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A cross-sectional study was conducted to assess the circulation and risk factors associated with West Nile virus (WNV) exposure in equine and wild bird populations following the largest epidemic outbreak ever reported in Spain. A total of 305 equids and 171 wild birds were sampled between November 2020 and June 2021. IgG antibodies against flaviviruses were detected by blocking enzyme-linked immunosorbent assay (bELISA) in 44.9% (109/243) and 87.1% (54/62) of unvaccinated and vaccinated equids, respectively. The individual seroprevalence in unvaccinated individuals (calculated on animals seropositive by both bELISA and virus microneutralization test [VNT]) was 38.3% (95%CI: 33.1-43.4). No IgM antibodies were detected in animals tested (0/243; 0.0%; 95%CI: 0.0-1.5) by capture-ELISA. The main risk factors associated with WNV exposure in equids were age (adult and geriatric), breed (crossbred) and the absence of a disinsection programme on the facilities. In wild birds, IgG antibodies against flaviviruses were found in 32.7% (56/171; 95%CI: 26.8-38.6) using bELISA, giving an individual WNV seroprevalence of 19.3% (95%CI: 14.3-24.3) after VNT. Seropositivity was found in 37.8% of the 37 species analysed. Species group (raptors), age (1-year old) and size (large) were the main risk factors related to WNV seropositivity in wild birds. Our results indicate high exposure and widespread distribution of WNV in equid and wild bird populations in Spain after the epidemic outbreak in 2020. The present study highlights the need to continue and improve active surveillance programmes for the detection of WNV in Spain, particularly in those areas at greatest risk of virus circulation.

On January 2020, an outbreak of acute mortality was detected in the capybara (Hydrochoerus hydrochaeris) population kept in the Cordoba Zoo Conservation Center (CZCC) in Spain. Morbidity and mortality rates were 71%, and the fatality rate was 100%. Macroscopic examination revealed multifocal nodular lesions with fibrinonecrotic exudate in lungs and mesenteric lymph nodes. Microscopic lesions were characterized as severe acute fibrinopurulent bronchopneumonia and generalized vascular changes. Cultures and biochemical analyses yielded Yersinia pseudotuberculosis (YPT). This species was confirmed by both PCR and MALDI-TOF. This report describes the first outbreak of fatal YPT infection in zoo animals in Spain and confirms that capybaras are highly susceptible to this pathogen, which could be of animal health, conservation, and public health concern. Future studies are warranted to determine potential sources of YTP infection in zoos to prevent future cases in susceptible species, including humans.