title: High-resolution melting-curve analysis of obg gene to differentiate the temperature-sensitive Mycoplasma synoviae vaccine strain MS-H from non-temperature-sensitive strains.
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.
authors.affiliation: Faculty of Veterinary Science, The University of Melbourne, Werribee, Victoria, Australia.
authors.forename: Muhammad A
authors.initials: MA
authors.lastname: Shahid
authors.affiliation: Asia-Pacific Centre for Animal Health, Faculty of Veterinary Science, The University of Melbourne, Victoria, Australia.
authors.forename: Philip F
authors.initials: PF
authors.lastname: Markham
authors.affiliation: Faculty of Veterinary Science, The University of Melbourne, Werribee, Victoria, Australia.
authors.forename: Marc S
authors.initials: MS
authors.lastname: Marenda
authors.affiliation: Faculty of Veterinary Science, The University of Melbourne, Werribee, Victoria, Australia.
authors.forename: Rebecca
authors.initials: R
authors.lastname: Agnew-Crumpton
authors.affiliation: Faculty of Veterinary Science, The University of Melbourne, Werribee, Victoria, Australia.
authors.forename: Amir H
authors.initials: AH
authors.lastname: Noormohammadi
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics*; D015373:Bacterial Typing Techniques / Q000379:methods*; D001428:Bacterial Vaccines / Q000008:administration & dosage / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D001483:Base Sequence; D002645:Chickens; D017931:DNA Primers / Q000737:chemistry; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics*; D019204:GTP-Binding Proteins / Q000235:genetics*; D005838:Genotype; D008969:Molecular Sequence Data; D009175:Mycoplasma Infections / Q000276:immunology / Q000382:microbiology / Q000517:prevention & control / Q000662:veterinary*; D045805:Mycoplasma synoviae / Q000235:genetics / Q000276:immunology*; D009691:Nucleic Acid Denaturation; D016133:Polymerase Chain Reaction; D011201:Poultry Diseases / Q000276:immunology / Q000382:microbiology / Q000517:prevention & control*; D013696:Temperature
chemical_list: D001426:Bacterial Proteins; D001428:Bacterial Vaccines; D017931:DNA Primers; D004269:DNA, Bacterial; C090542:Obg GTP-binding protein, Bacteria; D019204:GTP-Binding Proteins