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PCR-Detection of Hemophilus paragallinarum, Hemophilus somnus, Mannheimia (Pasteurella) hemolytica, Mannheimia spp., Pasteurella trehalosi, and Pasteurella multocida

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PCR Detection of Microbial Pathogens

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 216))

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Abstract

Most members of the bacterial family Pasteurellaceae are usually regarded as opportunistic secondary invaders, which under normal conditions might inhabit the mucosal membranes of the upper respiratory and lower genital tracts of mammals and birds (1). Out of the almost 100 species or species-like taxa that might be isolated from mammals, reptiles, and birds, only Pasteurella multocida, Actinobacillus pleuropneumoniae, and Hemophilus paragallinarum are regarded as major pathogens (1,2) while A. suis, H. somnus, and Mannheimia hemolytica are considered as potential animal pathogens. The pathogenic potential is incompletely known for most taxa, probably due to limitations in their classification and identification, and inappropriate detection methods.

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References

  1. Bisgaard, M. (1993) Ecology and significance of Pasteurellaceae in animals. Zbl. Bakt. 279, 7–26.

    CAS  Google Scholar 

  2. Bisgaard, M. ( 1995) Taxonomy of the family Pasteurellaceae Pohl 1981, in Haemophilus, Actinobacillus, and Pasteurella (Donachie, W., Lainson, F. A., and Hodgson, J. C., ), Plenum Press, New York, USA, pp. 1–7.

    Google Scholar 

  3. Blackall, P. J. (1999) Infectious coryza: overview of the disease and new diagnostic options. Clin. Microbiol. Rev. 12, 627–632.

    PubMed  CAS  Google Scholar 

  4. Harris, F. W. and Janzen, E. D. (1989). The Haemophilus somnus disease complex (Hemophilosis): a review. Can. Vet. J. 30, 816–822.

    PubMed  CAS  Google Scholar 

  5. Ward, A. C. S., Jaworski, M. D., Eddow, J. M., and Corbeil, L. B. (1995) A comparative study of bovine and ovine Haemophilus somnus isolates. Can. J. Vet. Res. 59, 173–178.

    PubMed  CAS  Google Scholar 

  6. Frank, G. H. ( 1989) Pasteurellosis of cattle, in Pasteurella and Pasteurellosis (Adlam, C. and Rutter, J. M., ), Academic Press, London, UK, pp. 197–222.

    Google Scholar 

  7. Gilmour, N. J. L. and Gilmour, J. S. (1989) Pasteurellosis of sheep, in Pasteurella and Pasteurellosis, (Adlam, C. and Rutter, J. M. ), Academic Press, London, UK, pp. 223–262.

    Google Scholar 

  8. Carter, G. R. and De Alwis, M. C. L. (1989) Haemorrhagic septicaemia, in Pasteurella and Pasteurellosis (Adlam, C. and Rutter, J. M. eds.), Academic Press, London, UK, pp. 131–160.

    Google Scholar 

  9. Chanter, N. and Rutter, J. M. ( 1989) Pasteurellosis in pigs and the determinants of virulence of toxigenic Pasteurella multocida, in Pasteurella and Pasteurellosis (Adlam, C., and Rutter, J. M. ), Academic Press, London, UK, pp. 161–195.

    Google Scholar 

  10. Nagai, S., Someno, S. and Yagihashi, T. (1994) Differentiation of toxigenic from nontoxigenic isolates of Pasteurella multocida by PCR. J. Clin. Microbiol. 32, 1004–1010.

    PubMed  CAS  Google Scholar 

  11. Baalsrud, K. J. (1987) Atrophicrhinitis in goats in Norway. Vet.Rec., 350–353.

    Google Scholar 

  12. DiGiacomo, R. F., Deeb, B. J., Giddens, W. E. Jr., Bernard, B.L., and Chengappa, M.M. (1989) Atrophic rhinitis in New Zealand white rabbits infected with Pasteurella multocida. Am. J. Vet. Res. 50, 1460–1465.

    PubMed  CAS  Google Scholar 

  13. Petersen, S. K. (1990) The complete nucleotide sequence of the Pasteurella multocida toxin gene and evidence for a transcriptional repressor, TxaR. Mol. Microbiol. 4, 821–830.

    Article  PubMed  CAS  Google Scholar 

  14. Christensen, J. P. and Bisgaard, M. (1997) Avian pasteurellosis: taxonomy of the organisms involved and aspects of pathogenesis. Avian Pathol. 26, 461–483.

