Detection of Mycoplasma anatis, M. anseris, M. cloacale and Mycoplasma sp. 1220 in waterfowl using species-specific PCR assays

Abstract

Mycoplasma anatis, M. anseris, M. cloacale and M. sp. 1220 colonise geese and ducks, and could be associated with infections of avian respiratory and nervous systems, cause mild to severe inflammation of cloaca and genital tracts, and embryo lethality. Co-occurrence of these Mycoplasma species in waterfowl is frequently detected and the identification of these mycoplasmas to the species level at a regular microbiology laboratory is difficult due to their similar morphological, cultural and biochemical properties. Moreover, species differentiation is only possible based on the sequence analysis of the product of a genus-specific PCR assay. Therefore, the aim of the current study was to develop an effective and robust method for the identification of these species in avian clinical specimens. Polymerase chain reaction (PCR) assays using species-specific primers, which target housekeeping genes in order to identify these species, were designed in the present study. The developed PCR assays can precisely identify these four mycoplasmas to the species level directly from DNA samples extracted from clinical specimens, and no cross-amplification was observed among these species and with other well-known avian mycoplasmas. The average sensitivity of the assays was 101-102 genomic equivalents per reaction. These conventional PCR assays can be run simultaneously at the same PCR cycling program, and the species can be differentiated directly (without sequence analysis) by gel electrophoresis due to the specific sizes of the amplicons. In conclusion, the presented species-specific assays were found to be suitable for routine use at regular veterinary diagnostic laboratories and promote the rapid, simple and cost-effective differentiation of these waterfowl Mycoplasma species.

Fig 1. Sizes of the PCR amplicons of Mycoplasma species generated by the species-specific PCR assays.

Abbreviations: m—molecular weight marker (GeneRuler 100 bp Plus DNA Ladder, Thermo Fisher Scientific, Waltham, USA); Mana—product from the M. anatis-specific assay; Mans—product from the M. anseris-specific assay; Mclo—product from the M. cloacale-specific assay; M1220—product from the M. sp. 1220-specific assay; nc—negative control.