TDK conference 2018

Mycoplasma anatis, M. anseris, M. cloacale and M. sp. 1220 are waterfowl pathogen bacteria. Mycoplasmas could cause serious diseases alone or in co-infections, which may lead to major economic losses. They are associated with inflammation of the cloaca and genital tracts, decreased egg production, embryo lethality and respiratory symtpoms. The identification depends on their culture and a genus-specific polymerase chain reaction (PCR). However, the co-occurrence of these species is frequent and they have similar biochemical properties and genomic similarities. These characteristics highlight the importance of the utilisation of molecular methods which could be applicable in routine diagnostics, and consequently promoting the targeted therapy. The aim of the present study was to develop and test species-specific PCR systems specific to M. anatis, M. anseris, M. cloacale and M. sp. 1220.

Genes from avian pathogen Mycoplasma species were collected and align in order to design specific primers. The DNA polymerase III subunits gamma/tau coding gene was found appropriate for the identification of M. anatis and M. cloacale, the ATP-dependent DNA helicase PcrA coding gene for the M. anseris-specific PCR and the DNA-directed RNA polymerase subunit beta coding gene was selected for the M. sp. 1220 species-specific primer design. The specificity of the assays was tested on 16 avian pathogen Mycoplasma type strains, 57 bacterial strains collected between 2011 and 2018 mostly from Hungary and 28 clinical samples originating from ducks and geese. The developed PCR assays can specifically identify the concerned species, as no cross-reactions were detected with other mycoplasmas infecting birds. The sensitivity of the PCR systems was 101-102 DNA copy template.

The assays can be performed simultaneously at the same annealing temperature; thus the parallel examinations of multiple samples are possible. The developed assays are applicable directly on clinical samples and promote the rapid, simple and cost-effective differentiation of the waterfowl pathogen mycoplasmas.