Development of Molecular Methods for Rapid Differentiation of Mycoplasma gallisepticum Vaccine Strains from Field Isolates

Abstract

Mycoplasma gallisepticum is among the most economically significant mycoplasmas causing production losses in poultry. Seven melt-curve and agarose gel-based mismatch amplification mutation assays (MAMAs) and one PCR are provided in the present study to distinguish the M. gallisepticum vaccine strains and field isolates based on mutations in the crmA, gapA, lpd, plpA, potC, glpK, and hlp2 genes. A total of 239 samples (M. gallisepticum vaccine and type strains, pure cultures, and clinical samples) originating from 16 countries and from at least eight avian species were submitted to the presented assays for validation or in blind tests. A comparison of the data from 126 samples (including sequences available at GenBank) examined by the developed assays and a recently developed multilocus sequence typing assay showed congruent typing results. The sensitivity of the melt-MAMA assays varied between 10 1 and 10 4 M. gallisepticum template copies/reaction, while that of the agarose-MAMAs ranged from 10 3 to 10 5 template copies/reaction, and no cross-reactions occurred with other Mycoplasma species colonizing birds. The presented assays are also suitable for discriminating multiple strains in a single sample. The developed assays enable the differentiation of live vaccine strains by targeting two or three markers/vaccine strain; however, considering the high variability of the species, the combined use of all assays is recommended. The suggested combination provides a reliable tool for routine diagnostics due to the sensitivity and specificity of the assays, and they can be performed directly on clinical samples and in laboratories with basic PCR equipment.

Mycoplasmas as Host Pantropic and Specific Pathogens: Clinical Implications, Gene Transfer, Virulence Factors, and Future Perspectives

“…In addition, its protection lasts for at least 57 weeks after a single vaccination at 3 weeks of age (Kulappu Arachchige et al, 2021). Since live vaccines are used in many parts of the world, Sulyok and his team have developed new highly specific molecular methods to rapidly differentiate MG vaccine strains from field virulent isolates using clinical samples (Sulyok et al, 2019).…”

Diagnosing Infectious Diseases in Poultry Requires a Holistic Approach: A Review

“…In some cases, such as MDV Rispens or CVI988 vaccine and virulent/oncogenic MDV strain, difference between the vaccine strain and wild-type/virulent strain is often minor, which introduces challenges in methodology. In the context of differentiating vaccinated and unvaccinated birds, method Mismatch Amplification Mutation Assay PCR (MAMA-PCR) has been applied for MDV, Mycoplasma gallisepticum and Mycoplasma synoviae [227][228][229]. The approach is based on the presence of a single nucleotide polymorphism (SNP) between vaccine and field/virulent strain, which is incorporated at 3 -end of one of the primers.…”

Current status of vaccine research, development, and challenges of vaccines for Mycoplasma gallisepticum

“…Control of MG infection needs investigation of MG-infection free flocks. For this reason, rapid and cost-effective PCR-based assays were developed for the simultaneous detection of ts-11, 6/85, and F vaccine strains from field isolates ( Sulyok et al., 2019 ). There are 2 main candidates of MG vaccine known as live-attenuated vaccine and killed vaccine/bacterins.…”