Seroprevalence of West Nile, Usutu and Tick borne encephalitis viruses in equids from southwestern France in 2023

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Blood samples were collected from 494 horses located in Gironde County divided into three zones: the Confluence, the Intermediate zone and the Arcachon basin and tested for WNV, USUV and TBEV specific antibodies. An overall seroprevalence of 14% (95% CI [11%-18%]) for orthoflavivirus antibody was detected in Gironde county. The highest seroprevalence rate for WNV and USUV were detected in the Confluence zone (9%, 95% CI, [6%-13%] and 5%, 95% CI, [3%-8%] respectively). The type of housing (animals kept in pasture only) and the distance to the nearest bird special protection area (SPA) were identified as risk factors for WNV seropositivity. This paper presents the first seroprevalence study investigating WNV, USUV and TBEV infections, conducted in equids located on the Atlantic coast of France and demonstrates an intense circulation of WNV in this region of France as well as equine USUV specific infection. Figures Figure 1 Figure 2 Introduction West Nile virus (WNV) is a reemerging zoonotic mosquito-borne orthoflavivirus ( Flaviviridae family) that represents a growing threat to human and animal health. It is one of the main viral agents causing encephalitis in humans and horses in Europe (Harris et al., 2006). It was identified for the first time in Uganda in 1937 (Smithburn et al., 1940), and its circulation is now evident on all five continents [ 1 ]. WNV is maintained in an enzootic cycle involving ornithophilic mosquitoes as vectors ( Culex spp.) and wild and resident birds as reservoirs and/or amplifying hosts. The virus can infect approximatively one hundred mammalian host species, which are considered epidemiologically dead-end hosts that do not participate in virus spreading [ 2 ]. Among them, only humans and horses are known to develop symptoms following WNV infection. WNV infection is predominantly asymptomatic or can cause flu-like syndrome. In 1 to 10% of cases, neurological signs, the most frequent being ataxia, weakness and muscle fasciculations, are encountered. Some horses develop signs of encephalitis, leading to death or euthanasia [ 3 – 5 ]. In addition, human-to-human transmission of WNV, during blood transfusions or organ transplants, has been extensively documented and remains a major concern in the transfusion context [ 6 , 7 ]. To reduce the risk of non-vector transmission, Europe introduced precautionary measures to control labile products, such as the exclusion of blood donors for 28 days after a visit to a high-risk area (European Directive 2004/33/EC) [ 7 ]. As WNV circulation is heterogeneous between Member States and is a compulsory notifiable disease for birds (since 2021), equines and humans (since 2009) to the European Commission [ 8 ], each country has implemented an appropriate surveillance and monitoring system to detect WNV cases in Equidae, avifauna and humans. Over the last decade, Europe has faced significant changes in the distribution of WNV, as well as two other mosquito-borne orthoflaviviruses, Usutu virus (USUV) and tick-borne encephalitis virus (TBEV) most likely caused by global change and increasing global warming. The three viruses actively cocirculate in most European countries. Infection are mainly subclinical, and accurate estimations of infection incidence rates are difficult. The surveillance of WNV infection relies heavily on neurological syndromic surveillance in humans and horses in most EU countries. Serosurveillance of equines is also commonly employed to detect virus circulation in a territory, and allows estimation of the intensity of circulation in that territory [ 1 , 9 – 15 ]. The outdoor lifestyle of equines and their proximity to humans are good indicators of WNV circulation and human exposure. In France, WNV was detected for the first time in the Camargue region in 1962. Between 2000 and 2021, several WNV outbreaks of varying intensity were reported, all of which were located in the Mediterranean region and Corsica [ 16 – 19 ]. The 2022 transmission season was marked by the emergence of WNV in southwestern France, on the Atlantic coast. In October 2022, three horses of three distinct stables in Gironde County (Fig. 1 ) exhibited neurological signs, including ataxia, weakness, muscle fasciculations, and encephalitis. Acute WNV infection was confirmed by serological diagnostic methods (ELISA IgM and IgG) carried out by the French National Reference Laboratory for WNV. To obtain an accurate assessment of orthoflavivirus circulation in Gironde County, the first seroprevalence survey was conducted among equids located near stables where WNV cases in horses were newly reported in 2022 by investigating possible seroconversion to WNV, USUV and TBEV (Fig. 1 ). We aimed to provide valuable insights into the intensity of WNV circulation in 2022 in this newly emerging West Nile virus zone and the associated risk factors for infection. We also evaluated the occurrence of USUV and TBEV in horses in this area. Material and methods 2.1. Geographical zones The survey was conducted in Gironde County, southwestern France, characterized by its contrasting landscape. The epidemiological parameters of the three defined zones were determined. The first zone, named Confluence, corresponds to the area where WNV-infected horses were reported in 2022 (Fig. 1 ). The confluence lies at the junction of two rivers, the Garonne and the Dordogne rivers, where large populations of mosquitoes can be expected. The second zone corresponds to the Arcachon basin. The Arcachon basin lies at the heart of the East Atlantic Flyway, one of the eight most important bird migration corridors on the planet. No cases of WNV infection were reported here. These two zones offer a protected habitat for resident and migratory bird species (represented in green in Fig. 1 ) that use wetlands as a stopover during their long journeys. These avian tranquility zones encourage interactions between local mosquitoes and competent avian hosts which promotes the establishment of the bird-mosquito-bird transmission cycle of WNV and USUV. The third zone covers the area between the Confluence and the Arcachon basin. 2.2. Study design and sample collection The study was a cross-sectional prospective study. It took place in April and May 2023, prior to the occurence of seasonal orthoflavivirus activity, to reflect the exposure of horses to WNV, USUV and TBEV during the summer 2022. Animals were recruited by the Clinique Equine de Conques (Saint Aubin de Branne, Gironde, France) through social media and client mailing, according to our three different zones. All owners signed a consent form and treating veterinarians were informed of the procedure. This study was approved by the Ethics Committee for Clinical Research (ComERC) of the Veterinary School of Alfort (EnVA) (Agreement number: 2023-06-23). Serum samples were collected from 494 adult (> 2-year-old) client-owned horses, ponies, and donkeys residing in 39 different stables of the three studied zones: 306 equids from 25 stables in the Confluence zone, 77 equids from 5 stables in the Arcachon zone, and 111 equids from 9 stables in the intermediate zone (Fig. 1 ). The animals were unvaccinated against WNV and had been living in the sampling area for the past 6 years throughout 2022 without traveling to the Mediterranean region. The number of equids sampled in a stable depended on the stable size: all horses were sampled in small stables (fewer than 10 animals), 10 horses were sampled in medium stables (between 10 and 15 animals), and 15 horses were sampled in large stables (more than 15 animals). All animals were asymptomatic at the time of sampling. The 3 cases of 2022 were not included in our cohort, although two of the three corresponding stables were included (Fig. 1 ). Serum samples were collected from each included animal. Blood samples were collected in vacutainer dry tubes and centrifuged at 5000 rpm for 5 minutes within 24 hours of collection. The serum was separated, stored at 4°C, and sent to the French National Reference Laboratory for West Nile virus, ANSES (Maisons-Alfort, France) for further analysis. In addition to sample collection, each stable manager/owner completed a survey consisting in a personal face-to-face interviews to gather information on individual characteristics and stable management. This information included age, sex, hair colour, and type of housing. We also collected information about the environment, such as the distance to the nearest water surface, and the distance to the nearest special protection areas. SPAs have been created for the protection of wild bird species listed in the Annex 1 of the EU bird directive (Directive 79/409/EEC), or that serve as breeding, moulting, wintering, or staging areas for migrating birds. Five SPAs have been defined in the study area around wetlands (Fig. 1 ). 