331 Veterinaria Italiana 2022, 58 (3), 331-337. doi: 10.12834/VetIt.2596.16323.2 Accepted: 15.11.2021 | Available on line: 31.12.2022 1Department of Veterinary Public Health and Preventive Medicine, University of Abuja, Nigeria. 2The Nigerian Police Force, Department of Operations, Veterinary Unit, Presidential State House, Abuja, Nigeria. *Corresponding author at: : Department of Veterinary Public Health and Preventive Medicine, University of Abuja, Nigeria. E‑mail: andrew.adamu@uniabuja.edu.ng. Rukaiya Musa-Gobe1,2, Gabriel Omeiza1, Wesley Nafarnda1 and Andrew Adamu1* Keywords West Nile virus, ELISA, Horses, Domestic chickens, Nigeria. Summary West Nile virus (WNV) is an emerging arbovirus which affects humans and horses. A cross sectional study was carried out on 106 local horses in Kaduna and 78 domestic chickens in Federal Capital Territory. A total of 184 sera were screened for West Nile virus anti Pr-E antibodies using ID Screen® West Nile competitive enzyme linked immunosorbent assay. For the horses, an overall prevalence of 92.45% was recorded while domestic chickens had a preponderance of 7.69%. From our study, there was a statistical significant difference between the occurrences of WNV in stallions than mares with p < 0.05. Comparing the occurrence of West Nile virus between species, horses were more likely to be infected by West Nile virus than domestic chickens (OR 147). This is the first seroprevalence study investigating West Nile virus infection in domestic chickens in Nigeria. The presence of the antibodies indicates the widespread circulation and the potential risk of infection in humans and animals. In order to understand the epidemiology of West Nile virus infection in Nigeria, there is need for surveillance to be implemented in human and animal sectors. Evidence of West Nile virus in chickens and horses in Nigeria: results from a serosurvey disease, which can be characterized by acute flaccid paralysis, encephalitis, and meningoencephalitis (De Fillet et al. 2012, Vilibic-Cavlek et al. 2014). From the 1990s, West Nile disease outbreaks have emerged across various continents and WNV is now recognized as one of the most prevalent flaviviruses worldwide (Vilibic-Cavlek et al. 2014, Cox et al. 2015, Fall et  al. 2016). In Nigeria, Culex species like Culex pipens, Culex quinquefasciatus have been reported in South West Nigeria (Motayo et  al. 2016). This is a clear indicator that most of the vectors in Nigeria are present and may predispose both humans and animal population to the WNV infection. West Nile Virus has been reported in horses and humans by different authors in Nigeria. Olaleye and colleagues (Olaleye et  al. 1989) reported a prevalence of 71% from horses in Southwest Nigeria, Sule and colleagues (Sule et  al. 2015) recorded 90.3% in the same geopolitical zone while Baba and colleagues (Baba et al. 2013) documented a prevalence of 25% from humans in Maiduguri North East Nigeria. Avian species have been categorized into four groups for the purpose of WNV surveillance: dead birds, trapped birds, captive and sentinel birds or domestic birds (Chintoutis et al. 2015). In the United Introduction West Nile virus (WNV) is a vector-borne virus belonging to the Japenese encephalitis complex within the Flaviviridae family. The complex includes Japanese encephalitis virus (JEV), Saint Louis encephalitis virus (SLEV), Murray Valley encephalitis virus (MVE) and Usutu virus (USUV) (Fall et al. 2016). Most of these flaviviruses, such as WNV, tick-borne encephalitis virus (TBEV), or JEV, are considered as emerging zoonoses that pose serious public health threats to animals and man (Cardinale et al. 2017). West Nile virus was first isolated from a febrile woman in Omogo region in the West Nile district of Uganda in 1937, from a set out survey to define the endemicity of yellow fever (Smithburn et al. 1940). WNV is chiefly transmitted by bite of infected Culex sp. mosquitoes. Birds are considered as a main reservoir host and migratory birds can play an important role in long distance viral dissemination (Hubalek and Halouzka 1999, Hayes et al. 2005, Ergunay et al. 2015). Humans and horses are known to be incidental hosts of WNV (Murray et  al. 2010). Clinical symptoms of WNV infection may range from asymptomatic or mild influenza-like illness to severe neurological