Centers for Disease Control and Prevention
http://www.cdc.gov/ncidod/EID/vol9no10/03-0222.htmWe report West Nile virus (WNV) activity in the
Dominican Republic for the first time. Specific anti-WNV
antibodies were detected in 5 (15%) of 33 resident birds
sampled at one location in November 2002. One seropositive
bird was <4 months old, indicating a recent infection

Blitvich, Bradley J.

Burkhalter, Kristen L.

Gonzálvez, Guillermo

Gubler, Duane J.

Klenk, Kaci

Komar, Nicholas

Komar, Oliver

Marlenee, Nicole L.

Peterson, A. Townsend

Peña, Carlos J.

Robbins, Mark B.

English

KU ScholarWorks

West Nile VirusTransmission inResident Birds,Dominican Republic Oliver Komar,* Mark B. Robbins,* Kaci Klenk,†Bradley J. Blitvich,‡ Nicole L. Marlenee,‡ Kristen L. Burkhalter,† Duane J. Gubler,†Guillermo Gonzálvez,§ Carlos J. Peña,§ A. Townsend Peterson,* and Nicholas Komar†We report West Nile virus (WNV) activity in theDominican Republic for the first time. Specific anti-WNVantibodies were detected in 5 (15%) of 33 resident birdssampled at one location in November 2002. One seroposi-tive bird was <4 months old, indicating a recent infection. The initial outbreak of West Nile virus (WNV; familyFlaviviridae, genus Flavivirus) in the WesternHemisphere took place in New York in 1999, with deathsobserved in humans, horses, and numerous species of wildbirds (1). Since then, this virus has spread rapidly acrossNorth America (2,3). Migratory birds are suspected ofbeing responsible for the rapid spread of WNV throughNorth America (4), and transport of WNV by Neotropicalmigratory birds throughout the New World has been antic-ipated (5).Although WNV has spread rapidly through continentalareas, its ability to spread across oceanic barriers is uncer-tain. The many islands of the West Indies represent thewintering grounds of numerous North American migratorybirds (6,7) that breed in or migrate through WNV transmis-sion foci in the United States. The Caribbean islands tendto have high human population density, and low popula-tions of many birds and other vertebrates are restricted tocertain islands. Introduction of WNV to the West Indieswould present a human and equine health concern andpotentially threaten numerous endangered and endemicbird species and perhaps other wild vertebrates. Given the speculation that WNV may be disseminatedby migrating birds (5,8,9), we hypothesized that the viruswould be introduced to the Dominican Republic.Accordingly, we sampled apparently healthy birds therefor evidence of locally acquired WNV infection. The StudyBirds were studied at two sites in the DominicanRepublic, on the island of Hispaniola (Figure): ParqueNacional Sierra de Baoruco (November 7–16, 2002; 18°12' N, 71° 32' W) and Parque Nacional Los Haitises(November 18–23, 2002; 19° 00' N, 69° 30' W). Birdswere collected by standard methods (10). Tissues (eye,spleen, and kidney) were removed from 89 birds of 29species (25 resident, 4 migratory) at Sierra de Baoruco andfrom 58 birds of 27 species (18 resident, 9 migratory) atLos Haitises; the tissues were tested for active WNV infec-tion. Blood samples were collected from a subsample ofthese birds, including 41 that represented 18 residentspecies at Sierra de Baoruco and 33 that represented 16resident species at Los Haitises. Blood was not collectedfrom migratory birds or from certain very small residentbirds, such as hummingbirds. Blood and tissue specimenswere frozen immediately in liquid nitrogen for transporta-tion and then stored at –70°C. Voucher specimens (includ-ing additional tissue samples) were prepared for all birdsand are deposited at the University of Kansas NaturalHistory Museum (KUNHM).The sex and breeding condition of each bird were deter-mined by examination and measurement of gonads. Birds’ages were assessed by plumage, skull