Use of polymerase chain reactions to detectMycoplasma gallisepticum,Mycoplasma imitans,Mycoplasma iowae,Mycoplasma meleagridisandMycoplasma synoviaein birds of prey

Abstract

Certain Mycoplasma spp. are pathogens of poultry, but little is known of the role of mycoplasmas in disease of birds of prey. Species-specific polymerase chain reactions (PCRs) for the detection of the poultry pathogens Mycoplasma gallisepticum, Mycoplasma imitans, Mycoplasma iowae, Mycoplasma meleagridis and Mycoplasma synoviae were therefore evaluated for use in birds of prey. The specificities of the PCR methods were established using avian and other mycoplasmas and also selected walled bacteria. The sensitivities of the different PCR assays varied between 100 fg and 10 pg DNA. Fifty-three tracheal swabs from healthy captive and free-ranging birds of prey were then investigated using these PCRs, and in no case was an amplicon obtained for M. gallisepticum/M. imitans, M. iowae or M. synoviae. Species-specific primers for M. meleagridis amplified a product from eight birds of prey but restriction enzyme analysis as well as sequencing of PCR products demonstrated these results to be false positives. Alignment studies of the sequenced products with the 16S rRNA gene sequence of various Mycoplasma species in GenBank demonstrated an identity of 91% to M. meleagridis but of 98% to Mycoplasma buteonis or Mycoplasma gallopavonis. Isolation and attempted identification of these mycoplasmas suggested it may be a previously unrecognized species.

Isolation and serological detection of mycoplasma gallisepticum and mycoplasma synoviae using a combined mg/ms enzyme-linked immunosorbent assay kit in indigenous chickens in Niger State, Nigeria

“…The local chickens appeared normal without any obvious clinical signs. This result was similar to the findings of previous authors (Carpenter et al, 1981;Lierz et al, 2000;Lierz et al, 2002;Lierz et al, 2008;Peebles et al, 2004) who found high seroprevalence of Mycoplasma in birds of prey even though the adult birds remained clinically normal. This can be attributed to the type of husbandry system practiced by the villagers and including lack of vaccination of local chickens and non-existence of mycoplasma control program in Nigeria.…”

A diagnostic polymerase chain reaction forMycoplasma iowaeusing primers located in the intergenic spacer region and the 23S rRNA gene

“…The complete genome sequence of M. iowae has been published recently (Wei et al, 2012) and reveals the presence of two rRNA operons, which may help to explain why the sensitivity can be less than one organism per reaction. This detection limit is similar to that obtained by Lierz et al (2008) who detected as little as 0.1 CFU, and is lower than the 3.2 CFUs per PCR reaction found by Cai et al (2008) or the 10 colour changing units per reaction reported by Laigret et al (1996). Zhao & Yamamoto (1993) could detect 1 pg of M. iowae DNA, while Boyle et al (1995) and Kempf et al (1994) did not assess the amount of DNA or count the CFUs.…”

“…Several of the PCR assays described for M. iowae have been based on the 16S rRNA gene (Kempf et al, 1994;Boyle et al, 1995;Kiss et al, 1997;Cai et al, 2008;Lierz et al, 2008) although two (Laigret et al, 1996;Raviv & Kleven, 2009) targeted a region upstream of the rRNA operon. The forward primer of the PCR assay described in this paper is based on the ISR between the 16S and 23S rRNA genes and the reverse primer on the 5?…”

“…Both conventional (Zhao & Yamamoto, 1993;Kempf et al, 1994;Boyle et al, 1995;Lierz et al, 2008) and real-time (Cai et al, 2008) M. iowae-specific PCRs have been described. Others have used nonspecific broad-range primers and have required further analysis such as restriction fragment length polymorphism (Fan et al, 1995;García et al, 1996;Kiss et al, 1997).…”

Development of a novel real-time polymerase chain reaction (PCR) assay by amplification of double target genes for quantitative detection of Mycoplasma gallisepticum

“…At present, some MG species-specific PCR, MG realtime PCR, PCR-RFLP and oligonucleotide probe techniques are available (Callison et al, 2006;Jarquin et al, 2009;Kahya et al, 2010;Lierz et al, 2008;Mardassi et al, 2005;Raviv et al, 2008;Sprygin et al, 2010). During the acute stage of the infection the number of MG in the upper respiratory tract is high, but in chronic infection the number of organisms is much lower and routine methods may not detect it (Levisohn and Kleven, 2000).…”