LPS-PCR typing of ovine Pasteurella multocida isolates from Iran based on (L1 to L8) outer core biosynthesis loci

Document Type: Original Articles

Authors

1 Pasteurella National Research Laboratory, Razi Vaccine and Serum Research Institute, Agricultural Research, Education, and Extension Organization, Karaj, Iran

2 assistant professor razi institute

Abstract

Pasteurella multocida isa gram-negative bacterial pathogen that is causative agent of a wide range of diseases in many animal species and humans. Lipopolysaccharides (LPS) are an important virulence factor, minor changes to structure of which can exert dramatic effects on pathogenicity of P. multocida in its host. LPS can be used for the identification and classification of strains with somatic typing systems.The aim of this study was to identify the LPS genotypes of the ovine P. multocida isolates obtained from pneumonia cases in Iran. The LPS genotype of the isolates was determined using eight specific primers for LPS outer core biosynthesis loci. The LPS genes were amplified by polymerase chain reaction (PCR), then they were sequenced and compared to the sequences registered in the GenBank. Of the 32 ovine P. multocida isolates tested, 21 (65.62%) isolates belonged to genotype L6, 9 (28.12%) isolates contained genotype L3, 1 (3.12%) isolate had both L3 and L6 loci, and 1 (3.12%) isolate remained untypeable. The LPS-PCR was able to type 31 of 32 field ovine isolates from Iran. According to the phylogenetic analysis, L3 genotype isolates were grouped into two distinct lineages. LPS gene sequences among L6 genotypes of ovine P. multocida isolates from Iran and the related sequences in the GenBank were highly similar (>99.5%). LPS-PCR is an accurate genotyping method that was able to classify P. multocida strains into one of the eight distinct LPS genotypes.

Keywords

Main Subjects


Article Title [French]

LPS-PCR typage des isolats iraniens de Pasteurella multocida de mouton sur la base des gènes externes (L1 à L8) LPS

Abstract [French]

Pasteurella multocida est une bactérie Gram négative pathogène chez de nombreuses espèces animales ainsi que pour l’homme. De toute évidence, les lipopolysaccharides (LPS) représentent un facteur pathogène important et le moindre changement dans leur structure peut fondamentalement affecter le pouvoir pathogène de Pasteurella multocida.De plus, les LPS peuvent être utilisés comme un facteur important dans le système de typage somatique pour l'identification et la classification des bactéries utilisées. Les génotypes des LPS des isolats de moutons ont été déterminés avec 8 paires d'amorces spécifiques à certains gènes exprimés par le noyau externe des LPS. Ensuite, les séquençages des gènes de LPS amplifiés par PCR ont été déterminées et ont été comparées aux séquences déjà existantes dans la banque de gènes. Parmi les 32 isolats de Pasteurella multocida de mouton testés, 21 isolats (62.65%) avaient le génotype L6, 9 isolats (12.28%) le génotype L3 , 1 isolat (3.12%) montraient les deux génotypes L3 et L6 ; et un isolat (3.12%) était inclassable. La méthode LPS-PCR a été en mesure de déterminer le génotype de 31 des 32 isolats étudiés. Sur la base de l'analyse phylogénétique, les isolats montrant le génotype L3 ont été regroupés en deux sous-catégories. Le pourcentage de similitude des séquences des gènes LPS entre les différents isolats génotypiques de mouton répertoriés dans la banque de gènes était considérablement élevé (>99.5%). Cette étude montre que la LPS-PCR est une méthode de génotypage précise qui peut classifier les isolats de Pasteurella multocida dans l'un des huit génotypes des LPS.

Keywords [French]

  • Pasteurella multocida
  • Mouton
  • noyau externe de LPS
  • le typage PCR
Brogden, K.A., Rebers, P.A., 1978. Serologic examination of the Westphal-type lipopolysaccharides of Pasteurella multocida. Am J Vet Res 39, 1680-1682.

