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Microorganisms2025; 13(8); 1910; doi: 10.3390/microorganisms13081910

Seroprevalence and Passive Clinical Surveillance of West Nile Virus in Horses from Ecological High-Risk Areas in Western Romania: Exploratory Findings from a Cross-Sectional Study.

Abstract: This cross-sectional study evaluated the seroprevalence and clinical impact of West Nile virus (WNV) infection in horses from three ecologically high-risk counties in western Romania (Timiș, Arad, and Bihor) between 2023 and 2025. A total of 306 unvaccinated horses were tested using a commercial ELISA, with 8.17% testing positive for WNV antibodies, indicating prior exposure. Passive surveillance for clinical signs during mosquito seasons identified 16 horses with acute neurological symptoms, four of which were confirmed as clinical cases based on WNV-specific IgM positivity, suggesting probable silent WNV circulation in the region. The overall case fatality rate among confirmed clinical cases was 25.0%. WNV seropositivity was highest in Bihor (8.85%), followed by Arad (8.57%) and Timiș (7.32%). Statistical comparisons using χ tests and binary logistic regression indicated no significant differences in seroprevalence between counties, sexes, or age groups, consistent with the overlapping 95% confidence intervals. These findings suggest the continued silent circulation of WNV in the region and support the integration of equine surveillance into the One Health framework as a potential tool for early detection and risk mitigation. However, in the absence of molecular confirmation (e.g., RT-PCR or virus isolation), these results should be interpreted as indicative of prior exposure rather than direct evidence of ongoing viral activity.
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Publication Date: 2025-08-16 PubMed ID: 40871414PubMed Central: PMC12388522DOI: 10.3390/microorganisms13081910Google Scholar: Lookup
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Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

Overview

  • This study assessed how widespread West Nile virus exposure is among horses in high-risk ecological areas of western Romania and investigated the presence of clinical illness related to the virus.
  • Results show evidence of past WNV infection in horses and identified some clinical cases, indicating ongoing but silent circulation of the virus in the region.

Background and Objective

  • West Nile virus (WNV) is a mosquito-borne virus that can infect horses, causing neurological disease.
  • The study was conducted in three western Romanian counties (Timiș, Arad, and Bihor), which are considered ecologically high-risk for WNV transmission due to environmental factors favorable for mosquitoes.
  • The main goal was to determine the seroprevalence (the proportion of horses with antibodies indicating prior exposure) and to monitor for clinical cases in horses during 2023–2025.

Methods

  • 306 unvaccinated horses were randomly sampled across the three counties.
  • Blood samples were analyzed using a commercial ELISA test to detect WNV-specific antibodies, specifically IgG, which indicates past exposure.
  • Passive surveillance for clinical signs involved monitoring horses during mosquito seasons to identify those showing neurological symptoms typical of WNV infection.
  • For horses showing clinical signs, detection of WNV-specific IgM antibodies was used to confirm recent/active infection.
  • Statistical analyses included chi-square (χ2) tests and binary logistic regression to compare seroprevalence across different counties, sex, and age groups.

Key Findings

  • Overall, 8.17% of tested horses were positive for WNV antibodies, signifying previous exposure to the virus.
  • Seroprevalence by county was:
    • Bihor: 8.85%
    • Arad: 8.57%
    • Timiș: 7.32%
  • No statistically significant differences were noted in seroprevalence among counties, sexes, or age groups, supported by overlapping 95% confidence intervals.
  • Passive surveillance identified 16 horses with acute neurological symptoms; among these, 4 horses were confirmed as recent WNV clinical cases by IgM positivity.
  • The case fatality rate among confirmed cases was 25%, meaning one in four infected horses with neurological signs died.
  • These results point toward a continued silent circulation of WNV in this geographical area.