SHORT COMMUNICATION 332 West Nile virus in Nigerian horses and chicken Musa-Gobe et al. Veterinaria Italiana 2022, 58 (3), 331-337. doi: 10.12834/VetIt.2596.16323.2 Study area, design and sample collection A cross sectional study was conducted with blood samples taken in 2019 from a total of 106 randomly selected local horses in three local government areas of Kaduna State with coordinate (10°30'36.7"N 7°25'01.6"E). The three local government areas were selected based on the concentration (Mshelia et  al. 2012) of local horse presence of polo fields, and their unvaccinated status: Zaria LGA (11°03'40.6"N 7°42'20.6"E) (n = 40), Sabon Gari LGA (11°08'01.0"N 7°43'02.0"E) (n = 30) and Igabi LGA (11°08'01.0"N 7°43'02.0"E) (n = 36) Figure 1 and Table  I. Similarly, blood samples from domestic chickens (n = 78) were collected from Federal Capital Territory (9°04'26.3"N 7°28'43.1"E) in commercially reared chickens (Isa Brown) (n = 40) and free range chickens (n = 38) in Kuje Area Council (8°52'49.0"N 7°13'37.7"E) to determine the spread of WNV. Blood samples were aseptically taken by jugular venipuncture and were allowed to clot and centrifuged at 10,000 g for fifteen minutes to allow for proper separation of serum from the clotted blood. Sera were harvested using a sterile pipette into 2 ml cryovial tubes, labeled and stored at -  80  °C at the National Veterinary Research Institute Laboratory, Vom, Nigeria for sample analysis. States of America and Italy, for decades, domestic birds have been used as living sentinels in arbovirus programs aimed at monitoring virus transmission (Komar et  al. 2003b). West Nile virus surveillance using domestic birds has been documented in America, Europe and Australia (Chintoutis et  al. 2015) and Africa (Fall et al. 2016, Amdouni et al. 2020). Chickens are frequently being used as sentinels for the surveillance of bird-transmitted arboviral encephalitides (Langevin et  al. 2005, Blackmore et  al. 2003). Furthermore, arbovirus surveillance systems based on testing of sentinel chickens provided evidence of WNV circulation prior to the occurrence of human cases (Healy et al. 2012). This study was therefore carried out to determine the presence of WNV infection serologically in domestic chickens and local horses in Nigeria. Materials and methods Ethical statement This study was approved by the University of Abuja Ethical Committee and Animal Care Committee UAECAU/2019/01. Horses and domestic chickens were selected with the consent of their owners after they were briefed on the objective of the study. Figure 1. Map of Nigeria with Kaduna state highlighted and sampling local government areas in red. 333 Musa-Gobe et al. West Nile virus in Nigerian horses and chicken Veterinaria Italiana 2022, 58 (3), 331-337. doi: 10.12834/VetIt.2596.16323.2 area recorded the highest prevalence (38/40; 95%), followed by Igabi local government area (34/36; 94.44%) and Sabon Gari LGA (26/40; 86.67%) (Table I). Horses in the sampled areas were divided into three age groups. The first group included animals 1-5 year old, the second included animals between 6 and 10 years, and the third animals aged between 11 and 15 years. The prevealence found in the first group was 80% (12/15), 96.83% (61/63) was the prevalence found in the second group while in the third group it was 89.29% (25/28). All males (100%; n = 94) were found positive to the WNV c-ELISA, while in only 66.67% of the sampled mares (n = 12) WNV antibodies were detected. There was a statistically significant difference (p < 0.05) between the occurrences of WNV in stallions and in mares (Table I). Prevalence of WNV antibodies in chickens in Kuje local government area A total of 78 chickens were sampled in Kuje local government area of Federal Capital Territory with an overall WNV prevalence of 7.69%. Free range chickens recorded a prevalence of 5.26% (2/38) while in exotic chickens (Isa Brown hybrid chicken) the prevalence found was 10% (4/40/) (Table II). A total of thirty eight (38) free range birds where sampled. All cocks (n = 4) were negative. A prevalence of 5.88% was detected in the 34 hens (Table II). Comparison of WNV antibodies among local horses and chickens in Kaduna and FCT From the sampled horses, 92.45% (98/106) prevalence was recorded in Kaduna State while 7.69% (6/78) was recorded from chickens