Monoclonal antibodies (MAbs) against were obtained by the fusion of SP2/0-Ag14 murine myeloma cells and spleen cells from BALB/c mice immunized with a whole-bacterial-cell suspension (WC) of strain SW124 (serotype 4). Two MAbs showing strong reactivity in ELISA were further characterized using SDS-PAGE and Western-blot assays. Different treatments of the WC indicated that MAbs 4D5 and 4G9 identified epitopes of proteinic and polysaccharidic nature, respectively. Electron microscopic examination revealed that, unlike the proteinic epitopes, the lipopolysaccharidic epitopes were exposed on the surface of the cell. Using coagglutination, Western-blot and dot-blot assays it was found that both MAbs recognized common epitopes of all the reference strains and field isolates of . None of the other bacteria tested reacted with the MAbs. These results indicated that both the proteinic and polysaccharidic antigens carried species-specific epitopes. It is suggested that these MAbs may potentially be useful for identification of isolates as well as for developing serological diagnostic tools. MAbs 4D5 and 4G9 were unable to kill in the presence of complement. However, an enhanced bacterial clearance from blood was observed in mice inoculated with either of the MAbs. Highly significant protection was observed in mice using MAb 4G9. This is believed to be the first report of MAbs capable of identifying common species-specific antigens of and of their implication in protection against challenge infection in mice.


Article metrics loading...

Loading full text...

Full text loading...



  1. Abul-Milh, M., Paradis, S.-É., Dubreuil, D. & Jacques, M.(1999). Binding of Actinobacillus pleuropneumoniae lipopolysaccharides to glycosphingolipids evaluated by thin-layer chromatography. Infect Immun 67, 4983–4987. [Google Scholar]
  2. Achacha, M. & Mittal, K. R.(1996). Identification and characterization of genus-specific epitopes of Serpulina species using monoclonal antibodies. Vet Microbiol 48, 73–85.[CrossRef] [Google Scholar]
  3. Amano, H., Shibata, M., Kajio, N. & Morozumi, T.(1994). Pathologic observations of pigs intranasally inoculated with serovar 1, 4 and 5 of Haemophilus parasuis using immunoperoxidase method. J Vet Med Sci 56, 639–644.[CrossRef] [Google Scholar]
  4. Beck, E. & Bremer, E.(1980). Nucleotide sequence of the gene ompA coding the outer membrane protein II of Escherichia coli K-12. Nucleic Acids Res 8, 3011–3027.[CrossRef] [Google Scholar]
  5. Bélanger, M., Dubreuil, D., Harel, J., Girard, C. & Jacques, M.(1990). Role of lipopolysaccharides in adherence of Actinobacillus pleuropneumoniae to porcine tracheal rings. Infect Immun 58, 3523–3530. [Google Scholar]
  6. Bélanger, M., Rioux, S., Foiry, B. & Jacques, M.(1992). Affinity for porcine respiratory tract mucus is found in some isolates of Actinobacillus pleuropneumoniae. FEMS Microbiolog Lett 76, 119–125. [Google Scholar]
  7. Bélanger, M., Dubreuil, D. & Jacques, M.(1994). Proteins found within porcine respiratory tract secretions bind lipopolysaccharides of Actinobacillus pleuropneumoniae. Infect Immun 62, 868–873. [Google Scholar]
