vet Microbiol 2000;72:97-109.