Development and use of novel real-time PCR assays for canine haemotropic mycoplasmas

Two canine haemoplasma species have been recognised; Mycoplasma haemocanis (Mhc) and “Candidatus M. haematoparvum” (CMhp). Both have been associated with haemolysis in immunocompromised or splenectomised dogs. The aims of this study were to develop quantitative real-time PCRs (qPCRs) to detect Mhc and CMhp in canine blood samples and to use these assays to determine prevalence of infection in groups of dogs from Trinidad and Tanzania. Both areas have a high prevalence of Rhipicephalus sanguineus, a putative vector of haemoplasma. Using 16S rDNA sequence data, qPCRs specific for each of Mhc and CMhp were designed, and each qPCR was duplexed with a canine glyceraldehyde-3-phosphate dehydrogenase (G3PDH)-specific qPCR to incorporate amplification of an endogenous control. These qPCRs were applied to DNA extracted from blood taken from pet dogs presenting to a veterinary hospital in Trinidad (n=185) and from pet dogs bled during a rabies monitoring programme in Northern Tanzania (n=100). Reaction efficiency for both haemoplasma qPCRs was >91%. The CMhp qPCR detected 10 copies/PCR whilst the Mhc qPCR detected 15 copies/PCR (both 10/10 replicates). Neither qPCR cross reacted with 106 copies of the non-target haemoplasma species sequence-specific plasmid. Inter-assay co-efficients of variation (CVs) for calculated copy number (and threshold cycle value) at high and low plasmid concentrations were 2.8 and 4.7% (0.13 and 0.43 %) respectively for the Mhc qPCR, and 5.1 and 13.5% (0.40 and 0.60 %) respectively for the CMhp qPCR, whilst intra-assay CVs were 9.2 and 19.8 % (0.87 and 0.98 %) respectively for the Mhc qPCR and 9.2 and 17.6 % (0.79 and 0.86 %) respectively for the CMhp qPCR. One of the Trinidadian samples was negative for canine G3PDH DNA and was excluded from further analysis. Of the 184 remaining Trinidadian samples 9 (4.9%) were positive for Mhc alone, 5 (2.7%) for CMhp alone, and 2 (1.1%) were co-infected with Mhc and CMhp. All 100 Tanzanian samples contained acceptable amounts of canine G3PDH DNA; of these 19 (19%) were positive for Mhc alone and 1 (1%) was co-infected. These qPCRs are the first for canine haemoplasmas to use an internal control to confirm the presence of amplifiable DNA in samples. They were successfully used in two prevalence studies which found that Mhc was the most common canine haemoplasma species in both Trinidad and Tanzania.