2.3. Serological testing ELISA tests Serum samples were, first, tested for IgG flaviviruses using the commercial Pan-Flavivirus ELISA “ID Screen Flavivirus competition” (Innovative Diagnostic, Montpellier, France). The protocol was performed according to the manufacturer's instructions. The results are expressed as %S/N. If a sample had a %S/N less than or equal to 40%, the sample was considered positive. If it is strictly greater than 50%, the sample is negative. If the result is between 40% and 50%, the sample is considered doubtful. Positive and doubtful results were confirmed for the presence of WNV, USUV and TBEV specific neutralizing antibodies using specific virus neutralization tests. Virus-neutralization test (VNT) To identify specifically against which flaviviruses the IgG antibodies detected by ELISA are directed, a virus neutralization test (VNT) was carried out for the three main flaviviruses circulating in France: WNV, USUV and TBEV. This test was performed in 96-well plates as described in Beck et al.[ 18 ] . Briefly, the sera were diluted in a cascade by a factor of 2, from 1:5 to 1:160, in a final volume of 100µL. Fifty microliters of virus was added at an infectious dose of 100 TCID50. After 1h30 of incubation at 37°C and 5% CO 2 , 100µL of Vero cells was added at a concentration of 2.10 5 cells/mL. The neutralizing antibody titer was obtained by observing the cytopathic effect (CPE) under a light microscope after 3-, 4- and 5-days of incubation at 37°C and 5% CO 2 for WNV, USUV and TBEV respectively. The last dilution of serum showing no CPE or a proportion of CPE less than a quarter of the well observed, was noted as the dilution of serum considered where there is still protection against the virus. The neutralizing antibody titer was therefore the inverse of the serum dilution. Sera neutralizing more than one virus were considered positive for the virus neutralized at a fourfold higher dilution than all other viruses. If a fourfold difference in neutralizing antibody titers was not reached, the serum was considered undifferentiated between the viruses. Sera that tested positive by competitive ELISA and negative by VNT were considered negative. Statistical analysis All collected variables were described in terms of frequency distribution (qualitative data) or median and range (quantitative data) classified by the serological status. Data were analysed using mixed effects logistic regression models, using the stable as a random effect. The dependent variable was the serological status of the animals, according to the seroneutralization test. Fixed effects were animal age (years), sex (male or female), hair colour (light or dark), the type of housing (always on pasture, or both on pasture and indoors), the distance in kilometres (km) to the nearest water surface (intermittent or permanent, of any type: river, canal, estuary, lake, marsh, reservoir etc.), and the distance in km to the nearest special protection area (SPAs). The exponentiated regression coefficients produced odds ratio (OR) as a measure of the effect of the variables. Starting from the model with all the fixed effects, we selected the most parsimonious model using backward model selection based on the likelihood ratio test, p-value < 0.05. We used the nonparametric approach proposed by Vaumourin et al. (2014) to test whether the association between positive VNT results against WNV, USUV and TBEV (i.e. the number of equids positive for 1, 2 or 3 of these viruses) significantly differed from the expected distribution under the null hypothesis of independent positive results. All the statistical analyses were performed using R 4.3.3 (R Core Team 2024). Mixed effects logistic regression models were fitted using the lme4 package [ 20 ]. Model selection was performed using the buildmer package [ 21 ]. Results This study included 494 adults: 334 horses, 151 ponies and 9 donkeys of which 271 were male and 223 were female. The majority (412 horses) were active at the time of sampling: 328 animals lived in pasture, whereas 166 had a mixed lifestyle (stable and pasture). Regarding hair coat color, 331 animals had a dark coat, while 163 had a light coat. 3.1 ELISA and VNT Of the 494 horses included in the study, 70 were positive for orthoflavivirus antibodies by competitive ELISA yielding a seroprevalence rate of 14% (95% CI [11%-18%]). Among them, 60 animals were located in 20 stables in the Confluence zone, 5 horses were housed in 4 stables in the Intermediate zone and 1 was based on 1 stall in the Arcachon area (Table 1 ). Table 1 Comparative serological results among selected horses in the Confluence, Intermediate and Arcachon zones in Gironde county Geographical zone Confluence Intermediate Arcachon Tested horses/stables 306/25 111/9 77/5 ELISA results: Positive horses/stables Animal prevalence (95% CI) 64/20 0.21 (0.16–0.26) 5/4 0.05 (0.02–0.10) 1/1 0.01 (0.00-0.07) VNT results in ELISA-positive animals: Positive horses/stables Animal prevalence (95% CI) WNV USUV TBEV WNV USUV TBEV 27/13 14/5 6/4 0.09 (0.06–0.13) 0.05 (0.03–0.08) 0.02 (0.01–0.04) 3/2 1/1 1/1 0.03 (0.01–0.08) 0.01 (0.0-0.05) 0.01 (0.0-0.05) 1/1 0/0 0/0 0.01 (0.0-0.07) 0.0 (0.0-0.05) 0.0 (0.0-0.05) Within the Confluence zone, 27 equids from 13 different stables were positive for WNV by VNT (9%, 95% CI, [6%-13%]). Within the same zone, 14 equids from 5 different stables were positive for USUV (5%, 95% CI, [3%-8%]), and 6 equids from 4 different stables were positive for TBEV (2%, 95% CI, [1%-4%]). In the intermediate zone, 3 equids housed on 2 stables were positive for WNV (3%, 95% CI, [1%-8%]), one horse was positive for TBEV and another horse was positive for USUV neutralising antibodies (1%, 95% CI, [0%-5%]). For the Arcachon zone, only one horse was positive for WNV (1%, 95% CI, [0%-7%]), and no horse was seropositive for the other viruses (Table 1 and Fig. 2 ). Undifferentiable VNT results were also observed. In total, 24 equids tested positive for WNV, 5 horses tested positive for both WNV and USUV, 2 horses tested positive for WNV and TBEV, and none tested positive for all three viruses simultaneously (WNV, USUV, TBEV). Additionally, 9 horses tested positive for USUV only, 1 horse tested positive for both USUV and TBEV, and 4 horses tested positive for TBEV only (Table 2 ). Table 2 Association between VNT serological status for WNV, USUV and TBEV WNV USUV TBEV Number of animals Positive Positive Positive 0 Positive Positive Negative 5 Positive Negative Positive 2 Positive Negative Negative 24 Negative Positive Positive 1 Negative Positive Negative 9 Negative Negative Positive 4 Negative Negative Negative 449 Total 494 The individual characteristics of the sampled animals as a function of their WNV and USUV serostatus are shown in Table 3 . The qualitative comparison showed that horses tested positive for WNV were mainly male kept on pasture only. This does not apply to horses seropositive for the Usutu virus. Table 3 Characteristics of horses as a function of the WNV and USUV serostatus. Individual horse characteristics WNV n (proportion) or median (range) USUV n (proportion) or median (range) Positive horses Negative horses Positive horses Negative horses Sex .Male .Female 19 (0.70) 8 (0.30) 162 (0.58) 117 (0.42) 8 (0.57) 6 (0.43) 173 (0.59) 119 (0.41) Age 13 (2–30) 11 (2–35) 10.5 (6–25) 11 (2–35) Hair color .Dark .Light 14 (0.52) 13 (0.48) 197 (0.29) 82 (0.71) 9 (0.64) 5 (0.36) 202 (0.69) 90 (0.31) Type of housing .Pasture only .Mixed pasture/stable 23 (0.85) 4 (0.15) 158 (0.57) 121 (0.43) 9 (0.64) 5 (0.36) 172 (0.59) 120 (0.41) 3.2 Risk factor analysis A mixed effects logistic regression model of VNT serostatus was fitted for WNV and USUV only, since for TBEV, the number of seropositive stables and equids was very low. Furthermore, we focused our analysis on the Confluence zone, where the vast majority of WNV and USUV seropositive equids were located (Table 3 ). For WNV, model selection allowed us to identify two risk factors of VNT seropositivity: the type of housing with a strongly increased risk in animals always kept on pasture (odds-ratio [OR] = 4.99) and the distance to the nearest SPA, with a decreasing risk when this distance increased ([OR] = 0.85 for a 1km increase) (Table 4 ). Other variables (age, sex, hair colour and distance to the nearest water surface) were not retained in the selected model. For USUV, model selection resulted in an empty model (containing the intercept only) and did not allow the identification of any seropositivity risk factors. Table 4 Mixed effects logistic regression model of the VNT WNV serological status of the horses sampled in the Confluence zone Variable Value Odds-ratio (95% CI) Intercept 0.10 (0.03–0.27) Type of housing Pasture and indoors Reference Pasture only 4.99 (1.83–17.47) Distance to the nearest SPA Increase by 1km 0.85 (0.78–0.93) Finally, we detected a significant association between WNV and USUV serological status (p = 0.006), with the number or number of equids positive for both viruses (5 animals) being significantly higher than the expected 95% interval under the null hypothesis (0–4 animals). Discussion Over the last decade, France has witnessed a changing epidemiology of WNV, USUV and TBEV orthoflaviviruses with increased incidence of diseases in vertebrate hosts [ 1 , 14 , 22 ]. From cohorts of equines located in the three defined areas of