Development of a toolkit for African horse sickness : identification of Culicoides vectors from Namibia and detection of African horse sickness virus

MSc (Environmental Sciences), North-West University, Potchefstroom Campus, 2017African horse sickness (AHS) is a non-contagious, viral, insect-borne disease of equids and this disease is caused by the African horse sickness virus (AHSV). The virus is part of the family Reoviridae of the genus Orbivirus. The virus has nine distinct serotypes. AHSV affects horses, mules, donkeys and zebras, resulting in severe animal health and welfare problems together with serious economic consequences. Main vectors of orbiviruses are haematophagous arthropods such as Culicoides Latreille midges, ticks, sand flies and mosquitoes. Female Culicoides biting midges are the primary vectors of AHSV. Culicoides midges (C. imicola Kieffer and C. bolitinos Meiswinkel) play a role in the abundance, prevalence and seasonal incidence of AHSV outbreaks. The aim of this study was to establish DNA barcodes for Culicoides species collected in Namibia and to develop a simplified nucleic acid diagnostic toolkit for the detection of AHSV. The first objectives of the study were to extract DNA from morphologically identified Culicoides specimens, sequence the mitochondrial cytochrome oxidase subunit I gene for DNA barcoding and align amplicons with sequences from databases for phylogenetic identification. A phylogenetic tree of Culicoides species was drawn and 11 new sequences of morphologically identified species were obtained along with five previously sequenced species. The second objective was to design specific primers for loop-mediated isothermal amplification (LAMP) assays of AHSV, optimise the reverse-transcription (RT)-LAMP method for AHSV detection and evaluate the assay with artificially infected Culicoides samples. Specific primers of the virus-protein-4 region of AHSV were designed for RT-LAMP assays. The RT-LAMP standard test was successful, with multiple band formation on an agarose gel indicating a positive result. The RT-LAMP method was optimised with virus RNA and evaluated through assays with artificially infected specimens to test sensitivity, but the primer set proved not to be sensitive enough. However, an RT-LAMP method can be used for AHSV detection in the near future, with additional research and better designed primers from different regions of the genome. A diagnostic toolkit will be helpful for the early detection of AHSV and will help manage and control epidemic outbreaks of AHSMaster