VECTOR-BORNE AND ZOONOTIC DISEASESVolume 9, Number 6, 2009© Mary Ann Liebert, Inc.DOI: 10.1089/vbz.2008.0130

Predicting West Nile Virus Seroprevalence in Wild Birds in Senegal

Veronique Chevalier,1 Pierre Reynaud,2 Thierry Lefrançois,3 Benoit Durand,4

Francois Baillon,5 Gilles Balança,1 Nicolas Gaidet,1 Bernard Mondet,5 and Renaud Lancelot6

Abstract

West Nile fever epidemiology is complex, and the role of birds in the maintenance, amplification, and dissemi-nation of the West Nile virus (WNV) remains partially unknown. In 2003, a serological study was performed inSenegal, where West Nile infection is considered endemic. The goal was to identify potential reservoirs of WNVamong bird species present in the Ferlo area (northern Senegal) and the Senegal River Valley, and to screen theecological factors possibly related to West Nile infection. Serological data were analyzed using a generalized lin-ear model. Statistical association between ecological factors and the risk of infection were then modeled to de-rive a species-specific risk. A cross-validation was conducted. The overall observed prevalence rate was 5.5%(n � 422). Thirteen bird species were found positive, from which five were migrating: Lanius senator, Anthus triv-ialis, Hippolais opaca, Jynx torquilla, and Cercotrichas galactotes. The nesting type in resident birds was positivelycorrelated with the risk of infection (odds ratio [OR] � 11, p 5 0.0003); the gregariousness level of birds appearedas a protective factor (OR � 0.3, p � 0.01). The predicted prevalence varied between 1% and 39% for residentspecies and between 1% and 7% for migrating species. Results of model internal validation were satisfactory atthe individual and species level. However, more field and experimental investigations are needed to confirmthese preliminary results and help target the future research and surveillance in Senegal.

Key Words: West Nile virus—Wild avifauna—Senegal—Risk factors—Prediction.

589

Introduction

West Nile fever is an emerging arbovirosis caused bythe West Nile virus (WNV) (Flavivirus, Flaviviridae).

The epidemiological cycle involves wild birds and Culicidaemosquitoes (mainly Culex genus). Horse and man are dead-end hosts but the disease is a veterinary and human public-health issue. The role of birds in the maintenance, amplifica-tion, and dissemination of the virus remains partiallyunknown. Recent studies suggested that the bird communitystructure, diversity, and presence of particular species maydetermine the mosquito infection rate (Kilpatrick et al. 2006).Nestling as well as hatch-year birds may be important factorsof amplification (Scott and Edman 1991, Hamer et al. 2008).

In Senegal, the WNV was isolated several times from Aedesand Culex mosquitoes in the Senegal River Valley (Traore-

Lamizana et al. 1994). Different serological surveys, under-taken in the Ferlo area (northern Senegal) in humans (Mur-gue et al. 2002), horses (Chevalier et al. 2006), and sentinelchickens (Chevalier et al. 2007), suggested that the WNV trans-mission regularly and intensively occurred in this country.

In Senegal, the avian biodiversity is high, especially in theSenegal River Valley, which is a major wintering area for Eu-ropean migratory birds.

To target future research and surveillance, we sought toidentify ecological factors linked to the risk of West Nile in-fection. A serological survey was conducted to estimate theserological point prevalence of anti-WNV antibodies in res-ident and local or Palearctic migratory bird species. Statisti-cal association between ecological factors and the risk of in-fection was then evaluated and modeled to derive aspecies-specific risk.

1International Centre of Research in Agronomy for Development (CIRAD, UR AGIRs), 34398 Montpellier Cedex 5, France.2Institut de Recherche pour le Développement (IRD), Département Société et Santé, les Orteaux, 05700, Trescléoux, France.3International Centre of Research in Agronomy for Development (CIRAD, UMR 15), Domaine de Duclos, Prise d’eau, 97170 Petit Bourg,

France.4Agence Française de Sécurité Sanitaire des Aliments (AFSSA), 94706 Maisons-Alfort Cedex, France.5Institut de Recherche pour le Développement (IRD), UR178- Conditions et Territoires d’Emergence des Maladies (CTEM), Unité des

Virus Emergents, Faculté de Médecine de Marseille, 13005 Marseille Cedex 05, France.6International Center of Research in Agronomy, (CIRAD, UMR 15), Montpellier Cedex 5, France.

Material and Methods

Study areas

Two study sites were selected based on previous studies(serology) and expert knowledge (ornithology). The first sitewas in the close vicinity of the Barkedji village, in the Ferloarea (Fig. 1). The Ferlo is a Sahelian region characterized bytemporary ponds that fill up in July and remain flooded un-til November–December. When flooded, ponds constitute afavorable biotope for Aedes and Culex mosquitoes. Manyspecies of endemic birds nest around these ponds, and mi-gratory birds are attracted by the water and abundant food(Morel and Morel 1978).

