The primary goal of the monitoring system was to obtain a large number of blood specimens from birds belonging to as many species as possible across the Caribbean. At seven study sites in Jamaica, Puerto Rico, and the Yucatan Peninsula of Mexico, we erected 15 mist nets (12-m) daily for 3 to 4 weeks during January through March 2002. We collected 1,619 avian blood specimens, which represented 98 species, 25 families, and eight orders. In Jamaica, 542 samples were collected from Westmoreland, Manchester, and St. Catherine Parishes; 649 samples were collected at Roosevelt Roads Naval Station in Puerto Rico; and 430 samples were collected from the states of Yucatan and Campeche in Mexico (Table 1). At the time of capture, all migratory birds were banded with an aluminum U.S. Fish and Wildlife Service band and 3–5 breast feathers were removed for isotope analysis. Resident birds had outer rectrices cut. All birds were evaluated for age and gender if possible, bled with microcapillary tubes from the brachial vein, and released. Blood was added immediately to BA-1 medium, consisting of M199 medium with Hank’s salts, 1% bovine albumin, TRIS base (tris [hydroxymethyl] aminomethane), sodium bicarbonate, 20% fetal bovine serum (FBS), and antibiotics. The samples were placed in a cooler on ice packs until storage in a –20°C freezer. Samples were sent on dry ice or hand-carried on ice packs to the Arbovirus Laboratories, Wadsworth Center, New York State Department of Health for serologic analysis and virus isolation attempts.

Specimens were screened at 1:100 for antibodies against flaviviruses by using an indirect enzyme-linked immunosorbent assay (ELISA) (13). Samples with a P/N ratio >2.0 were tested further by a plaque reduction neutralization test (PRNT) for Ilhéus virus (ILHV), St. Louis encephalitis virus (SLEV), and WNV, as described (14). The particular virus strains used for the PRNTs were ILHV (original), SLEV 59268 Parton, and WNV (3100365), an isolate from a pool of Culex sp. mosquitoes collected in Staten Island, New York. The indirect ELISA was chosen to screen the samples in order to take advantage of its ability to detect antibodies against a wide range of flaviviruses. PRNT was used as a confirmatory assay to differentiate among recognized flaviviruses (15,16). Briefly, serial dilutions of test samples were mixed with an equal amount of virus suspension containing 200 PFU/1 mL and incubated at 37°C for 1 h. We then added 0.05 mL of each virus-diluted blood sample onto 1 well of a 12-well tissue culture plate containing confluent monolayers of African green monkey kidney cells (Vero). The plate was incubated for 1 h at 37°C, after which an agarose overlay was added and incubation was continued. When virus plaques became visible, we added a second overlay containing neutral red and counted plaques. The antibody titer reported is the dilution of serum that inhibited 90% of the test virus inoculum. For virus isolation attempts using confluent Vero cell monolayers, 0.1 mL of each serum specimen was added onto one well of a six-well tissue culture plate, incubated for 1 h, rinsed with phosphate-buffered saline, and then the cells were refed with minimum essential medium containing 2% FBS. Cells were monitored once a day for 5 days for cytopathic effect. Cell cultures showing any abnormal cell morphology were then blind passaged after 5 days.

ELISA results indicated 34 of 1,413 serum specimens tested from the three study sites contained immunoglobulin G antibody against a flavivirus. Of the 34 reactive samples representing 20 bird species, 27 were collected in Jamaica (26 residents, 1 migrant), 5 in Puerto Rico (3 residents, 2 migrants), and 2 in Mexico (1 resident, 1 migrant). PRNTs on the Jamaican bird samples indicated 18 WNV infections, 3 SLEV infections, 5 undetermined flavivirus infections (positive results for two or more viruses without a fourfold difference in antibody titer); one additional reactive serum sample was negative for the three viruses tested. Results on the serum samples collected in Puerto Rico indicated one WNV infection in a migratory bird and one SLEV infection in a resident bird; three additional reactive serum samples were negative for all three viruses tested. In Mexico, we found evidence of WNV infection in one migrant bird and SLEV infection in one resident bird (Table 2). Virus isolation attempts were negative for all 1,603 specimens tested (16 samples destroyed). Negative isolation results were not entirely unexpected, considering that birds are viremic for a short period of time (17) and maintaining a proper cold chain (i.e., temperatures) to preserve virus is difficult when working in the tropics.

We detected neutralizing antibodies to WNV in resident birds from two parishes in Jamaica. This detection marks the earliest evidence of WNV introduction into the neotropics; WNV antibodies have been demonstrated in birds and horses in Mexico (late 2002, spring 2003) (18,19) and detected in resident birds from the Dominican Republic (spring 2003) (20). This evidence of WNV in the neotropics may be an important development in the spread of the virus. The tropics provide all the necessary components (i.e., high temperatures, dense avian populations, and large numbers of Culex sp. mosquitoes) to maintain an enzootic focus. Furthermore, the climates of Mexico and the Caribbean are suitable for year-round transmission of the virus. No dead birds have been reported in Jamaica, but surveillance activity there is less intensive than in the United States; the study sites, being rural in nature, are not conducive to observing dead birds. Another contributing factor to the lack of reports of dead birds may be the rapid decomposition of dead birds as a result of the heat, humidity, and detritivore foragers, such as ants.

Arbovirus activity, particularly of flaviviruses, is well documented in the Caribbean and Mexico. Dengue and yellow fever viruses are recurring public health threats in these areas. SLEV is endemic to Mexico and has been isolated from mosquitoes and one Northern mockingbird (Mimus polyglottos) nestling in Jamaica (21). This disease is still active in the region, and its known range may have expanded into Puerto Rico, considering the one seropositive Caribbean elaenia (Elaenia martinica) sampled during this study, although the antibody may have resulted from infection with yet another flavivirus. Neutralizing antibodies to WNV in migratory birds collected in Mexico and Puerto Rico, coupled with the apparent absence of antibody to WNV in the resident bird population, indicate that infection likely occurred in an enzootic area of the United States, but this observation shows that individual birds from at least three species of neotropical migratory birds have survived WNV infection and may serve as hosts for spreading the virus. The results from this study suggest that WNV now appears to be established in Jamaica, on the basis of the neutralizing antibodies to WNV found in the resident bird population.