Gironde County, we demonstrated WNV seroprevalence rates ranging from 9% (95% CI [ 6 – 13 ]) in the Confluence area to 3% (95% CI [ 1 – 8 ]) in the intermediate area and 1% (95% CI[0–7]) in the Arcachon basin. These findings are comparable to previous reports from Kosovo [ 23 ], Spain [ 24 – 27 ] Germany [ 28 – 31 ] and France [ 16 , 17 ]. In the Camargue region, which has been considered as the historical area of WNV circulation in France since the 1960s, the seroprevalence rate among equines estimated between 2016 and 2020 is slightly higher at approximatively 13% [ 17 ]. As for WNV, the highest USUV seroprevalence rate observed in horses was obtained in the Confluence area (5%). It was null near the Arcachon basin. Therefore, the Confluence area appears to be a hot spot for Culex-borne orthoflavivirus circulation. Our data showed a low percentage of seropositive individuals infected with TBEV (0,3%). Nevertheless, this low seroprevalence rate does not allow us to conclude that TBEV is circulating in southwestern France. The virus is known to circulate in the northeastern part of the country[ 32 – 34 ], with outbreaks detected further south in Jura and other regions [ 35 ]. Further investigations in ticks and reservoir animals such as rodents are needed to confirm the presence of TBEV in the Nouvelle Aquitaine region. In this study, we analysed the risk factors that might affect the susceptibility of horses to WNV infection. The risk of exposure to WNV increased when horses were kept outdoors, and thus were more exposed to vector bites, and when the stable was close to protected areas to preserve the avian biodiversity they harbor (SPAs). In and around these areas, bird and vector species assembly may favor local WNV circulation, as already suggested in southeastern France. This finding is in line with the association, at the European level, between West-Nile disease occurrence in humans and the local diversity of wild bird species [ 36 ] [ 37 ] . Age, sex, horse color and distance to the nearest water surface were not identified as risk factors associated with WNV and USUV seropositivity. Our data are consistent with those of García-Bocanegra et al [ 38 ]; Vanhomwegen et al [ 39 ] who showed no significant differences among age classes; of Durand et al [ 16 ] in France, Hassine et al in Jordania [ 40 ] and Bażanów et al in horses in Poland [ 41 ] who reported the absence of an effect of gender on WNV and USUV seropositivity. Analyses of the color of horse coats are rare in WNV seroprevalence studies in horses. The results from several research groups are disparate, with studies showing an association between darker color and a lower WNV seroprevalence rate [ 42 , 43 ] and others demonstrating no correlation between this variable and WNV and USUV seropositivity [ 44 ]. Interestingly, we did not find a correlation between the seroprevalence of mosquito-borne viruses and the nuisance caused by mosquitoes evaluated by owners. As it is a personal assessment and may vary from one individual to another, our data here do not allow conclusions to be drawn. The emergence of WNV in this new region of France raised several questions, particularly regarding the factors that favored its introduction into this area. Our main hypothesis is that the introduction of WNV into Nouvelle Aquitaine region occurred through migratory birds. Indeed, the region is located on a migratory corridor of different migratory bird species such as the grey crane, the red kite, the graylag goose and passerines. In addition, the large fire of July 2022, which burned more than 30,000 hectares of forest in Gironde (particularly in La Teste de Buch, Landiras, Landiras St Magne, Saumos and Arès) [ 45 ], may lead to the disruption of ecosystems and play a role in the new distribution of WNV. Forest fires can disrupt ecosystems and displace wildlife, potentially leading to increased interactions between humans and infected animals/mosquitoes [ 46 ]. Investigations are needed to identify the resident and amplifying avian species that may be involved in the endemicity of WNV in this region of France. The number of equine and avian cases recorded by the French National Reference Laboratory for West Nile during the 2023 transmission season suggests that the virus tends to establish itself and extend its range. Cases have been detected in departments bordering the Gironde especially in Charentes Maritime and Charente. Carrying out seroprevalence surveys such as the one conducted in this study is essential for strengthening monitoring of WNV circulation. This helps us to understand viral emergence and evaluate the intensity of circulation in new areas. The seroprevalence survey carried out close to syndromic cases diagnosed in 2022 identified areas with high viral circulation. In 2023, these data enabled mosquito traps to be set in mid-July, providing rapid evidence of active circulation of the West Nile and Usutu viruses in the Confluence area. These data have played a major part in the rapid implementation of the measures needed to ensure the safety of products derived from the human body (blood and organ donations) throughout the Gironde and Charente Maritime, a neighbouring department [ 47 ]. Horse owners located in the Confluence zone were encouraged to vaccinate their animals to prevent WNV infection in 2024 and the development of severe forms of the disease. Conclusion This first seroprevalence study conducted in equids located in Gironde County on the Atlantic coast of France showed that WNV, USUV and an orthoflavivirus belonging to the TBEV serocomplex circulate in this area and infect horses in 2022. Horses, known as sentinel of WNV infection, exhibited intense circulation of this virus in this region of France as well as equine USUV specific infection. This study confirmed the usefulness of serosurveys in horses for detecting the circulation of orthoflaviviruses, especially WNV, in specific regions. Abbreviations CPE CytoPathic Effect ELISA Enzyme-liked immunabsorbent assay IgG Immunoglobulin G IgM Immunoglobulin M OR Odd Ratio SPA Special Protection Area TBEV Tick-Borne Encephalitis Virus TCID50 Tissue Culture Infectious Dose 50 USUV Usutu Virus VNT Virus Neutralisation Test WNV West Nile Virus Declarations This study was approved by the Ethics Committee for Clinical Research (ComERC) of the Veterinary School of Alfort (EnVA) (Agreement number: 2023-06-23). Acknowledgements We would like to thank equine owners for taking part in the serological survey. This research was conducted in part under the French and European laboratory for West Nile Virus. Author contributions G.G., A.L., T.T., conceptualized the project.; C.V.M., G.G., N.C., C.M.P., E.Q., B.D., and C.L. contributed to the methodology; N.C., C.B., M.D., M.DM., collected the sera; C.V.M, M.D., T.H., M.C., did the laboratory tests; G.G., C.V.M, B.D., C.L. and N.C., analysed the data; , N.C, C.V.M., G.G., wrote the original draft; C.V.M, N.C., B.D., and G.G. reviewed and edited the original draft; G.G., M.D. and C.V.M., supervised the study; G.G., and C.V.M. contributed to the funding acquisition. All authors reviewed and approved the final draft. Fundings We acknowledge the French Agency for Food, Environmental and Occupational Health & Safety (ANSES) and the National Reference Laboratory for West Nile Virus for funding the research activities. Data availability The datasets of the current study are available from the corresponding author upon reasonable request. Consent for publication Not applicable. Competing interests The authors declare no competing interests. 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PLoS ONE 12(10):e0185962 García-Bocanegra I et al (2012) Monitoring of the West Nile virus epidemic in Spain between 2010 and 2011. Transbound Emerg Dis 59(5):448–455 Vanhomwegen J et al (2017) Circulation of Zoonotic Arboviruses in Equine Populations of Mallorca Island (Spain). Vector Borne Zoonotic Dis 17(5):340–346 Ben Hassine T et al (2014) First detection of co-circulation of West Nile and Usutu viruses in equids in the south-west of Tunisia. Transbound Emerg Dis 61(5):385–389 Bażanów B et al (2018) A Survey on West Nile and Usutu Viruses in Horses and Birds in Poland. Viruses, 10(2) Aharonson-Raz K et al (2014) Spatial and temporal distribution of West Nile virus in horses in Israel (1997–2013)--from endemic to epidemics. PLoS ONE 9(11):e113149 Azmi K et al (2017) West Nile Virus: Seroprevalence in Animals in Palestine and Israel. Vector Borne Zoonotic Dis 17(8):558–566 Guerrero-Carvajal F et al (2021) Serological evidence of co-circulation of West Nile and Usutu viruses in equids from western Spain. Transbound Emerg Dis 68(3):1432–1444 BUCCIO F (2022) INCENDIES ÉTÉ 2022 Gironde et Landes Retour d’expérience Moritz MA, Batllori E, Bolker BM (2023) The role of fire in terrestrial vertebrate richness patterns. Ecol Lett 26(4):563–574 publique HC (2022) .d.l.s., Avis sur la sécurisation des produits du corps humain dans un contexte de circul We would like to thank equine owners for taking part in the serological survey. ation du virus West Nile en France métropolitaine Cite Share Download PDF Status: Published