Epizootic hemorrhagic disease (EHD) is a Culicoides-borne disease of domestic and wild ruminants caused by EHD virus (EHDV). This virus circulates in multiple serotypes. In late September 2021, a novel strain belonging to EHDV-8 was reported in cattle farms in Central–Western Tunisia, and in the fall of 2022, the same virus was also detected in Italy and Spain. In the present study, we described EHDV-8 occurrence in deer and, a preliminary identification of the potential Culicoides species responsible for virus transmission in selected areas of Tunisia. EHDV-8 was identified in deer carcasses found in 2021 and 2022 in the national reserve of El Feidja, Jendouba, Northwestern Tunisia, and isolated on cell culture. Instead, insect vectors were collected in October 2021 only in the areas surrounding the city of Tozeur (Southern Tunisia) where EHDV-8 cases in cattle were confirmed. Morphological identification showed that 95% of them belonged to the Culicoides kingi and Culicoides oxystoma species and both species tested positive for EHDV-8 RNA. C. imicola was not detected in this collection and EHDV-8 RNA was not evidenced in vector pools collected in 2020, prior to official EHDV-8 emergence. EHDV whole genome sequences were also obtained directly from infected biological samples of deer and positive vectors. EHDV-8 sequences obtained from deer and vectors share a nucleotide identity ranging from 99.42 to 100% and amino acid identity from 99.18 to 100% across all genome segments with the EHDV-8/17 TUN2021 reference sequence.

Background: Culicoides kingi and Culicoides oxystoma belong to the Schultzei group of biting midges. These two species are vectors of disease in livestock of economic importance. As described in the literature, morphological identification for discrimination between them is still unclear. However, species-specific identification is necessary to solve taxonomic challenges between species and to understand their roles in disease transmission and epidemiology. This study aims to develop accurate tools to discriminate C. oxystoma from C. kingi using traditional morphometry and polymerase chain reaction-restriction fragment length polymorphism (PCR RFLP) assays for use in developing countries. Methods: Specimens were collected from the region of Kairouan in central Tunisia. A total of 446 C. oxystoma/C. kingi individuals were identified using traditional morphometric analyses combined with PCR–RFLP of the cytochrome c oxidase subunit I gene. Thirteen morphometric measurements were performed from the head, wings, and abdomen of slide-mounted specimens, and six ratios were calculated between these measurements. Multivariate analyses of the morphometric measurements were explored to identify which variables could lead to accurate species identification. Results: Four variables, namely antennae, wings, spermathecae, and palpus length, were suitable morphometric characteristics to differentiate between the species. Digestion with the SspI restriction enzyme of the PCR product led to good discriminative ability. Molecular procedures and phylogenetic analysis confirmed the efficiency of this simple and rapid PCR–RFLP method. Conclusions: This study highlights for the first time in Tunisia the presence of C. oxystoma and its discrimination from C. kingi using abdominal measurements and the PCR–RFLP method. This approach could be applied in future epidemiological studies at the national and international levels. Graphical