Rapid, Simple and Cost-Effective Molecular Method to Differentiate the Temperature Sensitive (ts+) MS-H Vaccine Strain and Wild-Type Mycoplasma synoviae Isolates

Mycoplasma synoviae infection in chickens and turkeys can cause respiratory disease, infectious synovitis and eggshell apex abnormality; thus it is an economically important pathogen. Control of M. synoviae infection comprises eradication, medication or vaccination. The differentiation of the temper...

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Published in:	PloS one Vol. 10; no. 7; p. e0133554
Main Authors:	Kreizinger, Zsuzsa, Sulyok, Kinga Mária, Pásztor, Alexandra, Erdélyi, Károly, Felde, Orsolya, Povazsán, János, Kőrösi, László, Gyuranecz, Miklós
Format:	Journal Article
Language:	English
Published:	United States Public Library of Science 24.07.2015 Public Library of Science (PLoS)
Subjects:	Animals Assaying Bacterial Vaccines - genetics Bacterial Vaccines - isolation & purification Base Pair Mismatch Base Sequence Birds Chickens Chromosomes Cost effectiveness Cost-Benefit Analysis Deoxyribonucleic acid DNA DNA polymerase Drugs Economic importance Egg shells Gel electrophoresis Genotypes Health aspects Infection Infections Medical research Methods Molecular Sequence Data Molecular Typing - economics Molecular Typing - methods Mutation Mycoplasma Mycoplasma Infections - diagnosis Mycoplasma Infections - immunology Mycoplasma Infections - microbiology Mycoplasma synoviae - genetics Mycoplasma synoviae - immunology Mycoplasma synoviae - isolation & purification Pathogens Polymerase chain reaction Polymorphism, Single Nucleotide Poultry Poultry Diseases - diagnosis Poultry Diseases - immunology Poultry Diseases - microbiology Real-Time Polymerase Chain Reaction Respiratory diseases Sensitivity Single nucleotide polymorphisms Single-nucleotide polymorphism Synovitis Temperature Temperature effects Time Factors Turkeys Vaccination Vaccines Vaccines, Attenuated - genetics Vaccines, Attenuated - isolation & purification
ISSN:	1932-6203, 1932-6203
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Abstract	Mycoplasma synoviae infection in chickens and turkeys can cause respiratory disease, infectious synovitis and eggshell apex abnormality; thus it is an economically important pathogen. Control of M. synoviae infection comprises eradication, medication or vaccination. The differentiation of the temperature sensitive (ts+) MS-H vaccine strain from field isolates is crucial during vaccination programs. Melt-curve and agarose gel based mismatch amplification mutation assays (MAMA) are provided in the present study to distinguish between the ts+ MS-H vaccine strain, its non-temperature sensitive re-isolates and wild-type M. synoviae isolates based on the single nucleotide polymorphisms at nt367 and nt629 of the obg gene. The two melt-MAMAs and the two agarose-MAMAs clearly distinguish the ts+ MS-H vaccine strain genotype from its non-temperature sensitive re-isolate genotype and wild-type M. synoviae isolate genotype, and no cross-reactions with other Mycoplasma species infecting birds occur. The sensitivity of the melt-MAMAs and agarose-MAMAs was 103 and 104 copy numbers, respectively. The assays can be performed directly on clinical samples and they can be run simultaneously at the same annealing temperature. The assays can be performed in laboratories with limited facilities, using basic real-time PCR machine or conventional thermocycler coupled with agarose gel electrophoresis. The advantages of the described assays compared with previously used methods are simplicity, sufficient sensitivity, time and cost effectiveness and specificity.