Journal Publication published 24 Apr, 2025 Read the published version in Veterinary Research → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4688411","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":326263175,"identity":"019712a7-dc30-460e-b434-ad217bfa0cbe","order_by":0,"name":"Noémie Chevalier","email":"","orcid":"","institution":"Clinique de Conques","correspondingAuthor":false,"prefix":"","firstName":"Noémie","middleName":"","lastName":"Chevalier","suffix":""},{"id":326263176,"identity":"a17bbdb7-1954-4c15-a4ad-2e0ade659f84","order_by":1,"name":"Camille Victoire Migné","email":"","orcid":"","institution":"Agence nationale de sécurité sanitaire de l\\'alimentation de l\\'environnement et du travail: Agence Nationale Securite Sanitaire Alimentaire Nationale","correspondingAuthor":false,"prefix":"","firstName":"Camille","middleName":"Victoire","lastName":"Migné","suffix":""},{"id":326263177,"identity":"4cad4e17-1a5e-4b2e-a77f-ddb61f1a427b","order_by":2,"name":"Teheipuaura Mariteragi-Helle","email":"","orcid":"","institution":"Agence nationale de sécurité sanitaire de l\\'alimentation de l\\'environnement et du travail: Agence Nationale Securite Sanitaire Alimentaire Nationale","correspondingAuthor":false,"prefix":"","firstName":"Teheipuaura","middleName":"","lastName":"Mariteragi-Helle","suffix":""},{"id":326263178,"identity":"1e782465-275c-410d-baa4-f2bb979fc6f0","order_by":3,"name":"Marine Dumarest","email":"","orcid":"","institution":"Agence nationale de sécurité sanitaire de l\\'alimentation de l\\'environnement et du travail: Agence Nationale Securite Sanitaire Alimentaire 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Agence Nationale Securite Sanitaire Alimentaire Nationale","correspondingAuthor":false,"prefix":"","firstName":"Benoit","middleName":"","lastName":"Durand","suffix":""},{"id":326263188,"identity":"8a02d182-83f3-472e-aa99-98e6d59b358d","order_by":13,"name":"Marianne Depecker","email":"","orcid":"","institution":"Clinique de Conques","correspondingAuthor":false,"prefix":"","firstName":"Marianne","middleName":"","lastName":"Depecker","suffix":""},{"id":326263189,"identity":"60ceb153-429f-4bd1-a5ee-fead1a06d29d","order_by":14,"name":"Gaëlle Gonzalez","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABC0lEQVRIie2RMUvEMBiGP5dOBddIof0LOQKiIHejf+MLgcvSw7XDcRSEu7Hrif4J/8EHwk39ARkcLgg6WrcKchildWrqKphnyUt5H96UAAQCf5Mj2vfRhbSL6WC3h7BPLoguisHqoCLL35Ts8oEIC5hWycY2WKx0dbuxtgV+5VMm9RwJa1A3d6QY1tFi+1gLEQM/L31KmXOSa1DcIDG5jhelyaME4IN7V6rXhuThS5HXrTwwnRn98u4u5lUylgO5X55yoyImS45u7hTiEYWzZ064Y3iynUdnuMPJvclFEvORlUrZfbO8mB0z/WSa5SpLjbZvbTGyQt8H65/j57tPcCtddeavBAKBwL/nE9cjWyv0ZwOIAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0003-4805-7316","institution":"Agence nationale de sécurité sanitaire de l\\'alimentation de l\\'environnement et du travail: Agence Nationale Securite Sanitaire Alimentaire Nationale","correspondingAuthor":true,"prefix":"","firstName":"Gaëlle","middleName":"","lastName":"Gonzalez","suffix":""}],"badges":[],"createdAt":"2024-07-04 20:02:28","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4688411/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4688411/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13567-025-01508-w","type":"published","date":"2025-04-24T15:57:14+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":61849794,"identity":"987b96a8-84db-471c-96a3-d0bd3dabe8bb","added_by":"auto","created_at":"2024-08-06 08:38:26","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":127101,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eMap of the study area in southwestern France with black dots representing the stables where horses were sampled, grouped in two main zones: the Confluence and Arcachon zones. The red circles indicate the stables where clinical cases were reported in 2022, and the green polygons indicate the special protection areas for the protection of wild birds.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4688411/v1/655e9fe8d05a41dd4e312e8b.png"},{"id":61849795,"identity":"435970dd-029d-4afc-9def-f948a5c2c5a3","added_by":"auto","created_at":"2024-08-06 08:38:26","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":225057,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eDistribution of WNV, USUV and/or TBEV seropositive equids in Confluence, Arcachon basin and intermediate area in Gironde county in 2023. Circles represent the stables selected for the study, the size of which is proportional to the number of sampled horses. The darker the color of the circles is, the greater the number of seropositive horses.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4688411/v1/5ad35a57c93b448e173f330e.png"},{"id":81569563,"identity":"81bbce92-7092-418e-b2f4-259b5ca6341d","added_by":"auto","created_at":"2025-04-28 16:07:26","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1045125,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4688411/v1/12ee1925-3bd4-472a-acf9-c5696b6d8877.pdf"}],"financialInterests":"","formattedTitle":"Seroprevalence of West Nile, Usutu and Tick borne encephalitis viruses in equids from southwestern France in 2023","fulltext":[{"header":"Introduction","content":"\u003cp\u003eWest Nile virus (WNV) is a reemerging zoonotic mosquito-borne orthoflavivirus (\u003cem\u003eFlaviviridae\u003c/em\u003e family) that represents a growing threat to human and animal health. It is one of the main viral agents causing encephalitis in humans and horses in Europe (Harris et al., 2006). It was identified for the first time in Uganda in 1937 (Smithburn et al., 1940), and its circulation is now evident on all five continents [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. WNV is maintained in an enzootic cycle involving ornithophilic mosquitoes as vectors (\u003cem\u003eCulex\u003c/em\u003e spp.) and wild and resident birds as reservoirs and/or amplifying hosts. The virus can infect approximatively one hundred mammalian host species, which are considered epidemiologically dead-end hosts that do not participate in virus spreading [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Among them, only humans and horses are known to develop symptoms following WNV infection. WNV infection is predominantly asymptomatic or can cause flu-like syndrome. In 1 to 10% of cases, neurological signs, the most frequent being ataxia, weakness and muscle fasciculations, are encountered. Some horses develop signs of encephalitis, leading to death or euthanasia [\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. In addition, human-to-human transmission of WNV, during blood transfusions or organ transplants, has been extensively documented and remains a major concern in the transfusion context [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. To reduce the risk of non-vector transmission, Europe introduced precautionary measures to control labile products, such as the exclusion of blood donors for 28 days after a visit to a high-risk area (European Directive 2004/33/EC) [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAs WNV circulation is heterogeneous between Member States and is a compulsory notifiable disease for birds (since 2021), equines and humans (since 2009) to the European Commission [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], each country has implemented an appropriate surveillance and monitoring system to detect WNV cases in Equidae, avifauna and humans.\u003c/p\u003e \u003cp\u003eOver the last decade, Europe has faced significant changes in the distribution of WNV, as well as two other mosquito-borne orthoflaviviruses, Usutu virus (USUV) and tick-borne encephalitis virus (TBEV) most likely caused by global change and increasing global warming. The three viruses actively cocirculate in most European countries. Infection are mainly subclinical, and accurate estimations of infection incidence rates are difficult.\u003c/p\u003e \u003cp\u003eThe surveillance of WNV infection relies heavily on neurological syndromic surveillance in humans and horses in most EU countries. Serosurveillance of equines is also commonly employed to detect virus circulation in a territory, and allows estimation of the intensity of circulation in that territory [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan additionalcitationids=\"CR10 CR11 CR12 CR13 CR14\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. The outdoor lifestyle of equines and their proximity to humans are good indicators of WNV circulation and human exposure.\u003c/p\u003e \u003cp\u003eIn France, WNV was detected for the first time in the Camargue region in 1962. Between 2000 and 2021, several WNV outbreaks of varying intensity were reported, all of which were located in the Mediterranean region and Corsica [\u003cspan additionalcitationids=\"CR17 CR18\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The 2022 transmission season was marked by the emergence of WNV in southwestern France, on the Atlantic coast. In October 2022, three horses of three distinct stables in Gironde County (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) exhibited neurological signs, including ataxia, weakness, muscle fasciculations, and encephalitis. Acute WNV infection was confirmed by serological diagnostic methods (ELISA IgM and IgG) carried out by the French National Reference Laboratory for WNV.