West Nile Virus Ecology in a Tropical Ecosystem in Guatemala

Maria E. Morales-Betoulle,† Nicholas Komar,*† Nicholas A. Panella, Danilo Alvarez, MarıáR.Lo´pez, Jean-Luc Betoulle, Silvia M. Sosa, MarıáL.Mu¨ ller, A. Marm Kilpatrick, Robert S. Lanciotti, Barbara W. Johnson, Ann M. Powers, Celia Cordo´ n-Rosales, and the Arbovirus Ecology Work Group‡ Center for Health Studies, Universidad del Valle de Guatemala, Guatemala; Centers for Disease Control and Prevention, Arbovirus Disease Branch, Fort Collins, Colorado; University of California, Santa Cruz, California; Fundacioń Mario Dary, Guatemala City, Guatemala; and Fundacioń para el Ecodesarrollo, Guatemala City, Guatemala

Abstract. West Nile virus ecology has yet to be rigorously investigated in the Caribbean Basin. We identified a transmission focus in Puerto Barrios, Guatemala, and established systematic monitoring of avian abundance and infection, seroconversions in domestic poultry, and viral infections in mosquitoes. West Nile virus transmission was detected annually between May and October from 2005 to 2008. High temperature and low rainfall enhanced the probability of chicken seroconversions, which occurred in both urban and rural sites. West Nile virus was isolated from Culex quinquefasciatus and to a lesser extent, from Culex mollis/Culex inflictus, but not from the most abundant Culex mosquito, Culex nigripalpus. A calculation that combined avian abundance, seroprevalence, and vertebrate reservoir competence suggested that great-tailed grackle (Quiscalus mexicanus) is the major amplifying host in this ecosystem. West Nile virus transmission reached moderate levels in sentinel chickens during 2007, but less than that observed during outbreaks of human disease attributed to West Nile virus in the United States.

INTRODUCTION amplify and transmit WNV in Guatemala, where WNV appears to have been circulating since 2003.9 Accordingly, we West Nile virus (WNV) is a mosquito-borne pathogen that 1 established a sentinel chicken surveillance network to detect began circulating in the Caribbean Basin in 2001. Ecological active WNV transmission foci for the development of further studies of WNV in Europe, Asia, the Middle East, and the ecological studies. Periodic sampling of resident poultry in United States have shown that Culex (Culex) mosquitoes several Guatemalan departments representing different eco- serve as vectors and that particular species of birds are the 2,3 regions was initiated in 2004. Once an active transmission principal amplifying hosts. Culex mosquitoes and passerine focus was detected, in the humid Atlantic coast eco-region, birds appeared to be responsible for WNV amplification in 4,5 we established systematic monitoring of seroconversions in Puerto Rico in 2007. By 2007, serologic evidence for WNV domestic poultry, seroprevalence in free-ranging birds, and circulation in free-ranging birds and/or horses was reported in viral infections in mosquitoes. Our principal objectives were numerous tropical locations around the rim of the Caribbean 6–8 9,10 11 to describe the vectors, amplifying hosts, and seasonality of Basin, including Mexico, Guatemala, Costa Rica, WNV transmission. We herein report the findings of our lon- Colombia,12 Venezuela,13 Guadaloupe,14 Puerto Rico,15 16,17 18 19 15 gitudinal investigation, including spatio-temporal patterns of Dominican Republic, Haiti, Jamaica, and Cuba. transmission, candidate vectors, and avian amplifying hosts. Ecological parameters of WNV transmission have yet to be clearly defined in tropical ecosystems typical of the Caribbean Basin countries.1 Serosurveys of free-ranging birds in several MATERIALS AND METHODS countries have identified infections in numerous species of Study sites. In 2004–2005, seven different departments of birds but with the exception of a recent report in Puerto Guatemala corresponding to different eco-regions were Rico,4 none of these studies were focused in time and place selected for initial prospective monitoring of free-ranging coincident with active transmission.7,8,16,17,19 Similarly, sev- domestic chickens for evidence of local WNV transmission eral isolates were derived from various species of Culex mos- (Figure 1). In 2006, we selected the municipality of Puerto quitoes in tropical America, but except for Puerto Rico,5 Barrios (15°50¢N and 88°28¢W), Department of Izabal, for insufficient data were available to incriminate them as WNV – follow-up longitudinal ecology studies of WNV. This Depart- vectors.20 22 To determine vectors and amplifying hosts, it is ment is located on the Caribbean coast of Guatemala within a necessary to study WNV ecology within a transmission focus. subtropical wet forest life zone. Climatic conditions are gen- No such studies have been reported from Central America. erally hot and humid without a well-defined dry season. Mean Presumably WNV uses similar hosts and vectors in the annual precipitation is 3,500 mm. Monthly rainfall and average tropics as in temperate regions. Therefore, we hypothesized temperature data from Puerto Barrios were obtained from the that certain passerine birds and Culex mosquitoes would Guatemalan Instituto Nacional de Sismologıá, Vulcanologıá, Metereologıá e Hidrologıá (NSIVUMEH). Ecological studies were conducted between March 2006 and * Address correspondence to Nicholas Komar, Arbovirus Diseases March 2009 in an 80 km2 geographic area within the Puerto Branch, CDC-NCEZID-DVBD, 3150 Rampart Road, Fort Collins, 2 CO 80521. E-mail: [email protected] Barrios municipality. Ten 1-km blocks were randomly †These authors contributed equally to this work. selected and a sampling site was selected within each of these ‡Arbovirus Ecology Work Group, in alphabetical order: Cristina blocks. The sampling sites were selected according to the Chaluleu, Carmen L. Contreras, Rebekah C. Kading, Eric Edwards, following criteria: 1) access to private property, 2) presence Marvin S. Godsey, Ana S. Gonza´lez, Kathryn P. Huyvaert, Kimberly M. Keene, Jeremy P. Ledermann, Luis Martıńez, Bernarda Molina, of backyard poultry, and 3) secure vehicular access (Figure 2). Marıá de Lourdes Monzoń, Janae L. Stovall, Mońica Santiago, Harry M. In the event of a change in access to a sampling site, the site Savage, and Ginger Young. was relocated to the nearest site that fulfilled the selection 116 WEST NILE VIRUS TROPICAL ECOLOGY 117

Figure 1. Guatemala departments where domestic chickens were serially sampled to detect West Nile virus (WNV) transmission, 2004–2005. Numbers 1–7 indicate the department, each representing a different eco-region (see text). criteria. At each site, we surveyed for seroconversion of domes- defined as having ³ 30% of road and human dwelling habitats. tic chickens, mosquito densities, and relative abundance of Rural habitats in the study area included predominantly ripar- bird populations. ian forest, pasture, and cropland. Each of the 10 sampling points was assigned a macrohabitat Wild birds were captured in two different sites located category (rural versus urban) according to visual estimates of within the 80 km2 study area as explained below. urbanization and vegetation in an area of ~3 hectares where Chicken monitoring. We placed uniquely numbered alumi- the bird population surveys were conducted. Urban sites were num leg bands on 5–10 chicks in each of the 10 sampling sites.

Figure 2. Sampling sites for assessment of West Nile virus (WNV) transmission in Puerto Barrios, Izabal, Guatemala, 2006–2009. Markers numbered 1–10 indicate locations of selected study sites where chickens and mosquitoes were sampled. A and B represent locations where wild birds were sampled. 118 MORALES-BETOULLE, KOMAR AND OTHERS