Implications and Limitations

  • The findings suggest that horses can serve as sentinel animals to monitor WNV circulation under the One Health approach, linking human, animal, and environmental health.
  • Equine surveillance may provide an early warning system to detect and mitigate WNV outbreaks in both animal and human populations.
  • However, interpretation of seroprevalence data must be cautious:
    • Seropositivity reflects past exposure, not necessarily current active infection.
    • There was no molecular confirmation through methods like RT-PCR or virus isolation to directly detect live virus in samples.
  • The study highlights the need for combined serological and molecular surveillance for comprehensive risk assessment.
  • Further longitudinal and molecular studies would help clarify the dynamics of WNV transmission and identify outbreaks promptly.

Cite This Article

APA
(2025). Seroprevalence and Passive Clinical Surveillance of West Nile Virus in Horses from Ecological High-Risk Areas in Western Romania: Exploratory Findings from a Cross-Sectional Study. Microorganisms, 13(8), 1910. https://doi.org/10.3390/microorganisms13081910

Publication

Microorganisms
ISSN: 2076-2607
NlmUniqueID: 101625893
Country: Switzerland
Language: English
Volume: 13
Issue: 8
PII: 1910

Researcher Affiliations

Conflict of Interest Statement

The authors declare no conflicts of interest.

References

This article includes 52 references
  1. Chancey C, Grinev A, Volkova E, Rios M. The Global Ecology and Epidemiology of West Nile Virus. BioMed Res. Int. 2015;2015:376230.
    doi: 10.1155/2015/376230pmc: PMC4383390pubmed: 25866777google scholar: lookup
  2. Paz S, Semenza JC. Environmental Drivers of West Nile Fever Epidemiology in Europe and Western Asia—A Review. Int. J. Environ. Res. Public Health 2013;10:3543–3562.
    doi: 10.3390/ijerph10083543pmc: PMC3774453pubmed: 23939389google scholar: lookup
  3. Dinu S, Stancu IG, Cotar AI, Ceianu CS, Pintilie GV, Karpathakis I, Fălcuță E, Csutak O, Prioteasa FL. Continuous and Dynamic Circulation of West Nile Virus in Mosquito Populations in Bucharest Area, Romania, 2017–2023. Microorganisms 2024;12:2080.
    doi: 10.3390/microorganisms12102080pmc: PMC11510486pubmed: 39458389google scholar: lookup
  4. Crivei LA, Moutailler S, Gonzalez G, Lowenski S, Crivei IC, Porea D, Anita DC, Ratoi IA, Zientara S, Oslobanu LE. Detection of West Nile Virus Lineage 2 in Eastern Romania and First Identification of Sindbis Virus RNA in Mosquitoes Analyzed using High-Throughput Microfluidic Real-Time PCR. Viruses 2023;15:186.
    doi: 10.3390/v15010186pmc: PMC9860827pubmed: 36680227google scholar: lookup
  5. Coroian M, Mihalca A, Dobler G, Euringer K, Girl P, Borșan S, Kalmár Z, Briciu V, Flonta M, Topan A. Seroprevalence Rates against West Nile, Usutu, and Tick-Borne Encephalitis Viruses in Blood-Donors from North-Western Romania. Int. J. Environ. Res. Public Health 2022;19:8182.
    doi: 10.3390/ijerph19138182pmc: PMC9266370pubmed: 35805850google scholar: lookup
  6. Epp T, Waldner C, West K, Townsend H. Factors Associated with West Nile Virus Disease Fatalities in Horses. Can. Vet. J. 2007;48:1137–1145.
