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

ABSTRACT: 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.

SUBMITTER: Kreizinger Z

PROVIDER: S-EPMC5395193 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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

Kreizinger Zsuzsa Z Sulyok Kinga Mária KM Grózner Dénes D Bekő Katinka K Dán Ádám Á Szabó Zoltán Z Gyuranecz Miklós M

PloS one 20170418 4

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 ...[more]

PMID: 28419134

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Project description:The temperature-sensitive (ts+) Mycoplasma synoviae vaccine strain MS-H harbors a non-synonymous mutation which results in Glycine to Arginine substitution at position 123 in the highly conserved glycine-rich motif of Obg-fold in the GTP-binding protein Obg. In-silico analysis of the wild-type and mutant Obgs of M. synoviae has indicated that this amino acid substitution affects structure of the protein, potentially leading to abrogation of Obg function in vivo. Present study was conducted to develop the first expression vector for M. synoviae and to investigate the potential effect(s) of complementation of MS-H vaccine with the wild-type obg from 86079/7NS, the parent strain of MS-H. An oriC vector, pKS-VOTL, harboring the 86079/7NS obg gene, downstream of the variable lipoprotein haemagglutinin (vlhA) gene promoter, also cloned from 86079/7NS, was used to transform MS-H. The plasmid was localised at the chromosomal oriC locus of MS-H without any detectable integration at the chromosomal obg locus. Analysis of the MS-H transformants revealed abundant obg transcripts as well as Obg protein, when compared to the MS-H transformed with a similar vector, pMAS-LoriC, lacking obg coding sequence. The MS-H transformants complemented with wild-type Obg maintained their original temperature-sensitivity phenotype (consistent with MS-H vaccine) but, when compared to the MS-H transformed with pMAS-LoriC, had significantly higher (p < 0.05) growth rate and viability at the permissive (33°C) and non-permissive temperature (39.5°C), respectively. Analysis of Obg expression in MS-H and its wild-type parent strain revealed comparatively lower levels of Obg in MS-H. These results indicate that not only the mutation in Obg, but also the level of Obg expression, can confer functional abnormalities in the bacterial host. Furthermore, with the construction of first expression vector for M. synoviae, this study has set foundation for the development of recombinant vaccine(s) based on MS-H.

Project description:Mycoplasma synoviae causes infectious synovitis and respiratory diseases in chickens and turkeys and may lead to egg shell apex abnormalities in chickens; hence possesses high economic impact on the poultry industry. Control of the disease consists of eradication, vaccination or medication. The aim of the present study was to determine the in vitro susceptibility to 14 different antibiotics and an antibiotic combination of M. synoviae strains originating from Hungary and other countries of Central and Eastern Europe.Minimal inhibitory concentration (MIC) values of a total of 41 M. synoviae strains were determined by the microbroth dilution method. The strains were collected between 2002 and 2016 and originated from Hungary (n?=?26), Austria (n?=?3), the Czech Republic (n?=?3), Slovenia (n?=?3), Ukraine (n?=?3), Russia (n?=?2) and Serbia (n?=?1). Tetracyclines (with MIC50 values of 0.078 ?g/ml, ?0.25 ?g/ml and 0.5 ?g/ml for doxycycline, oxytetracycline and chlortetracycline, respectively), macrolides (with MIC50 values of ?0.25 ?g/ml for tylvalosin, tylosin and tilmicosin), pleuromutilins (with MIC50 values of 0.078 ?g/ml and ?0.039 ?g/ml for tiamulin and valnemulin) and the combination of lincomycin and spectinomycin (MIC50 1 ?g/ml (0.333/0.667 ?g/ml)) were found to be the most effective antibiotic agents against M. synoviae in vitro. High MIC values were detected in numerous strains for fluoroquinolones (with MIC50 values of 1.25 ?g/ml and 2.5 ?g/ml for enrofloxacin and difloxacin), neomycin (MIC50 32 ?g/ml), spectinomycin (MIC50 2 ?g/ml), lincomycin (MIC50 0.5 ?g/ml) and florfenicol (MIC50 4 ?g/ml). Nevertheless, strains with elevated MIC values were detected for most of the applied antibiotics.In the medical control of M. synoviae infections the preliminary in vitro antibiotic susceptibility testing and the careful evaluation of the data are crucial. Based on the in vitro examinations doxycycline, oxytetracycline, tylvalosin, tylosin and pleuromutilins could be recommended for the therapy of M. synoviae infections in the region.

Dataset Information

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

Publications

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

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