Detection of Mixed Infections with “CandidatusMycoplasma Haemominutum” andMycoplasma HaemofelisUsing Real-Time TaqMan Polymerase Chain Reaction

Abstract

Abstract. The objective of this study was to evaluate the use of real-time TaqMan PCR assays for detection of coinfections with ''Candidatus Mycoplasma haemominutum'' (Mhm), and Mycoplasma haemofelis (Mhf), in vitro and over time in experimentally infected cats. First, the ability of each real-time PCR assay to detect and quantify mixed infections was determined in vitro by testing mixtures of plasmids containing Mhm and Mhf 16S rDNA with each assay. Subsequently, 4 specific pathogen-free (SPF) cats, 2 of which were splenectomized, were inoculated with blood from a cat infected with both Mhm and Mhf. Sixteen blood samples were then collected from each cat over a 55-day period. Each of the 64 postinoculation samples was tested using both conventional polymerase chain reaction (cPCR) and real-time PCR for the 16S rRNA gene of each organism. When applied to mixtures of plasmid DNA from each species, the results of quantitation with each of the real-time PCR assays approximately reflected the number of plasmid copies present. Forty-nine of 64 post-inoculation samples (77%) were positive using both cPCR and real-time PCR, 4 (6%) were positive using cPCR only, and 3 (5%) were positive using real-time PCR only. Both organisms were detected in 23 samples using real-time PCR. Mixed infections were not detected using cPCR. The size of the corresponding cPCR products suggested infection with Mhm in 4 and Mhf in 18 of these samples. The use of multiple separate real-time PCR assays rather than cPCR alone should thus be considered for epidemiologic studies of hemoplasmosis in cats.

Use of Conventional and Real‐Time Polymerase Chain Reaction to Determine the Epidemiology of Hemoplasma Infections in Anemic and Nonanemic Cats

“…However, interference between amplification of Mhf and Mhm was not expected based on results of validation using plasmid mixtures and in cats coinfected with Mhf and Mhm. 10 We recently have detected coinfections with Mhf and Mhm in 2% of 310 anemic cats in California using the same assays (Sykes et al, unpublished data). Geographic differences also may explain a difference in prevalence of coinfections.…”

“…9,10 The partial sequences amplified for Mhm and Mhf included the region of the target sequence for all 3 real-time PCR assays.…”

“…Quantification of the number of copies of organism DNA present in each sample was performed using the standard curve generated by serial dilutions of plasmid DNA template containing known copy numbers of either Mhf or Mhm and expressed as copy numbers of DNA template per milliliter of blood. 10 In addition, the real-time PCR system targeting the GAPDH pseudogene was used to quantify nucleated blood cells of cats by extrapolating the GAPDH cycle threshold value to a standard curve generated with genomic DNA extracted from known numbers of white blood cells of cats. The hemoplasma load and the number of nucleated cells then were combined into a unit hemoplasma load per million nucleated cells as previously described.…”

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

“…The failure of the previous qPCR assay to identify 25 the 12 (both dual and triple infected) samples infected with both Mhf and CMhm may 1 be due to one of the species being dominant in the reaction and thus inhibiting 2 amplification of the minor species through competition for reaction components 3 (Tasker, 2002). This phenomenon is a potential problem with assays, like those 4 previously described for detection of Mhf and CMhm (Sykes et al, 2007;Tasker et 5 al., 2003b;Willi et al, 2006a, ), which rely solely on the TaqMan probes to 6 differentiate the species present in the sample following amplification with universal 7 primers rather than on species-specific primers and probes. This phenomenon would 8 not be a problem with the previously described assay for the detection of CMt (Willi 9 et al, 2005) which uses species-specific primers and probes.…”

Uso da técnica de Southern Blot/HibridizaÇão associada à reaÇão em cadeia da polimerase para aumentar a sensibilidade no diagnóstico das infecÇões por hemoplasmas em gatos domésticos

“…Também se constitui em uma vantagem o fato desses ensaios serem realizados em sistemas de tubos fechados do início ao fim, o que reduz o risco de contaminação e de resultados falso-positivos, (WILLI et al, 2007c). Entretanto, Sykes et al (2007) encontraram uma sensibilidade similar entre as técnicas de PCR convencional e em tempo real.…”