Molecular Variability of the Adhesin-Encoding Gene pvpA among Mycoplasma gallisepticum Strains and Its Application in Diagnosis

Abstract

Mycoplasma gallisepticum is an important pathogen of chickens and turkeys that causes considerable economic losses to the poultry industry worldwide. The reemergence of M. gallisepticum outbreaks among poultry, the increased use of live M. gallisepticum vaccines, and the detection of M. gallisepticum in game and free-flying song birds has strengthened the need for molecular diagnostic and strain differentiation tests. Molecular techniques, including restriction fragment length polymorphism of genomic DNA (RFLP) and PCR-based random amplification of polymorphic DNA (RAPD), have already been utilized as powerful tools to detect intraspecies variation. However, certain intrinsic drawbacks constrain the application of these methods. The main goal of this study was to determine the feasibility of using an M. gallisepticum-specific gene encoding a phase-variable putative adhesin protein (PvpA) as the target for molecular typing. This was accomplished using a pvpA PCR-RFLP assay. Size variations among PCR products and nucleotide divergence of the C-terminus-encoding region of the pvpA gene were the basis for strain differentiation. This method can be used for rapid differentiation of vaccine strains from field isolates by amplification directly from clinical samples without the need for isolation by culture. Moreover, molecular epidemiology of M. gallisepticum outbreaks can be performed using RFLP and/or sequence analysis of the pvpA gene.

Use of molecular diversity of Mycoplasma gallisepticum by gene-targeted sequencing (GTS) and random amplified polymorphic DNA (RAPD) analysis for epidemiological studies

“…However, as previously reported (Boguslavsky et al, 2000;Liu et al, 2001;Pillai et al, 2003), the pvpA gene exhibited size polymorphism, with PCR products of 437, 578, 606 and 665 bp detected among M. gallisepticum reference strains and isolates examined (Table 3). The region of the pvpA gene amplified encodes the protein carboxy-terminus, where truncations of the pvpA protein have been reported to be located within the proline-rich direct repeat (DR) (Boguslavsky et al, 2000).…”

“…However, the full pvpA gene sequence of one Israeli isolate as compared to the USA atypical M. gallisepticum strain K703CK75 indicated that the latter has an additional 3 nt insertion near the DR region (Boguslavsky et al, 2000) not included by this GTS analysis. RAPD type B isolates, including vaccine strain F and house finch isolate K4409HF97 (RAPD type N), are among the other M. gallisepticum strains for which pvpA size polymorphism has been previously reported (Boguslavsky et al, 2000;Liu et al, 2001). …”

“…The mgc2 gene encodes a second cytadhesin protein also known to play a role in the attachment process (Hnatow et al, 1998) identified as genome CDS MGA_0932. The pvpA gene encodes a putative accessory cytadhesin that exhibits size variation among M. gallisepticum strains (Boguslavsky et al, 2000;Liu et al, 2001) and was identified as an interrupted CDS in the M. gallisepticum R low genome sequence by Papazisi et al (2003). A 37 nt internal duplication within the pvpA gene resulted in a reading frame shift predicting two coding sequence fragments identified as genome CDSs MGAL_0256 and MGAL_0258, encoding the C-terminal and N-terminal ends of the pvpA gene products, respectively (Papazisi et al, 2003).…”

Mycoplasma gallisepticumexperimental infection and tissue distribution in chickens, sparrows and pigeons

“…PCR was used to test for M. gallisepticum growth in the Frey's broth. DNA was extracted as previously described (Liu et al, 2001) and PCR was performed using M. gallisepticum-specific primers as previously described (Nascimento et al, 1991).…”

Bi-Antigenic Immunoassay Models Based on the Recombinant PvpA Proteins for Mycoplasma Gallisepticum Diagnosis in Chickens

“…3,8,16,17 Recently, the M. gallisepticum PvpA cytadhesin described as surface-exposed immunogenic protein 6,11,23 has been produced in Escherichia coli and used under purified form as a species-specific recombinant antigen (rPvpA336) to develop an individual rapid test for screening M. gallisepticum infections in the field and under limited laboratory conditions. 8 In the present study, a fragment of the pvpA structural gene corresponding to the central region was cloned to produce a recombinant antigen because recent studies 6,8,14,23 have demonstrated the size variability of the PvpA cytadhesin of different M. gallisepticum strains due to major deletions, particularly within the C-terminal and direct repeated regions (DR-1 and DR-2). Immunoreactivity of the recombinant protein consisting of 134 amino acids (rPvpA134) was determined using M. gallisepticumpositive and M. gallisepticum-negative chicken sera.…”