Monthly, blood samples (~1 mL) were collected from the bra- urban zone. Bird captures were carried out during 1- to 2-week chial or jugular vein of these marked domestic birds using 1-cc periods ~3 months apart from April 2006 to July 2009. Free- syringes with 26 g 1/2-inch sub-Q needles (Becton-Dickinson, ranging birds were captured using 18–20 mist nets of various Franklin Lakes, NJ), divided among two 0.6-mL Microtainer mesh sizes and lengths, monitored continuously from sunrise serum collection tubes (Becton-Dickinson), and centrifuged to sunset. Resident birds were identified and aged as juveniles for serum separation allowing at least 15 minutes for coagu- (< 1 year of age) or adults (> 1yearofage)whenpossible lation. Samples were frozen on dry ice for transport to the and marked with numbered leg bands. Blood (volume ~1% Universidad Del Valle de Guatemala (UVG) where they body mass up to maximum 0.65 mL) was collected from the were stored at −20°C. One tube per sample was thawed for jugular vein of birds that weighed > 10 g, using 1-cc syringes antibody detection assays described below. The second tube with 26 g 1/2-inch or 27 g 5/8-inch sub-Q needles (Becton- was available for additional testing if necessary. Initial blood Dickinson). Mass was measured using a handheld Pesola scale samples were tested to confirm seronegativity for WNV. (Avinet, Inc., Dryden, NY). Blood was processed identically as Results were expressed as the number of seroconversions in for chickens. Smaller birds and migratory species were released the numerator, and the number of chicken-weeks of expo- without sampling. sure in the denominator. One chicken exposed to mosquito Antibody detection assays. Serum samples from wild and bites for 1 week represents 1 chicken-week of exposure. Chickens domestic birds were tested using an epitope-blocking that either seroconverted (became positive for WNV-reactive enzyme-linked immunosorbent assay (B-ELISA) as described antibodies), disappeared, or died were replaced to maintain previously.26 Briefly, any sample that blocked both the non- 5–10 birds per sentinel flock. These chickens belonged to and specific flavivirus-reactive monoclonal antibody 6B6C-1 and were cared for by private property owners. Supplemental food the WNV-specific monoclonal antibody 3.1112g by ³ 30% and preventive veterinary care were provided as needed. was considered positive. The positive samples and a subset Mosquito surveys. Mosquito population densities were of negative samples were confirmed by the plaque reduction estimated from collections of adult mosquitoes in one CO2- neutralization test (PRNT) using Vero cells as previously baited Centers for Disease Control and Prevention (CDC) described.27 Reference virus strains used in the PRNT were light trap (John W. Hock Co., Gainesville, FL) and one gravid the NY99-4132 strain of WNV and the TBH-28 strain of trap23 placed for 1 night per month in each of the 10 sampling St. Louis encephalitis virus (SLEV). Serum samples were sites. In the morning, collection nets were placed in a box diluted 1:5 in BA1 media (Hanks M-199 salts, 0.05 M Tris, containing dry ice to kill the insects by CO2 asphyxiation. pH 7.6, 1% bovine serum albumin, 0.35 g/L of sodium bicar- Killed insects were poured onto plastic trays for manual sepa- bonate, 100 U/mL of penicillin, 100 mg/mL of streptomycin, ration of mosquitoes into Nalgene cryovials (Sigma-Aldrich 1 mg/mL of Fungizone) before incubation with an equal vol- Corp., St. Louis, MO) that were then frozen on dry ice for ume of virus suspension. To compare titers against both WNV transport to UVG. In the laboratory, collection cryovials were and SLEV, serum samples were tested in serial 2-fold dilu- stored at −70°C until thawed for mosquito identification. Adult tions in duplicate starting with 1:10. Samples that reduced the female mosquitoes were identified by their morphological char- number of plaques formed by 90% or more for at least one of acteristics viewed through a stereo microscope (on a chill table) the reference viruses were considered positive by PRNT, and using the Clark-Gil and Darsie key.24 Pools of no more than determined to contain anti-flavivirus antibodies. These anti- 50 mosquitoes were separated according to species, location, bodies were attributed to either WNV- or SLEV-infection if date, and trap type, and then replaced at −70°C until processed the reciprocal titer for WNV was at least 4-fold greater than for further testing. Beginning in 2007, supplemental mosquito that of SLEV or vice versa, respectively. collections were conducted within 6 weeks following chicken Arbovirus detection from mosquitoes. All pools of female seroconversions using 5–10 traps of each type in the sampling Culex mosquitoes were homogenized and tested in duplicate site where the seroconversion event was detected. for viral plaque growth or cytopathic effects on Vero cell Bird counts. To estimate bird abundance, point count sur- monolayers in 6-well plates or 1-dram shell vials.28 Cultured veys25 of birds (wild and domestic) were undertaken by one viruses were harvested in 1 mL of BA1 supplemented with ornithologist and an assistant at each of the 10 sampling sites. 20% fetal bovine serum, and 140 mL of the virus suspension At each site, birds were counted from four different stations was RNA-extracted and tested by reverse transcription- located at 150-m intervals along a walking route that began as polymerase chain reaction (RT-PCR) with group-specific close as possible to the sentinel chicken flock. At each station, flavivirus, alphavirus, and bunyavirus primers according to – the ornithologist reported visual and auditory observations of published protocols.29 31 Positive RT-PCR reactions were all individual birds by species within ~50 m during a 4-minute followed with specific primers and probes for WNV (and period. In 2006, point counts were conducted every 3 months. other regional arboviruses) in a real-time RT-PCR format Since May 2007, point counts were conducted monthly. when available.32 Relative abundance (no. observed individuals of a species To enhance the detection of WNV and other arboviruses, all divided by the no. of all birds observed) was calculated sea- Culex quinquefasciatus pools were also tested specifically for sonally for 119 species that were observed during the survey. WNV using WNV-specific primers.32 In addition, since 2007, Wild bird antibody surveys. Two locations (A and B; all pools of Culex (subgenus Culex)mosquitoeswerealsotested Figure 2) were selected within the 80 km2 study area to study with Flavivirus-consensus primers, including Culex nigripalpus, the prevalence of WNV-reactive antibodies in wild resident Culex chidesteri, and Culex mollis/Culex inflictus.29 birds. Both locations were comprised primarily of secondary Mosquito inoculation index. The mosquito inoculation forest and pastures. Location A was the Naval Base airport index (M) is a measure of the relative number of infectious about 1 km from the urban zone of Puerto Barrios. Location B vector mosquitoes derived from feeding on a vertebrate host was the rural village of Machacas del Mar about 7 km from the population.33 The value for M is derived from the product of WEST NILE VIRUS TROPICAL ECOLOGY 119 vertebrate host population (P), vertebrate infection rate (I), Five seroconversions (change in antibody status from sero- and vertebrate reservoir competence (C): negative to seropositive between two sampling time points) in chickens provided evidence of WNV transmission in the M = ðÞP ðÞI ðÞC : Department of Izabal. No evidence for active WNV transmission in chickens was detected in six other departments. Because the natural process of a vertebrate host infecting Temporal transmission of West Nile virus and relationship hematophagous vectors should incorporate host preference to temperature and rainfall. We monitored seasonality of twice (once for the process of infecting the vertebrate host, WNV transmission in Puerto Barrios, Izabal by sampling and once for the process of vectors acquiring the infection chicken flocks monthly at 10 sampling sites. Seroconversions to 34 from the vertebrate host ), we modified the formula to incor- WNV were detected annually, and only occurred during the porate the host selection of both infectious and uninfected period from May to October (Figure 3). Peak monthly incidence host-seeking mosquitoes by squaring the vertebrate infection reached 13.3/1,000 chicken-weeks in 2006, 60.7/1,000 chicken- rate term. weeks in 2007, and 6.9/1,000 chicken-weeks in 2008. Monthly rainfall in Puerto Barrios during the period of the 0 M = ðÞP ðÞI 2ðÞC : study ranged from a minimum of 29.1 mm in May 2007 to a maximum of 775.5 mm in June 2006 (Figure 4A). The temper- This approach has been used in several studies of WNV in ature fluctuated annually from cooler periods (November– Chicago, IL, Colorado, and the mid-Atlantic, and has been February; average monthly temperatures 23–25°C) to warmer shown to be useful in predicting WNV infection prevalence in periods (March–October; average monthly temperatures 26– – mosquitoes and the timing of WNV epidemics.35 38 It represents 29°C) (Figure 4B). The WNV seroconversions in sentinel a substantial advance over considering simply the abundance chickens only occurred following months when monthly aver- and seroprevalence because some abundant and frequently age temperatures were high (> 27.2°C) and increased with exposed species may be incompetent hosts, whereas others may decreasing rainfall at these warmer temperatures (Logistic be abundant but not frequently exposed, or vice versa. regression: Intercept: −83.5 ± (SE) 14.1; Temperature coef. = We used relative abundance values, A, from the bird sur- 2.82 ± 0.50, P < 0.001; Rainfall coef. = 0.11 ± 0.04, P = 0.005; veys for the population-based measure, P. Antibody sero- Temperature + Rainfall interaction coef. = −0.0041 ± 0.0014, prevalence, S, was used in place of I because in the absence P = 0.004). of pathogen-attributed mortality, seroprevalence is equal to Potential vectors. A total of 117,613 female mosquitoes infection rate, and there is no evidence for WNV-attributed corresponding to at least 37 species were captured (Supplemental mortality in Guatemalan birds as yet. For vertebrate reservoir Table S1). Focusing on Culex mosquitoes as potential WNV competence index values, we used data published for bird vectors, we tested these for virus plaque formation in Vero cell 39 species infected with WNV from southern Mexico. These culture. A subset of pools corresponding to Cx. quinquefasciatus, values are derived experimentally from the duration and Cx. nigripalpus and (since 2007) Cx. mollis/Cx. inflictus were infectiousness of viremia and describe a species’ innate poten- also tested for flavivirus- (N = 1,551) and/or WNV- (N = 40 tial for infecting mosquitoes. Thus, our equation for mos- 1,732) specific RNA by standard or real-time RT-PCR, respec- quito inoculation rate is tively. West Nile virus was isolated from supplemental collections of mosquitoes at locations with recent chicken seroconversions 0 = ðÞðÞ2ðÞ: M A S C to WNV from two pools of Cx. quinquefasciatus, two pools of Cx. mollis/Cx. inflictus, and three pools of undifferentiated Statistical analyses. Mosquito infection rates were calcu- Culex mosquitoes collected in July and August 2007. Infection lated by the maximum-likelihood estimate for WNV-infected rates in the Culex populations sampled at these points mosquito pools using the PooledInfRate version 3.0 program in space and time ranged from 0 in Cx. nigripalpus to 15.7/ in Excel.41 Logistic regression, Fisher’s exact and c2 tests were 1,000 mosquitoes in Cx. quinquefasciatus (Table 2). A phylo- used to analyze seroprevalence patterns among wild birds. genetic evaluation of these Guatemalan WNV isolates will be described elsewhere (CDC, unpublished data). RESULTS Population dynamics of Culex mosquitoes were assessed from standardized routine collections among the 10 sampling Identification of active West Nile virus transmission. We sites. Culex quinquefasciatus density peaked 1–4 months monitored WNV transmission in sentinel chickens from before the annual peaks of WNV infection incidence in 2004 to 2005 in seven departments of Guatemala (Table 1). chickens (Figure 3). In contrast, Cx. nigripalpus and Cx.

