Development of a multiplex real-time PCR to distinguish between Mycoplasma species found in South African poultry

Abstract

Avian mycoplasmosis is a serious and chronic bacterial disease caused by Mycoplasma species that can greatly impact the sustainability and profits of poultry production. The pathogens significant to poultry are Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS), thus molecular techniques that are readily available focus mainly on these pathogens. Previously, six mycoplasma species were identified from South African poultry flocks, viz. Mycoplasma gallisepticum, Mycoplasma synoviae, Mycoplasma gallinarum, Mycoplasma pullorum, Mycoplasma iners and Mycoplasma gallinaceum, and minimum inhibitory concentration tests performed demonstrated evidence of multidrug resistance in some of the non-pathogenic mycoplasma species. The project is aimed to develop, validate and test a multiplex real-time PCR that could detect and distinguish between four of the Mycoplasma species in circulation, for which whole Mycoplasma genomes are available. A pan genome analysis identified genes in conserved regions for primer and probe design and synthesis; and a literature review conducted to compare published primer and probe sequences for mycoplasma detection and differentiation. Oligonucleotide primers and probes for the PCR detection and differentiation of M. gallisepticum, M. synoviae, M. gallinaceum, and M. pullorum were successfully designed, tested and PCR conditions optimised. A multiplex real-time PCR assay using these oligonucleotides was developed, optimised, and used to test field samples (n=203) collected from farms known to have persisting Mycoplasma infections, in conjunction with cultivation and identification. The multiplex real-time PCR assay detected MG in 62 % of the samples tested, MS in 83 %, M. gallinaceum in 15 % and M. pullorum in 32 %; and coinfections observed in 68 % of the samples. Culture and identification yielded only 9 Mycoplasma species: MG, M. gallinaceum, M. pullorum (n=2), M. gallinarum, M. glycophilum, and M. iners (n=3); all of which are fast growing Mycoplasma species, excluding MG. The assay can accurately and simultaneously detect and differentiate between the four Mycoplasma species listed. The results obtained give an indication that although there are proportionately more MG and MS species circulating in poultry populations, non-pathogenic Mycoplasma species are exceedingly present and appear mostly in coinfections with either MG, MS, or both.