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Single nucleotide polymorphism genotyping of Erysipelothrix rhusiopathiae isolates from pigs affected with chronic erysipelas in Japan.

ABSTRACT: Over the past decades, Erysipelothrix rhusiopathiae strains displaying similar phenotypic and genetic profiles of the attenuated, acriflavine-resistant E. rhusiopathiae Koganei 65-0.15 strain (serovar 1a) have been frequently isolated from pigs affected with chronic erysipelas in Japan. In this study, using the conventional PCR assay that was designed to detect strain-specific single nucleotide polymorphism (SNP) sites found in the genome of the vaccine strain, we analyzed E. rhusiopathiae isolates from pigs with chronic disease in farms where the Koganei vaccine was used. Out of a total of 155 isolates, 101 isolates (65.2%) were determined to be the vaccine strain by SNP-based PCR. Among the 101 PCR-positive isolates, four isolates were found to be sensitive to acriflavine.

SUBMITTER: Shiraiwa K

PROVIDER: S-EPMC5402189 | biostudies-literature | 2017 Apr

REPOSITORIES: biostudies-literature

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Single nucleotide polymorphism genotyping of Erysipelothrix rhusiopathiae isolates from pigs affected with chronic erysipelas in Japan.

Shiraiwa Kazumasa K Ogawa Yohsuke Y Nishikawa Sayaka S Kusumoto Masahiro M Eguchi Masahiro M Shimoji Yoshihiro Y

The Journal of veterinary medical science 20170302 4

Over the past decades, Erysipelothrix rhusiopathiae strains displaying similar phenotypic and genetic profiles of the attenuated, acriflavine-resistant E. rhusiopathiae Koganei 65-0.15 strain (serovar 1a) have been frequently isolated from pigs affected with chronic erysipelas in Japan. In this study, using the conventional PCR assay that was designed to detect strain-specific single nucleotide polymorphism (SNP) sites found in the genome of the vaccine strain, we analyzed E. rhusiopathiae isola ...[more]

PMID: 28250289

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Project description:Erysipelothrix rhusiopathiae causes swine erysipelas, an important infectious disease in the swine industry. In Japan, the incidence of acute swine erysipelas due to E. rhusiopathiae serovar 1a has recently increased markedly. To study the genetic relatedness of the strains from the recent cases, we analyzed 34 E. rhusiopathiae serovar 1a swine isolates collected between 1990 and 2011 and further investigated the possible association of the live Koganei 65-0.15 vaccine strain (serovar 1a) with the increase in cases. Pulsed-field gel electrophoresis analysis revealed no marked variation among the isolates; however, sequencing analysis of a hypervariable region in the surface-protective antigen A gene (spaA) revealed that the strains isolated after 2007 exhibited the same spaA genotype and could be differentiated from older strains. Phylogenetic analysis based on genome-wide single-nucleotide polymorphisms (SNPs) revealed that the Japanese strains examined were closely related, showing a relatively small number of SNPs among them. The strains were classified into four major lineages, with Koganei 65-0.15 (lineage III) being phylogenetically separated from the other three lineages. The strains isolated after 2007 and the two older strains constituted one major lineage (lineage IV) with a specific spaA genotype (M203/I257-SpaA), while the recent isolates were further divided into two geographic groups. The remaining older isolates belonged to either lineage I, with the I203/L257-SpaA type, or lineage II, with the I203/I257-SpaA type. These results indicate that the recent increased incidence of acute swine erysipelas in Japan is associated with two sublineages of lineage IV, which have independently evolved in two different geographic regions.IMPORTANCE Using large-scale whole-genome sequence data from Erysipelothrix rhusiopathiae isolates from a wide range of hosts and geographic origins, a recent study clarified the existence of three distinct clades (clades 1, 2, and 3) that are found across multiple continents and host species, representing both livestock and wildlife, and an "intermediate" clade between clade 2 and the dominant clade 3 within the species. In this study, we found that the E. rhusiopathiae Japanese strains examined exhibited remarkably low levels of genetic diversity and confirmed that all of the Japanese and Chinese swine isolates examined in this study belong to clonal lineages within the intermediate clade. We report that spaA genotyping of E. rhusiopathiae strains is a practical alternative to whole-genome sequencing analysis of the E. rhusiopathiae isolates from eastern Asian countries.