Table 1 WNV seroconversions and incidence in free-ranging chickens from seven Departments of Guatemala, 2004–2005 No. Exposure Incidence Department Eco-region N seroconversions (chicken-weeks) (per 103 chicken-weeks) Pete´n Humid Forest 69 0 1,316 0.00 Alta Verapaz Humid Montane 24 0 432 0.00 Izabal Humid Atlantic 224 5 4,215 1.19 Zacapa Dry Forest 37 0 666 0.00 Chiquimula Agricultural 23 0 417 0.00 Santa Rosa Dry Pacific 56 0 1,145 0.00 Escuintla Humid Pacific 52 0 672 0.00 120 MORALES-BETOULLE, KOMAR AND OTHERS

Figure 3. Average mosquito abundance of candidate West Nile virus (WNV) mosquito vectors in relation to monthly incidence of chicken seroconversions across study sites in Puerto Barrios, Guatemala, 2006–2009. Mosquito densities were derived from monthly Centers for Disease Control and Prevention (CDC) gravid and light trap collections at 10 sampling points. Arrows indicate months in which WNV-infected mosquitoes were detected in supplemental collections. mollis/Cx. inflictus densities peaked inconsistently either isolated from Culex taeniopus, and group C bunyaviruses before or after the detection of WNV infection in chickens detected in Cx. taeniopus (data not shown). depending on the year. Vertebrate hosts. Bird relative abundance was assessed Additional arboviruses were detected and isolated includ- during different seasons of North American bird migration ing: Culex flavivirus, isolated from Cx. quinquefasciatus,28 (spring migration, breeding season, fall migration, non- enzootic strains of Venezuelan equine encephalitis virus breeding season). Almost 24,000 birds were recorded during

Figure 4. Climatic data recorded by the Guatemalan National Institute of Meteorology in Puerto Barrios, Guatemala, 2006–2008. (A) Rainfall (mm) and (B) monthly average temperature (°C). WEST NILE VIRUS TROPICAL ECOLOGY 121

Table 2 West Nile virus (WNV) infection rates (determined by maximum likelihood estimate) of Culex mosquitoes from supplemental collections in premises where WNV activity in chickens had been recently detected, Puerto Barrios, Guatemala, 2007 Site number (corresponding macrohabitat type)/collection date

# 7 (rural)/5-Jul-07 # 3 (urban)/3-Aug-07 # 5 (urban)/9-Aug-07

No. No. pos No. IR* No. No. pos No. IR No. No. pos No. IR Culex species pools pools individuals (95% CI) pools pools individuals (95% CI) pools pools individuals (95% CI) quinquefasciatus 6 1 176 5.7 (0.3–30.7) 3 1 62 15.7 (1.1–107.5) 2 0 26 – mollis/inflictus 6 1 212 4.7 (0.3–24.7) 2 0 88 – 7 1 267 3.5 (0.2–17.0) nigripalpus 10 0 456 – 5 0 236 – 12 0 590 – undifferentiated 9 1 342 2.9 (0.2–14.8) 1 1 50 20.0(NA) 3 1 106 9.0 (0.6–57.5) All other Cx. species 7 0 116 – 70 90 – 13 0 355 –

*IR = WNV infection rate expressed per 1,000 mosquitoes; NA = method to calculate 95% confidence interval for infection rate is not applicable. standardized surveys comprising 119 species belonging to 19 species tested positive for prior infection with WNV, as 41 families and 17 orders (including domestic and wild determined by the detection of WNV-specific antibodies birds) (Supplemental Table S2). About one-third of these (Supplemental Table S4). Seroconversions were detected species (N = 37) were migratory (transients and winter vis- among recaptures for the following species: clay-colored itors) and two-thirds (N = 82) were permanent residents, thrush (N = 4), great-tailed grackle (N = 1), ferruginous including domesticated species and captive birds. Great- pygmy-owl (Glaucidium brasilianum, N = 1) and great tailed grackle (Quiscalus mexicanus) was the most abundant kiskadee (Pitangus sulphuratus, N = 1). Seroprevalence species followed by domestic chicken (Gallus gallus) and clay- differed significantly among habitats (urban versus rural), colored thrush (Turdus grayi) in all seasons, representing species, year and age (juvenile versus adult) (Table 3). 30–40%, 15–20%, and 5–10%, respectively, of all resident birds Free-ranging birds were more likely to be seropositive for surveyed in Puerto Barrios (Figure 5). WNV if they were adult, captured near urban habitat, A total of 3,833 individual wild birds representing at sampled in 2007 or 2008, or one of the following species: least 136 different species were captured using mist nets melodious blackbird (Dives dives), ferruginous pygmy-owl (Supplemental Table S3). Blood samples (N = 1,799) were (G. brasilianum), gray-headed dove (Leptotila plumbeiceps), obtained from resident birds. A subset of those samples great-tailed grackle (Q. mexicanus), or clay-colored thrush (N = 291) corresponded to “recaptures” of individuals sam- (T. grayi). Among these, only the latter two species were pled in a previous season or year. A total of 120 birds of abundant throughout the study area.

Figure 5. Seasonal variation in percent of all avian detections during point counts for selected bird species in Puerto Barrios, Guatemala (2006–2008). “Spring” represents Spring migration, “Summer” breeding season, “Fall” fall migration and “Winter” non-breeding season. 122 MORALES-BETOULLE, KOMAR AND OTHERS

Table 3 Logistic regression analyses of West Nile virus (WNV)-antibody status of 1,150 birds from 17 species that had at least one seropositive and one seronegative individual, as required for logistic regression* Coef. Odds ratio Predictor Category (SE) Z or c2 P value (95% CI) Intercept −5.39 −8.36 < 0.001 (0.64) Age Juvenile (DF = 2) 15.92 < 0.001 1.0 Adult 0.89 3.18 0.001 2.45 (0.28) (1.44–4.37) Unknown 1.52 3.45 0.001 4.85 (0.44) (1.89–10.80) Habitat Rural (site B) (DF = 1) 2.80 0.005 1.0 Urban (Site A) 0.71 2.04 (0.26) (1.25–3.41) Year 2006 (DF = 3) 15.27 0.002 1.0 2007 0.82 2.83 0.005 2.27 (0.29) (1.31–4.08) 2008 1.01 3.15 0.002 2.74 (0.32) (1.48–5.22) 2009 0.13 0.32 0.751 1.14 (0.40) (0.50–2.47) Species† Columbina talpacoti (DF = 16) 54.6 < 0.001 1.0 Dives dives 2.90 4.13 < 0.001 18.23 (0.70) (4.61–76.20) Glaucidium brasilianum 2.38 3.14 0.002 10.81 (0.76) (2.32–48.60) Leptotila plumbeiceps 2.88 3.27 0.001 17.79 (0.88) (2.89–99.94) Quiscalus mexicanus 2.11 4.20 < 0.001 8.22 (0.50) (3.34–24.84) Turdus grayi 1.59 3.18 0.001 4.88 (0.50) (2.00–14.66) *The table shows the coefficients (Coef.), standard errors of coefficients (SE), and odds ratios for categorical predictors relative to the reference level, which is given on the row of the predictor name and underlined. The statistics on the predictor row use c2 when there are more than two categories for a predictor. DF = degrees of freedom †Only species with significantly different seroprevalence (P < 0.0029, a = 0.05) with respect to Columbina talpacoti (ruddy ground-dove) are shown. Eleven species for which no significant difference in seroprevalence was detected include Crotophaga sulcirostris (groove-billed ani), Euphonia hirundinacea (yellow-throated euphonia), Icterus pectoralis (spot-breasted oriole), Manacus candei (white-collared manakin), Momotus momota (blue-crowned motmot), Myiozetetes similis (social flycatcher), Pitangus sulphuratus (great kiskadee), Saltator atriceps (black-headed saltator), Saltator coerulescens (grayish saltator), Thraupis abbas (yellow-winged tanager), and Thraupis episcopus (blue-gray tanager).