ossification, andpresence or absence of a bursa of Fabricius. The migrato-ry or resident status of each bird was determined on thebasis of standard references (6,7). For species that had bothmigratory and resident populations, we based statusassessment on breeding conditions (breeding birds wereassumed to be resident). Serum samples were screened for flavivirus-neutraliz-ing antibodies by plaque-reduction neutralization test(PRNT) according to standard methods (11) and by usingchallenge inocula of 100 plaque-forming units (PFU)WNV strain NY99-4132 and Saint Louis encephalitis virus(SLEV) strain TBH-28. Testing for neutralizing antibodyEmerging Infectious Diseases • Vol. 9, No. 10, October 2003 1299DISPATCHES*University of Kansas, Lawrence, Kansas, USA; †Centers forDisease Control and Prevention, Fort Collins, Colorado, USA;‡Colorado State University, Fort Collins, Colorado, USA; and§Centro Nacional de Control de Enfermedades Tropicales, SantoDomingo, Dominican RepublicFigure. Hispaniola in the West Indies, on which are located Haiti(western third of the island) and the Dominican Republic. WestNile virus transmission occurred at Parque Nacional Los Haitisesbefore November 2002. Shades of gray are 500-m intervals (e.g.,0–500, 500–1000).Centers for Disease Control and Prevention http://www.cdc.gov/ncidod/EID/vol9no10/03-0222.htm to SLEV was important because this virus has been detect-ed in the Caribbean and isolated from wild birds (12) andcross-reacted to anti–WNV-neutralizing antibodies in 6%of seropositive birds sampled in New York (13). PRNTswere performed with Vero cells in 6-well plates and aserum dilution of 1:10 in BA1 buffer (Hanks M-199 salts,0.05 M Tris pH 7.6, 1% bovine serum albumin, 0.35 g/Lsodium bicarbonate, 100 U/mL penicillin, 100 mg/L strep-tomycin, 1 mg/L Fungizone). Specimens that neutralizedthe virus stocks by at least 80% were further titrated induplicate. To identify either virus as the causative agent,we used 90% neutralization as the criterion for a positivetest result, and a four fold greater titer to one of the fla-viviruses was considered diagnostic for that flavivirus. Serum samples that showed neutralizing antibody titersto WNV or SLEV were tested by epitope-blockingenzyme-linked immunosorbent assays (ELISAs) with theWNV-specific monoclonal antibody 3.1112G, which dis-criminates between WNV and SLEV infections in birds(14). Because of the current lack of information on fla-viviruses in the Dominican Republic, we required both thePRNT and the ELISA to test positive to consider a serumsample positive for WNV.Tissues from each individual bird were pooled andhomogenized in 2 mL of BA1 supplemented with 20%fetal bovine serum. Homogenates were clarified by cen-trifugation at 3,700 rpm for 10 min at 4°C. Four hundredmicroliters of each homogenate was screened for virus byVero plaque assay (11). Homogenates obtained from fla-vivirus-seropositive birds were assayed for WNV RNA byTaqMan reverse-transcription polymerase chain reactionwith WNV-specific primers (15). Serum samples from nine resident birds tested positivefor flavivirus-neutralizing antibodies (Table). Of these,five birds were positive for WNV antibodies by PRNT andblocking ELISA. All WNV antibody–positive birds weresampled at the Los Haitises study site. One serum samplewas collected from an immature bird (ruddy quail-dove,Geotrygon montana; KUNHM 94667) that was <4 monthsold, suggesting that virus transmission was recent. Viruswas not isolated from any of the tissues tested from 118resident and 29 migratory birds (all migrants wereParulidae), nor did we detect WNV RNA in any of the tis-sue homogenates from flavivirus-seropositive birds.ConclusionsOur finding of WNV-neutralizing antibodies in five res-ident birds represents the first evidence of WNV activity inthe Dominican