    pmc: PMC2034420pubmed: 18050794
  7. Vilibic-Cavlek T, Savic V, Petrovic T, Toplak I, Barbic L, Petric D, Tabain I, Hrnjakovic-Cvjetkovic I, Bogdanic M, Klobucar A. Emerging Trends in the Epidemiology of West Nile and Usutu Virus Infections in Southern Europe. Front. Vet. Sci. 2019;6:437.
    doi: 10.3389/fvets.2019.00437pmc: PMC6908483pubmed: 31867347google scholar: lookup
  8. Fehér OE, Fehérvári P, Tolnai CH, Cadar D, Bányai K, Varga-Kugler R, Bálint Á, Jakab F, Bányai T, Krisztalovics K. Epidemiology and Clinical Manifestation of West Nile Virus Infections of Equines in Hungary, 2007–2020. Viruses 2022;14:2551.
    doi: 10.3390/v14112551pmc: PMC9694158pubmed: 36423160google scholar: lookup
  9. Oslobanu L, Paslaru A, Savuta G. West Nile virus seroprevalence in horses from Romania: First step in the infection risk assessment. Bull. Univ. Agric. Sci. Vet. Med. Cluj-Napoca Vet. Med. 2015;72:34–36.
    doi: 10.15835/buasvmcn-vm:10505google scholar: lookup
  10. Kirov P, Iancu I, Panayotova E, Petrov R, Imre M, Herman V, Hristov H, Abudalleh A, Alexandrova R, Gligor A. First Trans-Border Serological Evidence of West Nile Virus Infection in Horses in Romania and Bulgaria. Int. J. Vet. Sci. 2025;14:777–781.
    doi: 10.47278/journal.ijvs/2025.019google scholar: lookup
  11. Vasić A, Oșlobanu LE, Marinov M, Dinu S, Pănculescu N, Ioniță M, Pistol A, Priori D, Monaco F, Savini G. Evidence of West Nile Virus (WNV) Circulation in Wild Birds and WNV RNA Negativity in Mosquitoes of the Danube Delta Biosphere Reserve, Romania, 2016. Trop. Med. Infect. Dis. 2019;4:116.
    doi: 10.3390/tropicalmed4030116pmc: PMC6789615pubmed: 31438608google scholar: lookup
  12. Institutul Național de Sănătate Publică. Raport Privind Infecțiile cu Virusul West Nile. Institutul Național de Sănătate Publică; Bucharest, Romania: 2023.
  13. Cleton N, Maanen K, Bergervoet SA, Bon N, Beck C, Godeke G, Lecollinet S, Bowen RA, Lelli D, Nowotny N. A serological protein microarray for detection of multiple cross-reactive flavivirus infections in horses for veterinary and public health surveillance. Transbound. Emerg. Dis. 2017;64:1801–1812.
    doi: 10.1111/tbed.12569pubmed: 27633257google scholar: lookup
  14. Beck C, Lowenski S, Durand B, Bahuon C, Zientara S, Lecollinet S. Improved Reliability of Serological Tools for the Diagnosis of West Nile Fever in Horses within Europe. PLoS Negl. Trop. Dis. 2017;11:e0005936.
    doi: 10.1371/journal.pntd.0005936pmc: PMC5617233pubmed: 28915240google scholar: lookup
  15. Thrusfield M. Veterinary Epidemiology. 4th ed. Wiley-Blackwell; Hoboken, NJ, USA: 2018.
  16. Fortin M.-J., Dale M. Spatial Analysis: A Guide for Ecologists. Cambridge University Press; Cambridge, UK: 2005.
  17. Joó K., Bakonyi T., Szenci O., Sárdi S., Ferenczi E., Barna M., Malik P., Hubálek Z., Fehér O., Kutasi O. Comparison of Assays for the Detection of West Nile Virus Antibodies in Equine Serum after Natural Infection or Vaccination. Vet. Immunol. Immunopathol. 2017;183:1–6.
    doi: 10.1016/j.vetimm.2016.10.015pubmed: 28063471google scholar: lookup
