Three distinct species of feline haemoplasmas are recognised: Mycoplasma haemofelis (Mhf), Candidatus Mycoplasma haemominutum (CMhm) and Candidatus Mycoplasma turicensis (CMt). These species differ in pathogenicity as Mhf and CMt can be associated with anaemia whereas CMhm usually results in few clinical signs. The purpose of this study was to develop quantitative real-time PCR assays for the detection of all three feline haemoplasma species combined with an endogenous internal control and to determine the prevalence of
infection, using these assays, in 1592
EDTA blood samples submitted to Langford Veterinary Diagnostics, University of Bristol for haemoplasma testing. Primers and TaqMan probes were designed against published 16S
rDNA sequences. These assays were combined with a feline 28S
rDNA-specific assay to produce three duplex assays. The assays detected 1-10 copies of a sequence-specific plasmid per PCR. None of the assays showed cross-reactivity with 10(6) copies of a sequence-specific plasmid from the non-target haemoplasma species. Real-time PCR was performed on all samples using the three assays. Seven samples were negative for feline 28S
rDNA and were excluded from the study. Of the remaining 1585 samples, 45 (2.8%), 177 (11.2%) and 27 (1.7%) samples were positive for Mhf, CMhm and CMt, respectively, including 11 Mhf/CMhm, 10 CMhm/CMt and two Mhf/CMt dual
infections and two triple
infections. The results of this study demonstrate the utility of these new duplex PCR assays for the detection of haemoplasma
infections. CMhm was the most common
infection and CMt
infections were often associated with
co-infection with other haemoplasma species, especially CMhm.