In addition, the seroprevalence of the two most well sam- mosquitoes during the period of peak WNV transmission pled species, great-tailed grackle and clay-colored thrush, from May to November of 2007 (Table 5). increased significantly coincident with the period of peak transmission observed in chickens (May–Nov 2007), and DISCUSSION remained high thereafter (Figure 6; Table 4). The force of transmission between vertebrate amplifying We sought to characterize ecological parameters of WNV host and mosquito vectors was quantified by calculating the transmission in a tropical ecosystem located in the municipal- modified mosquito inoculation index (M¢) for great-tailed ity of Puerto Barrios, on the Atlantic coast of Guatemala. grackle, clay-colored thrush, and domestic chicken. These The mosquito species found infected with WNV, Cx. calculations predicted that great-tailed grackles infected quinquefasciatus and Cx. mollis/Cx. inflictus, are both mem- ~6,000 vector mosquitoes for every one mosquito infected by bers of the subgenus Culex, which is known to include clay-colored thrushes, and that adult chickens infected no important vectors of WNV throughout the world.36,42 Culex quinquefasciatus, known commonly as the southern house mosquito, is frequently found in close association with human activity in urban and rural areas, and has been implicated as a vector for WNV in the United States.43,44 This is the first report of WNV infection in Cx. mollis/Cx. inflictus. Little is known about the bionomics of Cx. mollis or Cx. inflictus. Larval forms of Cx. mollis may be distinguished from Cx. inflictus, but adults are morphologically similar.24 Our collections probably refer to Cx. mollis,asCx. inflictus is associated with salt water habitats, where it breeds in crab holes.24 How- ever, such habitats are located just a few kilometers from our study sites; therefore, we could not eliminate this species from consideration. Another recognized WNV vector, Cx. nigripalpus, outnumbered other Culex (Culex) species mosqui- Figure 6. Seroprevalence of adult and juvenile clay-colored thrush toes in the transmission focus, yet was not found to be infected. (Turdus grayi), and great-tailed grackle (Quiscalus mexicanus) before, WNV-infected Cx. nigripalpus have been reported else- during, and after the peak transmission period (May–Nov 2007). where in the Caribbean Basin (Puerto Rico)45 and in Chiapas, WEST NILE VIRUS TROPICAL ECOLOGY 123