  18. Gothe L., Ganzenberg S., Ziegler U., Obiegala A., Lohmann K., Sieg M., Vahlenkamp T., Groschup M., Hörügel U., Pfeffer M. Horses as Sentinels for the Circulation of Flaviviruses in Eastern–Central Germany. Viruses 2023;15:1108.
    doi: 10.3390/v15051108pmc: PMC10222594pubmed: 37243194google scholar: lookup
  19. Girl P., Euringer K., Coroian M., Mihalca A., Borde J., Dobler G. Comparison of Five Serological Methods for the Detection of West Nile Virus Antibodies. Viruses 2024;16:788.
    doi: 10.3390/v16050788pmc: PMC11126072pubmed: 38793670google scholar: lookup
  20. Henneke D.R., Potter G.D., Kreider J.L., Yeates B.F. Relationship between Condition Score, Physical Measurements and Body Fat Percentage in Mares. Equine Vet. J. 1983;15:371–372.
    doi: 10.1111/j.2042-3306.1983.tb01826.xpubmed: 6641685google scholar: lookup
  21. World Organisation for Animal Health (WOAH). Manual of Diagnostic Tests and Vaccines for Terrestrial Animals: West Nile Fever. WOAH; Paris, France: 2023.
  22. IDVet. ID Screen® West Nile Competition—Instruction Manual. 2023.
  23. Magallanes S., Llorente F., Ruiz-López M.J., Martínez-de la Puente J., Soriguer R., Calderon J., Jímenez-Clavero M.Á., Aguilera-Sepúlveda P., Figuerola J. Long-Term Serological Surveillance for West Nile and Usutu Virus in Horses in South-West Spain. One Health 2023;17:100578.
    doi: 10.1016/j.onehlt.2023.100578pmc: PMC10665154pubmed: 38024263google scholar: lookup
  24. INGENASA. INgezim® West Nile IgM ELISA—Instruction Manual. 2023.
  25. Long M.T., Jeter W., Hernandez J., Sellon D.C., Gosche D., Gillis K., Bille E., Gibbs E.P. Diagnostic performance of the equine IgM capture ELISA for serodiagnosis of West Nile virus infection. J. Vet. Intern. Med. 2006;20:608–613.
    doi: 10.1111/j.1939-1676.2006.tb02904.xpubmed: 16734097google scholar: lookup
  26. American Association of Equine Practitioners (AAEP). West Nile Virus (WNV)—Infectious Disease Guidelines. AAEP; Lexington, KY, USA: 2024.
  27. Majeed A., Hwang S. When Poor-Quality Data Meet Anonymization Models: Threats and Countermeasures. IEEE Access 2025;13:49457–49475.
    doi: 10.1109/ACCESS.2025.3552412google scholar: lookup
  28. Brown L.D., Cai T.T., DasGupta A. Interval Estimation for a Binomial Proportion. Stat. Sci. 2001;16:101–133.
    doi: 10.1214/ss/1009213286google scholar: lookup
  29. Woodward M. Epidemiology: Study Design and Data Analysis. 3rd ed. CRC; Boca Raton, FL, USA: 2013.
  30. Nagy A., Horváth A., Mezei E., Henczkó J., Magyar N., Nagy O., Koroknai A., Csonka N., Takács M. West Nile Virus Infections in Hungary: Epidemiological Update and Phylogenetic Analysis of the Hungarian Virus Strains between 2015 and 2022. Acta Microbiol. Immunol. Hung. 2023;70:111–118.
    doi: 10.1556/030.2023.02040pubmed: 37130018google scholar: lookup
  31. Lupulović D., Martín-Acebes M.A., Lazić S., Alonso-Padilla J., Blázquez A.-B., Escribano-Romero E., Petrovic T., Saiz J.-C. First Serological Evidence of West Nile Virus Activity in Horses in Serbia. Vector Borne Zoonotic Dis. 2011;11:1303–1305.
    doi: 10.1089/vbz.2010.0249pubmed: 21438694google scholar: lookup
