High-Resolution Melting-Curve Analysis of obg Gene to Differentiate the Temperature-Sensitive Mycoplasma synoviae Vaccine Strain MS-H from Non-Temperature-Sensitive Strains

Muhammad A Shahid,Amir H Noormohammadi,Philip F Markham,Rebecca Agnew-Crumpton,Marc S Marenda,Mitchell F Balish

doi:10.1371/journal.pone.0092215

Muhammad A Shahid, Amir H Noormohammadi + Show 4 more

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https://doi.org/10.1371/journal.pone.0092215

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Journal: PloS one	Publication Date: Mar 18, 2014
Citations: 52	License type: CC BY 4.0

Affiliation: University of Melbourne

Abstract

Temperature-sensitive (ts +) vaccine strain MS-H is the only live attenuated M. synoviae vaccine commercially available for use in poultry. With increasing use of this vaccine to control M. synoviae infections, differentiation of MS-H from field M. synoviae strains and from rarely occurring non-temperature-sensitive (ts –) MS-H revertants has become important, especially in countries where local strains are indistinguishable from MS-H by sequence analysis of variable lipoprotein haemagglutinin (vlhA) gene. Single nucleotide polymorphisms (SNPs) in the obg of MS-H have been found to associate with ts phenotype. In this study, four PCRs followed by high-resolution melting (HRM)-curve analysis of the regions encompassing these SNPs were developed and evaluated for their potential to differentiate MS-H from 36 M. synoviae strains/isolates. The nested-obg PCR-HRM differentiated ts + MS-H vaccine not only from field M. synoviae strains/isolates but also from ts – MS-H revertants. The mean genotype confidence percentages, 96.9±3.4 and 8.8±11.2 for ts + and ts – strains, respectively, demonstrated high differentiating power of the nested-obg PCR-HRM. Using a combination of nested-obg and obg-F3R3 PCR-HRM, 97% of the isolates/strains were typed according to their ts phenotype with all MS-H isolates typed as MS-H. A set of respiratory swabs from MS-H vaccinated specific pathogen free chickens and M. synoviae infected commercial chicken flocks were tested using obg PCR-HRM system and results were consistent with those of vlhA genotyping. The PCR-HRM system developed in this study, proved to be a rapid and reliable tool using pure M. synoviae cultures as well as direct clinical specimens.

Highlights

Mycoplasma synoviae causes airsacculitis and infectious synovitis in chickens and turkeys [1]
Jeffery et al [11] described a combination of PCR and high-resolution melting (HRM) curve analysis of the variable lipoprotein haemagglutinin (vlhA) gene products to discriminate a large number of M. synoviae strains, their system did not differentiate MS-H from several Australian field strains as they shared the same vlhA gene sequence
We have recently developed a technique using a combination of differential growth at two different temperatures with a quantitative real-time PCR to determine ts phenotype of M. synoviae strains [16] this technique still requires culture of the organism and access to live cloned organism

Summary

Introduction

Mycoplasma synoviae causes airsacculitis and infectious synovitis in chickens and turkeys [1]. Differentiation of MS-H from field strains is an important step to establish whether a flock is free from wild-type M. synoviae. A number of PCR-based techniques have been reported for typing of M. synoviae strains, targeting the vlhA gene [4,5,6,7], 16S rRNA genes [8] or the 16S to 23S rRNA intergenic spacer region [9,10]. Pulsed-field gel electrophoresis (PFGE) using BlnI and BamHI digestions coupled with vlhA gene sequencing was useful in differentiating the MS-H from Japanese M. synoviae strains/isolates [12]. A PCR based cycling probe technology (CPT) developed by Ogino et al [13], targeting an ARG substitution at 365th nucleotide from the 59 conserved region of vlhA gene, has been claimed useful for MS-H differentiation from Japanese M. synoviae strains/isolates.

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