Table 4 Logistic regression analyses of West Nile virus (WNV)-antibody status of 624 Turdus grayi and Quiscalus mexicanus* Predictor Category Coef. (SE) Z or c2 P value Odds-Ratio (Intercept) −2.52 (0.44) −5.77 0.000 Species Q. mexicanus (DF = 1) 1.0 T. grayi −0.52 (0.28) −1.84 0.066 0.60 (0.34–1.04) Habitat Rural (Site B) (DF = 1) 1.0 Urban (Site A) 0.67 (0.29) 2.32 0.020 1.95 (1.12–3.49) Age Juvenile (DF = 2) 12.97 0.002 1.0 Adult 0.95 (0.34) 2.83 0.005 2.60 (1.39–5.25) Unknown 1.66 (0.54) 3.06 0.002 5.26 (1.75–15.11) Season Pre-Peak (DF = 1) 1.0 Peak/Post-Peak 0.70 (0.31) 2.24 0.025 2.01 (1.11–3.81) *The table shows the coefficients (Coef.), standard errors of coefficients (SE), and odds ratios for categorical predictors relative to the reference level, which is given on the row of the predictor name and underlined. The statistics on the predictor row use c2 when there are more than two categories for a predictor. The Peak and post-Peak samples were not significantly different (P = 0.77) and were combined into a single season in this analysis. the Mexican state that borders Guatemala to the west.46 serving as WNV vectors particularly in urban areas and repre- The population of Cx. nigripalpus from Honduras, which bor- sent a risk for WNV transmission to humans and horses. ders Guatemala to the east, has been shown to be competent Cx. mollis and/or Cx. inflictus may play a small role as a for WNV transmission in laboratory studies.47 To corroborate WNV vector in rural areas. that this species was not involved with WNV transmission in Avian hosts that are highly infectious and frequently Puerto Barrios, we found no association of Cx. nigripalpus fed upon by competent mosquitoes will be important in population fluctuations in relation to WNV transmission. We WNV amplification.36 We found that the great-tailed grackle did notice an increase of urban chicken seroconversions (Q. mexicanus), domestic chicken (G. gallus), and clay-colored immediately following a strong peak of Cx. quinquefasciatus thrush (T. grayi) were the three most abundant birds in the population density in May and June of 2007, but not following transmission focus. Although abundant, most chickens and smaller early summer peaks in 2006 and 2008. Instead, in clay-colored thrushes are incompetent as amplifying hosts these years, low-level transmission in rural sites appeared for WNV.39 On the other hand, the grackle was determined to follow late season (September) population spikes of to be highly competent in a study of Mexican birds.39 Using Cx. mollis/Cx. inflictus.Itislikelythatthispatternoccurred these data to calculate mosquito inoculation index values in 2007 as well, although it was somewhat obscured by the suggested that the great-tailed grackle in our study may have large amount of virus and mosquito activity in the urban infected 6,000-fold more vector mosquitoes than the clay- sections of the municipality. colored thrush. An alternative calculation derived from Determining the importance of mosquito species as vectors mosquito host selection studies also implicated the grackle of WNV (“vectorial capacity”) requires characterization of as the primary amplifying host among free-ranging birds.52 vector competence, and field estimates of virus infection, rela- The great-tailed grackle appears to be a key avian amplifying tive abundance, and host selection.48 Most studied populations host of WNV in Puerto Barrios, Guatemala. A different of Cx. quinquefasciatus have been found to be moderately grackle species, the Greater Antillean grackle (Quiscalus vector-competent for WNV in laboratory experiments.49,50 niger), was implicated as a putative amplifying host for WNV Although vector competence studies for Guatemalan mos- in Puerto Rico, also during a burst of WNV activity in 2007.4 quitoes have not been reported, a laboratory colony of In 2007, increased WNV activity was detected in diverse Cx. quinquefasciatus from Tegucigalpa, Honduras, was shown locations of the Caribbean Basin. In addition to Guatemala to be moderately competent with a Guatemalan strain of and Puerto Rico, transmission was detected in northern WNV.51 A study of the host selection of selected Culex mos- Colombia.12 The widespread activity suggests a possible quito species in urban and rural areas of the municipality of climatologic basis for the virus activity. The assessment of Puerto Barrios in 2008 found that Cx. quinquefasciatus fed rainfall and temperature factors in Puerto Barrios supported primarily on birds, and to a lesser degree on humans and previous findings from temperate locations that WNV trans- other mammals.52 Therefore, these mosquitoes appear to be mission is associated with high temperature and low rainfall. Although rainfall creates new mosquito breeding sites for Table 5 some mosquito species, it can also flush existing mosquito Mosquito inoculation index (M¢) values for three candidate amplifying larvae from their current, productive breeding sites. Low rain- hosts during a high West Nile virus (WNV) transmission period in fall may also favor population growth of Cx. quinquefasciatus Puerto Barrios, Guatemala, 2007 by increasing the organic content of the water available for − Host A* S† CM¢ ( +10 5)‡ mosquito breeding, and elevated temperature accelerates the G. gallus 0.15 0.23 0.00 0 viral infection kinetics in the vectors.53,54 Q. mexicanus 0.28 0.33 1.80 5,500 The impact of WNV on public health in Guatemala and T. grayi 0.07 0.12 0.01 1 throughout tropical America remains unresolved.1,36 Our study *Abundance (A) data used in this analysis was selected from surveys conducted in the provides an opportunity to compare the enzootic transmission summer of 2007. †Seroprevalence (S)usedforG. gallus was for the period May–August 2007; for intensity of WNV in a tropical setting with that in temperate Q. mexicanus and T. grayi combined seroprevalence of adult and juvenile birds captured in June–Nov 2007 was used. North America where relatively large outbreaks of human ‡M ¢ = (A)(S)2(C) with A = relative abundance; S = seroprevalence; C = vertebrate reservoir competence derived from studies conducted with a southern Mexico strain of neurological disease have been observed. The numbers of WNV, and calculated for the vector mosquito Culex quinquefasciatus. The units for M ¢ is chicken seroconversions per 1,000 chicken-weeks we observed the relative number of infectious Cx. quinquefasciatus mosquitoes derived from the specified vertebrate host population. (30–60 between June and August 2007) is comparable to 124 MORALES-BETOULLE, KOMAR AND OTHERS those seen in Coachella Valley, California in 2004 (17–67) Financial support: This research was supported by the Centers for where few human cases were observed,43 but substantially Disease Control and Prevention cooperative agreements #1U01/ lower than that seen in Kern County, California (100–300) GH000028, #U50/CCU021236-01, and #3U51/GH000011-02, Consejo – 55 Nacional de Ciencia y Tecnologıá (CONCYT) de Guatemala grant where the human incidence was 6 17/100,000. In Puerto #FD19-03, and Fondo de Ciencia y Tecnologıá (FODECYT) de Barrios, the density of infected mosquitoes (the product of Guatemala grant #03-2007. Dr. Kilpatrick’s salary was partly sup- mosquito abundance and WNV infection rates), summed ported by grants from the National Science Foundation, EF-0914866, across mosquito species during July and August, 2007, was and the National Institutes of Health, 1R01AI090159-01. 0.19 and 0.20 WNV-infected mosquitoes per trap night, Disclaimer: Animal care and use: All animal use for this study was in respectively. This is comparable to the density of infected compliance with the National Institutes of Health guidelines for the mosquitoes in July and August in Maryland in 2004 (0.16 and humane use of laboratory animals. The research was conducted under Institutional Animal Care and Use approval from CDC as well as 0.12, respectively) when there were 16 cases in a population ´ 56 permits from the Guatemala Ministerio de Agricultura, Ganaderıay of 6 million or a yearly incidence of 0.266/100,000. Given Alimentacio´ n and the Guatemala Consejo Nacional de Areas that the population of Puerto Barrios is about 60,000, then, Protegidas (permits # 36/2008 and I-029-04). using the observed levels of enzootic transmission in Puerto Authors’ addresses: Maria E. Morales-Betoulle, Danilo Alvarez, Barrios, the expected number of human cases in 2007 was MarıáR.Lo´ pez, Silvia M. Sosa, MarıáL.Mu¨ ller, and Celia Cordo´ n- fewer than 1. This analysis does not take into account biolog- Rosales, Center for Health Studies, Universidad del Valle de Guatemala, ical differences in the vector communities of temperate and Guatemala, E-mails: [email protected], [email protected] tropical ecosystems. Nonetheless, the intensity of enzootic .edu.gt, [email protected], [email protected], mmuller@ ces.uvg.edu.gt, and [email protected]. Nicholas Komar, Arbovirus transmission that we measured in Puerto Barrios suggests Diseases Branch, CDC-NCEZID-DVBD, Fort Collins, CO, E-mail: that human illness from WNV may be rare enough to be over- [email protected]. Nicholas A. Panella, Robert S. Lanciotti, Barbara looked by health practitioners, especially given the similarity W. Johnson, and Ann M. Powers, Centers for Disease Control and of the symptoms for febrile cases to other more common Prevention, Arbovirus Diseases Branch, Fort Collins, CO, E-mails: [email protected], [email protected], [email protected], and [email protected]. illnessessuchasdengueormalaria. Jean-Luc Betoulle, Fundacio´ n para el Ecodesarrollo, Office of the Periods of intense transmission of WNV are likely to Director, Guatemala City, Guatemala, E-mail: betoullejeanluc@ recur in Puerto Barrios and other WNV transmission foci in gmail.com. A. Marm Kilpatrick, University of California Santa Guatemala and throughout tropical regions of the Americas. Cruz, Ecology and Evolutionary Biology, Santa Cruz, CA, E-mail: [email protected]. Our results suggest that sentinel chicken surveillance is a sen- sitive system to detect WNV transmission in a tropical ecosystem and could thus be used as an early warning system to potentially mitigate risk of large outbreaks in humans or REFERENCES horses. Urban populations of Cx. quinquefasciatus were the best culicine indicators of WNV transmission activity among 1. Komar N, Clark GG, 2006. West Nile virus activity in Latin America and the Caribbean. Rev Panam Salud Publica 19: the mosquitoes sampled in Puerto Barrios. Although the 112–117. effort required for monitoring WNV transmission in free- 2. Hayes CG, 1989. West Nile fever. TP Monath, ed. The Arbovi- ranging birds is less cost effective for routine arbovirus sur- ruses: Epidemiology and Ecology. Volume V. Boca Raton, FL: – veillance, changes in seroprevalence among juvenile grackles CRC Press, 5 88. 3. 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– *Identifications and nomenclature were derived from a variety of publications.1 8 †The two species could not be differentiated. References for Table S1: 1.Carpenter SJ, La Casse WJ, 1955. Mosquitoes of North America (north of Mexico). Berkeley, CA: University of California Press. 2.Clark-Gil S, Darsie RF Jr, 1983. The mosquitoes of Guatemala. Their identification, distribution and bionomics, with keys to adult females and larvae in English and Spanish. Mosq Syst 15: 151–284. 3.Darsie RF Jr., 1986. Identification of Aedes albopictus in Guatemala (Diptera: Culicidae). Mosq Syst 18: 301–306. 4.Darsie RF Jr, 1994. A revised checklist of the mosquitoes of Guatemala including a new country record, Psorophora cyanescens. J Am Mosq Control Assoc 10: 511–514. 5.Darsie RF Jr, Ward RA, 2005. Identification and geographical distribution of the mos- quitos of North America, north of Mexico. Gainesville: University Press of Florida. 6.Lane J, 1953. Neotropical Culicidae. Sa˜o Paulo: University of Sa˜o Paulo. 7.Sallum MA, Forattini OP, 1996. Revision of the Spissipes section of Culex (Melanoconion) (Diptera:Culicidae). J Am Mosq Control Assoc 12: 517–600. 8.Wilkerson RC, Strickman D, Litwak TR, 1990. Illustrated key to the female anopheline mosquitoes of Central America and Mexico. J Am Mosq Control Assoc 6: 7–34. Supplemental TABLE S2 Bird species encountered during point counts in Puerto Barrios, Department of Izabal, Guatemala, 2006–2009, presented in taxonomic order,1 with residence status2* Species Common name Family Order No. observations Residence status Dendrocygna autumnalis Black-bellied Whistling-Duck Anatidae Anseriformes 22 Resident/Domestic Anser sp. Domestic Goose Anatidae Anseriformes 55 Domestic Anatidae sp. Domestic Duck Anatidae Anseriformes 205 Domestic Cairina moschata Muscovy Duck Anatidae Anseriformes 11 Resident/Domestic Ortalis vetula Plain Chachalaca Cracidae Galliformes 3 Resident Gallus gallus Chicken Phasianidae Galliformes 3626 Domestic Meleagris gallopavo Turkey Phasianidae Galliformes 61 Domestic Fregata magnificens Magnificent Frigatebird Fregatidae Suliformes 47 Resident Phalacrocorax brasilianus Neotropic Cormorant Phalacrocoracidae Suliformes 2 Resident Pelecanus occidentalis Brown Pelican Pelecanidae Pelecanifomes 7 Resident Ardea alba Great Egret Ardeidae Pelecaniformes 25 Resident Egretta thula Snowy Egret Ardeidae Pelecaniformes 24 Resident Egretta caerulea Little Blue Heron Ardeidae Pelecaniformes 14 Resident Egretta tricolor Tricolored Heron Ardeidae Pelecaniformes 1 Resident Bubulcus ibis Cattle Egret Ardeidae Pelecaniformes 485 Resident Butorides virescens Green Heron Ardeidae Pelecaniformes 6 Resident Coragyps atratus Black Vulture Cathartidae Accipitriformes 118 Resident Cathartes aura Turkey Vulture Cathartidae Accipitriformes 60 Resident Pandion haliaetus Osprey Pandionidae Accipitriformes 1 Winter Visitor Elanus leucurus White-tailed Kite Accipitridae Accipitriformes 1 Resident Buteo magnirostris Roadside Hawk Accipitridae Accipitriformes 7 Resident Falco rufigularis Bat Falcon Falconidae Falconiformes 7 Resident Laterallus ruber Ruddy Crake Rallidae Gruiformes 10 Resident Charadrius vociferus Killdeer Charadriidae Charadriiformes 6 Winter Visitor Jacana spinosa Northern Jacana Jacanidae Charadriiformes 38 Resident Actitis macularia Spotted Sandpiper Scolopacidae Charadriiformes 1 Winter Visitor Leucophaeus atricilla Laughing Gull Laridae Charadriiformes 23 Winter Visitor Columba livia Rock Pigeon Columbidae Columbiformes 1099 Resident/Exotic Patagioenas cayennensis Pale-vented Pigeon Columbidae Columbiformes 24 Resident Patagioenas flavirostris Red-billed Pigeon Columbidae Columbiformes 287 Resident Patagioenas nigrirostris Short-billed Pigeon Columbidae Columbiformes 1 Resident Streptopelia roseogrisea African Collared-Dove Columbidae Columbiformes 8 Captive Zenaida asiatica White-winged Dove Columbidae Columbiformes 11 Resident/Captive Columbina inca Inca Dove Columbidae Columbiformes 5 Resident Columbina talpacoti Ruddy Ground-Dove Columbidae Columbiformes 232 Resident Aratinga nana Olive-throated Parakeet Psittacidae Psittaciformes 293 Resident Pionus senilis White-crowned Parrot Psittacidae Psittaciformes 63 Resident Amazona autumnalis Red-lored Parrot Psittacidae Psittaciformes 202 Resident Psittacidae sp. Psittacine sp. Psittacidae Psittaciformes 586 Resident/Captive Piaya cayana Squirrel Cuckoo Cuculidae Cuculiformes 4 Resident Crotophaga sulcirostris Groove-billed Ani Cuculidae Cuculiformes 539 Resident Glaucidium brasilianum Ferruginous Pygmy-Owl Strigidae Strigiformes 47 Resident Streptoprocne zonaris White-collared Swift Apodidae Apodiformes 40 Resident Chaetura vauxi Vaux’s Swift Apodidae Apodiformes 18 Resident Florisuga mellivora White-necked Jacobin Trochilidae Apodiformes 1 Resident Anthracothorax prevostii Green-breasted Mango Trochilidae Apodiformes 9 Resident Amazilia candida White-bellied Emerald Trochilidae Apodiformes 2 Resident Amazilia tzacatl Rufous-tailed Hummingbird Trochilidae Apodiformes 15 Resident Amazilia rutila Cinnamon Hummingbird Trochilidae Apodiformes 31 Resident Archilochus colubris Ruby-throated Hummingbird Trochilidae Apodiformes 1 Transient Trogon melanocephalus Black-headed Trogon Trogonidae Trogoniformes 29 Resident Trogon caligatus Gartered Trogon Trogonidae Trogoniformes 5 Resident Megaceryle torquata Ringed Kingfisher Alcedinidae Coraciiformes 8 Resident Chloroceryle americana Green Kingfisher Alcedinidae Coraciiformes 1 Resident Chloroceryle aenea American Pygmy Kingfisher Alcedinidae Coraciiformes 2 Resident Pteroglossus torquatus Collared Aracari Ramphastidae Piciformes 6 Resident Melanerpes aurifrons Golden-fronted Woodpecker Picidae Piciformes 1062 Resident Sphyrapicus varius Yellow-bellied Sapsucker Picidae Piciformes 1 Winter Visitor Elaenia flavogaster Yellow-bellied Elaenia Tyrannidae Passeriformes 1 Resident Contopus sordidulus Western Wood-Pewee Tyrannidae Passeriformes 2 Transient Contopus virens Eastern Wood-Pewee Tyrannidae Passeriformes 1 Winter Visitor Empidonax traillii Willow Flycatcher Tyrannidae Passeriformes 1 Transient Myiarchus tuberculifer Dusky-capped Flycatcher Tyrannidae Passeriformes 9 Resident Myiarchus crinitus Great Crested Flycatcher Tyrannidae Passeriformes 2 Winter Visitor Pitangus sulphuratus Great Kiskadee Tyrannidae Passeriformes 558 Resident Megarynchus pitangua Boat-billed Flycatcher Tyrannidae Passeriformes 45 Resident Myiozetetes similis Social Flycatcher Tyrannidae Passeriformes 302 Resident

