High-resolution melting-curve analysis of obg gene to differentiate the temperature-sensitive Mycoplasma synoviae vaccine strain MS-H from non-temperature-sensitive strains

• Published in: PloS one Vol. 9; no. 3; p. e92215
• Main Authors: Shahid, Muhammad A, Markham, Philip F, Marenda, Marc S, Agnew-Crumpton, Rebecca, Noormohammadi, Amir H
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 18-03-2014; Public Library of Science (PLoS)
• Subjects: Analysis; Animals; Bacillus anthracis; Bacterial Proteins - genetics; Bacterial Typing Techniques - methods; Bacterial Vaccines - administration & dosage; Bacterial Vaccines - genetics; Bacterial Vaccines - immunology; Bacterial Vaccines - isolation & purification; Base Sequence; Biology and Life Sciences; Cell cycle; Chickens; DNA Primers - chemistry; DNA, Bacterial - chemistry; DNA, Bacterial - genetics; Genes; Genetic aspects; Genotype; Genotyping; GTP-Binding Proteins - genetics; Health aspects; Hemagglutinins; High resolution; Laboratories; Medicine and Health Sciences; Melting; Molecular Sequence Data; Mycoplasma; Mycoplasma Infections - immunology; Mycoplasma Infections - microbiology; Mycoplasma Infections - prevention & control; Mycoplasma Infections - veterinary; Mycoplasma synoviae - genetics; Mycoplasma synoviae - immunology; Nucleic Acid Denaturation; Polymerase Chain Reaction; Poultry; Poultry Diseases - immunology; Poultry Diseases - microbiology; Poultry Diseases - prevention & control; Research and Analysis Methods; Revertants; Science; Signal transduction; Single nucleotide polymorphisms; Single-nucleotide polymorphism; Specific pathogen free; Staphylococcus aureus; Staphylococcus infections; Strains (organisms); Temperature; Temperature effects; Vaccination; Vaccines
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0092215

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.