Republic. No cases of WNV infection inhumans, horses, or birds were known at the time of sam-pling. The birds in this study could have been infected withWNV in the Dominican Republic as recently as earlyNovember 2002; nonetheless, the virus probably arrivedearlier in the Caribbean region. Because no current infec-tions were detected, our results reflect past virus transmis-sion activity. Although we cannot determine when thisactivity began, the seropositive immature quail-dove pre-sumably was infected after mid-July 2002, when it was1300 Emerging Infectious Diseases • Vol. 9, No. 10, October 2003DISPATCHESTable. Laboratory results for flavivirus-seropositive birds collected in the Dominican Republic, 2002a Species KUNHM catalog no. Date sampled Age and sexb Locality SLEV PRNT90 WNV PRNT90 % inhibition by ELISAc Result Ruddy quail-dove (Geotrygon montana) 94667 21 Nov Immature male Los Haitises <10d 20d 40 WNV Mangrove cuckoo (Coccyzus minor) 94671 19 Nov Adult femalee Los Haitises <10 160 81 WNV Hispaniolan lizard cuckoo (Saurothera longirostris) 94669 18 Nov Adult female Los Haitises 160 640 73 WNV Hispaniolan lizard cuckoo (Saurothera longirostris) 94670 21 Nov Adult male Los Haitises <10 40 23 FLAV Hispaniolan trogon (Priotelus roseigaster) 94951 12 Nov Adult male Sierra de Baoruco <10 10 6 FLAV Red-legged thrush (Turdus plumbeus) 94956 20 Nov Adult femalee Los Haitises 10 160 86 WNV Red-legged thrush (Turdus plumbeus) 94689 19 Nov Adult malee Los Haitises <10 80 Not available FLAV Red-legged thrush (Turdus plumbeus) 94691 21 Nov Adult female Los Haitises 20 1280 61 WNV Greater Antillean grackle (Quiscalus niger) 94949 19 Nov Adult male Los Haitises 640 40 42 FLAVf aELISA, enzyme-linked immunosorbent assay; FLAV, undifferentiated flavivirus; KUNHM, University of Kansas Natural History Museum, Division of Ornithology; PRNT90, reciprocal 90% plaque reduction neutralization titer; SLEV, Saint Louis encephalitis virus; WNV, West Nile virus. bBirds were in nonbreeding condition unless otherwise indicated. cInhibition values >30% were considered significant. dValues represent reciprocal titers; threshold of detection was 1:10. eBreeding condition, as determined by size of gonads. fAlthough serologic results based upon PRNT would suggest that this specimen be identified as SLEV antibody–positive, the WNV antibody–positive result in the blocking ELISA indicates that this specimen was possibly positive for both SLEV and WNV. However, secondary flavivirus infections are notorious for heterologous reactivity, so infection by WNV or other flaviviruses causing these reactions could not be ruled out. Hence, the determination as FLAV. Centers for Disease Control and Prevention http://www.cdc.gov/ncidod/EID/vol9no10/03-0222.htm born, and before early November 2002, in order to havestimulated detectable antibody production by mid-November.The earliest evidence of WNV transmission in the WestIndies is a human case from the Cayman Islands in 2001(16). WNV-seropositive birds captured in Jamaica early in2002 may have been infected in 2001 or earlier (17).Additional evidence of WNV transmission in theCaribbean region includes the report of two seropositivehorses in Yucatán state, Mexico, sampled in July 2002(18). Although the seropositivity of vertebrates in CaymanIslands, Jamaica, Mexico, and now the DominicanRepublic is strong evidence for WNV activity in theregion, it is indirect evidence and does not entirely rule outthe possibility of cross-reactions with another flavivirus inlaboratory assays. WNV remains to be isolated from theregion.The presence of WNV at Los Haitises may have result-ed from transportation by viremic migratory birds fromNorth America, where WNV transmission foci are wide-spread (2). Several migratory bird species, in particular,parulid warblers (order