(Continued) (Continued) SSupplementalUPPLEMENTAL T TableABLE S2S2 Continued Species Common name Family Order No. observations Residence status Tyrannus melancholicus Tropical Kingbird Tyrannidae Passeriformes 128 Resident Tityra semifasciata Masked Tityra Tityridae Passeriformes 67 Resident Tityra inquisitor Black-crowned Tityra Tityridae Passeriformes 3 Resident Vireo griseus White-eyed Vireo Vireonidae Passeriformes 4 Winter Visitor Vireo flavifrons Yellow-throated Vireo Vireonidae Passeriformes 1 Winter Visitor Cyanocorax morio Brown Jay Corvidae Passeriformes 214 Resident Stelgidopteryx serripennis Northern Rough-winged Swallow Hirundinidae Passeriformes 2 Resident Hirundo rustica Barn Swallow Hirundinidae Passeriformes 16 Winter Visitor Campylorhynchus zonatus Band-backed Wren Troglodytidae Passeriformes 109 Resident Thryothorus maculipectus Spot-breasted Wren Troglodytidae Passeriformes 1 Resident Troglodytes aedon House Wren Troglodytidae Passeriformes 478 Resident Polioptila caerulea Blue-gray Gnatcatcher Polioptilidae Passeriformes 1 Winter Visitor Hylocichla mustelina Wood Thrush Turdidae Passeriformes 2 Winter Visitor Turdus grayi Clay-colored Thrush Turdidae Passeriformes 1433 Resident Dumetella carolinensis Gray Catbird Mimidae Passeriformes 297 Winter Visitor Parkesia noveboracensis Northern Waterthrush Parulidae Passeriformes 6 Winter Visitor Vermivora cyanoptera Blue-winged Warbler Parulidae Passeriformes 2 Winter Visitor Mniotilta varia Black-and-white Warbler Parulidae Passeriformes 29 Winter Visitor Oreothlypis peregrina Tennessee Warbler Parulidae Passeriformes 19 Winter Visitor Geothlypis formosa Kentucky Warbler Parulidae Passeriformes 3 Winter Visitor Geothlypis trichas Common Yellowthroat Parulidae Passeriformes 1 Winter Visitor Setophaga citrina Hooded Warbler Parulidae Passeriformes 6 Winter Visitor Setophaga ruticilla American Redstart Parulidae Passeriformes 58 Winter Visitor Setophaga cerulea Cerulean Warbler Parulidae Passeriformes 2 Transient Setophaga magnolia Magnolia Warbler Parulidae Passeriformes 147 Winter Visitor Setophaga castanea Bay-breasted Warbler Parulidae Passeriformes 1 Transient Setophaga fusca Blackburnian Warbler Parulidae Passeriformes 5 Transient Setophaga petechia Yellow Warbler Parulidae Passeriformes 610 Winter Visitor Setophaga pensylvanica Chestnut-sided Warbler Parulidae Passeriformes 10 Winter Visitor Setophaga coronata Yellow-rumped Warbler Parulidae Passeriformes 1 Winter Visitor Setophaga virens Black-throated Green Warbler Parulidae Passeriformes 2 Winter Visitor Ramphocelus passerinii Passerini’s Tanager Thraupidae Passeriformes 1 Resident Thraupis episcopus Blue-gray Tanager Thraupidae Passeriformes 523 Resident Thraupis abbas Yellow-winged Tanager Thraupidae Passeriformes 75 Resident Saltator coerulescens Grayish Saltator Genus incertae sedis Passeriformes 7 Resident Saltator maximus Buff-throated Saltator Genus incertae sedis Passeriformes 20 Resident Saltator atriceps Black-headed Saltator Genus incertae sedis Passeriformes 52 Resident Volatinia jacarina Blue-black Grassquit Emberizidae Passeriformes 128 Resident Sporophila americana Variable Seedeater Emberizidae Passeriformes 2 Resident Sporophila torqueola White-collared Seedeater Emberizidae Passeriformes 395 Resident Piranga rubra Summer Tanager Cardinalidae Passeriformes 78 Winter Visitor Piranga olivacea Scarlet Tanager Cardinalidae Passeriformes 1 Transient Pheucticus ludovicianus Rose-breasted Grosbeak Cardinalidae Passeriformes 2 Winter Visitor Dives dives Melodious Blackbird Icteridae Passeriformes 1168 Resident Quiscalus mexicanus Great-tailed Grackle Icteridae Passeriformes 6353 Resident Molothrus aeneus Bronzed Cowbird Icteridae Passeriformes 7 Resident Molothrus oryzivorus Giant Cowbird Icteridae Passeriformes 6 Resident Icterus spurius Orchard Oriole Icteridae Passeriformes 24 Winter Visitor Icterus pectoralis Spot-breasted Oriole Icteridae Passeriformes 20 Resident Icterus gularis Altamira Oriole Icteridae Passeriformes 156 Resident Icterus galbula Baltimore Oriole Icteridae Passeriformes 41 Winter Visitor Psarocolius montezuma Montezuma Oropendola Icteridae Passeriformes 748 Resident Euphonia affinis Scrub Euphonia Fringillidae Passeriformes 20 Resident Passer domesticus House Sparrow Passeridae Passeriformes 1 Resident *A total of 23,905 observations were recorded. References for Table S2: 1.American Ornithologists’ Union, 2011. Check-List of North American Birds. Seventh edition. Available at: http://www.aou.org/checklist/north/index.php. Accessed 11 July 2012. 2.Howell SNG, Webb S, 1995. A guide to the birds of Mexico and northern Central America. New York: Oxford University Press. Supplemental TABLE S3 Bird species (N = 136) captured by mist net in the municipality of Puerto Barrios, Department of Izabal, Guatemala, 2006–2009, presented in taxonomic order,1 with residence status2 Species Common name Family Order Number captured Residence status Butorides virescens Green Heron Ardeidae Pelecaniformes 3 Resident Falco rufigularis Bat Falcon Falconidae Falconiformes 2 Resident Laterallus ruber Ruddy Crake Rallidae Gruiformes 3 Resident Charadrius semipalmatus Semipalmated Plover Charadriidae Charadriiformes 1 Winter Visitor Actitis macularius Spotted Sandpiper Scolopacidae Charadriiformes 2 Winter Visitor Columba livia Rock Pigeon Columbidae Columbiformes 1 Resident Patagioenas cayennensis Pale-vented Pigeon Columbidae Columbiformes 1 Resident Patagioenas sp. Pigeon sp. Columbidae Columbiformes 1 Resident Zenaida asiatica White-winged Dove Columbidae Columbiformes 1 Resident Columbina inca Inca Dove Columbidae Columbiformes 1 Resident Columbina passerina Common Ground-Dove Columbidae Columbiformes 1 Resident Columbina minuta Plain-breasted Ground-Dove Columbidae Columbiformes 9 Resident Columbina talpacoti Ruddy Ground-Dove Columbidae Columbiformes 138 Resident Claravis pretiosa Blue Ground-Dove Columbidae Columbiformes 1 Resident Leptotila plumbeiceps Grey-headed Dove Columbidae Columbiformes 10 Resident Aratinga nana Olive-throated Parakeet Psittacidae Psittaciformes 4 Resident Piaya cayana Squirrel Cuckoo Cuculidae Cuculiformes 4 Resident Coccyzus erythropthalmus Black-billed Cuckoo Cuculidae Cuculiformes 2 Transient Crotophaga sulcirostris Groove-billed Ani Cuculidae Cuculiformes 157 Resident Glaucidium brasilianum Ferruginous Pygmy-Owl Strigidae Strigiformes 18 Resident Nyctidromus albicollis Common Pauraque Caprimulgidae Caprimulgiformes 1 Resident Chaetura pelagica Chimney Swift Apodidae Apodiformes 1 Transient Phaethornis longirostris Long-billed Hermit Trochilidae Apodiformes 5 Resident Phaethornis striigularis Stripe-throated Hermit Trochilidae Apodiformes 23 Resident Campylopterus curvipennis Wedge-tailed Sabrewing Trochilidae Apodiformes 1 Resident Florisuga mellivora White-necked Jacobin Trochilidae Apodiformes 2 Resident Anthracothorax prevostii Green-breasted Mango Trochilidae Apodiformes 46 Resident Hylocharis eliciae Blue-throated Sapphire Trochilidae Apodiformes 2 Resident Amazilia candida White-bellied Emerald Trochilidae Apodiformes 37 Resident Amazilia beryllina Berylline Hummingbird Trochilidae Apodiformes 2 Resident Amazilia tzacatl Rufous-tailed Hummingbird Trochilidae Apodiformes 77 Resident Amazilia rutila Cinnamon Hummingbird Trochilidae Apodiformes 59 Resident Archilochus colubris Ruby-throated Hummingbird Trochilidae Apodiformes 13 Transient Trogon melanocephalus Black-headed Trogon Trogonidae Trogoniformes 3 Resident Momotus momota Blue-crowned Motmot Momotidae Coraciiformes 5 Resident Megaceryle torquata Ringed Kingfisher Alcedinidae Coraciiformes 1 Resident Chloroceryle americana Green Kingfisher