    Article  PubMed  CAS  Google Scholar 

  15. Amigot, J. A., Torremorell, M. and Pijoan, C. (1998) Evaluation of techniques for the detection of toxigenic Pasteurella multocida strains from pigs. J. Vet. Diagn. Invest. 10, 169–173.

    PubMed  CAS  Google Scholar 

  16. Townsend, K. M., Hanh, T. X., O’Boyle, D., et al. (2000) PCR detection and analysis of Pasteurella multocida from the tonsils of slaughtered pigs in Vietnam. Vet. Microbiol. 72, 69–78.

    Article  PubMed  CAS  Google Scholar 

  17. Chen, X., Miflin, J. K., Zhang, P. and Blackall, P. J. (1996) Development and application of DNA probes and PCR tests for Haemophilusparagallinarum. Avian Dis. 40, 398–407.

    Article  PubMed  CAS  Google Scholar 

  18. Canto, G. J. and Biberstein, E. L. (1982) Serological diversity in Haemophilus somnus. J. Clin. Microbiol. 15, 1009–1015.

    CAS  Google Scholar 

  19. Angen, Ø., Ahrens, P., and Tegtmeier, C. (1998) Development of a PCR test for identification of Haemophilus somnus in pure and mixed cultures. Vet. Microbiol. 63, 39–48.

    Article  PubMed  CAS  Google Scholar 

  20. Tegtmeier, C., Uttenthal, Aa., Friis, N. F., Jensen, N. E., and Jensen H. E. (1999) Pathological and microbiological studies on pneumononic lungs from Danish calves. J. Vet. Med. B 46, 693–700.

    Article  CAS  Google Scholar 

  21. Townsend, K. M., Boyce, J. D., Chung, J. Y., Frost, A. J., and Adler, B. (2001) Genetic organization of Pasteurella multocida cap loci and development of a multiplex capsular PCR typing system. J. Clin. Microbiol. 39, 924–929.

    Article  PubMed  CAS  Google Scholar 

  22. Rimler, R. B. and Rhoades, K. R. ( 1989) Pasteurella multocida, in Pasteurella and Pasteurellosis (Adlam, C. and Rutter, J. M., ), Academic Press, London, UK, pp. 37–73.

    Google Scholar 

  23. Townsend, K. M., Frost, A. J., Le, C. W., Papadimitriou, J. M., and Dawkins, H. J. S. (1998) Development of PCR assays for species-and type-specific identification of Pasteurella multocida isolates. J. Clin. Microbiol. 36, 1096–1100.

    PubMed  CAS  Google Scholar 

  24. Fisher, M. A., Weiser, G. C., Hunter, D. L., Ward, A. C. S. (1999) Use of a polymerase chain reaction method to detect the leukotoxin gene lktA in biogroup and biovariant isolates of Pasteurella hemolytica and P. trehalosi. Am. J. Vet. Res. 60, 1402–1406.

    PubMed  CAS  Google Scholar 

  25. Green, A. L., DuTeau, N. M., Miller, M. W., Triantis, J. M., and Salman, M. D. (1999) Polymerase chain reaction techniques for differentiaitng cytotoxic and noncytotoxic Pasteurella terhalosi form Rocky Mountain bighorn sheep. Am. J. Vet. Res. 60, 583–588.

    PubMed  CAS  Google Scholar 

  26. Miflin, J. K. and Blackall, P. J. (2001) Development of a 23S rRNA based PCR Assay for the identification of Pasteurella multocida. Lett. Appl. Microbiol. 33, 216–221.

    Article  PubMed  CAS  Google Scholar 

  27. Kasten, R. W., Carpenter, T. E., Snipes, K. P. and Hirsh, D. C. (1997) Detection of Pasteurella multicida-specific DNA in turkey flocks by use of the polymerase chain reaction. Avian Dis. 41, 676–682.

    Article  PubMed  CAS  Google Scholar 

  28. Brickell, S. K., Thomas, L. M., Long, K. A., Panaccio, M. and Widders, P. R. (1998) Development of a PCR test based on a gene region associated with the pathogenicity of Pasteurella multocida serotype B:2, the causal agent of Haemorrhagic septicaemia in Asia. Vet. Microbiol. 59, 295–307.

    Article  PubMed  CAS  Google Scholar 

  29. Hotzel, H., Erler, W. und Schimmel D. (1997) Nachweis des Dermonekrotoxin-Gens in Pasteurella-multocida-Stämmen mittels Polymerase-Kettenreaktion (PCR). Berl. Münch. Tierärztl. Wschr. 110, 139–142.