  8. Bradford, M. M.(1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72, 248–254.[CrossRef] [Google Scholar]
  9. Carlone, G. M., Thomas, M. L., Rumschlag, H. S. & Sottnek, F. O.(1986). Rapid microprocedure for isolating detergent-insoluble outer membrane proteins from Haemophilus species. J Clin Microbiol 24, 330–332. [Google Scholar]
  10. Datta, D. B., Arden, B. & Henning, U.(1977). Major proteins of the Escherichia coli outer cell membrane as bacteriophage receptors. J Bacteriol 131, 821–829. [Google Scholar]
  11. Freudl, R. & Cole, S. T.(1983). Cloning and molecular characterization of the ompA gene from Salmonella typhimurium. Eur J Biochem 134, 497–502.[CrossRef] [Google Scholar]
  12. Haesebrouck, F., Chiers, K., Van Overbeke, I. & Ducatelle, R.(1997).Actinobacillus pleuropneumoniae infections in pigs: the role of virulence factors in pathogenesis and protection. Vet Microbiol 58, 239–249.[CrossRef] [Google Scholar]
  13. Hartmann, L., Schröder, W. & Lübke, A.(1995). Isolation of the major outer-membrane protein of Actinobacillus pleuropneumoniae and Haemophilus parasuis. J Vet Med B 42, 59–63.[CrossRef] [Google Scholar]
  14. Hewick, R. M., Hunkapiller, M. W., Hood, L. E. & Dreyer, W. J.(1981). A gas-liquid solid phase peptide and protein sequenator. J Biol Chem 256, 7990–7997. [Google Scholar]
  15. Jacques, M. & Paradis, S. E.(1998). Adhesin-receptor interactions in Pasteurellaceae. FEMS Microbiol Rev 22, 45–59. [Google Scholar]
  16. Jacques, M., Bélanger, M., Roy, G. & Foiry, B.(1991). Adherence of Actinobacillus pleuropneumoniae to porcine tracheal epithelial cells and frozen lung sections. Vet Microbiol 27, 133–143.[CrossRef] [Google Scholar]
  17. Jeannin, P., Magistrelli, G., Goetsch, L., Haeuw, J. F., Thieblemont, N., Bonnefoy, J. Y. & Delneste, Y.(2002). Outer membrane protein A (OmpA): a new pathogen-associated molecular pattern that interacts with antigen presenting cells–impact on vaccine strategies. Vaccine 20, A23–A27.[CrossRef] [Google Scholar]
  18. Kielstein, V. P.(1991). Zur Glässerschen Krankheit des Schweines Untersuchungen über Zusammenhänge zwischen serologischen Eigenschaften, Kapselausbilding und Virulenz von Haemophilus-parasuis-Stämmen. Mh Vet-Med 46, 137–142. [Google Scholar]
  19. Kielstein, P., Rosner, H. & Müller, W.(1991). Typing of heat-stable soluble Haemophilus parasuis antigen by means of agar gel precipitation and the dot-blot procedure. J Vet Med B 38, 315–320.[CrossRef] [Google Scholar]
  20. Kim, K. S.(2002). Strategy of Escherichia coli for crossing the blood-brain barrier. J Infect Dis 186, S220–224.[CrossRef] [Google Scholar]
  21. Koebnik, R.(1995). Proposal for a peptidoglycan-associating alpha-helical motif in the C-terminal regions of some bacterial cell-surface proteins. Mol Microbiol 16, 1269–1270.[CrossRef] [Google Scholar]
  22. Koebnik, R., Locher, K. P. & Van Gelder, P.(2000). Structure and function of bacterial outer membrane proteins: barrels in a nutshell. Mol Microbiol 37, 239–253.[CrossRef] [Google Scholar]
  23. Köhler, G. & Milstein, C.(1975). Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256, 495–497.[CrossRef] [Google Scholar]