Alcedinidae Coraciiformes 9 Resident Chloroceryle aenea American Pygmy Kingfisher Alcedinidae Coraciiformes 10 Resident Pteroglossus torquatus Collared Aracari Ramphastidae Piciformes 1 Resident Melanerpes aurifrons Golden-fronted Woodpecker Picidae Piciformes 98 Resident Sphyrapicus varius Yellow-bellied Sapsucker Picidae Piciformes 3 Winter Visitor Dendrocincla anabatina Tawny-winged Woodcreeper Furnariidae Passeriformes 3 Resident Myiopagis viridicata Greenish Elaenia Tyrannidae Passeriformes 2 Resident Elaenia flavogaster Yellow-bellied Elaenia Tyrannidae Passeriformes 14 Resident Mionectes oleaginus Ochre-bellied Flycatcher Tyrannidae Passeriformes 26 Resident Leptopogon amaurocephalus Sepia-capped Flycatcher Tyrannidae Passeriformes 1 Resident Todirostrum cinereum Common Tody-Flycatcher Tyrannidae Passeriformes 3 Resident Tolmomyias sulphurescens Yellow-olive Flycatcher Tyrannidae Passeriformes 8 Resident Contopus sordidulus Western Wood-Pewee Tyrannidae Passeriformes 6 Transient Contopus virens Eastern Wood-Pewee Tyrannidae Passeriformes 6 Winter Visitor Contopus cinereus Tropical Pewee Tyrannidae Passeriformes 1 Resident Contopus sp. Pewee sp. Tyrannidae Passeriformes 3 Undetermined Empidonax flaviventris Yellow-bellied Flycatcher Tyrannidae Passeriformes 9 Winter Visitor Empidonax trailii Willow Flycatcher Tyrannidae Passeriformes 6 Transient Empidonax albigularis White-throated Flycatcher Tyrannidae Passeriformes 1 Winter Visitor Empidonax minimus Least Flycatcher Tyrannidae Passeriformes 2 Winter Visitor Empidonax sp. Empidonax flycatcher sp. Tyrannidae Passeriformes 81 Transient Myiarchus tuberculifer Dusky-capped Flycatcher Tyrannidae Passeriformes 1 Resident Myiarchus crinitus Great Crested Flycatcher Tyrannidae Passeriformes 6 Winter Visitor Myiarchus tyrannulus Brown-crested Flycatcher Tyrannidae Passeriformes 7 Resident Pitangus sulphuratus Great Kiskadee Tyrannidae Passeriformes 50 Resident Megarynchus pitangua Boat-billed Flycatcher Tyrannidae Passeriformes 3 Resident Myiozetetes similis Social Flycatcher Tyrannidae Passeriformes 34 Resident Tyrannus melancholicus Tropical Kingbird Tyrannidae Passeriformes 6 Resident Tyrannus tyrannus Eastern Kingbird Tyrannidae Passeriformes 1 Transient Tityra semifasciata Masked Tityra Tityridae Passeriformes 5 Resident Pachyramphus polychopterus White-winged Becard Tityridae Passeriformes 2 Resident (Continued) (Continued) SSupplementalUPPLEMENTAL T TableABLE S3S3 Continued Species Common name Family Order Number captured Residence status Manacus candei White-collared Manakin Pipridae Passeriformes 14 Resident Pipra mentalis Red-capped Manakin Pipridae Passeriformes 2 Resident Vireo griseus White-eyed Vireo Vireonidae Passeriformes 18 Winter Visitor Vireo olivaceus Red-eyed Vireo Vireonidae Passeriformes 28 Transient Vireo flavoviridis Yellow-green Vireo Vireonidae Passeriformes 5 Summer Visitor Cyanocorax morio Brown Jay Corvidae Passeriformes 1 Resident Riparia riparia Bank Swallow Hirundinidae Passeriformes 1 Transient Petrochelidon pyrrhonota Cliff Swallow Hirundinidae Passeriformes 1 Transient Hirundo rustica Barn Swallow Hirundinidae Passeriformes 8 Winter Visitor Campylorhynchus zonatus Band-backed Wren Troglodytidae Passeriformes 4 Resident Thryothorus maculipectus Spot-breasted Wren Troglodytidae Passeriformes 9 Resident Troglodytes aedon House Wren Troglodytidae Passeriformes 11 Resident Catharus fuscescens Veery Turdidae Passeriformes 1 Transient Catharus minimus Gray-cheeked Thrush Turdidae Passeriformes 1 Transient Catharus ustulatus Swainson’s Thrush Turdidae Passeriformes 77 Winter Visitor Hylocichla mustelina Wood Thrush Turdidae Passeriformes 19 Winter Visitor Turdus grayi Clay-colored Thrush Turdidae Passeriformes 477 Resident Turdus assimilis White-throated Thrush Turdidae Passeriformes 1 Resident Dumetella carolinensis Gray Catbird Mimidae Passeriformes 365 Winter Visitor Mimus gilvus Tropical Mockingbird Mimidae Passeriformes 1 Resident Seiurus aurocapilla Ovenbird Parulidae Passeriformes 52 Winter Visitor Helmitheros vermivorum Worm-eating Warbler Parulidae Passeriformes 8 Winter Visitor Parkesia motacilla Louisiana Waterthrush Parulidae Passeriformes 6 Winter Visitor Parkesia noveboracensis Northern Waterthrush Parulidae Passeriformes 144 Winter Visitor Vermivora chrysoptera Golden-winged Warbler Parulidae Passeriformes 1 Winter Visitor Vermivora cyanoptera Blue-winged Warbler Parulidae Passeriformes 4 Winter Visitor Mniotilta varia Black-and-white Warbler Parulidae Passeriformes 18 Winter Visitor Protonotaria citrea Prothonotary Warbler Parulidae Passeriformes 9 Winter Visitor Oreothlypis peregrina Tennessee Warbler Parulidae Passeriformes 35 Winter Visitor Geothlypis poliocephala Gray-crowned Yellowthroat Parulidae Passeriformes 1 Resident Geothlypis philadelphia Mourning Warbler Parulidae Passeriformes 5 Transient Geothlypis formosa Kentucky Warbler Parulidae Passeriformes 29 Winter Visitor Geothlypis trichas Common Yellowthroat Parulidae Passeriformes 44 Winter Visitor Setophaga citrina Hooded Warbler Parulidae Passeriformes 48 Winter Visitor Setophaga ruticilla American Redstart Parulidae Passeriformes 27 Winter Visitor Setophaga magnolia Magnolia Warbler Parulidae Passeriformes 56 Winter Visitor Setophaga fusca Blackburnian Warbler Parulidae Passeriformes 1 Transient Setophaga petechia Yellow Warbler Parulidae Passeriformes 82 Winter Visitor Setophaga pensylvanica Chestnut-sided Warbler Parulidae Passeriformes 17 Winter Visitor Cardellina canadensis Canada Warbler Parulidae Passeriformes 4 Transient Cardellina pusiilla Wilson’s Warbler Parulidae Passeriformes 2 Winter Visitor Icteria virens Yellow-breasted Chat Parulidae Passeriformes 31 Winter Visitor Parulidae sp. Warbler sp. Parulidae Passeriformes 1 Undetermined Ramphocelus passerinii Passerini’s Tanager Thraupidae Passeriformes 17 Resident Thraupis episcopus Blue-gray Tanager Thraupidae Passeriformes 48 Resident Thraupis abbas Yellow-winged Tanager Thraupidae Passeriformes 4 Resident Saltator coerulescens Grayish Saltator Emberizidae* Passeriformes 14 Resident Saltator maximus Buff-throated Saltator Emberizidae* Passeriformes 10 Resident Saltator atriceps Black-headed Saltator Emberizidae* Passeriformes 10 Resident Volatinia jacarina Blue-black Grassquit Emberizidae Passeriformes 71 Resident Sporophila americana Variable Seedeater Emberizidae Passeriformes 55 Resident Sporophila torqueola White-collared Seedeater Emberizidae Passeriformes 460 Resident Oryzoborus funereus Thick-billed Seedfinch Emberizidae Passeriformes 52 Resident Piranga rubra Summer Tanager Cardinalidae Passeriformes 17 Winter Visitor Pheucticus ludovicianus Rose-breasted Grosbeak Cardinalidae Passeriformes 6 Winter Visitor Cyanocompsa cyanoides Blue-black Grosbeak Cardinalidae Passeriformes 1 Resident Passerina caerulea Blue Grosbeak Cardinalidae Passeriformes 4 Winter Visitor Passerina cyanea Indigo Bunting Cardinalidae Passeriformes 51 Winter Visitor Passerina ciris Painted Bunting Cardinalidae Passeriformes 14 Winter Visitor Spiza americana Dickcissel Cardinalidae Passeriformes 1 Transient Dives dives Melodious Blackbird Icteridae Passeriformes 18 Resident Quiscalus mexicanus Great-tailed Grackle Icteridae Passeriformes 155 Resident Molothrus aeneus Bronzed Cowbird Icteridae Passeriformes 4 Resident Molothrus oryzivorus Giant Cowbird Icteridae Passeriformes 5 Resident Icterus spurius Orchard Oriole Icteridae Passeriformes 15 Winter Visitor Icterus pustulatus Streak-backed Oriole Icteridae Passeriformes 1 Resident Icterus pectoralis Spot-breasted Oriole Icteridae Passeriformes 8 Resident Icterus gularis Altamira Oriole Icteridae Passeriformes 2 Resident

