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A robust PCR for the differentiation of potential virulent strains of Haemophilus parasuis.

ABSTRACT: Haemophilus parasuis is the etiological agent of Glässer's disease in swine. H. parasuis comprises strains with heterogeneous virulence capacity, from non-virulent to highly virulent. Determination of the pathogenic potential of the strains is important for diagnosis and disease control. The virulence-associated trimeric autotransporters (vtaA) genes have been used to predict H. parasuis virulence by PCR amplification of their translocator domains. Here, we report a new and improved PCR designed to detect a different domain of the vtaA genes, the leader sequence (LS) as a diagnostic tool to predict virulence.A collection of 360 ?H. parasuis ?strains was tested by PCR with LS specific primers. Results of the PCR were compared with the clinical origin of the strains and, for a subset of strains, with their phagocytosis and serum resistance using a Chi-square test.LS-PCR was specific to H. parasuis, and allowed the differential detection of the leader sequences found in clinical and non-clinical isolates. Significant correlation was observed between the results of the LS-PCR and the clinical origin (organ of isolation) of the strains, as well as with their phagocytosis and serum susceptibility, indicating that this PCR is a good predictor of the virulence of the strains. In addition, this new PCR showed a full correlation with the previously validated PCR based on the translocator domain. LS-PCR could be performed in a wide range of annealing temperatures without losing specificity.This newly described PCR based on the leader sequence of the vtaA genes, LS-PCR, is a robust test for the prediction of the virulence potential of H. parasuis strains.

SUBMITTER: Galofre-Mila N

PROVIDER: S-EPMC5422950 | biostudies-literature | 2017 May

REPOSITORIES: biostudies-literature

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A robust PCR for the differentiation of potential virulent strains of Haemophilus parasuis.

Galofré-Milà N N Correa-Fiz F F Lacouture S S Gottschalk M M Strutzberg-Minder K K Bensaid A A Pina-Pedrero S S Aragon V V

BMC veterinary research 20170508 1

<h4>Background</h4>Haemophilus parasuis is the etiological agent of Glässer's disease in swine. H. parasuis comprises strains with heterogeneous virulence capacity, from non-virulent to highly virulent. Determination of the pathogenic potential of the strains is important for diagnosis and disease control. The virulence-associated trimeric autotransporters (vtaA) genes have been used to predict H. parasuis virulence by PCR amplification of their translocator domains. Here, we report a new and im ...[more]

PMID: 28482900

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Project description:Haemophilus parasuis is a commensal bacterium of the upper respiratory tract of healthy pigs. However, in conjunction with viral infections in immunocompromised animals H. parasuis can transform into a pathogen that is responsible for causing Glasser's disease which is typically characterized by fibrinous polyserositis, polyarthritis, meningitis and sometimes acute pneumonia and septicemia in pigs. Haemophilus parasuis serovar 5 is highly virulent and more frequently isolated from respiratory and systemic infection in pigs. Recently a highly virulent H. parasuis serovar 4 was isolated from the tissues of diseased pigs. To understand the differences in virulence and virulence-associated genes between H. parasuis serovar 5 and highly virulent H. parasuis serovar 4 strains, a genomic library was generated by TruSeq preparation and sequenced on Illumina HiSeq 2000 obtaining 50 bp PE reads. A three-way comparative genomic analysis was conducted between two highly virulent H. parasuis serovar 4 strains and H. parasuis serovar 5. Haemophilus parasuis serovar 5 GenBank isolate SH0165 (GenBank accession number CP001321.1) was used as reference strain for assembly. Results of these analysis revealed the highly virulent H. parasuis serovar 4 lacks genes encoding for, glycosyl transferases, polysaccharide biosynthesis protein capD, spore coat polysaccharide biosynthesis protein C, polysaccharide export protein and sialyltransferase which can modify the lipopolysaccharide forming a short-chain LPS lacking O-specific polysaccharide chains often referred to as lipooligosaccharide (LOS). In addition, it can modify the outer membrane protein (OMP) structure. The lack of sialyltransferase significantly reduced the amount of sialic acid incorporated into LOS, a major and essential component of the cell wall and an important virulence determinant. These molecules may be involved in various stages of pathogenesis through molecular mimicry and by causing host cell cytotoxicity, reduced inflammatory and immunological response to infection with this organism. The mechanism by which sialyation of LPS contributes to virulence is a key to understanding the pathogenesis of this highly virulent H. parasuis serovar 4. This analysis also revealed the presence of virulence associated genes similar to the MerR family transcriptional regulators, macrophage infectivity potentiator protein, hemolysin, opacity associated protein, toxin antitoxin system, and virulence associated protein D and colicins. Haemophilus parasuis serovar 4 variants also possess extensive metal ion uptake and regulation mechanism which controls various virulence and virulence associated genes. A combination of virulence associated factors and/or genes and proteins with overlapping functions may be responsible for the apparent enhanced virulence of this organism. The extensive structural modification of LOS and OMP of variant H. parasuis serovar 4 strains appear to aid in nasal colonization, are associated with the organisms' ability to evade the host immune response and provide serum-resistance. In addition, the combination of capsule modification and phase variation due to LOS substitutions could help variant H. parasuis serovar 4 transform into a highly virulent pathogen. Based on these results, the variant H. parasuis serovar 4 strains harbor a diverse repertoire of virulence associated genes which have not been previously reported.

Dataset Information

A robust PCR for the differentiation of potential virulent strains of Haemophilus parasuis.

Publications

A robust PCR for the differentiation of potential virulent strains of Haemophilus parasuis.

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