Serologic Evidence of West Nile Virus Infection in Horses, Yucatan State, Mexico

Serum samples were obtained from 252 horses in the State of Yucatan, Mexico, from July to October 2002. Antibodies to West Nile virus were detected by epitope-blocking enzyme-linked immunosorbent assays in three (1.2%) horses and confirmed by plaque reduction neutralization test. We report the first West Nile virus activity in the State of Yucatan.

To determine whether WNV had already reached this part of Mexico, we obtained blood samples from 252 domestic horses in 14 study sites from July to October 2002 ( Table 1). The age distribution of the horses was 3 months to 25 years, and the mean age was 8.2 years. One hundred and fifty-one horses were male, and 101 were female. All study sites were on privately owned ranches, where the horses were primarily used to perform heavy labor and herd cattle. According to the owners, none of the horses had ever been outside the State of Yucatan. Furthermore, none of the horses had been vaccinated against WNV.
The climate and topography of the study sites are similar. The climate can be described as tropical. The average annual rainfall in each study site ranges from 600 to 1,100 mm, and the average annual temperature is 26°C. The average elevation is approximately 17 m.
All serum samples were screened for antibodies to flaviviruses by hemagglutination inhibition (HI) assays and epitope-blocking enzyme-linked immunosorbent assays (ELISAs) at the Universidad Autonoma de Yucatan in Merida. HI assays were performed by using Saint Louis encephalitis virus (SLEV) antigen as previously described (9). This antigen recognizes cross-reactive HI antibodies to WNV and to other flaviviruses. To preclude nonspecific HI reactions, samples were treated with kaolin, then adsorbed with goose erythrocytes, according to standard methods (9). Epitope-blocking ELISAs were performed by using the flavivirus group-reactive monoclonal antibody (MAb), 6B6C-1, or the WNV-specific MAb, 3.1112G as previously described (10). The ability of the Mexican horse serum samples to block the binding of MAbs to WNV antigen was compared to the blocking ability of horse serum without antibody to WNV (Vector Laboratories, Burlingame,  (Table 2). Serum samples from three of these horses (H-117, H-126, and H-252) were positive in the ELISA that used the WNV-specific MAb. H-117 (7-year-old stallion) and H-126 (2-year-old stallion) were both sampled at the Tizimin study site. Neither horse showed signs of illness at the time of serum collection or during the 7 months that followed. Furthermore, neither horse had a history of WNV-like illness. H-252 was a 3year-old stallion from Caucel that exhibited neurologic and muscular symptoms at the time of sampling; it was euthanized several hours later. We were not able to obtain tissue specimens from this horse postmortem. Of the 252 horses sampled, the only other horse to exhibit signs of clinical illness was H-60, which had signs consistent with gastrointestinal illness.
Serum samples positive for flavivirus antibodies by HI assay or ELISA were tested by plaque reduction neutralization assay (PRNT) to identify the infecting virus. PRNTs were conducted in the BSL-3 facilities at Colorado State University. Serum sample results shown to be negative by HI assay and ELISA were not tested. PRNTs were done by using WNV (strain NY99-35261-11), SLEV (strain TBH-28), Ilhéus virus (ILHV, original strain), and Bussuquara virus (BSQV, strain BeAn-4073). Virus stocks were obtained from the World Health Organization Center for Arbovirus Reference and Research, maintained at the Centers for Disease Control and Prevention, Division of Vector-Borne Infectious Diseases, Fort Collins, CO. We tested serum samples for neutralizing antibodies to SLEV because the virus is enzootic in the Americas and because antibodies to WNV and SLEV often cross-react.
Furthermore, horses are susceptible to natural SLEV infections, although clinical manifestations have not been reported (12). ILHV and BSQV are also present in the Americas, although neither virus is known to naturally infect horses (2). PRNTs were performed by using Vero cells. Serum samples were tested by using a starting dilution of 1:20. Titers were expressed as the reciprocal of serum dilutions yielding >90% reduction in the number of plaques (PRNT 90 ).
Neutralizing antibodies to WNV were detected in three horses ( Table 3). The PRNT-positive horses were H-117, H-126, and H-252, which exhibited PRNT 90 antibody titers of 320, >2,560, and 160, respectively. The SLEV, ILHV, and BSQV antibody titers of the three horses were all <20. Therefore, we considered H-117, H-126, and H-252 to be seropositive for WNV because the PRNT 90 antibody titers for WNV were more than fourfold higher than the other flaviviruses tested. Overall, the PRNT and ELISA data were in concordance; all serum samples that contained neutralizing antibodies to WNV were positive in the assay that used MAb 3.1112G (Tables 2 and 3). However, H-252 was negative in the assay that used MAb 6B6C-1, although the percent inhibition value was close to the diagnostic criterion. The three other horses (H-60, H-134, and H-141) that were positive for flavivirus antibodies by HI assay or ELISA did not have neutralizing antibodies to WNV. H-60 had a low SLEV PRNT 90 titer, suggesting it had been infected with SLEV or a closely related virus. H-134 exhibited a HI titer of 10 but was negative by the other serologic tests, suggesting that the HI antigen had reacted nonspecifically. H-141 was positive by HI assay and ELISA but had no neutralizing antibodies to any flavivirus tested; thus, the identity of the infecting virus was not determined.
We obtained serologic evidence for antibodies to WNV in Yucatan State, Mexico. The mode of entry of this virus into Yucatan State is not known; however, the virus may have been brought in by birds migrating from the north. We have also detected antibodies to WNV in certain species of migratory birds, which supports this hypothesis. Data from the avian surveillance studies conducted in Yucatan State will be described separately (J.A. Farfán-Ale, unpub. data). We plan to isolate and amplify viral sequences from migratory and resident birds, as well as from specimens from other seropositive animals, to determine the origin of the WNV strain in Yucatan State. We also provide serologic evidence for WNV infection in horses in Coahuila State (13). These two reports provide the first published evidence of WNV activity in horses in Mexico. Neutralizing antibodies to WNV have also been detected in a bovine in Chiapas, Mexico, in mid-2001, indicating that the animal had been infected with WNV or a closely related virus (14). WNV may become endemic in this country, which demonstrates the importance for continued WNV surveillance in Mexico, and elsewhere in the south. All material published in Emerging Infectious Diseases is in the public domain and may be used and reprinted without special permission; proper citation, however, is appreciated.