in the Federal Capital Territory. In this study the WNV prevalence found in horses was much (p < 0.05) than that found in chicken. Also, horses were 147 times more likely to be infected by WNV than chicken (OR 147) (Table III). Discussion The present study highlighted the circulation of West Nile specific IgG antibodies using competitive enzyme linked immunosorbent assay (c-ELISA) in Nigerian local horses and chickens (exotic and free range chickens) in Kaduna and Federal Capital Territory Abuja, respectively. A prevalence of 92.45% was recorded in horses. The result of this study was similar to other reports of WNV in horses in Tchad 90.3% or Senegal 92.5%. Conversely it was much higher than that found in Cote d’Ivoire 28%, Congo Democratic Republic 30%, Gabon 3% and Djibouti 9% (Cabre et  al. 2006, Sule et  al. 2015). The high Detection of WNV specific antibodies Serum samples (n = 184) were screened for the presence of anti-WNV IgG antibodies using the ID Screen® West Nile competitive ELISA kit (IDVet, Grabels, France) that detects WNV anti-Pr-E antibodies, according to the manufacturer’s instructions. Absorbance values were read at 450 nm wavelength, using a microplate spectrophotometer (Thermo Scientific Multiskan® EX; Thermo Fisher Scientific, Waltham, Massachusetts, USA). A presumptive positive diagnosis was made when the test samples produced an optical density (OD) less than or equal to 40%, they were considered as doubtful when the OD was between 40%-50%; and negative when the OD was greater than 50%. Data analysis Results obtained from serological tests were subjected to analysis by SPSS version 20.0 statistical packages for descriptive statistics. Chi square test and Fisher’s exact test were used to test for association between categorical variables; p value of less than < 0.05 was considered significant. Odd ratio was used to test for strength of association. Results Prevalence of WNV antibodies in local horses in selected local government areas of Kaduna State A total of 106 local horses were sampled with an overall prevalence of 92.45%. Zaria local government Table I. Distribution of West Nile Virus antibodies in local horses in three local government areas of Kaduna State according to demographic characteristics. Variables No. Sampled No. Positive % Prevalence (95% CI) p value Sex Male 94 94 100 (NaN - Infinity) 0.0001* Female 12 8 66.67 (37.69-88.39) Age (years) 1-5 15 12 80 (54.65-94.65) 0.065 6-10 63 61 96.83 (89.91-99.46) 11-15 28 25 89.29 (73.55-97.20) Location (LGA) Zaria 40 38 95 (84.45-99.15) 86.67 (70.90-95.62) 0.365 Sabon Gari 30 26 94.44 (82.84-99.06) Igabi 36 34 *NaN-Infinity = Not a number. 334 West Nile virus in Nigerian horses and chicken Musa-Gobe et al. Veterinaria Italiana 2022, 58 (3), 331-337. doi: 10.12834/VetIt.2596.16323.2 be attributed to the fact that they must have spent longer time in their stables thereby been exposed to several facilitating risk factors associated with the disease. This is contrary to the studies conducted by Zohaib and colleagues (Zohaib et  al. 2015) in Pakistan where antibodies to WNV decreased with age. The significant difference observed between sexes in our study where stallions recorded more seropositivity may be attributed to the availability of more stallions found in the location during sampling than mares and their use in ceremonial and sporting activities in northern part of the country. This is contrary to the studies of Sule and colleagues (Sule et al. 2015) in Southwest Nigeria where mares recorded a higher seropositivity than stallions. Birds are generally known to be host of WNV and other flaviviruses (Furlong et  al. 2023). Overall prevalence of 7.69% was recorded among domestic chickens in our study. A similar study was carried out by Yapici and colleagues (Yapici et  al. 2012) among domestic chickens with no seropositivity recorded. Several authors have worked on WNV infection in chickens with varying prevalence 0.63% by Komar and colleagues (Komar et al. 2003a) while Le Francois and colleagues (Le Francois et  al. 2006) recorded 1.7% and 0.6%, respectively, which were all lower than our study. However, Jozan and colleagues (Jozan et al. 2003) had 69% preponderance which is way higher compared to the recorded prevalence. From our study, exotic chickens had a higher preponderance than the