Molecular typing of Iranian field isolatesMycoplasma synoviaeand their differentiation from the live commercial vaccine strain MS-H usingvlhAgene

Abstract

1. The single-copy domain of the N-terminal region of the vlhA gene of Mycoplasma synoviae was sequenced, analysed and verified and used to type Iranian field isolates of M. synoviae and the MS-H live vaccine strain. In addition, a restriction fragment length polymorphism (RFLP) method was developed to differentiate between field isolates of Iranian and MS-H vaccine strains. 2. All sequences were analysed and aligned; the percentage similarity of the DNA was calculated and dendrograms were constructed. Based on single nucleotide polymorphism (SNP) that existed in all field isolates in Iran, the PCR-RFLP method allowed the differentiation of all M. synoviae field isolates from the vaccine strain. 3. Using phylogenetic analysis, the isolates were assigned to 8 unique genotypes and, within each group, DNA had a high level of similarity. 4. DNA sequence analysis and PCR-RFLP of the amplicon based on percent similarity and evolutionary relationship appeared to be useful tools for strain differentiation whether M. synoviae clinical isolates from infected chickens were derived from the vaccine strain or wild-type strains. 5. This study confirms the potential value of strain typing for epidemiological purposes and suggests that phylogenetic studies are essential to understand the true relationships between strains.

Variable lipoprotein haemagglutinin (vlhA) gene sequence typing of mainly DutchMycoplasma synoviaeisolates: comparison withvlhAsequences from Genbank and with amplified fragment length polymorphism analysis

“…Analysis of the vlhA gene has been utilized routinely for M. synoviae genotyping (Benčina et al, 2001;Hong et al, 2004;Jeffery et al, 2007;Hammond et al, 2009;Harada et al, 2009;Wetzel et al, 2010;Ogino et al, 2011;ElGazzar et al, 2012;Bayatzadeh et al, 2014). However, the results obtained with vlhA typing are not always consistent with the genogroups determined by conventional typing methods such as restriction fragment length polymorphism (Jeffery et al, 2007).…”

“…Typing based on the vlhA sequence has also been proposed for the identification of the vaccine strain MS-H (Jeffery et al, 2007;Harada et al, 2009;Ogino et al, 2011;Bayatzadeh et al, 2014) because the differentiation of this vaccine strain on its temperature-sensitivity characteristics is not always discriminative. The MS-H vaccine strain has a temperature-sensitive (ts+) phenotype, which was obtained by mutagenesis of a temperature-insensitive (ts-) Figure 2.…”

“…synoviae polymerase chain reactions (PCRs) based on the conserved 5′ region of the variable lipoprotein and haemagglutinin (vlhA) gene and subsequent DNA sequencing have been proposed more recently as suitable for molecular typing to study clonality (Benčina et al, 2001;Hong et al, 2004;Jeffery et al, 2007;Hammond et al, 2009;Harada et al, 2009;Wetzel et al, 2010;El-Gazzar et al, 2012;Bayatzadeh et al, 2014). In the present study, a real-time PCR based on the vlhA gene described by Jeffery et al (2007) including subsequent DNA sequencing was assessed for typing of mainly Dutch M. synoviae isolates collected from different organs of various categories of poultry, housed on different farms and obtained during a period of 10 years.…”

Development and evaluation of a multi-locus sequence typing scheme for Mycoplasma synoviae

“…This study showed that MLST based on five genes has a higher discriminatory power than vlhA sequence typing. Moreover, recent studies have shown that vlhA sequence typing can be used successfully for the differentiation between MS-H and M. synoviae field strains in specific geographical areas (Harada et al, 2009;Bayatzadeh et al, 2014), but is not always discriminatory enough (Jeffery et al, 2007;Ogino et al, 2011;Dijkman et al, 2014). Recently, it was shown that the MS-H vaccine strain could be distinguished from all M. synoviae field strains (Shahid et al, 2014) by high resolution melting curve analysis and sequencing of the obg gene making this gene a better target for vaccine strain differentiation.…”

“…In recent years sequencing of the variable lipoprotein and haemagglutinin (vlh) A gene (Benčina et al, 2001;Hong et al, 2004;Jeffery et al, 2007;Hammond et al, 2009;Harada et al, 2009;Wetzel et al, 2010;El-Gazzar et al, 2012;Dijkman et al, 2014) or restriction fragment length polymorphism of the vlhA gene (Bayatzadeh et al, 2014) had replaced several other molecular typing techniques, like restriction endonuclease analysis of DNA (Morrow et al, 1990b;Ley & Avakian, 1992), pulsed-field gel electrophoresis (Marois et al, 2001;Dufour-Gesbert et al, 2006;Harada et al, 2009), amplified fragment length polymorphism (Feberwee et al, 2005), analysis of the 16S to 23S rRNA intergenic spacer region of M. synoviae (Ramirez et al, 2006(Ramirez et al, , 2011 and random amplified polymorphic DNA analysis (Geary et al, 1994;Marois et al, 2001;Feberwee et al, 2005;Dufour-Gesbert et al, 2006) for typing M. synoviae isolates as these were too labour intensive and therefore costly for large scale application.…”