QPCRs specific for Mhc and CMhp were designed using 16S rRNA gene sequence data, and each was duplexed with an assay specific for canine glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The assays detected ≤10 copies of a sequence-specific haemoplasma plasmid per reaction and neither assay showed cross-reactivity with 106 copies of the sequence-specific plasmid from the non-target canine haemoplasma species.
Nineteen of the 100 Tanzanian samples (19 % ) were positive for Mhc alone and one (1 % ) was dually infected. One Trinidadian sample was negative for canine GAPDH DNA and was excluded from the study. Of the 184 remaining Trinidadian samples, nine (4.9 % ) were positive for Mhc alone, five (2.7 % ) for CMhp alone, and two (1.1 % ) dually infected.
This is the first report of canine haemoplasma qPCR assays that use an internal control to confirm the presence of amplifiable sample DNA, and their application to prevalence studies. Mhc was the most commonly detected canine haemoplasma species.