free range chickens. The low preponderance of WNV antibodies observed in free range chickens as compared to exotic chickens could be linked to location. Most of the poultry farms are sited far away from human settlements with only farm workers residing in the farm. This encroachment of the natural habitat of mosquitoes may have played a role in the difference between breed preponderance of the WNV infection. Exotic chickens are exposed several vectors such as mosquitoes and soft ticks which can be found in the crevices of walls and fruit bats that roost on trees and roofs of poultry pens and farm houses. Thereby, predisposing poultry workers in poultry farms and markets (those that sell and dress chickens) to WNV infection. On the other hand, free range chickens wander occasionally into nearby bush to scavenge for feed and return home for shelter around their prevalence of WNV recorded in this study could be due to factors such as tranboundary movement of horses from neighboring African countries into Nigeria and climate change. The vegetation of Kaduna is Sudan Savannah with short trees, shrubs and grasses and relatively low rainfall that provide conducive conditions for the proliferation and adaptations of vectors that are capable of transmitting the virus to horses which corroborate with the studies conducted by Hubalek and Halouzka and Chancey and colleagues (Hubalek and Halouzka 1999, Chancey et al. 2015). Kaduna State is characterized by high temperature which can cause an upsurge in growth rates of vector populations, decrease the interval between blood meals, shorten the incubation time from infection to infectiousness in mosquitoes, accelerate the virus evolution rate and increase virus transmission efficiency (Paz 2015). Local horses are used for different purposes ranging from sports (horse racing or polo), religious festivals and some are slaughtered for meat. horses are transported to different part of the country during competitions with the possibility of of introducing vectors along with them into naïve population further creating a complex dynamic in the transmission of WNV. Also, less attention is given to the health and management of local horses as they are believed to be resistant to disease pathogens. This is in sharp contrast with the exotic horses which are well groomed and vaccinated or treated against various infectious diseases including WNV from their country of purchased. Studies have documented that WNV infection risk increases during peak mosquito activity seasons (August-October) in temperate zones of the Northern hemisphere, however, this observation is different in tropical and subtropical regions where disease epizootic patterns does not conform to what is obtainable in temperate regions (Zohaib et al. 2015). Horses in the age range of 6-10  years were found to record the highest prevalence of WNV antibodies. This could Table II. Distribution of West Nile Virus antibodies among domestic chickens in Kuje Area Council, Federal Capital Territory. Variables No. Sampled No. Positive % Prevalence (95% CI) p value Breed Local 38 2 5.26 (0.89-16.32) 0.4326 Exotic 40 4 10 (3.26-22.38) Sex (Local) Male 4 0 0 0.618 Female 34 2 5.88 (1.00-18.10) Sex (Exotic) Male 0 0 0 Not determinedFemale 40 4 10 (3.26-22.38) Table III. Comparison of WNV antibodies among local horses and domestic chickens in the study areas. Species of animal No. sampled No. positive Proportion (%) (CI) 95% CI p value Horses 106 98 92.45 86.17-96.43 0.0001 Chickens 78 6 7.69 3.18-15.31 Total 184 104 56.52 49.99-63.24 335 Musa-Gobe et al. West Nile virus in Nigerian horses and chicken Veterinaria Italiana 2022, 58 (3), 331-337. doi: 10.12834/VetIt.2596.16323.2 In conclusion, this study was able to demonstrate the presence of WNV antibodies in local horses and domestic chickens (exotic and free range) in Nigeria, to the best of our knowledge; this is the first report in chickens. Since avian species play an important role in WNV life cycle, its close interaction with humans poses a risk of zoonosis as some of the febrile illness in man can be misjudged as malaria in communities with poor diagnostic capabilities. There is need to assess the clinical impact of WNV infection on humans, equine and domestic chicken populations. Further molecular studies are needed in order to characterize