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo obtain an accurate assessment of orthoflavivirus circulation in Gironde County, the first seroprevalence survey was conducted among equids located near stables where WNV cases in horses were newly reported in 2022 by investigating possible seroconversion to WNV, USUV and TBEV (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWe aimed to provide valuable insights into the intensity of WNV circulation in 2022 in this newly emerging West Nile virus zone and the associated risk factors for infection. We also evaluated the occurrence of USUV and TBEV in horses in this area.\u003c/p\u003e"},{"header":"Material and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Geographical zones\u003c/h2\u003e \u003cp\u003eThe survey was conducted in Gironde County, southwestern France, characterized by its contrasting landscape. The epidemiological parameters of the three defined zones were determined. The first zone, named Confluence, corresponds to the area where WNV-infected horses were reported in 2022 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The confluence lies at the junction of two rivers, the Garonne and the Dordogne rivers, where large populations of mosquitoes can be expected. The second zone corresponds to the Arcachon basin. The Arcachon basin lies at the heart of the East Atlantic Flyway, one of the eight most important bird migration corridors on the planet. No cases of WNV infection were reported here. These two zones offer a protected habitat for resident and migratory bird species (represented in green in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) that use wetlands as a stopover during their long journeys. These avian tranquility zones encourage interactions between local mosquitoes and competent avian hosts which promotes the establishment of the bird-mosquito-bird transmission cycle of WNV and USUV. The third zone covers the area between the Confluence and the Arcachon basin.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Study design and sample collection\u003c/h2\u003e \u003cp\u003eThe study was a cross-sectional prospective study. It took place in April and May 2023, prior to the occurence of seasonal orthoflavivirus activity, to reflect the exposure of horses to WNV, USUV and TBEV during the summer 2022. Animals were recruited by the Clinique Equine de Conques (Saint Aubin de Branne, Gironde, France) through social media and client mailing, according to our three different zones. All owners signed a consent form and treating veterinarians were informed of the procedure. This study was approved by the Ethics Committee for Clinical Research (ComERC) of the Veterinary School of Alfort (EnVA) (Agreement number: 2023-06-23).\u003c/p\u003e \u003cp\u003eSerum samples were collected from 494 adult (\u0026gt;\u0026thinsp;2-year-old) client-owned horses, ponies, and donkeys residing in 39 different stables of the three studied zones: 306 equids from 25 stables in the Confluence zone, 77 equids from 5 stables in the Arcachon zone, and 111 equids from 9 stables in the intermediate zone (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The animals were unvaccinated against WNV and had been living in the sampling area for the past 6 years throughout 2022 without traveling to the Mediterranean region.\u003c/p\u003e \u003cp\u003eThe number of equids sampled in a stable depended on the stable size: all horses were sampled in small stables (fewer than 10 animals), 10 horses were sampled in medium stables (between 10 and 15 animals), and 15 horses were sampled in large stables (more than 15 animals). All animals were asymptomatic at the time of sampling. The 3 cases of 2022 were not included in our cohort, although two of the three corresponding stables were included (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSerum samples were collected from each included animal. Blood samples were collected in vacutainer dry tubes and centrifuged at 5000 rpm for 5 minutes within 24 hours of collection. The serum was separated, stored at 4\u0026deg;C, and sent to the French National Reference Laboratory for West Nile virus, ANSES (Maisons-Alfort, France) for further analysis.\u003c/p\u003e \u003cp\u003eIn addition to sample collection, each stable manager/owner completed a survey consisting in a personal face-to-face interviews to gather information on individual characteristics and stable management. This information included age, sex, hair colour, and type of housing. We also collected information about the environment, such as the distance to the nearest water surface, and the distance to the nearest special protection areas. SPAs have been created for the protection of wild bird species listed in the Annex 1 of the EU bird directive (Directive 79/409/EEC), or that serve as breeding, moulting, wintering, or staging areas for migrating birds. Five SPAs have been defined in the study area around wetlands (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Serological testing\u003c/h2\u003e \u003c/div\u003e\n\u003ch3\u003eELISA tests\u003c/h3\u003e\n\u003cp\u003eSerum samples were, first, tested for IgG flaviviruses using the commercial Pan-Flavivirus ELISA \u0026ldquo;ID Screen Flavivirus competition\u0026rdquo; (Innovative Diagnostic, Montpellier, France). The protocol was performed according to the manufacturer's instructions. The results are expressed as %S/N. If a sample had a %S/N less than or equal to 40%, the sample was considered positive. If it is strictly greater than 50%, the sample is negative. If the result is between 40% and 50%, the sample is considered doubtful. Positive and doubtful results were confirmed for the presence of WNV, USUV and TBEV specific neutralizing antibodies using specific virus neutralization tests.\u003c/p\u003e\n\u003ch3\u003eVirus-neutralization test (VNT)\u003c/h3\u003e\n\u003cp\u003eTo identify specifically against which flaviviruses the IgG antibodies detected by ELISA are directed, a virus neutralization test (VNT) was carried out for the three main flaviviruses circulating in France: WNV, USUV and TBEV. This test was performed in 96-well plates as described in Beck et al.[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] .\u003c/p\u003e \u003cp\u003eBriefly, the sera were diluted in a cascade by a factor of 2, from 1:5 to 1:160, in a final volume of 100\u0026micro;L. Fifty microliters of virus was added at an infectious dose of 100 TCID50. After 1h30 of incubation at 37\u0026deg;C and 5% CO\u003csub\u003e2\u003c/sub\u003e, 100\u0026micro;L of Vero cells was added at a concentration of 2.10\u003csup\u003e5\u003c/sup\u003e cells/mL. The neutralizing antibody titer was obtained by observing the cytopathic effect (CPE) under a light microscope after 3-, 4- and 5-days of incubation at 37\u0026deg;C and 5% CO\u003csub\u003e2\u003c/sub\u003e for WNV, USUV and TBEV respectively. The last dilution of serum showing no CPE or a proportion of CPE less than a quarter of the well observed, was noted as the dilution of serum considered where there is still protection against the virus. The neutralizing antibody titer was therefore the inverse of the serum dilution.\u003c/p\u003e \u003cp\u003eSera neutralizing more than one virus were considered positive for the virus neutralized at a fourfold higher dilution than all other viruses. If a fourfold difference in neutralizing antibody titers was not reached, the serum was considered undifferentiated between the viruses. Sera that tested positive by competitive ELISA and negative by VNT were considered negative.\u003c/p\u003e\n\u003ch3\u003eStatistical analysis\u003c/h3\u003e\n\u003cp\u003eAll collected variables were described in terms of frequency distribution (qualitative data) or median and range (quantitative data) classified by the serological status.\u003c/p\u003e \u003cp\u003eData were analysed using mixed effects logistic regression models, using the stable as a random effect. The dependent variable was the serological status of the animals, according to the seroneutralization test. Fixed effects were animal age (years), sex (male or female), hair colour (light or dark), the type of housing (always on pasture, or both on pasture and indoors), the distance in kilometres (km) to the nearest water surface (intermittent or permanent, of any type: river, canal, estuary, lake, marsh, reservoir etc.), and the distance in km to the nearest special protection area (SPAs).\u003c/p\u003e \u003cp\u003eThe exponentiated regression coefficients produced odds ratio (OR) as a measure of the effect of the variables. Starting from the model with all the fixed effects, we selected the most parsimonious model using backward model selection based on the likelihood ratio test, p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003cp\u003eWe used the nonparametric approach proposed by Vaumourin et al. (2014) to test whether the association between positive VNT results against WNV, USUV and TBEV (i.e. the number of equids positive for 1, 2 or 3 of these viruses) significantly differed from the expected distribution under the null hypothesis of independent positive results.