(Continued) (Continued) SSupplementalUPPLEMENTAL T TableABLE S3S3 Continued Species Common name Family Order Number captured Residence status Icterus galbula Baltimore Oriole Icteridae Passeriformes 41 Winter Visitor Amblycercus holosericeus Yellow-billed Cacique Icteridae Passeriformes 5 Resident Psarocolius montezuma Montezuma’s Oropendola Icteridae Passeriformes 1 Resident Euphonia affinis Scrub Euphonia Fringillidae Passeriformes 13 Resident Euphonia hirundinacea Yellow-throated Euphonia Fringillidae Passeriformes 15 Resident *Genus incertae sedis. References for Table S3: 1.American Ornithologists’ Union, 2011. Check-list of North American Birds. Seventh edition. Available at: http://www.aou.org/checklist/north/index.php. Accessed 11 July 2012. 2.Howell SNG, Webb S, 1995. A guide to the birds of Mexico and northern Central America. New York: Oxford University Press.

Supplemental TABLE S4 West Nile virus (WNV) exposure among free-ranging resident bird species, expressed as the proportion serum antibody-positive (Prev), from Puerto Barrios, Guatemala, 2006–2009 Year Bird species No. pos No. sampled* Prev (95% CI) Mar–Dec 2006 Dives dives 2 3 0.66 (0.02–0.93) Euphonia hirundinacea 1 5 0.20 (0.03–0.62) Glaucidium brasilianum 2 6 0.33 (0.09–0.70) Saltator atriceps 1 3 0.33 (0.06–0.79) Saltator coerulescens 1 8 0.12 (0.02–0.47) Thraupis abbas 1 2 0.50 (0.09–0.90) Thraupis episcopus 1 13 0.07 (0.01–0.33) Turdus grayi 10 169 0.06 (0.03–0.10) 2007 Patagioenas cayennensis 1 1 1.00 (0.20–1.00) Columbina talpacoti 2 39 0.05 (0.01–0.16) Crotophaga sulcirostris 3 68 0.04 (0.01–0.12) Dives dives 1 7 0.14 (0.02–0.51) Glaucidium brasilianum 1 6 0.16 (0.03–0.56) Icterus pectoralis 1 4 0.25 (0.04–0.69) Leptotila plumbeiceps 1 2 0.50 (0.09–0.90) Manacus candei 1 11 0.09 (0.01–0.37) Momotus momota 1 3 0.33 (0.06–0.79) Myiozetetes similis 1 11 0.09 (0.01–0.37) Pitangus sulphuratus 2 18 0.11 (0.03–0.32) Quiscalus mexicanus 22 67 0.32 (0.22–0.44) Saltator coerulescens 1 2 0.50 (0.09–0.90) Thraupis episcopus 3 30 0.10 (0.03–0.25) Turdus grayi 20 171 0.11 (0.07–0.17) 2008 Columba livia 1 1 1.00 (0.20–1.00) Columbina talpacoti 1 37 0.02 (0.004–0.13) Crotophaga sulcirostris 2 21 0.09 (0.02–0.28) Dives dives 2 4 0.50 (0.15–0.85) Leptotila plumbeiceps 1 6 0.16 (0.03–0.56) Myiozetetes similis 1 7 0.14 (0.02–0.51) Pitangus sulphuratus 1 6 0.16 (0.03–0.56) Quiscalus mexicanus 9 48 0.18 (0.10–0.31) Turdus grayi 11 64 0.17 (0.09–0.28) Jan–Mar 2009 Columbina talpacoti 1 46 0.02 (0.003–0.11) Dives dives 1 4 0.25 (0.04–0.69) Glaucidium brasilianum 1 3 0.33 (0.06–0.79) Quiscalus mexicanus 3 27 0.11 (0.03–0.28) Turdus grayi 6 62 0.09 (0.04–0.19) *Individuals recaptured in the same period of analysis were counted only once.