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Twenty-two isolates of African horse sickness virus (AHSV), representing its distinct serotypes, geographical and historical origins, were fed to three populations of South African livestock-associated Culicoides spp. (Diptera, Ceratopogonidae). Infective blood meals included 12 recent isolates, nine historical reference strains and one live attenuated vaccine strain serotype 7 (AHSV-7) of the virus. Field-collected midges were fed through a chicken-skin membrane on sheep blood spiked with one of the viruses, which concentrations ranged from 5.4 to 8.8 log(10)TCID(50)/mL of blood. After 10 days incubation at 23.5 degrees C, AHSV was isolated from 11 Culicoides species. Standard in vitro passaging of AHSV-7, used for the preparation of live attenuated vaccine, did not reduce its ability to infect Culicoides species. Virus recovery rates in orally infected Culicoides midges differed significantly between species and populations, serotypes, isolates and seasons. Significant variations in oral susceptibility recorded in this study emphasize a complex inter-relationship between virus and vector, which is further influenced by multiple intrinsic and extrinsic factors. As it is not possible to standardize all these factors under laboratory conditions, conclusive assessment of the role of field-collected Culicoides midges in the transmission of orbiviruses remains problematic. Nevertheless, results of this study suggest the potential for multi-vector transmission of AHSV virus in South Africa.

Field-collected South African Culicoides species (Diptera, Ceratopogonidae) were fed on sheep blood containing bluetongue virus (BTV) represented by 13 low-passage reference serotypes: -1, -2, -4, -6, -7, -8, -9, -10, -11, -12, -13, -16 and -19. After 10 days of extrinsic incubation at 23.5 degrees C, of the 13 serotypes used, seven were recovered from C. (Avaritia) imicola Kieffer and 11 from C. (A.) bolitinos Meiswinkel. Virus recovery rates and the mean titres for most serotypes were significantly higher in C. bolitinos than in C. imicola. In addition, BTV was recovered from three non-Avaritia Culicoides species, namely C. (Remmia) enderleini Cornet & Brunhes (BTV-9), C. (Hoffmania) milnei Austen (BTV-4) and C. (H.) zuluensis de Meillon (BTV-16). No virus could be recovered from 316 individuals representing a further 14 Culicoides species. In Culicoides species fed on blood containing similar or identical virus titres of distinct BTV serotypes, significant differences were found in virus recovery rates. The results of this study confirm the higher vector competence of C. bolitinos compared with C. imicola.

The oral susceptibility of livestock-associated South African Culicoides midges (Diptera: Ceratopogonidae) to infection with the tissue culture-attenuated vaccine strains of African horse sickness virus (AHSV) currently in use is reported. Field-collected Culicoides were fed on horse blood-virus mixtures each containing one of the seven serotype-specific vaccine strains of AHSV, namely serotypes 1, 2, 3, 4, 6, 7 and 8. The mean titres of virus in the bloodmeals for the seven vaccine strains were between 6.8 and 7.6 log10TCID50/mL. All females (n = 3262) that survived 10 days extrinsic incubation (10 dEI) at 23.5 degrees C were individually assayed in microplate BHK-21 cell cultures. In midges tested immediately after feeding, AHSV was detected in 96.1% individuals; mean virus titre was 2.0 log10TCID50/midge. After 10 dEI virus recovery rates varied in Culicoides (Avaritia) imicola Kieffer from 1% (AHSV-2) to 11% (AHSV-7) and in Culicoides (A.) bolitinos Meiswinkel from 0% (AHSV-3) to 14.6% (AHSV-2). Although our results indicate that two major field vectors C. imicola and C. bolitinos are susceptible to oral infection with vaccine strains of AHSV, the level of viral replication for most of the vaccine strains tested was below the postulated threshold (=2.5 log10TCID50/midge) for fully disseminated orbivirus infection. In this study, for the first time AHSV has been recovered after 10 dEI from six non-Avaritia livestock-associated Old World species: C. engubandei de Meillon (AHSV-4), C. magnus Colaco (AHSV-3, -4), C. zuluensis de Meillon (AHSV-2, -4), C. pycnostictus Ingram & Macfie (AHSV-2), C. bedfordi Ingram & Macfie (AHSV-7), and C. dutoiti de Meillon (AHSV-7). As little is known about the virogenesis of AHSV in the southern African species of Culicoides, the epidemiological significance of our findings in relation to the potential for transmission of current AHSV vaccine strains by Culicoides requires further assessment.

