The prevalence of three species of feline haemoplasmas in samples submitted to a diagnostics service as determined by three novel real-time duplex PCR assays

Abstract

assays for the detection of all three feline haemoplasma species combined with an 7 endogenous internal control and to determine the prevalence of infection, using these 8 assays, in 1592 EDTA blood samples submitted to Langford Veterinary Diagnostics, 9University of Bristol for haemoplasma testing. 10Primers and TaqMan probes were designed against published 16S rDNA 11 sequences. These assays were combined with a feline 28S rDNA-specific assay to 12 produce three duplex assays. The assays detected 1-10 copies of a sequence-specific 13 plasmid per PCR. None of the assays showed cross-reactivity with 10 6 copies of a 14 sequence-specific plasmid from the non-target haemoplasma species. 15Real-time PCR was performed on all samples using the three assays. Seven 16 samples were negative for feline 28S rDNA and were excluded from the study.

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

“…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. 1,7,12,18 Additional prevalence data for other countries are needed, and no information exists regarding hemoplasma prevalence in Spain.…”

“…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.…”

Use of Dried Blood Smears for Detection of Feline Hemoplasmas Using Real-Time Polymerase Chain Reaction

“…2,4,8 Although uncommon, coinfections with multiple hemoplasma species were reported. 4,7,9,14 Cytologic examination of blood smears has low sensitivity and specificity for a diagnosis of hemoplasmosis, and ''Ca. M. turicensis'' has never been cytologically identified.…”

“…M. turicensis'', and M. haemofelis in whole-blood samples. 1,5,[7][8][9]11,15 The analytical sensitivity of these assays has been as low as between 1 and 10 copies of organism DNA.…”