    CAS  Google Scholar 

  30. Kamp, E. M., Bokken, G. C. A. M., Vermeulen, T. M. M., et al. (1996) A specific and sensitive PCR assay suitable for large-scale detection of toxigenic Pasteurella multocida in nasal and tonsillar swabs specimens of pigs. J. Vet. Diagn. Invest. 8, 304–309.

    PubMed  CAS  Google Scholar 

  31. Lichtensteiger, C. A., Steenbergen, S. M., Lee, R. M., Polson, D. D. and Vimr, E. R. (1996) Direct PCR analysis for toxigenic Pasteurella multocida. J. Clin. Microbiol. 34, 3035–3039.

    PubMed  CAS  Google Scholar 

  32. Lee, C. W., Wilkie, I. W., Townsend, K. M., and Frost, A. J. (2000) The demonstration of Pasteurella multocida in the alimentary tract of chickens after experimental oral infection. Vet. Microbiol. 72, 47–55.

    Article  PubMed  CAS  Google Scholar 

  33. Tegtmeier, C., Angen, Ø. and Ahrens, P. (2000) Comparison of bacterial cultivation, PCR, in situ hybridization and immunohistochemistry as tools for diagnosis of Haemophilus somnus pneumonia in cattle. Vet. Microbiol. 76, 385–394.

    Article  PubMed  CAS  Google Scholar 

  34. Dziva, F., Christensen, H., Olsen, J. E. and Mohan, K. (2002) Random amplification of polymorphic DNA and phenotypic typing of Zimbabwean isolates of Pasteurella multocida. Vet. Microbiol., in press.

    Google Scholar 

  35. Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed. CSH Laboratory Press, Cold Spring Harbor, NY, USA.

    Google Scholar 

  36. Leisner, J. J., Pot, B., Christensen, H., et al. (1999) Identification of lactic acid bacteria from Chili Bo, a Malaysian food ingredient. Appl. Environ. Microbiol. 65, 599–605.

    PubMed  CAS  Google Scholar 

  37. Chen, X., Chen, Q., Zhang, P., Feng, W. and Blackall, P. J. (1998) Evaluation of a PCR test for the detection of Haemophilus paragallinarum in China. Avian Pathol. 27, 296–300.

    Article  PubMed  CAS  Google Scholar 

  38. Appuhamy, S., Parton, R., Coote, J. G. and Gibbs, H. A. (1997) Genomic fingerprinting of Haemophilus somnus by a combination of PCR methods. J. Clin. Microbiol. 35, 288–291.

    PubMed  CAS  Google Scholar 

  39. Petras, S. F., Chidambaram, M., Illyes, E. F., Froshauer, S., Weinstock, G. M. and Reese, C. (1995) Antigenic and virulence properties of Pasteurella hemolytica leukotoxin mutants. Infect. Immunol. 63, 1033–1039

    CAS  Google Scholar 

  40. Highlander, S. K., Fedorova, N. D., Dusek, D. M., Panciera, R., Alvarez, L. E. and Rinehart, C. (2000) Inactivation of Pasteurella (Mannheimia) hemolytica leukotoxin causes partial attenuation of virulence in a calf challenge model. Infect. Immunol. 68, 3916–3922.

    Article  CAS  Google Scholar 

  41. Murphy, G. L., Whitworth, L. C., Clinkenbeard, K. D. and Clinkenbeard, P.A. (1995) Hemolytic activity of the Pasteurella hemolytica leukotoxin. Infect. Immunol. 63, 3209–3212.

    CAS  Google Scholar 

  42. Fedorova, N. D., Highlander, S. K. (1997) Generation of targeted nonpolar gene insertions and operon fusions in Pasteurella hemolytica and creation of a strain that produces and secretes inactive leukotoxin. Infect. Immunol. 65, 2593–2598.

    CAS  Google Scholar 

  43. Burrows, L.L., Olah-Winfield, E. and Lo, R. Y. (1993) Molecular analysis of the leukotoxin determinants from Pasteurella hemolytica serotypes 1 to 16. Infect. Immunol. 61, 5001–5007.

    CAS  Google Scholar 

  44. Chang, Y. F., Ma, D. P., Shi, J. and Chengappa, M. M. (1993) Molecular characterization of a leukotoxin gene from a Pasteurella hemolytica-like organism, encoding a new member of the RTX toxin family. Infect. Immunol. 61, 2089–2095.

