Development of a multi-locus sequence typing scheme for avian isolates of Pasteurella multocida.
ABSTRACT 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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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. · 1.73 Impact Factor
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ABSTRACT: The aims of the present study were to use multilocus sequence typing (MLST) of a diverse collection of Pasteurella multocida as to animal source, geography and time, including all available serovars of Carter, Heddleston, Little & Lyon, Namioka, Cornelius and Roberts, to further investigate the evolution of this species with a focus on two lineages, A (P. multocida subsp. multocida and P. multocida subsp. gallicida) and B (P. multocida subsp. septica), previously reported. Isolates of P. multocida (n = 116) including reference strains of major serotyping systems were investigated by MLST based on partial sequences of the genes adk, est, gdh, mdh, pgi, pmi, and zwf, and 67 sequence types (ST) were observed. Phylogenetic analysis of these concatenated sequences confirmed the separation of groups A (41 ST, 71 isolates) and B (22 ST, 38 isolates) out of the 67 STs. All Carter's serovars, 12 Heddleston serovars, all three Little-Lyon types, six out of seven Namioka serovars, all five of Robert's types and all four Cornelius' serovars were allocated in the A group, while group B included the remaining four Heddleston serovars 6, 7, 8 and 13, in addition to Namioka type 8:A. The overrepresentation of reference strains of serotyping systems in the A group contrasts the high number of isolates obtained from diseased birds in the B group the impact of which should be addressed in future vaccine development. Isolates from birds (25) dominated the B group which also included four isolates from Felidae, whereas group A included isolates from all types of hosts. The evolutionary implications of the lack of capsular type D, pig and bovine isolates in group B as well as its association with Aves and Felidae that also applied to the whole RIRDC MLST database, need further investigation. The combination of rpoB and 16S rRNA gene sequence comparison as well as the developed PCR test assigned isolates to lineage A represented by the type strain of P. multocida subsp. multocida or lineage B represented by the type strain of P. multocida subsp. septica. It was not possible to circumscribe either the A or B lineages with a set of conserved phenotypic characters, questioning the valid status of subspecies within P. multocida. Phylogenetic analysis carried out on individual MLST genes showed deviations as to single or multiple genes for 17 % of group A and 43 % of group B indicating that lineage A probably developed from lineage B, and that major changes are on-going. From a genotypical point of view, we conclude that P. multocida subsp. gallicida represents an artificial unit.Microbiology 01/2013; · 3.06 Impact Factor
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ABSTRACT: Two serovars of Pasteurella multocida, B:2 and E:2, have been reportedly associated with haemorrhagic septicaemia (HS), a peracute and devastating disease mainly affecting cattle and water buffaloes. We multilocus sequence typed (MLST) 64 isolates of P. multocida including 55 associated with HS and found that they mainly included sequence type (ST) 122 (n=50) and rarely ST63 (n=1), ST147 (n=2) and ST162 (n=2) compared to other members of the species isolated from other lesion types and hosts. Single-nucleotide polymorphisms suitable for specific detection of STs associated with HS were detected in the est gene. A new HS-est-RT-PCR (est indicating the target gene) specifically detected ST122, ST63, ST147 and ST162 associated with HS. The new HS-est-RT-PCR did not detect strains of ST151 with capsular type D isolated from pigs that were found positive with a previously published HS PCR detection method. The new HS-est-RT-PCR represents a fast and specific detection of the specific types of P. multocida involved in HS. The HS-est-RT-PCR developed in the current study seems to more accurately identify isolates of P. multocida associated with HS compared to PCR detection methods previously published.Veterinary Microbiology 02/2014; · 3.13 Impact Factor