\u003c/p\u003e \u003cp\u003eAll the statistical analyses were performed using R 4.3.3 (R Core Team 2024). Mixed effects logistic regression models were fitted using the lme4 package [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Model selection was performed using the buildmer package [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eThis study included 494 adults: 334 horses, 151 ponies and 9 donkeys of which 271 were male and 223 were female. The majority (412 horses) were active at the time of sampling: 328 animals lived in pasture, whereas 166 had a mixed lifestyle (stable and pasture). Regarding hair coat color, 331 animals had a dark coat, while 163 had a light coat.\u003c/p\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.1 ELISA and VNT\u003c/h2\u003e \u003cp\u003eOf the 494 horses included in the study, 70 were positive for orthoflavivirus antibodies by competitive ELISA yielding a seroprevalence rate of 14% (95% CI [11%-18%]). Among them, 60 animals were located in 20 stables in the Confluence zone, 5 horses were housed in 4 stables in the Intermediate zone and 1 was based on 1 stall in the Arcachon area (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComparative serological results among selected horses in the Confluence, Intermediate and Arcachon zones in Gironde county\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c5\" namest=\"c3\"\u003e \u003cp\u003eGeographical zone\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eConfluence\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eIntermediate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eArcachon\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTested horses/stables\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e306/25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e111/9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e77/5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eELISA results:\u003c/p\u003e \u003cp\u003ePositive horses/stables\u003c/p\u003e \u003cp\u003eAnimal prevalence (95% CI)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e64/20\u003c/p\u003e \u003cp\u003e0.21 (0.16\u0026ndash;0.26)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5/4\u003c/p\u003e \u003cp\u003e0.05 (0.02\u0026ndash;0.10)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1/1\u003c/p\u003e \u003cp\u003e0.01 (0.00-0.07)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVNT results in ELISA-positive animals:\u003c/p\u003e \u003cp\u003ePositive horses/stables\u003c/p\u003e \u003cp\u003eAnimal prevalence (95% CI)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWNV\u003c/p\u003e \u003cp\u003eUSUV\u003c/p\u003e \u003cp\u003eTBEV\u003c/p\u003e \u003cp\u003eWNV\u003c/p\u003e \u003cp\u003eUSUV\u003c/p\u003e \u003cp\u003eTBEV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e27/13\u003c/p\u003e \u003cp\u003e14/5\u003c/p\u003e \u003cp\u003e6/4\u003c/p\u003e \u003cp\u003e0.09 (0.06\u0026ndash;0.13)\u003c/p\u003e \u003cp\u003e0.05 (0.03\u0026ndash;0.08)\u003c/p\u003e \u003cp\u003e0.02 (0.01\u0026ndash;0.04)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3/2\u003c/p\u003e \u003cp\u003e1/1\u003c/p\u003e \u003cp\u003e1/1\u003c/p\u003e \u003cp\u003e0.03 (0.01\u0026ndash;0.08)\u003c/p\u003e \u003cp\u003e0.01 (0.0-0.05)\u003c/p\u003e \u003cp\u003e0.01 (0.0-0.05)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1/1\u003c/p\u003e \u003cp\u003e0/0\u003c/p\u003e \u003cp\u003e0/0\u003c/p\u003e \u003cp\u003e0.01 (0.0-0.07)\u003c/p\u003e \u003cp\u003e0.0 (0.0-0.05)\u003c/p\u003e \u003cp\u003e0.0 (0.0-0.05)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eWithin the Confluence zone, 27 equids from 13 different stables were positive for WNV by VNT (9%, 95% CI, [6%-13%]). Within the same zone, 14 equids from 5 different stables were positive for USUV (5%, 95% CI, [3%-8%]), and 6 equids from 4 different stables were positive for TBEV (2%, 95% CI, [1%-4%]). In the intermediate zone, 3 equids housed on 2 stables were positive for WNV (3%, 95% CI, [1%-8%]), one horse was positive for TBEV and another horse was positive for USUV neutralising antibodies (1%, 95% CI, [0%-5%]). For the Arcachon zone, only one horse was positive for WNV (1%, 95% CI, [0%-7%]), and no horse was seropositive for the other viruses (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eUndifferentiable VNT results were also observed. In total, 24 equids tested positive for WNV, 5 horses tested positive for both WNV and USUV, 2 horses tested positive for WNV and TBEV, and none tested positive for all three viruses simultaneously (WNV, USUV, TBEV). Additionally, 9 horses tested positive for USUV only, 1 horse tested positive for both USUV and TBEV, and 4 horses tested positive for TBEV only (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAssociation between VNT serological status for WNV, USUV and TBEV\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eWNV\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eUSUV\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eTBEV\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eNumber of animals\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ePositive\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ePositive\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003ePositive\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003e0\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ePositive\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ePositive\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eNegative\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003e5\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ePositive\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eNegative\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003ePositive\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003e2\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ePositive\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eNegative\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eNegative\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003e24\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eNegative\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ePositive\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003ePositive\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003e1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eNegative\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ePositive\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eNegative\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003e9\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eNegative\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eNegative\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003ePositive\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003e4\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eNegative\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eNegative\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eNegative\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003e449\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eTotal\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003e494\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe individual characteristics of the sampled animals as a function of their WNV and USUV serostatus are shown in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. The qualitative comparison showed that horses tested positive for WNV were mainly male kept on pasture only. This does not apply to horses seropositive for the Usutu virus.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCharacteristics of horses as a function of the WNV and USUV serostatus.