Detection of African horse sickness virus in Culicoides imicola using real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR)

Abstract

African horse sickness (AHS) is an infectious, non-contagious arthropod-borne disease of equids. The disease is caused by the African horse sickness virus (AHSV), a member of the genus Orbivirus, of the Reoviridae family. It is endemic to sub-Saharan and East Africa and thought the most lethal viral disease of horses. Previous research focused on the diagnosis of host samples rather than detection in the vector. This study focused on detection of AHSV in Culicoides imicola pools by the application of real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR). The aim of this study was the detection of AHSV in field-collected C. imicola complex pools in Namibia. The first part of this dissertation focuses on, the performance of the RT-qPCR methodology was determined. Next, the optimal pool for the limit of detection (LOD) of AHSV in C. imicola pools was determined by assaying midges in different pool sizes. Midges were fed with AHSV-infected blood and sorted into different pool sizes, with one infected individual per pool. RNA was extracted and prepared for RT-qPCR. The virus was successfully detected and the optimal pool size for the LOD of the virus was determined. A guideline was suggested on the size of pools for accurate and sensitive detection. The second part of the dissertation focused on the application of the RT-qPCR methodology on field collected Culicoides in Namibia. Culicoides were collected at different sites in Namibia, based on AHS incidence (low, medium and high) over a two-year period (2013–2014), coinciding with the AHS season. Culicoides species and abundance at each site were determined. Culicoides imicola, the principal vector, was the most abundant species overall. Other implicated AHSV vectors were present at all sites. Collected, sorted and pooled C. imicola were assayed with the RT-qPCR methodology based on the pool size determined above. AHSV was detected at all incidence sites and comparisons between sites were made. Windhoek, the medium AHSV incidence site, had the highest number of positives across all sites. There seemed to be a high AHS-incidence time period across sites as well. The importance and application of these results are relevant for AHSV vector identification, vector and virus ecology and for future research on locality-based preventive AHS management.