Development and validation of a duplex real-time PCR assay for the diagnosis of equine piroplasmosis

Abstract

BackgroundEquine piroplasmosis (EP) is an economically significant infection of horses and other equine species caused by the tick-borne protozoa Theileria equi and Babesia caballi. The long-term carrier state in infected animals makes importation of such subclinical cases a major risk factor for the introduction of EP into non-enzootic areas. Regulatory testing for EP relies on screening of equines by serological methods. The definitive diagnosis of EP infection in individual animals will benefit from the availability of sensitive direct detection methods, for example, when used as confirmatory assays for non-negative serological test results. The objectives of this study were to develop a real-time quantitative polymerase chain reaction (qPCR) assay for simultaneous detection of both agents of EP, perform comprehensive evaluation of its performance and assess the assay’s utility for regulatory testing.ResultsWe developed a duplex qPCR targeting the ema-1 gene of T. equi and the 18S rRNA gene of B. caballi and demonstrated that the assay has high analytical sensitivities for both piroplasm species. Validation of the duplex qPCR on samples from 362 competitive enzyme-linked immunosorbent assay (cELISA)-negative horses from Canada and the United States yielded no false-positive reactions. The assay’s performance was further evaluated using samples collected from 430 horses of unknown EP status from a highly endemic area in Brazil. This set of samples was also tested by a single-target 18S rRNA qPCR for T. equi developed at the OIE reference laboratory for EP in Japan, and a previously published single-target 18S rRNA qPCR for B. caballi whose oligonucleotides we adopted for use in the duplex qPCR. Matching serum samples were tested for antibodies to these parasites using cELISA. By the duplex qPCR, T. equi-specific 18S rRNA qPCR and cELISA, infections with T. equi were detected in 87.9% (95% confidence interval, CI: 84.5–90.7%), 90.5% (95% CI: 87.3–92.3%) and 87.4% (95% CI: 84.0–90.2%) of the horses, respectively. The B. caballi prevalence estimates were 9.3% (95% CI: 6.9–12.4%) by the duplex qPCR and 7.9% (95% CI: 5.7–10.9%) by the respective single-target qPCR assay. These values were markedly lower compared to the seroprevalence of 58.6% (95% CI: 53.9–63.2%) obtained by B. caballi-specific cELISA. The relative diagnostic sensitivity of the duplex qPCR for T. equi was 95.5%, as 359 of the 376 horses with exposure to T. equi confirmed by cELISA had parasitemia levels above the detection limit of the molecular assay. In contrast, only 39 (15.5%) of the 252 horses with detectable B. caballi-specific antibodies were positive for this piroplasm species by the duplex qPCR.ConclusionsThe duplex qPCR described here performed comparably to the existing single-target qPCR assays for T. equi and B. caballi and will be more cost-effective in terms of results turnaround time and reagent costs when both pathogens are being targeted for disease control and epidemiological investigations. These validation data also support the ...

Molecular Identification and Phylogenetic Analysis of Theileria equi and Babesia caballi Infections in Equids from Erbil Province, North of Iraq

“…Equine piroplasmosis has been previously reported in Iraq using several direct and indirect methods that detected B. caballi and T. equi including Giemsa stained blood smears (Alssad, 2009), cELISA (Alsaad and Al-Obaidi, 2012; Aziz and Al-Barwary, 2019). However these methods are suitable only for the detection in the acute stage of infection, but they are not applicable in identifying presymptomatic and carrier animals with low parasetemia (Lobanov et al, 2018). Therefore, this study aimed to investigate the frequency rate of piroplasms infection in different species of equids from Erbil province, North Iraq by molecular technique and to document the phylogenic sequencing of PCR products for the first time.…”

Twenty Years of Equine Piroplasmosis Research: Global Distribution, Molecular Diagnosis, and Phylogeny

“…Currently, these methods are more often used for research than in clinical practice [2,3,5,20,68,[85][86][87][88][89][90][91][92]. Numerous assays targeting one or multiple EP parasites, including conventional PCR [86,87], nested PCR (nPCR) [90,93], real-time PCR (rtPCR) [82,[94][95][96][97][98][99], multiplex PCR (mPCR) [87,89], reverse line blot (RLB) [100,101], and loop mediated isothermal amplification (LAMP) [85,88,91], have been developed. Several of these assays were determined to have high sensitivity, with a detection limit of 10 −7 % parasitized erythrocytes (PE) [2,3,5,20,68,[85][86][87][88][89][90][91][92].…”

“…Several of these assays were determined to have high sensitivity, with a detection limit of 10 −7 % parasitized erythrocytes (PE) [2,3,5,20,68,[85][86][87][88][89][90][91][92]. Quantitative methods, such as rtPCR (qPCR), have also been developed, but are mostly applied to increase the sensitivity of parasite detection, and are rarely used to evaluate parasite loads [94][95][96][97][98][99]102].…”

Rapid isothermal duplex real-time recombinase polymerase amplification (RPA) assay for the diagnosis of equine piroplasmosis

“…Real-time PCR is the widely-recognized gold standard of genomic DNA identification and quantification due to its good specificity and high sensitivity. In this study, we employed the reported PCR primers and probes to assay pUC57-Bc18S and pUC57-ema DNA 26,28 and the results showed that 2.0 fg pUC57-Bc18S DNA (Figs. 4e) and 2.0 fg of pUC57-ema DNA (Fig.…”

“…As a comparison, real-time PCR assay was also performed. Primers and probes reported in a previous duplex qPCR assay of EP 26,28 (Table 4) were synthesized by Sangon Biotech (Shanghai, China). The PCR reactions were run on a Light Cycler 480 (Roche, USA) using a TaqMan Gene Expression Master Mix (Thermo Fisher Scientific, USA).…”