Development of a multi-locus sequence typing scheme for avian isolates of Pasteurella multocida

Department of Microbiology and Parasitology, School of Molecular and Microbial Sciences, The University of Queensland, St Lucia, Australia.
Veterinary Microbiology (Impact Factor: 2.51). 03/2010; 141(3-4):354-61. DOI: 10.1016/j.vetmic.2010.01.017
Source: PubMed


A total of 63 isolates of Pasteurella multocida from Australian poultry, all associated with fowl cholera outbreaks, and three international reference strains, representing the three subspecies within P. multocida were used to develop a multi-locus sequence typing scheme. Primers were designed for conserved regions of seven house-keeping enzymes -adk, est, gdh, mdh, pgi, pmi and zwf - and internal fragments of 570-784 bp were sequenced for all isolates and strains. The number of alleles at the different loci ranged from 11 to 20 and a total of 29 allelic profiles or sequence types were recognised amongst the 66 strains. There was a strong concordance between the MLST data and the existing multi-locus enzyme electrophoresis and ribotyping data. When used to study a sub-set of isolates with a known detailed epidemiological history, the MLST data matched the results given by restriction endonuclease analysis, pulsed-field gel electrophoresis, ribotyping and REP-PCR. The MLST scheme provides a high level of resolution and is an excellent tool for studying the population structure and epidemiology of P. multocida.

8 Reads
  • Source
    • "Twenty-three other Pasteurellaceae genomes were identified and downloaded (Table S2). Genomes were searched for the presence of the housekeeping genes of the MLST schemes of H. influenzae (Meats et al., 2003) and Pasteurella multocida (Subaaharan et al., 2010), as well as the phylogenetic marker genes atpD, infB, recN and rpoA (Zeigler, 2003). Eleven genes (adk, atpD, atpG, frdB, infB, mdh, pgi, recA, recN, rpoA and zwf) were identified in all genomes, and the genes from each strain were concatenated using CLC Main Workbench (CLC Bio) and used for phylogenetic analysis. "
    [Show abstract] [Hide abstract]
    ABSTRACT: The newly described species Haemophilus sputorum has been cultured from the upper respiratory tract of humans and appears to have little pathogenic potential. The species encode a capsular biosynthesis locus of approximately 12 kb composed of three distinct regions. Region I and III genes, involved in export and processing of the capsular material, show high similarity to the corresponding genes in capsulate lineages of the pathogenic species Haemophilus influenzae; indeed, standard bexA and bexB PCRs for detection of capsulated strains of H. influenzae give positive results with strains of H. sputorum. Three open reading frames are present in region II of the sequenced strain of H. sputorum, of which a putative phosphotransferase showed homology with corresponding genes from H. influenzae serotype c and f. Phylogenetic analysis of housekeeping genes from 24 Pasteurellaceae species showed that H. sputorum was only distantly related to H. influenzae. In contrast to H. influenzae, the capsule locus in H. sputorum is not associated with transposases or other transposable elements. Our data suggest that the capsule locus of capsulate lineages of H. influenzae may relatively recently have been recruited from the commensal species H. sputorum by horizontal gene transfer.
    Microbiology 03/2015; 161(6). DOI:10.1099/mic.0.000081 · 2.56 Impact Factor
    • "All the isolates were subjected to MLST scheme developed by Subaaharan et al. (2010) for P. multocida. PCR amplification and sequencing was carried out for seven housekeeping genes using primers and protocols available at RIRDC MLST Database ( of PUBMLST developed by Jolley et al. (2004) and sited at University of Oxford. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Multilocus sequence typing (MLST), a sequence-based typing method for bacterial pathogens, is currently the best method for long-term epidemiological study and to understand the population structure of the bacteria. This investigation was carried out to study the diversity of Pasteurella multocida isolates circulating in India. Ten different sequence types (ST) identified in this study are ST 122 from cattle, goat, mithun and pig; ST 50 from pig; ST 9 from cattle and sheep; ST 229 from cattle and goat; ST 71 and ST 277 from cattle; and ST 129, ST 280, ST 281 and ST 282 from avian species. Of these, ST 277, ST 280, ST 281 and ST 282 were identified for the first time. The analysis of results provides novel epidemiological information on the circulation of multiple STs across India. The majority of STs or their variants identified in this study have already been reported from different parts of the globe. This suggests that probably transboundary spread of strains across countries and continents has occurred across evolutionary time and is still happening. The isolation of ST 122 from small ruminants and pigs suggests that these species may be included in the preventive vaccination policy for effective control of haemorrhagic septicaemia in India.
    Transboundary and Emerging Diseases 09/2014; DOI:10.1111/tbed.12270 · 2.94 Impact Factor
  • Source
    • "Genotyping. All isolates were examined by the RIRDC MLST scheme as described by Subaaharan et al. (2010). This MLST scheme is based on sequencing of an internal fragment of each of seven housekeeping genes, with 450 to 500 base pair internal fragments of each gene being used. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Pasteurella multocida was isolated from poultry on six farms (one free-range duck farm, one free-range turkey farm, one conventional enclosed turkey farm, and three free-range layer farms) suffering fowl cholera outbreaks. In addition, historical isolates from previous outbreaks were available for the conventional turkey farm and the three free-range layer farms. The isolates were serotyped using the Heddleston scheme and genotyped using multi-locus sequencing typing. In the current outbreaks, two of the farms had two different sequence types (STs) of P. multocida in the investigated outbreak (the free-range turkey farm and one of the free-range layer farms). The remaining four farms had one ST within the investigated outbreak. In looking at the historical isolates, two of the four farms had multiple genotypes involved. On the four farms with historical isolates from previous outbreaks, at least one new genotype was present in the investigated outbreak as compared with the historical isolates. On one layer farm, one genotype persisted over a 10-year period. Serotyping revealed the presence of multiple serovars in the current and historical outbreaks, with serovars sometimes changing over time. This study has shown that several STs of P. multocida can be present during some outbreaks of fowl cholera, although other outbreaks involve a single ST. Also, the STs present on a property suffering repeated fowl cholera can both persist and change over time.
    Avian Pathology 12/2013; 42(6):581-5. DOI:10.1080/03079457.2013.854861 · 1.64 Impact Factor
Show more