  24. Laemmli, U. K.(1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.[CrossRef] [Google Scholar]
  25. Li, Z. S., Jensen, N. S., Bélanger, M., L'Espérance, M. C. & Jacques, M.(1992). Molecular characterization of Serpulina (Treponema) hyodysenteriae isolates representing serotypes 8 and 9. J Clin Microbiol 30, 2941–2947. [Google Scholar]
  26. Lugtenberg, B., van Boxtel, R., Evenberg, D., de Jong, M., Storm, P. & Frik, J.(1986). Biochemical and immunological characterization of cell surface proteins of Pasteurella multocida strains causing atrophic rhinitis in swine. Infect Immun 52, 175–182. [Google Scholar]
  27. Matsudaira, P.(1987). Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes. J Biol Chem 262, 10035–10038. [Google Scholar]
  28. Miniats, O. P., Smart, N. L. & Rosendal, S.(1991). Cross protection among Haemophilus parasuis strains in immunized gnotobiotic pigs. Can J Vet Res 55, 37–41. [Google Scholar]
  29. Mittal, K. R. & Ingram, D. G.(1975). Factors involved in bactericidal activity of sheep serum. Am J Vet Res 36, 1183–1187. [Google Scholar]
  30. Mittal, K. R., Higgins, R. & Larivière, S.(1983). Identification and serotyping of Haemophilus pleuropneumoniae by coagglutination test. J Clin Microbiol 18, 1351–1354. [Google Scholar]
  31. Møller, K., Fussing, V., Grimont, P. A. D., Paster, B. J., Dewhirst, F. E. & Kilian, M.(1996).Actinobacilus minor sp. nov., Actinobacillus porcinus sp. nov., and Actinobacillus indolicus sp. nov., three new V factor-dependent species from the respiratory tract of pigs. Int J Syst Bacteriol 46, 951–956.[CrossRef] [Google Scholar]
  32. Morozumi, T. & Nicolet, J.(1986a). Morphological variations of Haemophilus parasuis strains. J Clin Microbiol 23, 138–142. [Google Scholar]
  33. Morozumi, T. & Nicolet, J.(1986b). Some antigenic properties of Haemophilus parasuis and a proposal for serological classification. J Clin Microbiol 23, 1022–1023. [Google Scholar]
  34. Mutharia, L. M. & Hancock, R. E. W.(1985). Characterization of two surface-localized antigenic sites on porin protein F of Pseudomonas aeruginosa. Can J Microbiol 31, 381–386.[CrossRef] [Google Scholar]
  35. Nicolet, J., Paroz, P. H. & Krawinkler, M.(1980). Polyacrylamide gel electrophoresis of whole-cell proteins of porcine strains of Haemophilus. Int J Syst Bacteriol 30, 69–76.[CrossRef] [Google Scholar]
  36. Nikaido, H. & Vaara, M.(1985). Molecular basis of bacterial outer membrane permeability. Microbiol Rev 49, 1–32. [Google Scholar]
  37. Oliveira, S. & Pijoan, C.(2002). Diagnosis of Haemophilus parasuis in affected herds and use of epidemiologial data to control disease. J Swine Health Prod 10, 221–225. [Google Scholar]
  38. Oliveira, S., Galina, L. & Pijoan, C.(2001). Development of a PCR test to diagnose Haemophilus parasuis infections. J Vet Diagn Invest 13, 495–501.[CrossRef] [Google Scholar]
  39. Oliveira, S., Pijoan, C. & Morrison, R.(2002). The role of Haemophilus parasuis in nursery mortality. In Proceedings Allen D Leman Swine Conference, pp. 111–113. University of Minnesota, USA: Veterinary Outreach Programs.
