West Nile Virus Seroprevalence in Wild Birds and Equines in Madrid Province, Spain

Simple Summary West Nile virus (WNV) is a flavivirus that circulates among birds and mosquitoes and can cause outbreaks in people and horses, sometimes leading to serious brain-related illness and death. This study aimed to investigate WNV circulation in birds and horses in Madrid, Spain. Through serological testing (cELISA), a proportion of birds were positive for WNV antibodies, indicating potential exposure. Four birds and one horse were confirmed positive for WNV antibodies with a second test, while four other birds showed antibodies to an undetermined flavivirus. Because birds positive for WNV antibodies were adults, they might have been exposed elsewhere. The horse had previously lived in a WNV endemic zone. The birds positive for flavivirus included two young birds that had not traveled outside Madrid. The presence of antibodies in two juvenile birds that could hardly fly suggests local circulation of flavivirus in birds in Madrid. The study addresses the potential circulation of WNV or related flaviviruses in birds in Madrid, emphasizing the need for increased surveillance to understand transmission dynamics and the principal species involved. Given the growing incidence and spread of WNV in Spain, continued research is vital for risk assessment and implementing effective control measures.Abstract West Nile virus (WNV) is a re-emerging flavivirus, primarily circulating among avian hosts and mosquito vectors, causing periodic outbreaks in humans and horses, often leading to neuroinvasive disease and mortality. Spain has reported several outbreaks, most notably in 2020 with seventy-seven human cases and eight fatalities. WNV has been serologically detected in horses in the Community of Madrid, but to our knowledge, it has never been reported from wild birds in this region. To estimate the seroprevalence of WNV in wild birds and horses in the Community of Madrid, 159 wild birds at a wildlife rescue center and 25 privately owned equines were sampled. Serum from thirteen birds (8.2%) and one equine (4.0%) tested positive with a WNV competitive enzyme-linked immunosorbent assay (cELISA) designed for WNV antibody detection but sensitive to cross-reacting antibodies to other flaviviruses. Virus-neutralization test (VNT) confirmed WNV antibodies in four bird samples (2.5%), and antibodies to undetermined flavivirus in four additional samples. One equine sample (4.0%) tested positive for WNV by VNT, although this horse previously resided in a WN-endemic area. ELISA-positive birds included both migratory and resident species, juveniles and adults. Two seropositive juvenile birds suggest local flavivirus transmission within the Community of Madrid, while WNV seropositive adult birds may have been infected outside Madrid. The potential circulation of flaviviruses, including WNV, in birds in the Madrid Community raises concerns, although further surveillance of mosquitoes, wild birds, and horses in Madrid is necessary to establish the extent of transmission and the principal species involved.