the WNV in various livestock and human populations. This will be able to rule out any cases of cross reactions with other arboviruses. Acknowledgements We will like to thank all the animal owners and the livestock attendants that gave us their cooperation during the sample collection. Our gratitude goes to Mr. Dan Marwa Agom of Viral Division, National Veterinary Research Institute Vom, Plateau State for his support during the laboratory work. pens or some sleep outside by perching on trees, thereby exposed to adverse weather conditions and mosquitoes. At least, there is an effective mosquito control around homes where these chickens reside. From the free range chickens, hens were the only chickens that tested positive. Hens are mostly kept in rural areas with only few cocks that have good reproductive pedigrees are left to stay longer in the population. Cocks are either sold to meet immediate economic challenges or consumed during festivals and ceremonies. Horses and chickens seropositivity were compared. Horses were highly likely to be infected with WNV than chickens. This was attributed to the different age and exposure time to the WNV. Vitek and colleagues (Vitek et  al. 2005) remarked that horses experience deleterious effect when they are infected with WNV infection. However, horses are considered incidental host because they do not develop sufficient viremia to infect mosquito vectors. Conversely, morbidity and mortality among avian species varies (Vilibic et  al. 2014), birds are thought to play a vital role as host for WNV life cycle due to the fact that they develop viremia adequately enough to infect mosquitoes. 336 West Nile virus in Nigerian horses and chicken Musa-Gobe et al. Veterinaria Italiana 2022, 58 (3), 331-337. doi: 10.12834/VetIt.2596.16323.2 Amdouni J., Monaco F., Portanti O., Sghaier S., Conte A., Hassine T.B., Polci A., Valleriani F., Gennaro A.D., Zoueri M., Savini G. & Hammami S. 2020. Detection of enzootic circulation of a new strain of West Nile virus lineage 1 in sentinel chickens in the north of Tunisia. Acta Trop, 202, 105223. Baba M., Logue C.H., Oderinde B., Abdulmaleek H., Williams J., Lewis J., Laws T.R., Hewson R., Marcello A. & D'Agaro P. 2013. Evidence of arbovirus co-infection in suspected febrile malaria and typhoid patients in Nigeria. J Infect Dev Ctries, 7 (1), 51-59. Blackmore C.G., Stark L.M., Jeter W.C., Oliveri R.L., Brooks R.G., Conti L.A. & Wiersma S.T. 2003. Surveillance results from the first West Nile virus transmission season in Florida, 2001. Am J Trop Med Hyg, 69 (2), 141-150. Cabre O., Grandadam M. & Marié J.L. 2006. West Nile Virus in horses, sub-Saharan Africa. Emerg Infect Dis, 12 (12), 1958-1960. Cardinale E., Bernard C., Lecollinet S., Rakotoharinome V.M., Ravaomanana J., Roger M., Olive M.M., Meenowa D., Jaumally M.R., Melanie J., Héraud J.M., Zientara S. & Cêtre-Sossah C. 2017. West Nile virus infection in horses, Indian Ocean. Comp Immunol Microbiol Infect Dis, 53, 45-49. Chaintoutis S.C., Dovas C.I., Danis K., Gewehr S., Mourelatos S., Hadjichristodoulou C. & Papanastassopoulou M. 2015. Surveillance and early warning of West Nile virus lineage 2 using backyard chickens and correlation to human neuroinvasive cases. Zoonoses Public Health, 62 (5), 344-355. Chancey C., Grinev A., Volkova E. & Rios M. 2015. The global ecology and epidemiology of West Nile virus. Bio Med Res Int, 2015, 376230. Cox S. L., Campbell G.D. & Nemeth N.M. 2015. Outbreaks of West Nile virus in captive waterfowl in Ontario, Canada. Avian Pathol, 30, 1-27. De Filette M., Ulbert S., Diamond M. & Sanders N.N. 2012. Recent progress in West Nile virus diagnosis and vaccination. Vet Res, 43 (1), 16. Ergunay K., Bakonyi T., Nowotny N. & Ozkul A. 2015. Close relationship between West Nile virus from Turkey and lineage 1 strain from Central African Republic. Emerg Infect Dis, 21(2), 352-355. Fall G., Faye M., Weidmann M., Kaiser M., Dupressoir A., Ndiaye E.H., Ba Y., Diallo M., Faye O. & Sall A.A. 2016. Real-time RT-PCR assays for detection and genotyping of West Nile virus lineages circulating in Africa. Vector Borne Zoonotic Dis, 16 (12), 781-789. Furlong M., Adamu A.M., Hoskins A., Russell T.L., Gummow B., Golchin M., Hickson R.I. & Horwood P.F. 2023. Japanese encephalitis enzootic and epidemic risks across Australia. Viruses, 15 (2), 450. Hayes