AbstractList	Mycoplasma synoviae infection in chickens and turkeys can cause respiratory disease, infectious synovitis and eggshell apex abnormality; thus it is an economically important pathogen. Control of M. synoviae infection comprises eradication, medication or vaccination. The differentiation of the temperature sensitive (ts+) MS-H vaccine strain from field isolates is crucial during vaccination programs. Melt-curve and agarose gel based mismatch amplification mutation assays (MAMA) are provided in the present study to distinguish between the ts+ MS-H vaccine strain, its non-temperature sensitive re-isolates and wild-type M. synoviae isolates based on the single nucleotide polymorphisms at nt367 and nt629 of the obg gene. The two melt-MAMAs and the two agarose-MAMAs clearly distinguish the ts+ MS-H vaccine strain genotype from its non-temperature sensitive re-isolate genotype and wild-type M. synoviae isolate genotype, and no cross-reactions with other Mycoplasma species infecting birds occur. The sensitivity of the melt-MAMAs and agarose-MAMAs was 103 and 104 copy numbers, respectively. The assays can be performed directly on clinical samples and they can be run simultaneously at the same annealing temperature. The assays can be performed in laboratories with limited facilities, using basic real-time PCR machine or conventional thermocycler coupled with agarose gel electrophoresis. The advantages of the described assays compared with previously used methods are simplicity, sufficient sensitivity, time and cost effectiveness and specificity. Mycoplasma synoviae infection in chickens and turkeys can cause respiratory disease, infectious synovitis and eggshell apex abnormality; thus it is an economically important pathogen. Control of M. synoviae infection comprises eradication, medication or vaccination. The differentiation of the temperature sensitive (ts.sup.+) MS-H vaccine strain from field isolates is crucial during vaccination programs. Melt-curve and agarose gel based mismatch amplification mutation assays (MAMA) are provided in the present study to distinguish between the ts.sup.+ MS-H vaccine strain, its non-temperature sensitive re-isolates and wild-type M. synoviae isolates based on the single nucleotide polymorphisms at nt367 and nt629 of the obg gene. The two melt-MAMAs and the two agarose-MAMAs clearly distinguish the ts.sup.+ MS-H vaccine strain genotype from its non-temperature sensitive re-isolate genotype and wild-type M. synoviae isolate genotype, and no cross-reactions with other Mycoplasma species infecting birds occur. The sensitivity of the melt-MAMAs and agarose-MAMAs was 10.sup.3 and 10.sup.4 copy numbers, respectively. The assays can be performed directly on clinical samples and they can be run simultaneously at the same annealing temperature. The assays can be performed in laboratories with limited facilities, using basic real-time PCR machine or conventional thermocycler coupled with agarose gel electrophoresis. The advantages of the described assays compared with previously used methods are simplicity, sufficient sensitivity, time and cost effectiveness and specificity. Mycoplasma synoviae infection in chickens and turkeys can cause respiratory disease, infectious synovitis and eggshell apex abnormality; thus it is an economically important pathogen. Control of M. synoviae infection comprises eradication, medication or vaccination. The differentiation of the temperature sensitive (ts+) MS-H vaccine strain from field isolates is crucial during vaccination programs. Melt-curve and agarose gel based mismatch amplification mutation assays (MAMA) are provided in the present study to distinguish between the ts+ MS-H vaccine strain, its non-temperature sensitive re-isolates and wild-type M. synoviae isolates based on the single nucleotide polymorphisms at nt367 and nt629 of the obg gene. The two melt-MAMAs and the two agarose-MAMAs clearly distinguish the ts+ MS-H vaccine strain genotype from its non-temperature sensitive re-isolate genotype and wild-type M. synoviae isolate genotype, and no cross-reactions with other Mycoplasma species infecting birds occur. The sensitivity of the melt-MAMAs and agarose-MAMAs was 103 and 104 copy numbers, respectively. The assays can be performed directly on clinical samples and they can be run simultaneously at the same annealing temperature. The assays can be performed in laboratories with limited facilities, using basic real-time PCR machine or conventional thermocycler coupled with agarose gel electrophoresis. The advantages of the described assays compared with previously used methods are simplicity, sufficient sensitivity, time and cost effectiveness and specificity.Mycoplasma synoviae infection in chickens and turkeys can cause respiratory disease, infectious synovitis and eggshell apex abnormality; thus it is an economically important pathogen. Control of M. synoviae infection comprises eradication, medication or vaccination. The differentiation of the temperature sensitive (ts+) MS-H vaccine strain from field isolates is crucial during vaccination programs. Melt-curve and agarose gel based mismatch amplification mutation assays (MAMA) are provided in the present study to distinguish between the ts+ MS-H vaccine strain, its non-temperature sensitive re-isolates and wild-type M. synoviae isolates based on the single nucleotide polymorphisms at nt367 and nt629 of the obg gene. The two melt-MAMAs and the two agarose-MAMAs clearly distinguish the ts+ MS-H vaccine strain genotype from its non-temperature sensitive re-isolate genotype and wild-type M. synoviae isolate genotype, and no cross-reactions with other Mycoplasma species infecting birds occur. The sensitivity of the melt-MAMAs and agarose-MAMAs was 103 and 104 copy numbers, respectively. The assays can be performed directly on clinical samples and they can be run simultaneously at the same annealing temperature. The assays can be performed in laboratories with limited facilities, using basic real-time PCR machine or conventional thermocycler coupled with agarose gel electrophoresis. The advantages of the described assays compared with previously used methods are simplicity, sufficient sensitivity, time and cost effectiveness and specificity.