The oral susceptibility of 22 South African livestock associated Culicoides species to infection with bluetongue virus serotype 1 (BTV-1) and its replication rate in C. imicola Kieffer and C. bolitinos Meiswinkel (Diptera: Ceratopogonidae) over a range of different incubation periods and temperatures are reported. Field-collected Culicoides were fed on sheep blood containing 7.5 log10TCID50/mL of BTV-1, and then held at constant different temperatures. Virus replication was measured over time by assaying individual flies in BHK-21 cells using a microtitration procedure. Regardless of the incubation temperatures (10, 15, 18, 23.5 and 30 degrees C) the mean virus titre/midge, infection rates (IR) and the proportion of infected females with transmission potential (TP = virus titre/midge > or = 3 log10 TCID50) were found to be significantly higher in C. bolitinos than in C. imicola. Results from days 4-10 post-infection (dpi), at 15-30 degrees C, shows that the mean IR and TP values in C. bolitinos ranged from 36.7 to 87.8%, and from 8.4 to 87.7%, respectively; in C. imicola the respective values were 11.0-13.7% and 0-46.8%. In both species the highest IR was recorded at 25 degrees C and the highest TP at 30 degrees C. The time required for the development of TP in C. bolitinos ranged from 2 dpi at 25 degrees C to 8 dpi at 15 degrees C. In C. imicola it ranged from 4 dpi at 30 degrees C to 10 dpi at 23.5 degrees C; no individuals with TP were detected at 15 degrees C. There was no evidence of virus replication in flies held at 10 degrees C. When, at various points of incubation, individual flies were transferred from 10 degrees C to 23.5 degrees C and then assayed 4-10 days later, virus was recovered from both species. The mean virus titres/midge, and proportion of individuals with TP and IR, were again significantly higher in C. bolitinos than in C. imicola. Also the infection prevalence in C. magnus Colaco was higher than in C. imicola. Low infection prevalences were found in C. bedfordi Ingram & Macfie, C. leucostictus Kieffer, C. pycnostictus Ingram & Macfie, C. gulbenkiani Caeiro and C. milnei Austen. BTV-1 was not detected in 14 other Culicoides species tested; however, some of these were tested in limited numbers. The present study indicates a multivector potential for BTV transmission in South Africa. In C. imicola and C. bolitinos the replication rates are distinct and are significantly influenced by temperature. These findings are discussed in relation to the epidemiology of bluetongue in South Africa.

Summary Equine encephalosis virus (EEV) was recognized and described in the Republic of South Africa in 1967 and subsequent serological studies have shown this orbivirus to be both widespread and prevalent in southern Africa. In the present study it was shown that wild-caught Culicoides (Avaritia) imicola Kieffer (Diptera: Ceratopogonidae) can become infected with and permit the replication of the Bryanston serotype of EEV following membrane-feeding on infective blood containing 5.0 log10 plaque-forming-units (PFU)/ml. The mean prevalence of Bryanston virus infection in C. imicola after 10 days extrinsic incubation at 23.5°C was 22.3% (23/103). The mean infectivity of Bryanston virus in the infected C. imicola increased from 1.3 log10 PFU/midge, in insects assayed immediately after feeding on the blood-virus mixture, to 2.6 log10 PFU/midge in insects assayed after incubation. The virus concentration in individual C. imicola infected with the Bryanston serotype of EEV ranged from 0.7 to 3.6 log10 PFU/midge. Bryanston virus titres higher than 2.5 log10 TCID50, found in individual C. imicola, suggest that this species may be able to transmit this virus to susceptible hosts. Prevalence of virus infection in C. imicola was determined by PFU and microtitration assays on both BHK and Vero cells and confirmation of the Bryanston serotype of EEV was determined by plaque inhibition. No virus replication could be demonstrated in 102 C. nivosus tested after the incubation period, suggesting that not all Culicoides species are equally susceptible to Bryanston virus infection. Other Culicoides species that survived the incubation period and that were negative for the presence of Bryanston virus were C. pycnostictus (42), C. leucostictus (7), C. magnus (2), C. bolitinos (1) and C. bedfordi (1).

The susceptibility of field-collected Culicoides bolitinos to infection by oral ingestion of bluetongue virus serotypes 1, 3 and 4 (BLU 1, 3 and 4) was compared with that of field-collected C. imicola and laboratory reared C. variipennis sonorensis. The concentration of the virus per millilitre of bloodmeal was 10(5.0) and 10(6.0)TCID50 for BLU 4 and 10(7.2)TCID50 for BLU 1 and 3. Of 4927 C. bolitinos and 9585 C. imicola fed, 386 and 287 individual midges survived 10 days extrinsic incubation, respectively. Midges were assayed for the presence of virus using a microtitration assay on BHK-21 cells and/or an antigen capture ELISA. Infection prevalences for the different serotypes as determined by virus isolation ranged from 22.7 to 82.0% in C. bolitinos and from 1.9 to 9.8% in C. imicola; infection prevalences were highest for BLU 1, and lowest for BLU 4 in both species. The mean log10 TCID50 titre of the three BLU viruses per single fly was higher in C. bolitinos than in C. imicola. The results suggested that C. bolitinos populations are capable vectors of the BLU viruses in South Africa. A high correlation was found between virus isolation and ELISA results for the detection of BLU 1, and less for BLU 4; the ELISA failed to detect the presence of BLU 3 in infected flies. The C. v. sonorensis colonies had a significantly lower susceptibility to infection with BLU 1, 3 and 4 than C. bolitinos and C. imicola. However, since infection prevalence of C. v. sonorensis was determined only by ELISA, this finding may merely reflect the insensitivity of this assay at low virus titres, compared to virus isolation.

The susceptibility of Culicoides (Avaritia) imicola Kiefer (Diptera: Ceratopogonidae) to 21 isolates representing all nine known serotypes of African horse sickness virus (AHSV), recovered from clinical cases of the disease in South Africa during 1998-2004, was compared with its susceptibility to approximately 40-year-old isolates stored at the Agricultural Research Council-Onderstepoort Veterinary Institute. Field-collected C. imicola were fed through a chicken skin membrane on sheep blood spiked with one of the virus isolates to a concentration in the range of 5.6-7.5 log (10)TCID(50)/mL. After 10 days incubation at 23.5 degrees C, five of the nine historical serotypes (AHSV-1, -2, -3, -7 and -9) could not be isolated from C. imicola. All nine serotypes were recovered for the 21 recent isolates, for 16 of which the virus recovery rates were higher than for the corresponding historical isolates. These results emphasize the need to assess the oral susceptibility of local Culicoides populations to viruses in circulation during outbreaks in order to estimate their vector potential.