Passeriformes), were observed atthis site. At least some passerine birds are capable of trans-mitting virus during their few days of viremia (19).Therefore, transmission to mosquitoes or predators fromviremic migrants would be possible for a brief period (afew days, at most) after arrival at a site. Virus introductioninto Caribbean ecosystems is therefore likely to occur atcoastal sites where transoceanic migrants make first land-fall. We found no evidence of active virus in bird tissues ofboth resident and migratory species. We did not test serumsamples from migrants because the presence of antibodieswould not be informative, given the history of these birdstraveling through areas of WNV transmission in or nearNorth American breeding grounds. We presume that thefive seropositive resident birds were infected locallybecause the four species involved are not migratory (7).Although young birds may disperse several kilometersfrom natal sites (20), adults probably live entirely withinbreeding territories. Although only five birds (15%, 95% confidence inter-val [CI] 5% to 32%) at Los Haitises were found to beseropositive, the results suggest that transmission of WNVamong bird populations at that site was widespread. Forcomparison, seroprevalence of WNV-neutralizing antibod-ies in resident birds was 50% (CI 44% to 57%) in Queens,New York City, after the 1999 outbreak (13), and 23% (CI18% to 29%) in Staten Island after the 2000 outbreak (21).We used conservative criteria for determining a positiveresult because the background diversity of flaviviruses inthe Dominican Republic has not been studied recently. Ifonly the PRNT had been used (as was the case in the NewYork studies), then seven (21%, CI 7% to 35%) of the birdsfrom Los Haitises would have been reported as positive forantibodies to WNV.The evidence for local WNV transmission in theDominican Republic indicates risk for West Nile fever andmeningoencephalitis in the human, equine, and avian pop-ulations of Hispaniola. We suggest that WNV be consid-ered in the differential diagnosis of humans and other ver-tebrates with central nervous system disease in Hispaniola. AcknowledgmentsWe thank Bolivar Cabrera, Tristan Davis, Juan CepedesHidalgo, José Dolores Jiménez, Esteban López-Medrano, andDomingo Sirí for assistance in the field; José Ramón Albaine andSimón Guerrero for logistical assistance and assistance with per-mits; John Roehrig for assisting with the interpretation of sero-logic test results; and the two anonymous reviewers who provid-ed comments on the manuscript.This study was supported in part by National ScienceFoundation Grant #0211388, and in part by the Centers forDisease Control and Prevention contract U50/CCU 820510-02.Mr. Komar is an ornithologist working on a Ph.D. in biolo-gy at the University of Kansas Natural History Museum andDepartment of Ecology and Evolutionary Biology in Lawrence,Kansas. His primary research interests are avian ecology andconservation in the Neotropical region. References1. Lanciotti RS, Roehrig JT, Deubel V, Smith J, Parker M, Steele K, etal. Origin of the West Nile virus responsible for an outbreak ofencephalitis in the northeastern United States. Science1999;286:2333–7. 2. Chow CC, Montgomery SP, O’Leary DR, Nasci RS, Campbell GL,Kipp AM, et al. Provisional surveillance summary of the West Nilevirus epidemic—United States, January–November 2002. MMWRMorb Mortal Wkly Rep 2002;51:1129–33.3. Health Canada. Population and Public Health Branch WNV surveil-lance updates, December 4, 2002. Available from: URL:http://www.hc-sc.gc.ca/pphb-dgspsp/wnv-vwn/mon_e.html#sitrep4. McLean RG, Ubico SR, Bourne D, Komar N. West Nile virus in live-stock and wildlife. Curr Top Microbiol Immunol 2002;267:271–308.5. Rappole JH, Derrickson SR, Hubalek Z. Migratory birds and spreadof West Nile virus in the Western Hemisphere. Emerg Infect Dis2000;6:319–28.6. American Ornithologists’ Union. Check-list of North American birds.7th ed. Washington: American Ornithologists’ Union; 1998.7. Raffaele H, Wiley J, Garrido O, Keith A, Raffaele J. A guide to thebirds of the West Indies. Princeton (NJ): Princeton Univ. Press; 1998. 