    CAS  Google Scholar 

  45. Davies, R. L., Whittam, T. S., and Selander, R. K. (2001) Sequence diversity and molecular evolution of the leukotoxin (lktA) gene in bovine and ovine strains of Mannheimia (Pasteurella) hemolytica. J. Bacteriol. 183, 1394–1404

    Article  PubMed  CAS  Google Scholar 

  46. Biberstein, E. L. and Gills, M. G. (1962) The relationship of the antigenic types of the A and T types of Pasteurella hemolytica. J. Comp. Pathol. 72, 316–320.

    PubMed  CAS  Google Scholar 

  47. Jaworski, M. D., Hunter, D. L. and Ward, A. C. S. (1998) Biovariants of isolates of Pasteurella from domestic and wild ruminants. J. Vet. Diagn. Invest. 10, 49–55.

    PubMed  CAS  Google Scholar 

  48. Veit, H. P., Wise, D. J., Carter, G. R. and Chengappa, M. M. (1998) Toxin production by Pasteurella granulomatis. Ann. N. Y. Acad. Sci. 849, 479–484.

    Article  PubMed  CAS  Google Scholar 

  49. Dewhirst, F. E., Paster, B. J., Olsen, I., and Fraser, G. J. (1993) Phylogeny of the Pasteurellaceae as determined by comparison of 16S ribosomal ribonucleic acid sequences. Zbl. Bakt. 279, 35–44.

    CAS  Google Scholar 

  50. Townsend, K., Gunawardana, G. A., and Briggs, R. E. (2002) PCR Identification and Typing of Pasteurella Multocida: An Update. Poster 13. International Pasteurellaceae Society Conference. Banff, AB, Canada. May 5-10, 2002.

    Google Scholar 

  51. Jong, M. F. De, Kamp, E., van der Schoot, A., and Banniseth, T. von (1996) Elimination of AR toxinogenic Pasteurella from infected sow herds by a combination of ART vaccination and testing sows with a PCR and ELISA test. Proceedings of the 14th IPVS Congress, Bologna, Italy, 7-10 July 1996. 274

    Google Scholar 

  52. Hunt, M. L., Adler, B. and Townsend, K. M. (2000) The molecular biology of Pasteurella multocida. Vet. Microbiol. 72, 3–25.

    Article  PubMed  CAS  Google Scholar 

  53. Angen, Ø., Ahrens, P. and Leser, T. D. (2001) Development of a PCR test for detection of Haemophilus parasuis. Abstract Z-43. ASM 101 st General Meeting Orlando Florida. ASM, Washington DC, USA.

    Google Scholar 

  54. Van Ostaaijen, J., Frey, J., Rosendal, S., and MacInnes, J. I. (1997). Actinobacillus suis strains isolated from healthy and diseased swine are clonal and carry apxICABD var.suis toxin genes. J. Clin. Microbiol. 35, 1131–1137.

    PubMed  Google Scholar 

  55. Paul, J. H., and Myers, B. (1982) Fluorometric determination of DNA in aquatic microorganisms by use of Hoechst 33258. Appl. Environ. Microbiol. 43, 1393–1399.

    PubMed  CAS  Google Scholar 

  56. Kasten, R. W., Hansen, L. M., Hjinojoza, J., Biebger, D., Ruehl, W. W. and Hirsch, D. C. (1995) Pasteurella multocida produces a protein with homology to the P6 outer membrane protein of Haemophilus influenzae. Infect. Immunol. 63, 989–993.

    CAS  Google Scholar 

  57. Christensen, H. Jørgensen, K. and Olsen, J. E. (1999) Differentiation of Campylobacter coli and C. jejuni by length and DNA sequence of the 16S-23S rRNA internal spacer region. Microbiology 145, 99–105.

    Article  PubMed  CAS  Google Scholar 

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Christensen, H., Bisgaard, M., Larsen, J., Elmerdahl Olsen, J. (2003). PCR-Detection of Hemophilus paragallinarum, Hemophilus somnus, Mannheimia (Pasteurella) hemolytica, Mannheimia spp., Pasteurella trehalosi, and Pasteurella multocida. In: Sachse, K., Frey, J. (eds) PCR Detection of Microbial Pathogens. Methods in Molecular Biology™, vol 216. Humana Press. https://doi.org/10.1385/1-59259-344-5:257

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  • DOI: https://doi.org/10.1385/1-59259-344-5:257

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