Project description:The aim of this study was to characterize Erysipelothrix sp. isolates from clinically affected pigs and their environment and compare them to the Erysipelothrix sp. vaccines used at the sites. Samples were collected during swine erysipelas outbreaks in vaccinated pigs in six Midwest United States swine operations from 2007 to 2009. Pig tissue samples were collected from 1 to 3 pigs from each site. Environmental samples (manure, feed, central-line water, oral fluids, and swabs collected from walls, feed lines, air inlets, exhaust fans, and nipple drinkers) and live vaccine samples were collected following the isolation of Erysipelothrix spp. from clinically affected pigs. All Erysipelothrix sp. isolates obtained were further characterized by serotyping. Selected isolates were further characterized by PCR assays for genotype (E. rhusiopathiae, E. tonsillarum, Erysipelothrix sp. strain 1, and Erysipelothrix sp. strain 2) and surface protective antigen (spa) type (A, B1, B2, and C). All 26 isolates obtained from affected pigs were E. rhusiopathiae, specifically, serotypes 1a, 1b, 2, and 21. From environmental samples, 56 isolates were obtained and 52/56 were E. rhusiopathiae (serotypes 1a, 1b, 2, 6, 9, 12, and 21), 3/56 were Erysipelothrix sp. strain 1 (serotypes 13 and untypeable), and one was a novel species designated Erysipelothrix sp. strain 3 (serotype untypeable). Four of six vaccines used at the sites were commercially available products and contained live E. rhusiopathiae serotype 1a. Of the remaining two vaccines, one was an autogenous live vaccine and contained live E. rhusiopathiae serotype 2 and one was a commercially produced inactivated vaccine and was described by the manufacturer to contain serotype 2 antigen. All E. rhusiopathiae isolates were positive for spaA. All Erysipelothrix sp. strain 1 isolates and the novel Erysipelothrix sp. strain 3 isolate were negative for all currently known spa types (A, B1, B2, and C). These results indicate that Erysipelothrix spp. can be isolated from the environment of clinically affected pigs; however, the identified serotypes in pigs differ from those in the environment at the selected sites. At one of the six affected sites, the vaccine strain and the isolates from clinically affected pigs were of homologous serotype; however, vaccinal and clinical isolates were of heterologous serotype at the remaining five sites, suggesting that reevaluation of vaccine efficacy using recent field strains may be warranted.

Project description:BACKGROUND: Erysipelothrix rhusiopathiae is the causative agent of animal erysipelas and, to a fewer occurrences, human erysipeloid. It is ubiquitous in nature and commensal in diverse species of animals, wild or domestic, from mammals and birds to reptiles and fish. Mechanisms of its virulence and pathogenicity are poorly understood. RESULTS: Making use of the complete genome sequencing of E. rhusiopathiae strain SY1027 and comparative genome analysis between the three highly pathogenic strains (SY1027, Fujisawa and ATCC19414), the genomic structure and putative functional elements, such as pathogenicity island (PAI)-like regions, potential virulence factors and horizontal transferring genes of the bacteria are identified. Strain SY1027 genome is 1,752,910 base pairs long, just 30 kilobases smaller than strain Fujisawa, with the same GC level of 36.36%. It contains 1,845 open reading frames (ORF) predicted by GLIMMER 3.02, of which 1,775 were annotated by PGAAP, 1,757 (~95.23%) were annotated by NCBI nr blast, 1,209 by COG database and 1,076 by KEGG database. 37 potential virulence factors were annotated in strain SY1027 by VFDB, while 19 (~51.35%) of them are common in the 2 strains, 7 of which are potentially related to antibiotic resistance and highly conserved (~98-100% match identity (ID)) amongst the three strains of E. rhusiopathiae and modestly homologous to other gastrointestinal tract-inhabiting Firmicutes (~40% match ID), e.g. Clostridium spp., Enterococcus spp. Genomic island- and pathogenicity island-like regions were also predicted, in which some showed association with tRNA and potential virulence factors. CONCLUSION: Complete genome sequencing of Erysipelothrix rhusiopathiae, the causative agent of animal erysipelas, was performed. Molecular identification of various genomic elements pave the way to the better understanding of mechanisms underlying metabolic capabilities, pathogenicity of swine erysipelas and prospective vaccine targets besides the widely used SpaA antigens.

Dataset Information

Single nucleotide polymorphism genotyping of Erysipelothrix rhusiopathiae isolates from pigs affected with chronic erysipelas in Japan.

Publications

Single nucleotide polymorphism genotyping of Erysipelothrix rhusiopathiae isolates from pigs affected with chronic erysipelas in Japan.

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