Christensen, J.P., Bisgaard, M., 2000. Fowel cholera. Rev Sci Tech OIE 19, 626-637.

Ewers, C., Lübke-Becker, A., Bethe, A., Kießling, S., Filter, M., Wieler, L.H., 2006. Virulence genotype of Pasteurella multocida strains isolated from different hosts with various disease status. Veterinary Microbiology 114, 304-317.

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

Harper, M., Cox, A.D., Adler, B., Boyce, J.D., 2011a. Pasteurella multocida lipopolysaccharide: the long and the short of it. Vet Microbiol 153, 109-115.

Harper, M., Michael, F., Vinogradov, E., John, M., Steen, J.A., Van Dorsten, L., et al., 2013a. Structure and biosynthetic locus of the lipopolysaccharide produced by pasteurella multocida serovars 8 and 13 and the identification of a novel phosphor-glycero moiety. Glycobiology 23, 286-294.

Harper, M., St Michael, F., John, M., Steen, J., van Dorsten, L., Parnas, H., et al., 2014. Structural analysis of lipopolysaccharide produced by Heddleston serovars 10, 11, 12 and 15 and the identification of a new Pasteurella multocida lipopolysaccharide outer core biosynthesis locus, L6. Glycobiology 24, 649-659.

Harper, M., St Michael, F., John, M., Vinogradov, E., Adler, B., Boyce, J.D., et al., 2011b. Pasteurella multocida Heddleston serovars 1 and 14 express different lipopolysaccharide structures but share the same lipopolysaccharide biosynthesis outer core locus. Vet Microbiol 150, 289-296.

Harper, M., St Michael, F., John, M., Vinogradov, E., Steen, J.A., van Dorsten, L., et al., 2013b. Pasteurella multocida Heddleston serovar 3 and 4 strains share a common lipopolysaccharide biosynthesis locus but display both inter- and intrastrain lipopolysaccharide heterogeneity. J Bacteriol 195, 4854-4864.

Harper, M., St Michael, F., Vinogradov, E., John, M., Boyce, J.D., Adler, B., et al., 2012. Characterization of the lipopolysaccharide from Pasteurella multocida Heddleston serovar 9: identification of a proposed bi-functional dTDP-3-acetamido-3,6-dideoxy-alpha-D-glucose biosynthesis enzyme. Glycobiology 22, 332-344.

Heddleston, K.L., Gallagher, J.E., Rebers, P.A., 1972. Fowl cholera: gel diffusion precipitin test for serotyping Pasteruella multocida from avian species. Avian Dis 16, 925-936.

Raetz, C.R.H., Whitfield, C., 2002. Lipopolysaccharide Endotoxins. Annual Review of Biochemistry 71, 635-700.

Shayegh, J., Dolgari-Sharaf, J., Mikaili, P., Namvar, H., 2009. Pheno- and genotyping of Pasteurella multocida isolated from goat in Iran. Af J Biotechnol 8, 3707-3710.

Singh, R., Blackall, P.J., Remington, B., Turni, C., 2013. Studies on the presence and persistence of Pasteurella multocida serovars and genotypes in fowl cholera outbreaks. Avian Pathol 42, 581-585.

St Michael, F., Harper, M., Parnas, H., John, M., Stupak, J., Vinogradov, E., et al., 2009. Structural and genetic basis for the serological differentiation of Pasteurella multocida Heddleston serotypes 2 and 5. J Bacteriol 191, 6950-6959.

Townsend, K.M., Boyce, J.D., Chung, J.Y., Frost, A.J., 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.

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

Wilkie, I.W., Harper, M., Boyce, J.D., Adler, B., 2012. Pasteurella multocida: diseases and pathogenesis. Curr Top Microbiol Immunol 361, 1-22.

Wilson, M.A., Morgan, M.J., Barger, G.E., 1993. Comparison of DNA fingerprinting and serotyping for identification of avian Pasteurella multocida isolates. J Clin Microbiol 31, 255-259.