Serological evidence for West Nile virus infection in horses in Croatia

WEST Nile virus (WNV) is an arthropod-borne virus belonging to the family Flaviviridae, genus Flavivirus. WNV is included in the Japanese encephalitis virus group, together with the Japanese, St Louis and Murray Valley encephalitis viruses (Heinz and others 2000). Its natural life cycle involves birds and mosquitoes, particularly Culex species and Aedes/Ochlerotatus species (Hubalek and Halouzka 1999, Kulasekera and others 2001). Many species of wild birds may act as vertebrate hosts (Komar and others 2001); human beings, horses and other mammals are only incidental hosts. Although infection of human beings in endemic areas is common, such infection usually causes only an asymptomatic or mild illness. However, severe neurological diseases, mostly associated with people over 50 years of age, as well as high rates of mortality among birds and horses have recently been observed (Makar and Stowell 2004). Bridge vectors for equine infection are primary Aedes/ Ochlerotatus species. Since the mid-1990s, outbreaks of WNV infection have been reported with increasing frequency in human beings, birds and horses in many countries such as Romania (1996), Morocco (1996), Tunisia (1997), Italy (1998), Russia (1999), USA (from 1999), Israel (1999), France (2000, 2004), Canada (2002) and Mexico (2002) (Hubalek and Halouzka 1999, Murgue and others 2001, Ostlund and others 2001, Petersen and Roehrig 2001, Estrada-Franco and others 2003). No data on the prevalence and spread of WNV in horses in Croatia are available, although haem-inhibition antibodies in human beings have been detected (VesenjakHirjan and others 1991). This short communication describes the survey of a population of horses in Croatia for the presence of WNV antibodies in an attempt to provide information for veterinary epidemiologists. Between May 2001 and October 2002, 980 equine serum samples were collected by the Croatian Veterinary Services. Horses were randomly selected from eight Croatian regions: 119 from Buje, 81 from Ðakovo, 69 from Garesnica, 46 from Labin, 66 from Nasice, 398 from Sisak, 82 from Sibenik and 119 from Pula (Fig 1). Sampling was carried out at the time of the equine infectious anaemia national monitoring plan. Serum samples were first screened using a home-made immunoglobulin G (IgG) ELISA, according to the method described by Tsai and others (1998). Plates were coated with WNV antigen prepared on Vero E6 cells, ATCC CRL-1586 VERO C1008 (Early and Johnson 1988) according to the method used by the Unite des Arbovirus et des Fievres Hemorragiques, Institut Pasteur, and IgG antibodies were revealed by peroxidase antibody (Sigma). Sera were considered positive if the optical density was more than three standard deviations above the negative mean, and the positive results were confirmed using serum neutralisation (Anon 2004). The serology was performed at the Instituto Zooprofilattico Sperimentale ‘G. Caporale’, Teramo, Italy. In the statistical analysis of the results, the total prevalence of WNV in horses was calculated by a Bayesian approach using the beta (s+1, n–s+1) distribution, where s is the total number of positives and n is the total number of tested animals. The probability distribution of the percentage of positive animals shows not only the most probable value of prevalence, but also the level of uncertainty due to sample size. Of the 980 serum samples tested, four were found to be positive for WNV antibody by the home-made IgG ELISA. All four were confirmed by serum neutralisation. Neutralising antibody titres ranged from 1:10 to 1:80. The overall prevalence was 0·4 per cent (95 per cent confidence interval 0·17 to 1·06). All positive animals came from a stud farm located in the Ðakovo region, which was home to 160 animals. The stud farm considered animals from different locations under the guidance of a selection centre. A total of 81 randomly selected horses from the Ðakovo region were examined in the present study, and the four positive animals identified were the only animals positive for WNV antibody (Table 1). Fig 2 shows the most probable values of WNV prevalence in horses from Croatia and the Ðakovo region. No clinical signs were observed in horses at the time of sampling. In laboratory diagnosis, WNV infection can be detected by IgG ELISA; however, the assay cannot readily differentiate between WNV and other members of the Flavivirus genus. Serological confirmation of WNV infection is possible only by the detection of WNV-specific neutralising antibodies by the serum neutralisation assay or plaque reduction neutralisation test (PRNT) in which titres equal to or greater than 1:10 in a single serum sample are considered positive (Ostlund and others 2001). Horses with subclinical infections developed neutralising antibody titres of at least 1:10 (Bunning and others 2002). Thus, as demonstrated in other similar studies, the combination of both ELISA and PRNT or the serum neutralisation assay, the tests applied in this study, were the most reliable methods of detecting and confirming cases of WNV (Ostlund and others 2001). FIG 1: Locations of equine serum sampling for West Nile virus detection in Croatia. Inset: Migratory routes of birds from northern Europe to South Africa, modified from Vuilleumier (2001)