Bluetongue Virus Serotype 3 Follow-Up of the 2024 Outbreak in Two Belgian Zoos

In summer 2024, Western Europe experienced extensive outbreaks of bluetongue virus Serotype 3 (BTV-3), a Culicoides-borne orbivirus. Clinical disease was first detected in August 2024 in one of two Belgian zoos, predominantly affecting European bison (Bison bonasus) and American bison (Bison bison). These species showed high morbidity, two fatalities, and severe multisystemic lesions at necropsy. This outbreak coincided with reemergence of BTV-8 and increasing reports of emerging BTV-12 and epizootic hemorrhagic disease virus (EHDV) circulation in Europe. Following the outbreak, samples from 116 animals representing 33 species were analyzed across two Belgian zoos (urban and rural), spanning a 20-year period (2005-2025). Species belonged to Artiodactyla, Perissodactyla, Proboscidea, Rodentia, Diprodontia, and Carnivora. Archived sera predating 2024 (retrospective), samples collected during the 2024 outbreak (outbreak), and follow-up samples obtained through June 2025 (follow-up) were tested at the Belgian National Reference Laboratory (NRL). Antibodies were screened using a pan-BTV ELISA, and ELISA-positive samples were further analyzed by virus neutralization tests (VNTs) to differentiate BTV-3 and BTV-8. Viral RNA detection was performed using RT-qPCR assays targeting pan-BTV, BTV-3, BTV-12, and EHDV. Serological reactivity was confined to Artiodactyla and Perissodactyla. In total, 46 ruminants (67% of tested ruminants) and one greater one-horned (GOH) rhinoceros (Rhinoceros unicornis) were ELISA positive. VNT confirmed BTV-3 infection in 62% and BTV-8 in 9% of seropositive ruminants. Viral RNA was detected in whole blood from 28 individuals, representing 42% of the tested ruminants. Marked epidemiological differences were observed between zoos, with limited detection in the urban collection and widespread infection in the rural collection, likely reflecting differences in vector exposure. No evidence of BTV-12 or EHDV was detected. This study provides comprehensive documentation of BTV-3 in zoo-housed species, integrating clinical, pathological, and longitudinal surveillance data. It underscores pronounced interspecies variability in susceptibility and immune response, providing critical insights for outbreak preparedness.