Abstract
Epizootic haemorragic disease (EHD) is an important disease of white-tailed deer and can cause a bluetongue-like illness in cattle. A definitive diagnosis of EHD relies on molecular assays such as real-time RT-qPCR or conventional PCR. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) is a cost-effective, specific, and sensitive technique that provides an alternative to RT-qPCR. We designed two sets of specific primers targeting segment-9 of the EHD virus genome to enable the detection of western and eastern topotypes, and evaluated their performance in singleplex and multiplex formats using cell culture isolates (n = 43), field specimens (n = 20), and a proficiency panel (n = 10). The limit of detection of the eastern and western RT-LAMP assays was estimated as ~24.36 CT and as ~29.37 CT in relation to real-time RT-qPCR, respectively, indicating a greater sensitivity of the western topotype singleplex RT-LAMP. The sensitivity of the western topotype RT-LAMP assay, relative to the RT-qPCR assay, was 72.2%, indicating that it could be theoretically used to detect viraemic cervines and bovines. For the first time, an RT-LAMP assay was developed for the rapid detection of the EHD virus that could be used as either a field test or high throughput screening tool in established laboratories to control the spread of EHD.
Highlights
Epizootic haemorrhagic disease (EHD), caused by the EHD virus (EHDV), is an infectious, non-contagious disease that is transmitted by Culicoides biting midges
It was not possible to design one primer set for the detection of all-known EHDV strains due to several mismatches in the alignment; two separate primer sets targeting eastern (s9.E) and western (s9.W) topotypes were designed and evaluated in this study in two formats: singleplex and multiplex
The limit of detection (LOD) of eastern and western Reverse transcription loop-mediated isothermal amplification (RT-LAMP) assays was estimated as ~24.36 CT and ~29.37 CT, respectively (Tables 3 and 4), indicating a greater sensitivity of the western topotype singleplex RTLAMP assay
Summary
Epizootic haemorrhagic disease (EHD), caused by the EHD virus (EHDV), is an infectious, non-contagious disease that is transmitted by Culicoides biting midges. EHD affects wild and domestic ruminants and causes a severe haemorrhagic disease in whitetailed deer with high morbidity and mortality, and is considered one of the most important diseases affecting deer [1,2]. EHD resembles a bluetongue (BT)-like illness and can cause significant production losses due to decreased milk production, abortions, and malformations. (Ibaraki virus) caused large-scale outbreaks of disease in cattle in Japan, in 1959 [4]. Additional serotypes (EHDV-6 and -7), have been reported to cause severe clinical signs of EHD in dairy and beef cattle in countries neighboring the European Union (EU), such as Turkey, Morocco, Tunisia, Algeria, Israel, and Jordan [2].
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