Serologic evidence of West Nile virus infection in horses, Coahuila State, Mexico - Dispatches

Emerging Infectious Diseases, July, 2003 by Bradley J. Blitvich, Ildefonso Fernandez-Salas, Juan F. Contreras-Cordero, Nicole L. Marlenee, Jose I. Gonzales-Rojas, Nicholas Komar, Duane J. Gubler, Charles H. Calisher, Barry J. Beaty

Serum samples were obtained from 24 horses in the State of Coahuila, Mexico, in December 2002. Antibodies to West Nile virus were detected by epitope-blocking enzyme-linked immunosorbent assay and confirmed by plaque reduction neutralization test in 15 (62.5%) horses. We report the first West Nile virus activity in northern Mexico.

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West Nile virus (WNV; family Flaviviridae, genus Flavivirus) is a member of the Japanese encephalitis virus complex, which also includes Japanese encephalitis virus, Saint Louis encephalitis virus (SLEV), and Murray Valley encephalitis virus (1). These viruses are maintained in cycles between mosquitoes and birds (2). The principal vectors for WNV are Culex species mosquitoes, and many species of wild birds act as vertebrate hosts (3). Humans, horses, and other mammals usually serve as dead-end hosts. In humans and equines, WNV infection is usually asymptomatic or characterized by a mild febrile illness, although fatal meningoencephalitis or encephalitis may occur (3-5). WNV has a broad geographic distribution, recently including North America (4,5). The initial outbreak of WNV in North America was recognized in New York City in August 1999. Since then, WNV's geographic range has increased. WNV activity has now been reported in 44 states in the United States, the District of Columbia, and 5 of the 10 Canadian provinces (6,7).

In anticipation of the possible emergence of WNV into Mexico, we conducted equine infection surveillance in the northeastern states of Mexico. Coahuila State is bordered on the north by Texas. WNV activity has been detected in 204 (80%) of 254 Texas counties, including most counties that border Coahuila State (8). Therefore, Coahuila State was considered to be a likely point of incursion of WNV into Mexico from the United States.

Case Study

We present data from a small equine serosurvey conducted in Coahuila State in December 2002. A more extensive equine serosurvey is currently under way and will be described in detail elsewhere (B.J. Beaty, unpub, data). In the present study, blood samples were taken from 24 domestic horses at study sites located in Ciudad Acuna, Jimenez, and Saltillo (Figure). The Ciudad Acuna and Jimenez sites are approximately 40 km apart, and both are <15 km from the Texas border. Saltillo is located in the southeast region of Coahuila State and is approximately 220 km from Texas. All study sites are privately owned ranches.

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The climatic conditions of the three study sites are similar and can be described as hot, dry, and arid. The average annual temperature ranges from 18[degrees]C to 22[degrees]C. The average rainfall is from 100 to 300 mm per year. The sites in Ciudad Acuna and Jimenez are approximately 300 m above sea level. The Saltillo site is situated at an elevation of approximately 1500 m.

A horse from the Ciudad Acuna ranch died October 17, 3 days after being observed with neurologic signs. The case was not reported immediately, and we were unable to obtain a tissue specimen postmortem. On December 19, with the assistance of a local veterinary practitioner, we sampled 14 horses at this site, 5 of which had developed neurologic disease in mid- to late October. Clinical symptoms included ataxia, weakness of limbs, trembling, and anxiety. All five horses survived. The horses with clinical signs were from 1 to 5 years of age; three were male, and two were female. Ages and sexes of horses without clinical symptoms were not documented. The veterinarian reported a great abundance of mosquitoes in the area. Another six horses were sampled in Jimenez and four in Saltillo, none of which had signs of illness. According to the owners, none of the horses had ever been outside the State of Coahuila, and none of the horses had been vaccinated against WNV.

All serum samples were tested for antibodies to WNV by epitope-blocking enzyme-linked immunosorbent assay (ELISA). Blocking ELISAs were performed by using the WNV-specific monoclonal antibody (MAb) 3.1112G, as previously described (9). The ability of the Mexican horse serum samples to block the binding of the MAb to WNV antigen was compared to the blocking ability of horse serum without antibody to WNV (Vector Laboratories, Burlingame, CA). Data were expressed as relative percentages by using the formula of Hall et al., (10). Previously, we considered an inhibition value [greater than or equal to] 30% to indicate the presence of viral antibodies (9). Recently, we have shown that ELISAs performed with MAb 3.1112G detect WNV antibodies in various vertebrate species, including horses (9,11).

Fourteen serum samples were positive in blocking ELISA that utilized MAb 3.1112G (Table). Serum from another horse (H-16) inhibited the binding of MAb by 25%, which is close to the diagnostic criterion. Previously, we observed that the nonspecific inhibition values for serum samples from noninfected control birds ranged from 0% to 24.3% (9). Therefore, if we used a less stringent threshold value of [greater than or equal to] 25%, this serum could be considered positive for WNV antibodies.