A total of 20 horses belonging to the Pasteur Institute of Tunis used for the production of therapeutic serum antiscorpion venom were tested for the presence of antibodies anti-West Nile virus (WNV) during the 2023 outbreak of West Nile disease that affected humans and horses in Tunisia. Of the 20 samples tested in November 2023 by enzyme-linked immuno-sorbent assay (ELISA), five were positive, leading to a seroprevalence of 25%. Since the number of horses is not substantial, all samples were tested also by virus microneutralization test (MNT) using the Tunisian strain of WNV lineage 1 isolated in 2014 from a pool of field-collected Culex pipiens from Central Tunisia. WNV-neutralizing antibodies were detected in two horses, yielding a seroprevalence of 10%. Of the five horses tested positive by ELISA, only two were positive by MNT. The follow-up of the serological analysis performed in December 2023 and January 2024 did not show any seroconversion in the remaining horses. No clinical cases were reported during the investigation. While molecular blood analysis failed to detect viral RNA, MNT-based seroprevalence provided strong evidence of the circulation of WNV during this outbreak. Taking into account that humans and horses share similar clinical symptoms and antibody responses following WNV infection, equid surveillance could provide an accurate and timely detection of WNV outbreaks.
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