  32. Abbas I., Ahmed F., Muqaddas H., Alberti A., Varcasia A., Sedda L. Epidemiology and Surveillance of West Nile Virus in the Mediterranean Basin during 2010–2023: A Systematic Review. Curr. Res. Parasitol. Vector Borne Dis. 2025;7:100277.
    doi: 10.1016/j.crpvbd.2025.100277pmc: PMC12205625pubmed: 40586111google scholar: lookup
  33. Calzolari M., Pautasso A., Montarsi F., Albieri A., Bellini R., Bonilauri P., Defilippo F., Lelli D., Moreno A., Chiari M. West Nile Virus Surveillance in 2013 via Mosquito Screening in Northern Italy and the Influence of Weather on Virus Circulation. PLoS ONE 2015;10:e0140915.
    doi: 10.1371/journal.pone.0140915pmc: PMC4619062pubmed: 26488475google scholar: lookup
  34. Calzolari M., Gaibani P., Bellini R., Defilippo F., Pierro A., Albieri A., Maioli G., Luppi A., Rossini G., Balzani A.. Mosquito, Bird and Human Surveillance of West Nile and Usutu Viruses in Emilia-Romagna Region (Italy) in 2010. PLoS ONE 2012;7:e38058.
    doi: 10.1371/journal.pone.0038058pmc: PMC3364206pubmed: 22666446google scholar: lookup
  35. Bruno L., Nappo M.A., Frontoso R., Perrotta M.G., Di Lecce R., Guarnieri C., Ferrari L., Corradi A. West Nile Virus (WNV): One-Health and Eco-Health Global Risks. Vet. Sci. 2025;12:288.
    doi: 10.3390/vetsci12030288pmc: PMC11945822pubmed: 40266979google scholar: lookup
  36. Sejvar J.J. Clinical Manifestations and Outcomes of West Nile Virus Infection. Viruses 2014;6:606–623.
    doi: 10.3390/v6020606pmc: PMC3939474pubmed: 24509812google scholar: lookup
  37. Papa A., Xanthopoulou K., Tsioka K., Kalaitzopoulou S., Mourelatos S. West Nile Virus in Mosquitoes in Greece. Parasitol. Res. 2013;112:1551–1555.
    doi: 10.1007/s00436-013-3302-xpubmed: 23371497google scholar: lookup
  38. Autorino G.L., Battisti A., Deubel V., Ferrari G., Forletta R., Giovannini A., Lelli R., Murri S., Scicluna M.T. West Nile virus epidemic in horses, Tuscany region, Italy. Emerg. Infect. Dis. 2002;8:1372–1378.
    doi: 10.3201/eid0812.020234pmc: PMC2738505pubmed: 12498650google scholar: lookup
  39. García-Bocanegra I., Belkhiria J., Napp S., Cano-Terriza D., Jiménez-Ruiz S., Martínez-López B. Epidemiology and spatio-temporal analysis of West Nile virus in horses in Spain between 2010 and 2016. Transbound. Emerg. Dis. 2018;65:567–577.
    doi: 10.1111/tbed.12742pubmed: 29034611google scholar: lookup
  40. Calistri P., Giovannini A., Savini G., Monaco F., Bonfanti L., Ceolin C., Terregino C., Tamba M., Cordioli P., Lelli R. West Nile virus transmission in 2008 in north-eastern Italy. Zoonoses Public Health 2010;57:211–219.
    doi: 10.1111/j.1863-2378.2009.01303.xpubmed: 20042066google scholar: lookup
  41. de Heus P., Kolodziejek J., Camp J.V., Dimmel K., Bagó Z., Hubálek Z., van den Hoven R., Cavalleri J.-M.V., Nowotny N. Emergence of West Nile virus lineage 2 in Europe: Characteristics of the first seven cases of West Nile neuroinvasive disease in horses in Austria. Transbound. Emerg. Dis. 2020;67:1189–1197.
    doi: 10.1111/tbed.13452pmc: PMC7317211pubmed: 31840920google scholar: lookup
  42. Veljović L., Maksimović-Zorić J., Radosavljevic V., Stanojević S., Žutić J., Kureljušić B., Pavlović I., Jezdimirovic N., Milićević V. Seroprevalence of West Nile fever virus in horses in the Belgrade epizootiological area. Vet. Glasnik. 2020;74:194–201.