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIndividual horse characteristics\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eWNV\u003c/p\u003e \u003cp\u003en (proportion) or median (range)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eUSUV\u003c/p\u003e \u003cp\u003en (proportion) or median (range)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePositive horses\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNegative horses\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePositive horses\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNegative horses\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSex\u003c/p\u003e \u003cp\u003e.Male\u003c/p\u003e \u003cp\u003e.Female\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e19 (0.70)\u003c/p\u003e \u003cp\u003e8 (0.30)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e162 (0.58)\u003c/p\u003e \u003cp\u003e117 (0.42)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8 (0.57)\u003c/p\u003e \u003cp\u003e6 (0.43)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e173 (0.59)\u003c/p\u003e \u003cp\u003e119 (0.41)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAge\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e13 (2\u0026ndash;30)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e11 (2\u0026ndash;35)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10.5 (6\u0026ndash;25)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e11 (2\u0026ndash;35)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHair color\u003c/p\u003e \u003cp\u003e.Dark\u003c/p\u003e \u003cp\u003e.Light\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e14 (0.52)\u003c/p\u003e \u003cp\u003e13 (0.48)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e197 (0.29)\u003c/p\u003e \u003cp\u003e82 (0.71)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e9 (0.64)\u003c/p\u003e \u003cp\u003e5 (0.36)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e202 (0.69)\u003c/p\u003e \u003cp\u003e90 (0.31)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eType of housing\u003c/p\u003e \u003cp\u003e.Pasture only\u003c/p\u003e \u003cp\u003e.Mixed pasture/stable\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e23 (0.85)\u003c/p\u003e \u003cp\u003e4 (0.15)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e158 (0.57)\u003c/p\u003e \u003cp\u003e121 (0.43)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e9 (0.64)\u003c/p\u003e \u003cp\u003e5 (0.36)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e172 (0.59)\u003c/p\u003e \u003cp\u003e120 (0.41)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Risk factor analysis\u003c/h2\u003e \u003cp\u003eA mixed effects logistic regression model of VNT serostatus was fitted for WNV and USUV only, since for TBEV, the number of seropositive stables and equids was very low. Furthermore, we focused our analysis on the Confluence zone, where the vast majority of WNV and USUV seropositive equids were located (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). For WNV, model selection allowed us to identify two risk factors of VNT seropositivity: the type of housing with a strongly increased risk in animals always kept on pasture (odds-ratio [OR]\u0026thinsp;=\u0026thinsp;4.99) and the distance to the nearest SPA, with a decreasing risk when this distance increased ([OR]\u0026thinsp;=\u0026thinsp;0.85 for a 1km increase) (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Other variables (age, sex, hair colour and distance to the nearest water surface) were not retained in the selected model. For USUV, model selection resulted in an empty model (containing the intercept only) and did not allow the identification of any seropositivity risk factors.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMixed effects logistic regression model of the VNT WNV serological status of the horses sampled in the Confluence zone\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVariable\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eValue\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eOdds-ratio (95% CI)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIntercept\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.10 (0.03\u0026ndash;0.27)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eType of housing\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePasture and indoors\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eReference\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePasture only\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.99 (1.83\u0026ndash;17.47)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDistance to the nearest SPA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIncrease by 1km\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.85 (0.78\u0026ndash;0.93)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eFinally, we detected a significant association between WNV and USUV serological status (p\u0026thinsp;=\u0026thinsp;0.006), with the number or number of equids positive for both viruses (5 animals) being significantly higher than the expected 95% interval under the null hypothesis (0\u0026ndash;4 animals).\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eOver the last decade, France has witnessed a changing epidemiology of WNV, USUV and TBEV orthoflaviviruses with increased incidence of diseases in vertebrate hosts [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFrom cohorts of equines located in the three defined areas of Gironde County, we demonstrated WNV seroprevalence rates ranging from 9% (95% CI [\u003cspan additionalcitationids=\"CR7 CR8 CR9 CR10 CR11 CR12\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]) in the Confluence area to 3% (95% CI [\u003cspan additionalcitationids=\"CR2 CR3 CR4 CR5 CR6 CR7\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]) in the intermediate area and 1% (95% CI[0\u0026ndash;7]) in the Arcachon basin. These findings are comparable to previous reports from Kosovo [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], Spain [\u003cspan additionalcitationids=\"CR25 CR26\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] Germany [\u003cspan additionalcitationids=\"CR29 CR30\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e] and France [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. In the Camargue region, which has been considered as the historical area of WNV circulation in France since the 1960s, the seroprevalence rate among equines estimated between 2016 and 2020 is slightly higher at approximatively 13% [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAs for WNV, the highest USUV seroprevalence rate observed in horses was obtained in the Confluence area (5%). It was null near the Arcachon basin.\u003c/p\u003e \u003cp\u003eTherefore, the Confluence area appears to be a hot spot for \u003cem\u003eCulex-borne\u003c/em\u003e orthoflavivirus circulation. Our data showed a low percentage of seropositive individuals infected with TBEV (0,3%). Nevertheless, this low seroprevalence rate does not allow us to conclude that TBEV is circulating in southwestern France. The virus is known to circulate in the northeastern part of the country[\u003cspan additionalcitationids=\"CR33\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], with outbreaks detected further south in Jura and other regions [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Further investigations in ticks and reservoir animals such as rodents are needed to confirm the presence of TBEV in the Nouvelle Aquitaine region.\u003c/p\u003e \u003cp\u003eIn this study, we analysed the risk factors that might affect the susceptibility of horses to WNV infection. The risk of exposure to WNV increased when horses were kept outdoors, and thus were more exposed to vector bites, and when the stable was close to protected areas to preserve the avian biodiversity they harbor (SPAs). In and around these areas, bird and vector species assembly may favor local WNV circulation, as already suggested in southeastern France. This finding is in line with the association, at the European level, between West-Nile disease occurrence in humans and the local diversity of wild bird species [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e] [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e] .\u003c/p\u003e \u003cp\u003eAge, sex, horse color and distance to the nearest water surface were not identified as risk factors associated with WNV and USUV seropositivity. Our data are consistent with those of Garc\u0026iacute;a-Bocanegra et al [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]; Vanhomwegen et al [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e] who showed no significant differences among age classes; of Durand et al [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] in France, Hassine et al in Jordania [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e] and Bażan\u0026oacute;w et al in horses in Poland [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e] who reported the absence of an effect of gender on WNV and USUV seropositivity. Analyses of the color of horse coats are rare in WNV seroprevalence studies in horses. The results from several research groups are disparate, with studies showing an association between darker color and a lower WNV seroprevalence rate [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e] and others demonstrating no correlation between this variable and WNV and USUV seropositivity [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eInterestingly, we did not find a correlation between the seroprevalence of mosquito-borne viruses and the nuisance caused by mosquitoes evaluated by owners. As it is a personal assessment and may vary from one individual to another, our data here do not allow conclusions to be drawn.