Molecular diagnosis of African horse sickness.

Abstract

African horse sickness (AHS) is a viral disease of equids caused by the African horse sickness virus (AHSV), a double-stranded RNA virus of the genus Orbivirus. The disease had a devastating effect on the sporting horse industry, and many communities who rely on equids for subsistence farming. In this study, which started in 2016, blood samples were collected from 81 horses in the KwaZulu Natal province of South Africa. Six horses (7%) were identified as AHSV positive and had their viral load quantified using a standard RT-qPCR assay. A semi-quantitative method, using digital gel image analysis, was evaluated as an alternative to qPCR in resource-limited settings. A significant correlation was found in the quantification data obtained using qPCR and digital gel analysis (P < 0.01), demonstrating the potential for its use in under-equipped diagnostic laboratories. Vaccination is currently the main tool for control of AHS in endemic areas. Live attenuated vaccines (LAV) have the potential to disrupt effective disease monitoring by being a source of the detectable virus. The sensitivity of nucleic acid diagnostic assays allows for the detection of low levels of AHSV prior to the onset of clinical signs. A study was conducted on the effect that this vaccine has on viraemia levels and the disruptive effect this may have is discussed. Routinely vaccinated miniature mares (n=9) had their AHSV viraemia levels monitored following the use of a commercial polyvalent LAV. No AHSV was detected in the horses following vaccination. The absence of AHSV in the mares supports the concept that multiple vaccinations reduce the risk of vaccine-induced viraemia. Flinders Technology Associates® filter paper cards (FTA® cards) are designed to store nucleic acid from fresh sample material in a stable matrix and may be a practical solution to disease surveillance in resource-limited settings, where conventional sampling methods are not always suitable. Storage and elution conditions of AHSV RNA from these cards have yet to be investigated. This study compared the efficacy of two elution reagents for AHSV RNA extraction from blood applied to FTA® cards. Cards with AHSV positive blood applied were stored at room temperature for one week. Amplified products were extracted using TE-buffer with an elution time of 24 hours. This study shows that FTA® cards are feasible for AHSV nucleic acid assays but further investigation into standardised procedures for the storage and recovery of AHSV RNA from FTA® cards is required. The AHSV genome is made up of ten double-stranded RNA segments with nine antigenically different serotypes of AHSV identified so far. However, there is limited information on the serotype prevalence of AHSV in endemic areas. Therefore, multivalent vaccines remain the mainstay for disease control in these regions. Improved epizootiological information can improve vaccination programs and control mechanisms. In this experiment, a novel approach was investigated for the assessment of AHSV nucleic acid variations using high-resolution melt analysis (HRMA). Blood infected with a wild strain of AHSV was used in a PCR that targeted regions of Segment 10 (S10) and Segment 2 (S2). Following this, high-resolution melting curves were generated from these PCR products. S10 primers amplified the expected 181 bp product but the other primer sets failed to amplify the S2 gene segment, warranting further evaluation and optimisation of the primer sets used to detect currently circulating AHSV strains.