    doi: 10.2298/VETGL200207011Vgoogle scholar: lookup
  43. European Centre for Disease Prevention and Control (ECDC). West Nile Virus Infection: Annual Epidemiological Report for 2018. ECDC; Stockholm, Sweden: 2019.
  44. Faverjon C., Vial F., Andersson M., Lecollinet S., Leblond A. Early detection of West Nile virus in France: Quantitative assessment of syndromic surveillance system using nervous signs in horses. Epidemiol. Infect. 2016;145:1044–1057.
    doi: 10.1017/S0950268816002946pmc: PMC9507807pubmed: 27938434google scholar: lookup
  45. Humblet M., Vandeputte S., Fecher-Bourgeois F., Léonard P., Gosset C., Balenghien T., Durand B., Saegerman C. Estimating the economic impact of a possible equine and human epidemic of West Nile virus infection in Belgium. Eurosurveillance 2016;21:17–30.
    doi: 10.2807/1560-7917.ES.2016.21.31.30309pmc: PMC4998509pubmed: 27526394google scholar: lookup
  46. Ziegler U., Seidowski D., Angenvoort J., Fast C., Schulz J., Scheuerlein A., Essbauer S., Nawrocki S., Eiden M., Groschup M.H. Monitoring of West Nile Virus Infections in Germany. Zoonoses Public Health 2012;59:95–101.
    doi: 10.1111/zph.12015pubmed: 22958253google scholar: lookup
  47. Schvartz G., Tirosh-Levy S., Erester O., David D., Davidson I., Steinman A., King R., Gelman B., Katz Y., Kenigsberg D.. Exposure of Horses in Israel to West Nile Virus and Usutu Virus. Viruses 2020;12:1099.
    doi: 10.3390/v12101099pmc: PMC7650752pubmed: 32998459google scholar: lookup
  48. Williams R., Valencia H., López I., González F., Llorente F., Jiménez-Clavero M., Busquets N., Mateo M., Diez G., Ayllón T. West Nile Virus Seroprevalence in Wild Birds and Equines in Madrid Province, Spain. Vet. Sci. 2024;11:259.
    doi: 10.3390/vetsci11060259pmc: PMC11209238pubmed: 38922006google scholar: lookup
  49. Țârdei G., Ruță S., Chiţu V., Rossi C., Tsai T., Cernescu C. Evaluation of immunoglobulin M (IgM) and IgG enzyme immunoassays in serologic diagnosis of West Nile Virus infection. J. Clin. Microbiol. 2000;38:6.
    doi: 10.1128/JCM.38.6.2232-2239.2000pmc: PMC86770pubmed: 10834982google scholar: lookup
  50. Bergmann F., Trachsel D.S., Stoeckle S.D., Bernis Sierra J., Lübke S., Groschup M.H., Gehlen H., Ziegler U. Seroepidemiological Survey of West Nile Virus Infections in Horses from Berlin/Brandenburg and North Rhine-Westphalia, Germany. Viruses 2022;14:243.
    doi: 10.3390/v14020243pmc: PMC8877243pubmed: 35215837google scholar: lookup
  51. Patz J.A., Campbell-Lendrum D., Holloway T., Foley J.A. Impact of Regional Climate Change on Human Health. Nature 2005;438:310–317.
    doi: 10.1038/nature04188pubmed: 16292302google scholar: lookup
  52. Rwego I.B., Babalobi O.O., Musotsi P., Nzietchueng S., Tiambo C.K., Kabasa J.D., Naigaga I., Tchouassi D.P. One Health: A Concept Led by Africa, with Global Benefits. Front. Public Health 2017;5:236.
    doi: 10.3389/fpubh.2017.00236pmc: PMC5591825pubmed: 28929098google scholar: lookup

Citations

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