Development and use of real-time PCR to detect and quantify Mycoplasma haemocanis and “Candidatus Mycoplasma haematoparvum” in dogs

Abstract

Two canine haemoplasma species have been recognised to date; Mycoplasma haemocanis (Mhc), which has been associated with anaemia in splenectomised or immunocompromised dogs, and “Candidatus Mycoplasma haematoparvum” (CMhp), recently described in an anaemic splenectomised dog undergoing chemotherapy. The study aim was to develop quantitative real-time PCR assays (qPCRs) incorporating an endogenous internal control to detect Mhc and CMhp and to apply these assays to DNA samples extracted from canine blood collected in Northern Tanzania (n = 100) and from dogs presented to a Trinidadian veterinary hospital (n = 185).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.

Prevalence of Hemotropic Mycoplasmas in Healthy and Unhealthy Cats and Dogs in Spain

“…"content-markup(./author-grp [1]/aff|./author-grp [1]/dept-list)> Three feline hemoplasma species exist: Mycoplasma haemofelis, 'Candidatus Mycoplasma haemominutum', and 'Candidatus Mycoplasma turicensis', 8,10,15,20 whereas dogs can be infected by 2 hemoplasma species: Mycoplasma haemocanis and 'Candidatus Mycoplasma haematoparvum'. 1,7,14,18 The pathogenicity of hemoplasmas species varies from being asymptomatic to inducing severe hemolysis. The most pathogenic feline species is regarded as M. haemofelis.…”

“…6,14 Worldwide geographical variation in the prevalence of the different feline and canine hemoplasma species has been reported. 1,15,22 In Europe, prevalence studies with the use of polymerase chain reaction (PCR) for the diagnosis of hemoplasmas in cats have only been performed on domestic cats in Switzerland, Germany, northern Italy, and the United Kingdom. 2,5,9,19 Moreover, prevalence studies that use PCR for the diagnosis of M. haemocanis and 'Candidatus M. haematoparvum' have only been performed on domestic dogs in France, Switzerland, Japan, northern Tanzania, and Trinidad.…”

“…2,5,9,19 Moreover, prevalence studies that use PCR for the diagnosis of M. haemocanis and 'Candidatus M. haematoparvum' have only been performed on domestic dogs in France, Switzerland, Japan, northern Tanzania, and Trinidad. 1,7,12,18 Additional prevalence data for other countries are needed, and no information exists regarding hemoplasma prevalence in Spain. The aims of the present study were to determine the prevalence of hemoplasmas in cats and dogs presenting to the Veterinary Teaching Hospital of the Universitat Autònoma de Barcelona in Spain with the use of real-time quantitative PCR (real-time qPCR) assays and to evaluate any possible associations between hemoplasma infection, clinical presentation, and other vector-borne infections.…”

“…All qPCR assays incorporated an internal amplification control (feline 28S ribosomal [r]DNA or canine glyceraldehyde-3-phosphate dehydrogenase [GAPDH]) to confirm the presence of amplifiable DNA and an absence of PCR inhibitors in the qPCR. 1,9 Positive control DNA samples obtained from cats and dogs infected with each of the hemoplasma species, and a negative control sample (water), were included in each PCR run. The PCR was performed in a real-time PCR detection system b as described previously.…”

“…The PCR was performed in a real-time PCR detection system b as described previously. 1,9 Statistical analyses were performed with the use of a statistical package. c Chi-square analysis or Fisher's exact test was used.…”