The second site was located in the Djoud’j National Park(PNOD; Fig. 1). The PNOD is a large wetland located in theSenegal River delta, made of a large lake surrounded bystreams, ponds, and backwaters.

Bird trapping

Birds nesting in Barkedji leave in December when pondsdry up. Birds were trapped from September 28 to October 7,2003 in Barkedji to guarantee the trapping of both residentand migrating birds. The trapping period was similar in thePNOD. Ground-level mist nets (12 � 2.8 m, 36-mm mesh)were used to focus on passerine birds, which are supposedto play a prominent role in the epidemiological cycle of WNV(Komar et al. 2003). Nets were operated from sunrise to 11A.M. and from 5 p.m. to sunset. Birds were sampled from

the wing or jugular vein. Sera were centrifuged and storedat 4°C until they were transported to Dakar.

Serological analysis

Sera were analyzed using an epitope-blocking enzyme-linked immunosorbent assay using the antigen used byBlitvich et al. (2003).

This test was performed using the WNV-specific mono-clonal antibody (MAb) 3.1112G (Chemicon, Rosemount, IL).The original technique was slightly modified: WNV antigen(100 mL, 1/4000 in 50 mM NaHCO3, 50 mM Na2CO3, pH 9.6)was coated overnight at 4°C, followed by 1 hour at 37°C, onMaxisorp 96-well plates (NUNC, Thermo Fisher Scientific,Roskilde, Denmark). Plates were washed four times withphosphate-buffered saline (PBS; pH 7.4) containing 0.1%Tween 20, blocked with 200 mL of blocking buffer (PBS con-taining 0.1% Tween 20 and 0.2% bovine serum albumin) at37°C for 45 minutes, and washed again. Fifty microliters ofsera diluted 1/10 in blocking buffer were added to each well,and incubated at 37°C for 2 hours. After four washes, 50 mLof MAb, diluted 1/4000 in blocking buffer, was added toeach well and incubated for 1 hour at 37°C, followed by 1hour at 4°C. After four more washes, 50 mL of horseradishperoxidase labeled rabbit anti mouse immunoglobulin G(Serotec; Oxford, UK), diluted 1/500 in blocking buffer, wasadded to each well and incubated for 1 hour at 37°C. Afterfour washes, 200 mL of tetramethylbenzidine substrate(Sigma Aldrich, St. Louis, MO) were added. After 30 min-

CHEVALIER ET AL.590

FIG. 1. Location of the Barkedji village and the Djoud’j National Park (PNOD) in Senegal.

utes, optical densities were measured at 630 nm. The abilityof the test sera to block the binding of the MAbs to WNVantigen was compared to the blocking ability of chickenserum without antibody to WNV (Vector Laboratories,Burlingame, CA). Data were expressed as relative percent-ages. An inhibition value �30% was considered to indicatethe presence of viral antibodies.

Data analysis

Seven factors possibly related with the exposition of birdsto mosquito bites were included as explanatory variables: thetrapping location (Barkedji, PNOD), the migratory status (res-ident in Africa, or migratory breeding outside Africa), thefeeding behavior (flying or settled), the resting site (ground,bush, canopy), the type of nest (on the ground, platform, cupor cavity nest—so-called “medium, or bulky” nest), the herdinstinct level (high, low), and the affinity with urban areas(high, moderate, low). Among these variables, the nest typewas treated specifically because of its particular meaning: asthe exposure level of birds during the breeding season de-pends on nesting location, the nest type effect cannot be dis-sociated from the nesting site effect (i.e., the migrating sta-tus). Therefore in the analysis, the nesting type variable wasstudied separately in resident and in migratory birds.

A bivariate analysis using Fisher exact test was performedto assess the relation between the observed prevalence rateand each variable. Variables associated with serologicalpoint prevalence (p value � 0.20) were selected for inclusionin a logistic regression model where the individual serolog-ical status (positive or negative serum) was the response vari-able. For the reasons detailed above, the nest type variablewas not included alone but only in interaction with the mi-grating status variable. A stepwise procedure was performedto select the best model according to the Akaike informationcriterion (Burnham and Anderson 2002). Odds ratios (ORs)and their confidence intervals (CIs) were computed for theexplanatory variables of the resulting model, and the modelwas used to predict the prevalence rate and its CI for eachcombination of ecological characteristics corresponding tothe tested species.

A cross-validation was conducted (Stone 1974): half of thetested birds were randomly chosen for fitting the model,which was then used to predict the serological status of theremaining birds. Prediction error was evaluated at the indi-vidual level and at the species level. Individual-level errorwas the usual proportion of incorrect predictions. Forspecies-level cross-validation, two prevalence classes wereconsidered to distinguish low prevalence species (�10%)and high prevalence species (�10%). Prediction error wasthe proportion of misclassified species, weighted by thenumber of tested animals.

