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 , 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 . 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 20c 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 . based on screening enzyme - linked immunosorbent assay ( elisa ) results . results based on 441 samples suitable for elisa ( 206 samples were blood clots only ) . 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 ilhus 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 . 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 37c 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 37c , 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 . 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 . 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 . 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 .
in spring 2002 , an intensive avian serosurvey was initiated in jamaica , puerto rico , and mexico . 
 we collected > 1,600 specimens from resident and nonresident neotropical migratory birds before their northerly migrations . 
 plaque reduction neutralization test results indicated specific neutralizing antibodies to west nile virus in 11 resident species from jamaica .