The increasing threat of vector-borne diseases (VBDs) represents a great challenge to those who manage public and animal health. Determining the spatial distribution of arthropod vector species is an essential step in studying the risk of transmission of a vector-borne pathogen (VBP) and in estimating risk levels of VBD. Risk maps allow better targeting surveillance and help in designing control measures. We aimed to study the geographical distribution of Culicoides imicola, the main competent vector of Bluetongue virus (BTV) in sheep in Tunisia. Fifty-three records covering the whole distribution range of C.imicola in Tunisia were obtained during a 2-year field entomological survey (August 2017 - January 2018 and August 2018 - January 2019). The ecological niche of C. imicola is described using ecological-niche factor analysis (ENFA) and Mahalanobis distances factor analysis (MADIFA). An environmental suitability map (ESM) was developed by MaxEnt software to map the optimal habitat under the current climate background. The MaxEnt model was highly accurate with a statistically significant area under curve (AUC) value of 0.941. The location of the potential distribution of C. imicola is predicted in specified regions of Tunisia. Our findings can be applied in various ways such as surveillance and control program of BTV in Tunisia.

Bluetongue (BT), a disease that affects mainly sheep, causes economic losses owing to not only its deleterious effects on animals but also its associated impact on the restriction of movement of livestock and livestock germplasm. The causative agent, bluetongue virus (BTV), can occur in the semen of rams and bulls at the time of peak viraemia and be transferred to a developing foetus. The risk of the transmission of BTV by bovine embryos is negligible if the embryos are washed according to the International Embryo Transfer Society (IETS) protocol. Two experiments were undertaken to determine whether this holds for ovine embryos that had been exposed to BTV. Firstly, the oestrus cycles of 12 ewes were synchronised and the 59 embryos that were obtained were exposed in vitro to BTV-2 and BTV-4 at a dilution of 1 x 102.88 and 1 x 103.5 respectively. In the second experiment, embryos were recovered from sheep at the peak of viraemia. A total of 96 embryos were collected from BTV-infected sheep 21 days after infection. In both experiments half the embryos were washed and treated with trypsin according to the IETS protocol while the remaining embryos were neither washed nor treated. All were tested for the presence of BTV using cell culture techniques. The virus was detected after three passages in BHK-21 cells only in one wash bath in the first experiment and two unwashed embryos exposed to BTV-4 at a titre of 1 x 103.5. No embryos or uterine flush fluids obtained from viraemic donors used in the second experiment were positive for BTV after the standard washing procedure had been followed. The washing procedure of the IETS protocol can thus clear sheep embryos infected with BTV either in vitro or in vivo.

An antigen-capture ELISA was used to detect bluetongue virus (BTV) from blood of infected sheep. A rabbit-origin capture antibody and a mouse-origin detection antibody combined with biotin-avidin amplification were used for the assay. The antigen-capture ELISA could not detect virus directly from the blood of infected sheep because of low virus titer. To enhance detection, virus from infected blood was amplified in cell culture. Virus could then be detected from cell culture supernatant fluids, using the ELISA. This amplification step increased the sensitivity of the assay comparable to that of assays performed in cell culture measuring cytopathic effects. The ELISA procedure was specific for BTV and did not mistakenly identify the antigenically related epizootic hemorrhagic disease virus. The antigen-capture ELISA permitted indirect quantitation and identification of BTV from the blood of infected sheep.

Thirty-two bovine field isolates of bluetongue virus (BTV), 6 field isolates of epizootic hemorrhagic disease virus (EHDV) from deer, 4 BTV prototype serotypes (10, 11, 13, and 17), and 2 EHDV prototype serotypes (1 and 2) were coelectrophoresed, using polyacrylamide gels. Field isolates were obtained from various regions of the United States. Analysis of polyacrylamide gels and scattered plots generated for comparison of migration patterns for different isolates within each serotype of BTV revealed wide variation among the individual segments. The BTV serotypes 10 and 11 had more variation, compared with BTV serotypes 13 and 17, especially for migration of genome segment 5. A definitive correlation was not seen between the double-stranded RNA migration profiles on polyacrylamide gel electrophoresis, geographic origin, herd of origin, or year of collection. One BTV field isolate contained more than 1 electropherotype, with 2 bands at the segment-7 position, and it was further characterized as BTV serotype 11. Segments 2 and 5 of EHDV isolates were more variable in their migration than were the other gene segments. Generally, migration profiles for EHDV double-stranded RNA were more variable, compared with those of BTV isolates. Although a correlation was found between migration profiles and serotype of 2 isolates of EHDV, a study of additional EHDV isolates is required before the diversity of electrophoretic patterns of EHDV can be determined.

