Rapid Preclinical Detection of Sheeppox Virus by a Real-Time PCR Assay

Abstract

Sheeppox virus (SPPV) is a member of the Capripoxvirus (CaPV) genus of the Poxviridae family. Members of this genus, which also include goatpox and lumpy skin disease viruses, cause economically significant disease in sheep, goats, and cattle. A rapid diagnostic assay for CaPV would be useful for disease surveillance as well as for detection of CaPV in clinical samples and for outbreak management. Here we describe a fluorogenic probe hydrolysis (TaqMan) PCR assay designed for rapid detection of CaPV and tested on sheep experimentally infected with a virulent strain of SPPV. This assay can detect SPPV in buffy coats, nasal swabs, oral swabs, scabs, and skin lesions as well as in lung and lymph nodes collected at necropsy. This single-tube diagnostic assay can be performed in 2 h or less and can detect viral DNA in preclinical, clinical, and postmortem samples.The Capripoxvirus (CaPV) genus of the Poxviridae family of viruses comprises sheeppox (SPPV), goatpox (GTPV), and lumpy skin disease (LSDV) viruses, which cause disease in sheep, goats, and cattle, respectively. These viruses are considered reportable agents to the World Organization for Animal Health due to their potential for significant economic impact. Members of the CaPV genus are closely related, with genomic identities ranging from 96% between viral species to 99% between isolates of the same species (27). They have complete open reading frame (ORF) colinearity (13,26,27) and are indistinguishable via serological methods (14)(15)(16). CaPVs tend to be host specific; however, incidences where SPPV and GTPV have crossed species, into goats and sheep, respectively, have been documented (8,20).Transmission of SPPV is thought to occur through exposure to aerosols or respiratory droplets produced by acutely infected animals or by direct or indirect contact with lesions or oronasal secretions (8,17). Virus can be detected in nasal secretions of animals infected by aerosol or contact exposure (17) as well as in lesions found in the upper and lower respiratory tracts of infected animals (1c, 11). Transmission may also occur through the dermis, after contact exposure with cuts or abrasions (1c, 21, 22, 25), or via mechanical transmission by arthropod vectors (19).Clinical signs of SPPV infection include fever, anorexia, depression, conjunctivitis, rhinitis, respiratory distress, generalized skin lesions, and enlargement of lymph nodes (9,22). A transient viremia develops but may not be detectable throughout the entire course of infection (14). Transient viremias have also been described for cattle infected with LSDV (7, 28). Internal lesions are often seen at necropsy, especially in the lungs, although lesions in the trachea, rumen, tongue, kidney, nasal turbinates, and reproductive organs have also been reported (1c, 11, 14).SPPV is endemic throughout much of Africa, southwest and central Asia, and the Indian subcontinent (3,4,23). Young animals are most susceptible, where mortality rates can be as high as 50 to 70% (10, 12). Outbreaks are controlled by ring v...

Real-Time PCR Assays for the Specific Detection of Field Balkan Strains of Lumpy Skin Disease Virus

“…Biopsy samples had the lowest Ct values which makes them the most suitable for testing. A number of real-time PCR methods for the detection of capripoxviruses have recently been described [3,11,18,22,23]. However, to our knowledge, until now no papers have been published describing TaqMan real Time PCR assays for the specifi c detection of the virulent fi eld strain of LSDV.…”

Contagious Pustular Dermatitis (Orf Disease) - Epidemiology, Diagnosis, Control and Public Health Concerns

“…Studies demonstrated that TaqMan probe based duplex real-time PCR (drt-PCR) has been optimized with two different sets of primers and probes targeting the highly conserved DNA pol gene of capripox, sheeppox and orf virus genome. In comparison to real-time PCR for detection of orf virus, drt-PCR assay has proved to be more specific (100% diagnostic specificity) and highly sensitive (diagnostic sensitivity of 98.7%) (Balinsky et al, 2008;Balamurugan et al, 2009;Bora et al, 2011;Venkatesana et al, 2014a, b). Schmidt et al (2013) performed partial nucleotide sequencing and phylogenetic analysis of B2L gene of seventeen Brazilian orf viruses and concluded that discrete nucleotide changes in B2L gene may help in grouping orf viruses according to host species.…”

Outbreak of sheeppox in farmed sheep in Kyrgystan: Histological, eletron microscopical and molecular characterization

“…Detection of Capripoxvirus by PCR assay utilised genus specific primers and probe (Balinsky et al, 2008) and was performed on a 7900HT Fast Real-Time PCR System (Thermo Fisher Scientific, Waltham MA, USA) with standard ramping conditions. The sample analysed reacted positively for Capripoxvirus.…”