First Serological Evidence of West Nile Virus Activity in Horses in Serbia Diana Lupulovic, 1 Miguel A. Martı ´n-Acebes, 2 Sava Lazic, 1 Julio Alonso-Padilla, 2 Ana-Bele ´n Bla ´ zquez, 2 Estela Escribano-Romero, 2 Tamas Petrovic, 1 and Juan-Carlos Saiz 2 Abstract West Nile virus (WNV), the most widely distributed flavivirus worldwide, has lately reemerged in Europe, causing worrisome outbreaks in humans and horses. Serological analysis by enzyme-linked immunoassay and plaque reduction neutralization test showed for the first time in Serbia that 12% of 349 horses presented specific neutralizing WNV antibodies, which in one case also cross-neutralized Usutu virus (USUV). This is the first time that anti-USUV high neutralizing antibody titers are reported in horses. All these data indicate that WNV and USUV are circulating in the region and advise on the convenience of implementing surveillance programs. Key Words: Horses—Serbia—Usutu virus—Vector-borne—West Nile virus. Introduction W est Nile virus (WNV) is the most worldwide distrib- uted flavivirus. Its transmission cycle involves mos- quito vectors and birds, but equines and humans are also susceptible to infection (Komar 2000, Kramer et al. 2007, Blitvich 2008). Human WNV infections are usually asymp- tomatic; however, approximately 1 of 150 cases can progress to encephalitis or meningoencephalitis, which can lead to a fatal outcome (Komar 2000, Kramer et al. 2007, Blitvich 2008). In horses, WNV infection is also frequently clinically unap- parent, but around 10% of cases develop neurological disor- ders with up to 50% mortality rates (Blitvich 2008, Calistri et al. 2010). In Europe, until the 1990s, WNV had caused sporadic outbreaks with rare reports of encephalitis, but its epidemi- ological behavior changed when it reemerged with virulence in Romania, Russia, and the Mediterranean basin, causing dozens of humans and horses deaths (Komar 2000, Kramer et al. 2007, Blitvich 2008, Calistri et al. 2010). In North America, since its first detection in 1999, WNV has already caused in humans over 1100 fatalities, over 12,000 cases of meningitis/encephalitis, and more than 30,000 diagnosed infections, and over 25,000 accumulated cases have been re- ported in horses (www.cdc.gov). Lately, human cases of WN disease (WND) have been described in Italy, Hungary, and Romania, and along 2010 new WNV outbreaks have been reported in Greece and neighboring countries, with more than 250 laboratory-confirmed cases and 27 deaths (www.ecdc .europa.eu). Likewise, an increasing number of severe out- breaks in horses have been reported in Europe, including a large one that took place in northeast Italy in 2008 involving 251 stables with 794 cases and 5 deaths (Calistri et al. 2010). During summer 2010, the first outbreaks of severe WND in horses have been reported in southern Spain, with 41 diag- nosed cases and 10 deaths (www.oie.int/wahis/). All these data suggest that new epidemiological scenarios are being developed in Europe and, thus, that assessment of WNV ac- tivity and implementation of surveillance programs are nec- essary across the continent. The objective of the present study was to analyze, for the first time, the presence of specific anti- WNV antibodies in Serbian horses. Materials and Methods Samples Sera from 349 horses were randomly collected during 2009– 2010 (29.5%, 39.2%, 28.4%, and 2.9% during winter, spring, summer, and autumn, respectively) in stables of the Belgrade district area, of the municipality of Sabac and of 26 munici- palities of the Vojvodina province of northern Serbia, which is bordered to Croatia, Hungary, and Romania (Fig. 1). Vojvo- dina presents more than 50% of Serbian water surfaces and raises more than one-third of the country’s horses (webrzs .stat.gov.rs/axd/en/god.htm). Almost half of the animals (48.4%) analyzed were racing horses, 36% Lipizzaner breed 1 Scientific Veterinary Institute ‘‘Novi Sad,’’ Novi Sad, Serbia. 2 Departamento de Biotecnologı ´a, Instituto Nacional de Investigaciones Agrarias, Madrid, Spain. VECTOR-BORNE AND ZOONOTIC DISEASES Volume 11, Number 9, 2011 ª Mary Ann Liebert, Inc. DOI: 10.1089/vbz.2010.0249 1303