E.B., Sejvar J.J., Zaki S.R., Lanciotti R.S., Bode A.V. & Campbell G.L. 2005. Virology, pathology, and clinical manifestations of West Nile virus disease. Emerg Infect Dis, 11 (8), 1174-1179. References Healy J., Reisen W.K., Kramer V. & Barker C.M. 2012. Do current surveillance methods provide adequate warning for human infections with West Nile virus? Proc Mosq Control Assoc Calif, 80, 17-21. Hubálek Z. & Halouzka J. 1999. West Nile fever - a reemerging mosquito-borne viral disease in Europe. Emerg Infect Dis, 5, 643-650. Jozan M., Evans R., McLean R., Hall R., Tangredi B., Reed L. & Scott J. 2003. Detection of West Nile virus infection in birds in the United States by blocking ELISA and immunohistochemistry. Vector Borne Zoonotic Dis, 3 (3), 99-110. Komar N., Langevin S., Hinten S., Nemeth N., Edwards E., Hettler D., Davis B., Bowen R. & Bunning M. 2003a. Experimental infection of North American birds with the New York 1999 strain of West Nile virus. Emerg Infect Dis, 9 (3), 311-322. Komar O., Robbins M.B. & Klenk K. 2003b. West Nile virus transmission in resident birds, Dominican Republic. Emerg Infect Dis, 9 (10), 1299-1302. Langevin S.A., Brault A.C., Panella N.A., Bowen R.A. & Komar N. 2005. Variation in virulence of West Nile virus strains for house sparrows (Passer domesticus). Am J Trop Med Hyg, 72, 99-102. Lefrancois T., Blitvich B.J., Pradel J., Molia S., Vachiery N. & Martinez D. 2006. West Nile virus in Guadeloupe: introduction, spread, and decrease in circulation level: 2002-2005. Ann New York Acad Sci, 1081, 206-215. Motayo B.O., Onoja B.A., Faneye A.O. & Adeniji J.A. 2016. Seasonal abundance and molecular identification of West Nile virus vectors, Culex pipens and Culex quinquefasciatus (Diptera: Culicidae) in Abeokuta, South-West, Nigeria. Afr Health Sci, 16 (1), 135-140. Mshelia W.P., Onoja I.I., Saidu Q.V. & Edeh E.R. 2012. Husbandry and management practices in domestic horses (Equus caballus) in Northern Nigeria and their clinical implications. Proceedings of the 2nd International Equine Science Meeting Xenophon Verlag, Wald, March 16-19. Murray K.O., Mertens E. & Despres P. 2010. West Nile virus and its emergence in the United States of America. Vet Res, 41 (6), 67. Olaleye O.D., Omilabu S.A., Ilomechina E.N. & Fagbami A.H. 1989. A survey for haemagglutination-inhibiting antibody to West Nile virus in human and animal sera in Nigeria. Comp Immunol Microbiol Infect Dis, 13, 35-39. Onoja A.B., Maina M.M. & Opayele A.V. 2018. Molecular identification and phylogenetic analysis of acetylcholinesterase-2 gene from Culex pipiens and Culex quinquefasciatus (Diptera: Culicine). Nigerian Vet J, 39 (4), 285-294. Paz S. 2015. Climate change impacts on West Nile virus transmission in a global context. Philos Trans R Soc Lond B Biol Sci, 370 (1665), 20130561. Smithburn K.C., Hughes T.P., Burke A.W. & Paul J.H. 1940. A 337 Musa-Gobe et al. West Nile virus in Nigerian horses and chicken Veterinaria Italiana 2022, 58 (3), 331-337. doi: 10.12834/VetIt.2596.16323.2 Vitek C.J., Richards S.L., Mores C.N., Day J.F. & Lord C.C. 2005. Arbovirus transmission by Culex nigripalpus in Florida, 2005. J Med Entomol, 45 (3), 483-493. Yapici O., Kale M., Gur S., Mamak N., Yavru S., Hasircioglu S. & Bulut O. 2012. Serologic investigation for West Nile virus infection in commercial domestic chickens (Gallus gallus domesticus). J Anim Vet Advs, 11 (13), 2211-2214. Zohaib A., Saqib M., Beck C., Hussain M.H., Lowenski S., Lecollinet S., Sial A., Asi M.N., Mansoor M.K., Saqalein M., Sajid M.S., Ashfaq K., Muhammad G. & Cao S. 2015. High prevalence of West Nile virus in equines from the two provinces of Pakistan. Epidemiol Infect, 143, 1931-1935. neurotropic virus isolated from the blood of a native of Uganda. American J Trop Med Hyg, 20, 471-492. Sule W.F., Oluwayelu D.O. & Adedokun R.A. 2015. High seroprevelance of West Nile virus antibodies observed in horses from southwestern Nigeria. Vector Borne Zoonotic Dis, 15 (3), 218-220. Vilibic-Cavlek T., Kaic B., Barbic L., Pem-Novosel I., Slavic-Vrzic V., Lesnikar V., Kurecic-Filipovic S., Babic-Erceg A., Listes E., Stevanovic V., Gjenero-Margan I. & Savini G. 2014. First evidence of simultaneous occurrence of West Nile virus and Usutu virus neuroinvasive disease in humans in Croatia during the 2013 outbreak. Infection, 42 (4), 689-695.