Evaluation of the Capacity of PCR and High-Resolution Melt Curve Analysis for Identification of Mixed Infection with Mycoplasma gallisepticum Strains.

Pathogenicity and presentation of Mycoplasma gallisepticum (MG) infection may differ from one strain to another and this may have implications on control measures. Infection of individual birds with more than one MG strain has been reported. A PCR followed by high resolution melt (HRM) curve analysis has been developed in our laboratory and routinely used for detection and differentiation of MG strains. However the potential of this test for identification of MG strains in a mixed specimen has not been evaluated. In the present study, the capability of PCR-HRM curve analysis technique, targeting vlhA and pvpA genes was assessed for identification of individual MG strains in a mixed population. Different DNA ratios of two MG strains from 1 to 10(-4) ng were tested with some generated conventional and normalized curves distinct from those of individual strains alone. Using genotype confidence percentages (GCP) generated from HRM curve analysis, it was found that vlhA PCR-HRM was more consistent than pvpA PCR-HRM for the detection of MG ts-11 vaccine strain mixed with any of the MG strains 6/85, F, S6 or a field isolate. The potential of vlhA PCR-HRM to detect mixed MG strains in a specimen was found to be primarily dependent on quantity and proportion of the target DNAs in the mixture. This is the first study examining the capacity of PCR-HRM technique for identification of individual MG strains in a mixed strain population

Conventional and normalized melt curves analysis of PCR products of <i>vlhA</i> (a and b) and <i>pvpA</i> (c and d) genes from choanal swab samples.

<p>Mixed specimens A, B and C contain equal DNA concentrations (1 ng) of ts-11 and AP3AS strains.</p

Percentage sequence identity of MG strains in <i>vlhA</i> and <i>pvpA</i> genes.

<p>Percentage sequence identity of MG strains in <i>vlhA</i> and <i>pvpA</i> genes.</p

<i>Mycoplasma gallisepticum</i> cultures used in this study and their origin.

<p><i>Mycoplasma gallisepticum</i> cultures used in this study and their origin.</p

Conventional and normalized melt curve analysis of mixed strains using <i>pvpA</i>-PCR.

<p>Mixture of ts-11 and F strain (a and b), ts-11 and 6/85 strain (c and d), ts-11 and S6 strain (e and f), ts-11 and 86026 field isolate (g and h). Specimens A, B, C, D and E each contains 1 ng of ts-11 DNA and 1-10^-4 ng of contaminant MG DNA and specimens F, G, H and I each contains 1-10^-4 ng of ts-11 DNA and 1 ng of contaminant MG DNA, respectively.</p

Homologue of Macrophage-Activating Lipoprotein in Mycoplasma gallisepticum Is Not Essential for Growth and Pathogenicity in Tracheal Organ Cultures

While the genomes of a number of Mycoplasma species have been fully determined, there has been limited characterization of which genes are essential. The surface protein (p47) identified by monoclonal antibody B3 is the basis for an enzyme-linked immunosorbent assay for serological detection of Mycoplasma gallisepticum infection and appears to be constitutively expressed. Its gene was cloned, and the DNA sequence was determined. Subsequent analysis of the p47 amino acid sequence and searches of DNA databases found homologous gene sequences in the genomes of M. pneumoniae and M. genitalium and identity with a gene family in Ureaplasma urealyticum and genes in M. agalactiae and M. fermentans. The proteins encoded by these genes were found to belong to a family of basic membrane proteins (BMP) that are found in a wide range of bacteria, including a number of pathogens. Several of the BMP family members, including p47, contain selective lipoprotein-associated motifs that are found in macrophage-activating lipoprotein 404 of M. fermentans and lipoprotein P48 of M. agalactiae. The p47 gene was predicted to encode a 59-kDa peptide, but affinity-purified p47 had a molecular mass of approximately 47 kDa, as determined by polyacrylamide gel analysis. Analysis of native and recombinant p47 by mass peptide fingerprinting revealed the absence of the carboxyl end of the protein encoded by the p47 gene in native p47, which would account for the difference seen in the predicted and measured molecular weights and indicated posttranslational cleavage of the lipoprotein at its carboxyl end. A DNA construct containing the p47 gene interrupted by the gene encoding tetracycline resistance was used to transform M. gallisepticum cells. A tetracycline-resistant mycoplasma clone, P2, contained the construct inserted within the genomic p47 gene, with crossovers occurring between 73 bp upstream and 304 bp downstream of the inserted tetracycline resistance gene. The absence of p47 protein in clone P2 was determined by the lack of reactivity with rabbit anti-p47 sera or monoclonal antibody B3 in Western blots of whole-cell proteins. There was no difference between the p47(−) mutant and wild-type M. gallisepticum in pathogenicity in chicken tracheal organ cultures. Thus, p47, although homologous to genes that occur in many prokaryotes, is not essential for growth in vitro or for attachment and the initial stages of pathogenesis in chickens

GapA+ Mycoplasma gallisepticum ts-11 has improved vaccine characteristics

Mycoplasma gallisepticum (MG) is an important poultry pathogen that causes respiratory disease and loss of production worldwide, and is currently controlled with live attenuated vaccines. These vaccines have limitations as they vary in their pathogenicity, the protection afforded and their transmissibility, but have been shown to effectively reduce losses associated with challenge in the field. A live attenuated vaccine, ts-11, has been used for the control of M. gallisepticum in several countries. This vaccine is highly dose-dependent and the flock antibody response is weak. GapA is the primary cytadherence molecule in M. gallisepticum, and the absence of GapA expression has been observed in the vast majority of cells in the ts-11 vaccine strain. In this study the immunogenicity of a GapA+ M. gallisepticum ts-11 vaccine was investigated in specific-pathogen-free chickens. Birds vaccinated with GapA+ M. gallisepticum ts-11 were protected against clinical signs of disease following challenge with virulent M. gallisepticum, and GapA+ M. gallisepticum ts-11 was shown to be non-pathogenic and more immunogenic at a lower dose than the currently available M. gallisepticum ts-11 vaccine. Thus, GapA+ M. gallisepticum ts-11 appears to have improved potential as a vaccine candidate.</jats:p