Audience	Academic
Author	Kreizinger, Zsuzsa Sulyok, Kinga Mária Pásztor, Alexandra Erdélyi, Károly Felde, Orsolya Povazsán, János Gyuranecz, Miklós Kőrösi, László
AuthorAffiliation	1 Institute for Veterinary Medical Research, Centre for Agricultural Research, Hungarian Academy of Sciences, Budapest, Pest, Hungary 3 Rhone-Vet Kft., Herceghalom, Pest, Hungary 2 Veterinary Diagnostic Directorate, National Food Chain Safety Office, Budapest, Hungary Universidad Nacional de La Plata, ARGENTINA
AuthorAffiliation_xml	– name: 2 Veterinary Diagnostic Directorate, National Food Chain Safety Office, Budapest, Hungary – name: 1 Institute for Veterinary Medical Research, Centre for Agricultural Research, Hungarian Academy of Sciences, Budapest, Pest, Hungary – name: Universidad Nacional de La Plata, ARGENTINA – name: 3 Rhone-Vet Kft., Herceghalom, Pest, Hungary
Author_xml	– sequence: 1 givenname: Zsuzsa surname: Kreizinger fullname: Kreizinger, Zsuzsa – sequence: 2 givenname: Kinga Mária surname: Sulyok fullname: Sulyok, Kinga Mária – sequence: 3 givenname: Alexandra surname: Pásztor fullname: Pásztor, Alexandra – sequence: 4 givenname: Károly surname: Erdélyi fullname: Erdélyi, Károly – sequence: 5 givenname: Orsolya surname: Felde fullname: Felde, Orsolya – sequence: 6 givenname: János surname: Povazsán fullname: Povazsán, János – sequence: 7 givenname: László surname: Kőrösi fullname: Kőrösi, László – sequence: 8 givenname: Miklós surname: Gyuranecz fullname: Gyuranecz, Miklós
BackLink	https://www.ncbi.nlm.nih.gov/pubmed/26207635$$D View this record in MEDLINE/PubMed
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CitedBy_id	crossref_primary_10_3390_pathogens13010078 crossref_primary_10_1016_j_psj_2025_105011 crossref_primary_10_1016_j_vetmic_2018_10_012 crossref_primary_10_1017_S0043933916000830 crossref_primary_10_1016_j_psj_2024_103874 crossref_primary_10_1080_03079457_2023_2267022 crossref_primary_10_1016_j_vetmic_2020_108840 crossref_primary_10_1016_j_vetmic_2017_08_021 crossref_primary_10_1080_03079457_2021_1944605 crossref_primary_10_1186_s12917_018_1669_8 crossref_primary_10_1080_03079457_2017_1296105 crossref_primary_10_3382_ps_pew228 crossref_primary_10_3389_fvets_2024_1354548 crossref_primary_10_1128_JCM_01084_18 crossref_primary_10_1128_spectrum_03591_23 crossref_primary_10_1371_journal_pone_0175969
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Notes	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 14 content type line 23 Competing Interests: The authors have declared that no competing interests exist. Conceived and designed the experiments: MG. Performed the experiments: ZK KMS AP OF. Analyzed the data: ZK KE MG. Contributed reagents/materials/analysis tools: MG. Wrote the paper: ZK KE MG. Collected strains and trachea swabs: ZK LK JP MG.
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References_xml	– volume: 55 start-page: 148 year: 2014 ident: ref8 article-title: Molecular typing of Iranian field isolates Mycoplasma synoviae and their differentiation from the live commercial vaccine strain MS-H using vlhA gene publication-title: Br Poult Sci doi: 10.1080/00071668.2013.878781 – volume: 54 start-page: 961 year: 2010 ident: ref3 article-title: Treatment of eggshell abnormalities and reduced egg production caused by Mycoplasma synoviae infection publication-title: Avian Dis doi: 10.1637/9121-110309-Case.1 – volume: 73 start-page: 311 year: 2000 ident: ref5 article-title: Detection of Mycoplasma synoviae in poultry environment samples by culture and polymerase chain reaction publication-title: Vet Microbiol doi: 10.1016/S0378-1135(00)00178-4 – volume: 9 start-page: e92215 year: 2014 ident: ref7 article-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 publication-title: PLoS One doi: 10.1371/journal.pone.0092215 – volume: 7 start-page: e32866 year: 2012 ident: ref14 article-title: Melt analysis of mismatch amplification mutation assays (Melt-MAMA): a functional study of a cost-effective SNP genotyping assay in bacterial models publication-title: PLoS One doi: 10.1371/journal.pone.0032866 – volume: 153 start-page: 2679 year: 2007 ident: ref9 article-title: Classification of Mycoplasma synoviae strains using single-strand conformation polymorphism and high-resolution melting-curve analysis of the vIhA gene single-copy region publication-title: Microbiology doi: 10.1099/mic.0.2006/005140-0 – volume: 53 start-page: 538 year: 2009 