  40. Paradis, S.-É., Dubreuil, D. & Jacques, M.(1996). Examination of surface polysaccharides of Actinobacillus pleuropneumoniae serotype 1 grown under iron-restricted condition. FEMS Microbiology Lett 137, 201–206.[CrossRef] [Google Scholar]
  41. Pautsch, A. & Schulz, G. E.(1998). Structure of the outer membrane protein A transmembrane domain. Nature Struct Biol 5, 1013–1017.[CrossRef] [Google Scholar]
  42. Pautsch, A. & Schulz, G. E.(2000). High-resolution structure of the OmpA membrane domain. J Mol Biol 298, 273–282.[CrossRef] [Google Scholar]
  43. Prasadarao, N. V., Wass, C. A., Weiser, J. N., Stins, M. F., Huang, S. H. & Kim, K. S.(1996). Outer membrane protein A of Escherichia coli contributes to invasion of brain microvascular endothelial cells. Infect Immun 64, 146–153. [Google Scholar]
  44. Prasadarao, N. V., Wass, C. A., Stins, M. F., Shimada, H. & Kim, K. S.(1999). Outer membrane protein A-promoted actin condensation of brain microvascular endothelial cells is required for Escherichia coli invasion. Infect Immun 67, 5775–5783. [Google Scholar]
  45. Rapp-Gabrielson, V. J., Ross, R. F. & Nicolet, J.(1986). Characterization of the outer membrane proteins of Hemophilus parasuis. In Proceedings of the 9th International Pig Veterinary Society Congress, p. 262. Barcelona: Departamento de Prensa y Publicaciones
  46. Rebers, P. A., Phillips, M., Rimler, R., Boykins, R. A. & Rhodes, K. R.(1980). Immunizing properties of Westphal lipopolysaccharide from an avian strain of Pasteurella multocida. Am J Vet Res 41, 1650–1654. [Google Scholar]
  47. Schweizer, M. & Henning, U.(1977). Action of a major outer cell envelope membrane protein in conjugation of Escherichia coli K-12. J Bacteriol 129, 1651–1652. [Google Scholar]
  48. Spinola, S. M., Griffiths, G. E., Shanks, K. L. & Blake, M. S.(1993). The major outer membrane protein of Haemophilus ducreyi is a member of the OmpA family of proteins. Infect Immun 61, 1346–1351. [Google Scholar]
  49. Sugawara, E. & Nikaido, H.(1992). Pore-forming activity of OmpA protein of Escherichia coli. J Biol Chem 267, 2507–2511. [Google Scholar]
  50. Tadjine, M., Mittal, K. R., Bourdon, S. & Gottschalk, M.(2004). Development of a new serological test for serotyping Haemophilus parasuis isolates and determination of their prevalence in North America. J Clin Microbiol 42, 839–840.[CrossRef] [Google Scholar]
  51. Tagawa, Y., Haritani, M., Ishikawa, H. & Yuasa, N.(1993). Characterization of a heat-modifiable outer membrane protein of Haemophilus somnus. Infect Immun 61, 1750–1755. [Google Scholar]
  52. Tascon, R. I., Vazquez-Boland, J. A., Gutiérrez-Martin, C. B., Rodriguez-Barbosa, J. I. & Rodriguez-Ferri, E. F.(1996). Virulence factors of the swine pathogen Actinobacillus pleuropneumoniae. Microbiologia 12, 171–184. [Google Scholar]
  53. Towbin, H., Staehelin, T. & Gordon, J.(1979). Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A 76, 4350–4354.[CrossRef] [Google Scholar]
  54. Tsai, C. M. & Frasch, C. E.(1982). A sensitive silver stain for detection of lipopolysaccharides in polyacrylamide gels. Anal Biochem 119, 115–119.[CrossRef] [Google Scholar]
  55. Vasfi Marandi, M. & Mittal, K. R.(1996). Characterization of an outer membrane protein of Pasteurella multocida belonging to the OmpA family. Vet Microbiol 53, 303–314.[CrossRef] [Google Scholar]
  56. Weiser, J. N. & Gotschlich, E. C.(1991). Outer membrane protein A (OmpA) contributes to serum resistance and pathogenicity of Escherichia coli K-1. Infect Immun 59, 2252–2258. [Google Scholar]
  57. Wilson, M. E.(1991). The heat-modifiable outer membrane protein of Actinobacillus actinomycetemcomitans: relationship to OmpA proteins. Infect Immun 59, 2505–2507. [Google Scholar]
  58. Woodward, M. P., Young, W. W. & Bloodgood, R. A.(1985). Detection of monoclonal antibodies specific for carbohydrate epitopes using periodate oxidation. J Immunol Methods 78, 143–153.[CrossRef] [Google Scholar]

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error