A slot blot hybridization technique was applied detection of bluetongue virus (BTV) in blood mononuclear cells (BMNC) obtained from cattle with experimentally induced infection. This technique lacked sensitivity to detect the viral nucleic acid directly in clinical specimens. When aliquots of mononuclear cells from these cattle were cultivated in vitro for 10 days to amplify virus titer, only 33.3% of the samples collected during viremia gave a positive signal in the slot blot hybridization format. By contrast results for 34.3% of noncultured and 63.3% of cultured mononuclear cell samples collected during viremia were positive by immunofluorescence. The average number of infected cells, as detected by immunofluorescence in the noncultured mononuclear cell samples, was 1 to 5/300,000, and was usually > 10/300,000 in the cultured cell samples. Virus was isolated from all postinoculation blood samples obtained from 4 heifers that were seronegative at the time of inoculation, but was not isolated from any of the preinoculation samples, or from any of the postinoculation samples obtained from 2 heifers that were seropositive at the time of inoculation. When virus isolation was attempted from separated mononuclear cells in 2 heifers, 43.7% of the noncultured and 87.5% of the cultured samples had positive results.

A recombinant cDNA probe from genome segment 5 obtained from a virulent US bluetongue virus strain (BTV-11 strain UC8) was hybridized to US and Israeli BTV prototypes and field isolates. The cloned genetic probe hybridized with US BTV prototype 10, but not with US prototypes 2, 11, 13, and 17; with the avirulent BTV-11 strain UC2; and with the Israeli prototype 10. When the probe was hybridized to field isolates from the US serotypes, it hybridized to 12 of 14 BTV-10 isolates and 4 of 17 BTV-11 samples, but not to the BTV-13 and BTV-17 samples tested. Hybridization was not observed with the Israeli field isolates studied. Results indicate that a reassortant event occurred between a strain of US BTV-10 and US BTV-11 that originated the BTV-11 strain UC8.

The double-stranded RNA genome from 117 field isolates of bluetongue virus (BTV) serotypes 10, 11, 13, and 17 was blotted onto nitrocellulose paper and hybridized with a radioactively labeled cloned copy of DNA genome segment 2 of BTV-17. Viral RNA from BTV prototype strains 2, 10, 11, 13, and 17 were used as controls. The probe hybridized only with the viral RNA from prototype BTV-17 virus and field isolates of BTV-17. There was no cross hybridization with field isolates of BTV serotypes 10, 11, and 13. A complementary DNA probe developed from genes coding for BTV serotype specificity was effectively used in a slot-blot hybridization system for efficiently characterizing the viral serotype.

The increasing threat of vector-borne diseases (VBDs) represents a great challenge to those who manage public and animal health. Determining the spatial distribution of arthropod vector species is an essential step in studying the risk of transmission of a vector-borne pathogen (VBP) and in estimating risk levels of VBD. Risk maps allow better targeting surveillance and help in designing control measures. We aimed to study the geographical distribution of Culicoides imicola, the main competent vector of Bluetongue virus (BTV) in sheep in Tunisia. Fifty-three records covering the whole distribution range of C.imicola in Tunisia were obtained during a 2-year field entomological survey (August 2017 – January 2018 and August 2018 – January 2019). The ecological niche of C. imicola is described using ecological-niche factor analysis (ENFA) and Mahalanobis distances factor analysis (MADIFA). An environmental suitability map (ESM) was developed by MaxEnt software to map the optimal habitat under the current climate background. The MaxEnt model was highly accurate with a statistically significant area under curve (AUC) value of 0.941. The location of the potential distribution of C. imicola is predicted in specified regions of Tunisia. Our findings can be applied in various ways such as surveillance and control program of BTV in Tunisia.

Replication of bluetongue virus (BTV) in leukocytes from the blood of sheep, cattle, elk, and mule deer inoculated with BTV serotype 10 or 17 was assessed by immunocytochemical staining and dot blot northern hybridization to determine if differences in the prevalence of infection in this blood fraction might account for the differences in clinical disease among these species. Viremia was confirmed by virus isolation in all inoculated animals. Analysis of leukocytes with monoclonal antibodies specific for BTV proteins revealed low numbers of infected leukocytes in only 2 sheep 8 days after inoculation with BTV serotype 10. Most of the cells expressing BTV were identified morphologically as monocytes; approximately 10% of infected cells were lymphocytes. Bluetongue virus was not detected by use of dot-blot hybridization on samples of blood. Our results suggest that differential infection of leukocytes does not account for the pronounced differences in clinical signs and pathologic changes among ruminants.

Three independent 1-year studies were conducted during 3 consecutive years to better define the prevalence of bluetongue virus (BTV) infection in Mexico. Serologic data were obtained by use of agar-gel immunodiffusion for identification of BTV group-reactive antibodies, and virologic data were obtained by virus isolation. Samples were obtained from sheep in 6 states over a 1-year period, with 9% seropositive; samples were obtained from cattle in 11 states during the same 1-year period, with 35% seropositive. Two years later, samples were obtained from cattle in 4 additional states, with 69% seropositive. Virus isolation was conducted on pooled blood samples obtained from cattle in 7 states. Six virus isolates were recovered and included 2 isolates each of BTV serotypes 11 and 13 and 1 isolated each of serotypes 10 and 17. All virus isolates were partially characterized by electrophoretic analysis of genomic RNA migration profiles (electropherotypes) in polyacrylamide gels. All Mexican isolates of BTV differed considerably in electropherotype profile, as compared with their respective US prototype strain of the same serotype. Such differences appeared to be much more extensive than those described to exist between numerous California isolates of the same serotype.