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.

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    • "The MLST scheme was performed on 20 isolates according to the protocol of Subaaharan et al. (2010). All purified products were sequenced at the DNA Core Facility at the Centre for Applied Molecular Biology (CAMB), Ministry of Science and Technology, Lahore, Pakistan. "
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    ABSTRACT: A comparative genetic study of 23 field isolates and vaccine strains of Pasteurella multocida associated with haemorrhagic septicaemia cases from Pakistan and Thailand was done using pulsed field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). The MLST sequence type (ST) for all 20 of the 23 isolates tested was 122. The PFGE results showed one band difference between the Pakistani and the Thai isolates. Sequence type 122 is the dominant associated profile with haemorrhagic septicaemia (HS) cases in South Asia. The study supports the concept of using PFGE for short-term epidemiology and MLST for long-term epidemiology.
    Research in Veterinary Science 08/2013; 95(3). DOI:10.1016/j.rvsc.2013.07.003 · 1.41 Impact Factor
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    • "The DNA was used to amplify loci from 7 housekeeping genes. The primers and conditions were as per the MLST (RIRDC) scheme [18,19] As specified, 7 loci (adk, est, pmi, pgi, zwf, gdh, mdh) were used and gene fragments of lengths 570-808 bp were amplified. For the zwf locus, both sets of primers were used on all samples (ZWF-F1/ZWF-R1 and ZWF-F2/ZWF-R2). "
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    ABSTRACT: Pasteurella multocida causes disease in many host species throughout the world. In bovids, it contributes to bovine respiratory disease (BRD) and causes haemorrhagic septicaemia (HS). Previous studies have suggested that BRD-associated P. multocida isolates are of limited diversity. A multilocus sequence typing (MLST) scheme for P. multocida was used to determine whether the low levels of diversity reported are due to the limited discriminatory power of the typing method used, restricted sample selection or true niche association. Bovine respiratory isolates of P. multocida (n = 133) from the UK, the USA and France, collected between 1984 and 2008 from both healthy and clinically affected animals, were typed using MLST. Isolates of P. multocida from cases of HS, isolates from other host species and data from the MLST database were used as comparison. Bovine respiratory isolates were found to be clonal (I(S)(A) 0.45) with 105/128 belonging to clonal complex 13 (CC13). HS isolates were not related to bovine respiratory isolates. Of the host species studied, the majority had their own unique sequence types (STs), with few STs being shared across host species, although there was some cross over between porcine and bovine respiratory isolates. Avian, ovine and porcine isolates showed greater levels of diversity compared to cattle respiratory isolates, despite more limited geographic origins. The homogeneity of STs of bovine respiratory P. multocida observed, and the differences between these and P. multocida subpopulations from bovine non-respiratory isolates and non-bovine hosts may indicate niche association.
    BMC Microbiology 05/2011; 11(1):115. DOI:10.1186/1471-2180-11-115 · 2.73 Impact Factor
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    • "Pulsed-field gel electrophoresis (PFGE) and PCRbased methods such as random-amplified polymorphic DNA (RAPD) typing are highly discriminatory and useful for investigations of disease outbreaks (Blackall and Miflin, 2000; Dziva et al., 2008). Multilocus sequence typing (MLST) (Subaaharan et al., 2010) has advantages over PFGE and RAPD typing in terms of reproducibility and portability , allowing global analysis of strains. However, the use of housekeeping genes may limit discriminatory power and reduce its utility for investigations of disease outbreaks or in farm level epidemiology. "
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    ABSTRACT: The molecular epidemiology of Pasteurella multocida has rarely been studied at the farm level in cattle. The aim of this study was to determine whether single or multiple strains of P. multocida tend to exist within farms. Molecular characterisation was carried out on isolates obtained from nasal swabs from 105 calves from 32 randomly selected beef and dairy farms located throughout Scotland, and from 131 calves from 20 farms in the Mayenne region of France, where sampling occurred in response to respiratory disease outbreaks. P. multocida isolates were characterised by random-amplified polymorphic DNA (RAPD) typing and pulsed-field gel electrophoresis (PFGE) using restriction enzyme ApaI. In addition, isolates representative of each farm/RAPD profile combination were typed by multilocus sequence typing (MLST). Among 105 Scottish isolates, 15 RAPD profiles were distinguished. The majority of farms (27/32) had indistinguishable profiles in all positive animals. Five farms had two profiles. Among 140 French isolates, 23 RAPD profiles were distinguished. More within-farm heterogeneity was observed although 10/20 farms had just one profile (E4) in sampled calves. Profile E4 accounted for 60% (84/140) of French isolates. PFGE was more discriminatory than RAPD but confirmed results with respect to within farm homogeneity or heterogeneity of strains, whereas MLST was not discriminatory enough for farm level epidemiology. As in other host species, either several strains or one dominant strain of P. multocida may exist within farms, with evidence for a role of management factors such as movements onto the farm in the number of strains detected.
    Veterinary Microbiology 03/2011; 151(3-4):329-35. DOI:10.1016/j.vetmic.2011.03.018 · 2.51 Impact Factor
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