\u003c/p\u003e \u003cp\u003eThe emergence of WNV in this new region of France raised several questions, particularly regarding the factors that favored its introduction into this area. Our main hypothesis is that the introduction of WNV into Nouvelle Aquitaine region occurred through migratory birds. Indeed, the region is located on a migratory corridor of different migratory bird species such as the grey crane, the red kite, the graylag goose and passerines. In addition, the large fire of July 2022, which burned more than 30,000 hectares of forest in Gironde (particularly in La Teste de Buch, Landiras, Landiras St Magne, Saumos and Ar\u0026egrave;s) [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e], may lead to the disruption of ecosystems and play a role in the new distribution of WNV. Forest fires can disrupt ecosystems and displace wildlife, potentially leading to increased interactions between humans and infected animals/mosquitoes [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Investigations are needed to identify the resident and amplifying avian species that may be involved in the endemicity of WNV in this region of France. The number of equine and avian cases recorded by the French National Reference Laboratory for West Nile during the 2023 transmission season suggests that the virus tends to establish itself and extend its range. Cases have been detected in departments bordering the Gironde especially in Charentes Maritime and Charente.\u003c/p\u003e \u003cp\u003eCarrying out seroprevalence surveys such as the one conducted in this study is essential for strengthening monitoring of WNV circulation. This helps us to understand viral emergence and evaluate the intensity of circulation in new areas. The seroprevalence survey carried out close to syndromic cases diagnosed in 2022 identified areas with high viral circulation. In 2023, these data enabled mosquito traps to be set in mid-July, providing rapid evidence of active circulation of the West Nile and Usutu viruses in the Confluence area. These data have played a major part in the rapid implementation of the measures needed to ensure the safety of products derived from the human body (blood and organ donations) throughout the Gironde and Charente Maritime, a neighbouring department [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Horse owners located in the Confluence zone were encouraged to vaccinate their animals to prevent WNV infection in 2024 and the development of severe forms of the disease.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis first seroprevalence study conducted in equids located in Gironde County on the Atlantic coast of France showed that WNV, USUV and an orthoflavivirus belonging to the TBEV serocomplex circulate in this area and infect horses in 2022. Horses, known as sentinel of WNV infection, exhibited intense circulation of this virus in this region of France as well as equine USUV specific infection. This study confirmed the usefulness of serosurveys in horses for detecting the circulation of orthoflaviviruses, especially WNV, in specific regions.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eCPE CytoPathic Effect\u003c/p\u003e\n\u003cp\u003eELISA \u0026nbsp;Enzyme-liked immunabsorbent assay\u003c/p\u003e\n\u003cp\u003eIgG Immunoglobulin G\u003c/p\u003e\n\u003cp\u003eIgM Immunoglobulin M\u003c/p\u003e\n\u003cp\u003eOR Odd Ratio\u003c/p\u003e\n\u003cp\u003eSPA Special Protection Area\u003c/p\u003e\n\u003cp\u003eTBEV Tick-Borne Encephalitis Virus\u003c/p\u003e\n\u003cp\u003eTCID50 Tissue Culture Infectious Dose 50\u003c/p\u003e\n\u003cp\u003eUSUV Usutu Virus\u003c/p\u003e\n\u003cp\u003eVNT Virus Neutralisation Test\u003c/p\u003e\n\u003cp\u003eWNV West Nile Virus\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eThis study was approved by the Ethics Committee for Clinical Research (ComERC) of the Veterinary School of Alfort (EnVA) (Agreement number: 2023-06-23). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank equine owners for taking part in the serological survey. This research was conducted in part under the French and European laboratory for West Nile Virus.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eG.G., A.L., T.T., conceptualized the project.; C.V.M., G.G., N.C., C.M.P., E.Q., B.D., and C.L. contributed to the methodology; N.C., C.B., M.D., M.DM., collected the sera; C.V.M, M.D., T.H., M.C., did the laboratory tests; G.G., C.V.M, B.D., C.L. and N.C., analysed the data; , N.C, C.V.M., G.G., wrote the original draft; C.V.M, N.C., B.D., and G.G. reviewed and edited the original draft; G.G., M.D. and C.V.M., supervised the study; G.G., and C.V.M. contributed to the funding acquisition. All authors reviewed and approved the final draft.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFundings\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe acknowledge the French Agency for Food, Environmental and Occupational Health \u0026amp; Safety (ANSES) and the National Reference Laboratory for West Nile Virus for funding the research activities.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets of the current study are available from the corresponding author upon reasonable request.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eConsent for publication\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003eCompeting interests\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFootnotes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePublisher\u0026rsquo;s Note\u003c/p\u003e\n\u003cp\u003eSpringer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eContributor Information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCamille V. Mign\u0026eacute;, Email: [email protected]\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eGa\u0026euml;lle Gonzalez, Email: [email protected]\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSimonin Y (2024) Circulation of West Nile Virus and Usutu Virus in Europe: Overview and Challenges. Viruses, 16(4)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRoot J (2013) West Nile virus associations in wild mammals: a synthesis. Arch Virol 158(4):735\u0026ndash;752\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLecollinet S et al (2019) Viral Equine Encephalitis, a Growing Threat to the Horse Population in Europe? Viruses, 12(1)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAngenvoort J et al (2013) West Nile viral infection of equids. 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Vector Borne Zoonotic Dis 17(8):558\u0026ndash;566\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGuerrero-Carvajal F et al (2021) Serological evidence of co-circulation of West Nile and Usutu viruses in equids from western Spain. Transbound Emerg Dis 68(3):1432\u0026ndash;1444\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBUCCIO F (2022) \u003cem\u003eINCENDIES \u0026Eacute;T\u0026Eacute; 2022 Gironde et Landes Retour d\u0026rsquo;exp\u0026eacute;rience\u003c/em\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMoritz MA, Batllori E, Bolker BM (2023) The role of fire in terrestrial vertebrate richness patterns. Ecol Lett 26(4):563\u0026ndash;574\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003epublique HC (2022) .d.l.s., \u003cem\u003eAvis sur la s\u0026eacute;curisation des produits du corps humain dans un contexte de circul We would like to thank equine owners for taking part in the serological survey. ation du virus West Nile en France m\u0026eacute;tropolitaine\u003c/em\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-4688411/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4688411/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe circulation of West Nile virus (WNV), Usutu virus (USUV) and Tick-borne encephalitis virus (TBEV) was investigated in southwestern France within the first six months of 2023 following the emergence of WNV in equids in Gironde county in 2022. Blood samples were collected from 494 horses located in Gironde County divided into three zones: the Confluence, the Intermediate zone and the Arcachon basin and tested for WNV, USUV and TBEV specific antibodies. An overall seroprevalence of 14% (95% CI [11%-18%]) for orthoflavivirus antibody was detected in Gironde county. The highest seroprevalence rate for WNV and USUV were detected in the Confluence zone (9%, 95% CI, [6%-13%] and 5%, 95% CI, [3%-8%] respectively). The type of housing (animals kept in pasture only) and the distance to the nearest bird special protection area (SPA) were identified as risk factors for WNV seropositivity. This paper presents the first seroprevalence study investigating WNV, USUV and TBEV infections, conducted in equids located on the Atlantic coast of France and demonstrates an intense circulation of WNV in this region of France as well as equine USUV specific infection.\u003c/p\u003e","manuscriptTitle":"Seroprevalence of West Nile, Usutu and Tick borne encephalitis viruses in equids from southwestern France in 2023","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-06 08:38:21","doi":"10.21203/rs.3.rs-4688411/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"977c9dab-fde9-45eb-b89a-944b2224e6a5","owner":[],"postedDate":"August 6th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-04-28T16:00:11+00:00","versionOfRecord":{"articleIdentity":"rs-4688411","link":"https://doi.org/10.1186/s13567-025-01508-w","journal":{"identity":"veterinary-research","isVorOnly":false,"title":"Veterinary Research"},"publishedOn":"2025-04-24 15:57:14","publishedOnDateReadable":"April 24th, 2025"},"versionCreatedAt":"2024-08-06 08:38:21","video":"","vorDoi":"10.1186/s13567-025-01508-w","vorDoiUrl":"https://doi.org/10.1186/s13567-025-01508-w","workflowStages":[]},"version":"v1","identity":"rs-4688411","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4688411","identity":"rs-4688411","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}