8. Malkinson M, Banet C, Weisman Y, Pokamunski S, King R, DrouetMT, et al. Introduction of West Nile virus in the Middle East bymigrating white storks. Emerg Infect Dis 2002;8:392–7.9. Peterson AT, Vieglais DA, Andreasen JK. Migratory birds modeled ascritical transport agents for West Nile virus in North America. VectorBorne Zoonotic Dis 2003;3:27–37.10. Gaunt AS, Oring LW, editors. Guidelines to the use of wild birds inresearch. The Ornithological Council; 1997. Available from: URL:www.nmnh.si.edu/BIRDNET/GuideToUseEmerging Infectious Diseases • Vol. 9, No. 10, October 2003 1301DISPATCHESCenters for Disease Control and Prevention http://www.cdc.gov/ncidod/EID/vol9no10/03-0222.htm 11. Beaty BJ, Calisher CH, Shope RE. Arboviruses. In: Lennette EH,Lennette DA, Lennette ET, editors. Diagnostic procedures for viral,rickettsial, and chlamydial infections, 7th ed. Washington: AmericanPublic Health Association; 1995. p. 189–212.12. Tsai TF, Mitchell CJ. St. Louis encephalitis. In: Monath TP, editor.Vol. IV, The arboviruses: epidemiology and ecology. Boca Raton(FL): CRC Press; 1988. p. 113–43.13. Komar N, Panella NA, Burns JE, Dusza SW, Mascarenhas TM,Talbot TO. Serologic evidence for West Nile virus infection in birdsin the New York City vicinity during an outbreak in 1999. EmergInfect Dis 2001;7:621–5.14. Blitvich BJ, Marlenee NL, Hall RA, Calisher CH, Bowen RA,Roehrig JT, et al. Epitope-blocking enzyme-linked immunosorbentassays for the detection of serum antibodies to West Nile virus in mul-tiple avian species. J Clin Microbiol 2003;41:1041–7.15. Lanciotti RS, Kerst AJ, Nasci RS, Godsey MS, Mitchell CJ, SavageHM, et al. Rapid detection of West Nile virus from human clinicalspecimens, field-collected mosquitoes, and avian samples by aTaqMan reverse transcriptase-PCR assay. J Clin Microbiol2000;38:4066–71.16. O’Leary DR, Nasci RS, Campbell GL, Marfin AA. West Nile virusactivity—United States, 2001. MMWR Morb Mortal Wkly Rep2002;51:497–501.17. Dupuis II AP, Marra PP, Kramer LD. Serologic evidence for WestNile virus transmission in Jamaica, West Indies. Emerg Infect Dis2003;9:860–3.18. Loroño-Pino MA, Blitvich BJ, Farfán-Ale JA, Puerto FI, Blanco JM,Marlenee NL, et al. Serologic evidence of West Nile virus infectionin horses, Yucatan State, Mexico. Emerg Infect Dis 2003;9:857–9.19. Komar N, Langevin S, Hinten S, Nemeth N, Edwards E, Hettler D, etal. Experimental infection of North American birds with the NewYork 1999 strain of West Nile virus. Emerg Infect Dis2003;9:311–22.20. Greenwood PJ. Mating systems, philopatry and dispersal in birds andmammals. Animal Behavior 1980;28:1140–62.21. Komar N, Burns J, Dean C, Panella NA, Dusza S, Cherry B.Serologic evidence for West Nile virus infection in birds in StatenIsland, New York, after an outbreak in 2000. Vector Borne ZoonoticDis 2001;1:191–6. Address for correspondence: Oliver Komar, Natural History Museum andBiodiversity Research Center, University of Kansas, 1345 JayhawkBoulevard, Lawrence, KS 66045-7561, USA; fax: 785-864-5335; email:okomar@ku.edu1302 Emerging Infectious Diseases • Vol. 9, No. 10, October 2003DISPATCHESSearch past issues of EID at www.cdc.gov/eidCenters for Disease Control and Prevention http://www.cdc.gov/ncidod/EID/vol9no10/03-0222.htm 

West Nile virus transmission in resident birds, Dominican Republic

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West Nile virus transmission in resident birds, Dominican Republic

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