Epidemiology of bluetongue virus with special reference to the Mnisi area, Mpumalanga Province, South Africa

Abstract

Bluetongue virus (BTV) is the prototype member of the genus Orbivirus in the family Reoviridae. This virus causes an economically important infectious, non-contagious disease, bluetongue, rendering it a notifiable disease at the OIE. Twenty six serotypes of the virus have been identified that are transmitted primarily by certain species of biting midges that belong to the genus Culicoides (Diptera: Ceratopogonidae). The distribution of BTV is determined by the occurrence of competent Culicoides midge species, climatic conditions and susceptible ruminant hosts. During the last decade BTV has become a major concern worldwide as well as the focus point of many epidemiological studies and surveillance programmes. The first experimental study was based in Mnisi, a rural area located in Mpumalanga. This area is adjacent to the Kruger National Park and ideally represents the interphase in a wildlife-domestic animal interaction. Cattle farming is the major source of income of the local community. Chapter 2 focused on determining the prevalent Culicoides spp. in the area as well as to determine whether BTV is circulating among cattle in the area. The epidemiology of bluetongue virus is very complex due to the involvement of several mammalian hosts and vector species. The role of cattle in the epidemiology of BT in SA is not well understood. Light traps were used to collect midges over 16 trap nights during autumn and winter. Culicoides midges were identified to species level and pooled (200 midges/ pool). Midge pools were subjected to real-time RT-qPCR to test for the presence of BTV RNA. Serum samples were randomly collected from 1 260 cattle and screened for antibodies specific to bluetongue virus using a BTV-specific cELISA. Blood samples were collected from seronegative cattle and screened for the presence of BTV RNA with a real-time RT-qPCR. Twenty-five different Culicoides spp. were identified of which C. imicola were found to be the most abundant. Of the 25 species collected, 19 species yielded parous females with 16 Culicoides species demonstrating a vector rating higher than 25%. Bluetongue virus RNA was detected in 51.2% and 75.9% of midge pools collected during autumn and winter, respectively resulting in an infection prevalence of 0.3% and 0.7%. Antibodies specific to BTV were detected in 1 206 (95.7%) of the sera samples tested with significant differences (p < 0.05) in seroprevalence between age groups and between villages. No significant differences in seroprevalence were observed between different breeds or sex. A total of 16 out of 45 (35.5%) blood samples from seronegative cattle tested positive for BTV RNA. These results demonstrate that C. imicola is the most abundant midge species and that BTV is highly prevalent in autumn as well as throughout winter in Mnisi. The second part of the study focused on the overwintering of BTV in Culicoides populations at the ARC-OVI. The exact method(s) of overwintering of BTV is unknown. Recent studies have suggested that the virus is overwintering within the midge vector. Climatic conditions in South Africa are suitable for adult Culicoides midges to remain active throughout much of the year with only certain areas where temperatures can become unsuitable. During such periods ambient temperature might be too low for midge emergence or viral replication within the vector. It is hypothesised that during summer, BTV infection becomes more prevalent in the Culicoides population and therefore disease only occurs in late summer to early autumn. Real-time RT-qPCR was used to detect BTV in Culicoides midges collected from July 2010 to August 2011. Bluetongue virus RNA was detected in 52 out of 57 (91.2%) midge pools tested. The results obtained strengthen the findings of chapter 2 demonstrating that BTV is present throughout winter in Culicoides populations at various temperatures. It is therefore clear Culicoides are present throughout the year and that BTV are capable of overwintering within the midge even though temperatures fall well below the normal activity range of these midges. This also forms part of a study focussing on overwintering of AHSV in the Culicoides vector. In the last research chapter of the dissertation, segment 10 of BTV isolates previously used as reference strains in the Department of Veterinary Tropical Diseases, from 1972 - 2000 were compared to establish the topotypes of these strains. Bluetongue virus can be divided into five topotypes based on segment 10 i.e. western 1, 2 and 3 and eastern 1 and 2. The groupings are based on evolutionary distinct geographical variants and phylogenetic analysis. It is hypothesised that the segment 10 genes have co-evolved with respect to the specific Culicoides species found in a geographical area. Comparing the highly conserved NS3 gene region to newly isolated strains in South Africa as well as to representative global strains could give us an indication of the degree of variability between strains and serotypes. Segment 10 from these samples were sequenced and compared to global NS3 sequences. A total of 11 sequences were obtained and submitted to GenBank. All sequences demonstrated the conserved cysteine regions as well as the tryptophan residue. Both hydrophobic regions and the proline-rich regions were also conserved throughout all 11 isolates as well as the two glycosylation sights. Both a neighbour-joining and a neighbour-net tree were compiled using MEGA 6 and SplitsTree 4 respectively. Three lineages groups were established with i.e. western group 1 and 2 and eastern group 1. The phylogenetic trees coincide with previous studies done on segment 10 of the BTV genome. These results also confirm that BTV-20, BTV-21 and BTV-23 are exotic to South Africa. BTV-2 demonstrated the ability of strains to cluster together, irrespective of their serotype providing evidence of genetic differences within serotypes. The presence of both competent midges and amplifying host, e.g. cattle and wildlife, contribute in the epidemiology of the disease, especially in episystems where distinct grouping of viruses co-evolved with respect to specific Culicoides species. The epidemiology of BTV is therefore likely to be largely influenced by the level of contact between livestock (and wildlife) and BTV-infected Culicoides species that influences both the distribution and the genetics of the virus.