Combination of differential growth at two different temperatures with a quantitative real-time polymerase chain reaction to determine temperature-sensitive phenotype of<i>Mycoplasma synoviae</i>

Shahid, Muhammad Akbar; Ghorashi, Seyed A.; Agnew-Crumpton, Rebecca; Markham, Philip F.; Marenda, Marc S.; Noormohammadi, Amir H.

doi:10.1080/03079457.2013.779363

Cited by 15 publications

(21 citation statements)

References 23 publications

(21 reference statements)

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“…The origin/source and ts phenotype of these strains have been described in our previous study [31] and included in this paper as Table S1.…”

Section: Methodsmentioning

confidence: 99%

“…However, it is not precisely known whether temperature sensitivity is the cause of attenuation or just a coincidental phenotype in bacterial ts vaccines. In our previous study, the ts phenotype of 23 Mycoplasma synoviae strains/isolates, including various reisolates of the MS-H vaccine, was determined [31]. In this study, comparative genomic analysis of ts + M. synoviae vaccine strain MS-H with its non-temperature-sensitive ( ts – ) parent strain 86079/7NS and two ts – reisolates of MS-H (MS-H 4 and MS-H 5 ) revealed a single mutation (G→A) in obg associated with ts + phenotype.…”

Section: Introductionmentioning

confidence: 99%

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Mutations in GTP Binding Protein Obg of Mycoplasma synoviae Vaccine Strain MS-H: Implications in Temperature-Sensitivity Phenotype

Shahid

Markham

et al. 2013

PLoS ONE

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Mycoplasma synoviae strain MS-H, developed by chemical mutagenesis of the Australian field strain 86079/7NS, is a live temperature-sensitive (ts +) vaccine used for control of M. synoviae infection in poultry worldwide. Genetic basis of temperature sensitivity and attenuation of MS-H has not been revealed thus far. Comparison of the complete genome sequence of MS-H, its parent strain 86079/7NS and two non-temperature sensitive (ts –) reisolates of MS-H revealed a mutation in a highly conserved domain of GTP binding protein Obg of MS-H, with reversion in ts – MS-H reisolates. Nucleotide change from G to A at position 369 of the obg gene resulted in an alteration of glycine to arginine at position 123 in Obg fold. Further analysis of the complete obg gene sequence in several MS-H reisolates revealed that a Gly123Arg substitution was associated with alteration in temperature sensitivity phenotype of MS-H. A second mutation, C to T at position 629, in obg gene was found in some of the MS-H reisolates and appeared to suppress the effects of the Gly123Arg substitution. In silico analysis of point mutations revealed that Gly123Arg has highly destabilizing effect on the MS-H Obg structure that can potentially abolish its biological functions in vivo especially at non-permissive temperature. Findings of this study implicate Obg alteration (Gly123Arg) as one of the possible causes of MS-H attenuation/temperature sensitivity and warrant further investigations into exploring the role of Obg-like proteins, an evolutionarily conserved protein from human to bacteria, in the biology of mycoplasmas.

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“…The origin/source and ts phenotype of these strains have been described in our previous study [31] and included in this paper as Table S1.…”

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Mutations in GTP Binding Protein Obg of Mycoplasma synoviae Vaccine Strain MS-H: Implications in Temperature-Sensitivity Phenotype

Shahid

Markham

et al. 2013

PLoS ONE

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“…The MS1 vaccine strain has been commercially released recently, it was obtained by spontaneous mutation during in vitro passages and it does not require NAD for its growth [9]. The re-isolation of the vaccine strains and the examination of the phenotypic characteristics are time-consuming [17]. Genotyping based on the vlhA gene is used worldwide for the molecular differentiation of M .…”

Section: Discussionmentioning

confidence: 99%

Development of mismatch amplification mutation assays for the differentiation of MS1 vaccine strain from wild-type Mycoplasma synoviae and MS-H vaccine strains

et al. 2017

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Mycoplasma synoviae is an economically significant pathogen in the poultry industry, inducing respiratory disease and infectious synovitis in chickens and turkeys, and eggshell apex abnormality in chickens. Eradication, medication and vaccination are the options for controlling M. synoviae infection. Currently there are two commercial, live, attenuated vaccines available against M. synoviae: the temperature sensitive MS-H vaccine strain and the NAD independent MS1 vaccine strain. Differentiation of vaccine strains from field isolates is essential during vaccination and eradication programs. The present study provides melt-curve and agarose gel based mismatch amplification mutation assays (MAMA) to discriminate the MS1 vaccine strain from the MS-H vaccine strain and wild-type M. synoviae isolates. The assays are based on the A/C single nucleotide polymorphism at nt11 of a HIT family protein coding gene. The melt- and agarose-MAMAs reliably distinguish the MS1 vaccine strain genotype from the MS-H vaccine strain and wild-type M. synoviae isolate genotype from 102 template number/DNA sample. No cross-reactions with other avian Mycoplasma species were observed. The assays can be performed directly on clinical samples and they can be run simultaneously with the previously described MAMAs designed for the discrimination of the MS-H vaccine strain. The developed assays are applicable in laboratories with limited facilities and promote the rapid, simple and cost effective differentiation of the MS1 vaccine strain.

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“…The collection of MS-H reisolates examined in this study (Table 1) is a unique collection prepared in our laboratory through extensive monitoring of the MS-H vaccinated flocks. For 23 out of 36 M. synoviae strains/isolates, the ts phenotype was determined in a previous study [16]. For MS-H P5, WVU-1853, 94036/5-5a and 94036/6-3a, the ts phenotype was determined in this study using microtitration as described before [16].…”

Section: Methodsmentioning

confidence: 99%

“…We have recently developed a technique using a combination of differential growth at two different temperatures with a quantitative real-time PCR ( vlhA Q-PCR) to determine ts phenotype of M. synoviae strains [16] however this technique still requires culture of the organism and therefore access to live cloned organism.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2014

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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.

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Combination of differential growth at two different temperatures with a quantitative real-time polymerase chain reaction to determine temperature-sensitive phenotype ofMycoplasma synoviae

Cited by 15 publications

References 23 publications

Mutations in GTP Binding Protein Obg of Mycoplasma synoviae Vaccine Strain MS-H: Implications in Temperature-Sensitivity Phenotype

Mutations in GTP Binding Protein Obg of Mycoplasma synoviae Vaccine Strain MS-H: Implications in Temperature-Sensitivity Phenotype

Development of mismatch amplification mutation assays for the differentiation of MS1 vaccine strain from wild-type Mycoplasma synoviae and MS-H vaccine strains

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

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