Abstract
This study reports the development of multiplex real-time PCR assays for differential detection of capripoxvirus (CaPV), parapoxvirus (PaPV), and foot-and-mouth disease virus (FMDV) in sheep, goats and cattle. Three multiplex assays were developed, a capripox (CaP) rule-out assay for simultaneous detection and differentiation of CaPV and PaPV, a FMD rule-out assay for simultaneous detection and differentiation of FMDV and PaPV, and a FMD/CaP rule-out assay for simultaneous detection and differentiation of CaPV, PaPV and FMDV. All multiplex assays included β-actin gene ACTB as an internal positive control to monitor PCR inhibition and accuracy of nucleic acid extractions. The optimized assays were highly specific to the target viruses (CaPV, PaPV, and FMDV) with no cross-reactivity against other differential viruses. Using positive control plasmids as template, the limit of detection (LOD) of the multiplex assays were estimated as 2 (CaPV), 7 (PaPV), and 15 (FMDV) copies per assay. The amplification efficiency (AE) and correlation co-efficient (R2), estimated from the standard curves (Ct vs. log10 template dilution), were 94-106% and >0.99, respectively, for CaP and FMD rule-out assays, 96-116% (AE) and >0.98 (R2), respectively, for CaP/FMD rule-out assays and 91-102% and >0.99, respectively, for the corresponding singleplex assays. The diagnostic sensitivity (DSe) of the multiplex assays was assessed on 35 (CaPV), 36 (PaPV) and 39 (FMDV) clinical specimens collected from experimentally (CaPV and FMDV) and naturally (PaPV) infected animals, and all tested positive (DSe 100%) except two FMDV specimens that were tested negative (37/39; DSe 95%). The newly developed multiplex assays offer a valuable tool for differential detection of clinically indistinguishable CaPV, PaPV, and FMDV in suspected animals and animals with mixed infections.
INTRODUCTION
Capripoxvirus (CaPV), parapoxvirus (PaPV), and foot-and-mouth disease virus (FMDV) are etiological agents of capripox (CaP), parapox (PaP), and foot-and-mouth disease (FMD), respectively. These viruses are common pathogens infecting ruminants including sheep, goats and cattle. PaP is endemic in many countries worldwide including the United States, while CaP and FMD are endemic in many regions of Asia and most of Africa. Both CaP and FMD are listed as economically important foreign animal disease (FAD) in the US and reportable to the World Organization for Animal Health, formerly the Office International des Epizooties (OIE).
CaPVs belong to the genus Capripoxvirus within the familyPoxviridae , subfamily Chordopoxvirinae and comprised of three genetically similar species:Sheeppoxvirus(SPV), Goatpoxvirus (GPV), and Lumpy skin disease virus(LSDV) (Tuppurainen et al. 2017). Animals susceptible to SPV, GPV, and LSDV are sheep, goats, and cattle, respectively. There has never been a CaP outbreak in the Western hemisphere; however, the disease is endemic in many parts of the world, including many regions of Asia (SP, GP and LSD), Africa (SP, GP, and LSD) with recent outbreaks in Southeast Europe (LSD) that were successfully controlled through vaccination. In endemic countries, the diseases cause large economic losses in production and trade. The clinical presentations of CaPV infections include fever, depression, reduced milk production (cow), and skin lesions (nodule, papules, and macules) (Babiuk et al. 2008). Disease is mainly transmitted by insect vectors (LSDV), direct contact of infected animals and/or through aerosols (SPV and GPV) (Tuppurainen et al. 2017). LSDV infections in cattle have a moderate morbidity rate (5-45%) and a low mortality rate (below 10%) while SPV and GPV infections (sheep and goats) have high morbidity rate (70-90%) and a high mortality rate (up to 50%).
PaPVs belong to the family Poxviridae and genusParapoxvirus and consists of four species: Orf virus(ORFV), Bovine papular stomatitis virus (BPSV),Pseudocowpox virus (PCPV) and Parapoxvirus of red deer in New Zealand (PVNZ) (Buttner and Rziha 2002). Ruminants are the main hosts of PaPVs worldwide, including sheep and goats (infected by ORFV) and cattle (infected by BPSV or PCPV). All PaPVs are closely related genetically and antigenically, exhibit similar morphology, and have virulence mechanisms that are indistinguishable (Fleming et al. 1993). PaPV infections are often mild; and the clinical disease is characterized by fever, sores and proliferative dermatitis including raised/elevated skin, and ulcerative lesions on leg, mouth, teats, lips, nostrils, gums and tongue. In most cases, the infected animals recover completely by themselves within 1-2 months without any post-infection treatment (Haig and Mercer 1998; McKeever et al. 1988). PaPVs are also zoonotic, infecting humans with lesions characterized by large, painful nodules (milker’s nodule) on the hands after close contact with skin lesions of infected animals or handling virus-contaminated materials (Memer & Tyring 1995; Handler et al. 2017). Although PaP is not a reportable disease to OIE, its clinical resemblance to CaP and FMD requires a differential diagnosis of the disease to rule-out CaP and FMD.
FMDVs belong to the family Picornaviridae and genusAphthovirus (Grubman and Baxt 2004) and consists of seven distinct serotypes: A, Asia1, C, O, and SAT (Southern African Territories) -1, -2, and -3 (Alexandersen et al., 2003; Bachrach 1968). It is a highly contagious viral disease affecting wild and domestic cloven-hoofed animals, including pigs and ruminants (Alexandersen et al., 2003; Arzt et al., 2011b) and is a reportable disease to OIE. FMD has a substantial impact on the economy of endemic countries due to international trade restrictions in animal products. The disease spreads rapidly by direct and indirect contact with infected animals or an infected environment (Alexandersen et al., 2003). The disease is endemic in many regions of the world including Africa, most of Asia and South America with sporadic outbreaks in free areas. An outbreak of FMD in countries that are free from the disease can result in massive economic losses due to culling of large numbers of animals and restrictions in the movement of livestock and trade in animal products. The clinical presentation of FMD is characterized by fever, reduced milk production, lameness and vesicular lesions on tongue, muzzle, feet and teats (Artz et al. 2011a & 2011b). Infected sheep and goats can shed the virus with mild or no clinical disease which is often overlooked allowing FMDV to be undetected (Barnett & Cox 1999; Callens et al. 1998; Hughes et al. 2002).
Mixed infections with multiple viral pathogens are common and it can be devastating if any of the co-infecting pathogens is highly contagious, such as FMDV or CaPV. Mixed infections of FMDV or CaPV with other viruses in ruminants have been reported, including PaPV (Xu et al. 2019; He et al. 2017; Venkatesan et al. 2014a & 2014b; Chu et al. 2011), peste des petits ruminant virus (PPRV) (Kumar et al. 2016), and blue tongue virus (BTV) (Malik et al. 2011). Clinical diagnosis of mixed infections involving CaPV, PaPV, and FMDV can be challenging due to very similar clinical signs associated with these diseases. In countries free of CaP and FMD but endemic to PaP, it is extremely important to detect and differentiate PaPV from FMDV and CaPV in suspected animals. Differential diagnosis of these viruses is more challenging in countries where all three viruses are endemic.
In this study, three multiplex real-time PCR (qPCR) and RT-PCR (RT-qPCR) assays were developed for the differential detection of CaPV, PaPV and FMDV. The performance of the newly developed assays were compared against the corresponding singleplex assays to evaluate their sensitivity, specificity, and compatibility.