ident: ref10 article-title: Molecular typing of Japanese field isolates and live commercial vaccine strain of Mycoplasma synoviae using improved pulsed-field gel electrophoresis and vlhA gene sequencing publication-title: Avian Dis doi: 10.1637/8934-052309-Reg.1 – volume: 55 start-page: 187 year: 2011 ident: ref11 article-title: Genotyping of Japanese field isolates of Mycoplasma synoviae and rapid molecular differentiation from the MS-H vaccine strain publication-title: Avian Dis doi: 10.1637/9461-071310-Reg.1 – start-page: 845 year: 2008 ident: ref1 article-title: Diseases of poultry – volume: 42 start-page: 185 year: 2013 ident: ref15 article-title: Combination of differential growth at two different temperatures with a quantitative real-time polymerase chain reaction to determine temperature-sensitive phenotype of Mycoplasma synoviae publication-title: Avian Pathol doi: 10.1080/03079457.2013.779363 – volume: 8 start-page: e73954 year: 2013 ident: ref13 article-title: Mutations in GTP binding protein Obg of Mycoplasma synoviae vaccine strain MS-H: implications in temperature-sensitivity phenotype publication-title: PLoS One doi: 10.1371/journal.pone.0073954 – volume: 38 start-page: 77 year: 2009 ident: ref2 article-title: Induction of eggshell apex abnormalities by Mycoplasma synoviae: field and experimental studies publication-title: Avian Pathol doi: 10.1080/03079450802662772 – volume: 52 start-page: 367 year: 2008 ident: ref4 article-title: Control of avian mycoplasma infections in commercial poultry publication-title: Avian Dis doi: 10.1637/8323-041808-Review.1 – volume: 43 start-page: 465 year: 2014 ident: ref12 article-title: Variable lipoprotein haemagglutinin (vlhA) gene sequence typing of mainly Dutch Mycoplasma synoviae isolates: comparison with vlhA sequences from Genbank and with amplified fragment length polymorphism analysis publication-title: Avian Pathol doi: 10.1080/03079457.2014.958980 – volume: 42 start-page: 667 year: 1998 ident: ref6 article-title: Production of temperature-sensitive clones of Mycoplasma synoviae for evaluation as live vaccines publication-title: Avian Dis doi: 10.2307/1592700 – reference: 24069254 - PLoS One. 2013;8(9):e73954 – reference: 19156584 - Avian Pathol. 2009 Feb;38(1):77-85 – reference: 23581447 - Avian Pathol. 2013 Apr;42(2):185-91 – reference: 25189763 - Avian Pathol. 2014;43(5):465-72 – reference: 21793432 - Avian Dis. 2011 Jun;55(2):187-94 – reference: 20608549 - Avian Dis. 2010 Jun;54(2):961-4 – reference: 17660432 - Microbiology. 2007 Aug;153(Pt 8):2679-88 – reference: 24643035 - PLoS One. 2014;9(3):e92215 – reference: 22438886 - PLoS One. 2012;7(3):e32866 – reference: 24405029 - Br Poult Sci. 2014;55(2):148-56 – reference: 20095154 - Avian Dis. 2009 Dec;53(4):538-43 – reference: 9876833 - Avian Dis. 1998 Oct-Dec;42(4):667-70 – reference: 18939621 - Avian Dis. 2008 Sep;52(3):367-74 – reference: 10781729 - Vet Microbiol. 2000 May 11;73(4):311-8
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SubjectTerms	Animals Assaying Bacterial Vaccines - genetics Bacterial Vaccines - isolation & purification Base Pair Mismatch Base Sequence Birds Chickens Chromosomes Cost effectiveness Cost-Benefit Analysis Deoxyribonucleic acid DNA DNA polymerase Drugs Economic importance Egg shells Gel electrophoresis Genotypes Health aspects Infection Infections Medical research Methods Molecular Sequence Data Molecular Typing - economics Molecular Typing - methods Mutation Mycoplasma Mycoplasma Infections - diagnosis Mycoplasma Infections - immunology Mycoplasma Infections - microbiology Mycoplasma synoviae - genetics Mycoplasma synoviae - immunology Mycoplasma synoviae - isolation & purification Pathogens Polymerase chain reaction Polymorphism, Single Nucleotide Poultry Poultry Diseases - diagnosis Poultry Diseases - immunology Poultry Diseases - microbiology Real-Time Polymerase Chain Reaction Respiratory diseases Sensitivity Single nucleotide polymorphisms Single-nucleotide polymorphism Synovitis Temperature Temperature effects Time Factors Turkeys Vaccination Vaccines Vaccines, Attenuated - genetics Vaccines, Attenuated - isolation & purification
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Title	Rapid, Simple and Cost-Effective Molecular Method to Differentiate the Temperature Sensitive (ts+) MS-H Vaccine Strain and Wild-Type Mycoplasma synoviae Isolates
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