Results

Serological results

A total of 422 birds were sampled, belonging to 16 fami-lies and 49 species. One hundred seventy birds were trappedin Barkedji, belonging to 8 families and 16 species, most ofthem being resident (89%). In the PNOD, 252 birds were sam-pled, belonging to 13 families and 33 species. Most of themwere migratory (80%). The most frequently trapped birds be-

longed to the Muscicapidae family (29%, n � 122), followedby the Ploceidae (24%, n � 100), the Columbidae (12%, n � 51),and the Alcedinidae (8%, n � 35).

The overall observed prevalence rate was 5.5% (n � 422).Anti-WNV antibodies were detected in 23 birds from 7 fam-ilies and 13 species, among which five were migrating:Woodchat Shrike (Lanius senator), Tree Pipit (Anthus trivialis),Western Olivaceus Warbler (Hippolais opaca), Eurasian Wry-neck (Jynx torquilla), and Rufous Scrub-robin (Cercotrichasgalactotes).

For 19 species, the number of trapped birds was �5, rep-resenting 87% of the totality of tested birds (n � 369). Amongthese species, the prevalence rate was �10% for each of thethree Columbidae species, as well as in two Muscicapidaespecies. The prevalence rate was �10% for H. opaca and Plo-ceus cucullarus. No positive bird was found for the 13 otherspecies with �5 trapped birds (Table 1).

The 13 remaining species were weakly represented, with�5 trapped animals per species. Positive birds were foundin six of these species (Table 2): both tested Laniidae species,Urocolius macrourus (Coliidae), Jynx torquilla (Picidae), Ploceusvelatus (Ploceidae), and Anthus trivialis (Motacillidae).

Bivariate analysis

Prevalence rate was higher in resident (8.4%, n � 203) thanin migrating birds (2.7%, n � 219; p � 0.02), whereas theserates were similar for birds trapped in the PNOD (4.8%, n �252) and in Barkedji (6.5%, n � 170; p � 0.51).

The prevalence rate was higher in species showing a lowherd instinct (11.5%, n � 78) than in gregarious species (4.1%,n � 344; p � 0.02).

In resident birds, a high prevalence rate was observed inspecies building medium nests (19.2%, n � 73), whereas lowrates were observed for the two other types: 2.9% for birdsbuilding bulky nests (n � 102) and 0.0% for birds nesting onthe ground (n � 28; p � 0.0001). In migratory birds, nestingtype was not associated with prevalence rate; none of the mi-gratory birds belonged to species building bulky nests;prevalence rate was similar for the two other nesting types:medium nests (3.6%, n � 138) and nests built on the ground(1.2%, n � 81; p � 0.41).

Birds feeding on flight were as frequently infected (4.8%,n � 167) as birds feeding settled, in trees, or on the ground(5.9%, n � 255; p � 0.67). Prevalence rate was high in birdsresting on the ground (10.2%, n � 88), with lower values be-ing observed in birds resting in bush (4%, n � 125) or in thecanopy (4.3%, n � 209; p � 0.11). Last, affinity with urban ar-eas was not associated with the prevalence rate, ranging from3.7% (n � 107) for birds living in villages, 8% (n � 160) forperidomestic birds, and 3.9% (n � 154) for birds living farfrom urban areas (p � 0.25).

Logistic model

The explanatory variables selected for inclusion in the lo-gistic regression model were: the migration status, the herdinstinct, the resting site, and the interaction between the mi-gration status and the nest type. As none of the migratorybirds belonged to a species building bulky nests and as sim-ilar prevalence rates had been observed in species buildingbulky nests and in species nesting on the ground, the nesttype variable was recoded in two categories: medium nests

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and other nest types (bulky nests and nests built on theground). The stepwise model selection procedure led to theexclusion of the resting site variable, and the resulting modelthus included the migration status and its interaction withthe nest type, and the herd instinct (Table 3). The largest ef-fect was attributed to the nest type in resident birds with anOR of 11.0 (p � 0.0003) for medium nests (reference: bulkynests or nests built on soil), whereas the nesting type effectwas not significant in migratory birds. High herd instinct(reference: low) had a protective effect with OR of 0.3 (p �0.01).

Predicted prevalence varied between 1% and 39% for res-ident species and between 1% and 7% for migratory species.This predicted prevalence (and its CI) refers to combinationsof ecological characteristics (herd instinct and interaction be-tween nest type and migration status); therefore, speciessharing the same ecological characteristics have identicalpredicted prevalence and CIs (Tables 1 and 2). Cross-vali-dation procedure yielded an individual prediction error of5%: when half of the data set was left out when fitting themodel, predicted serological status for the other half was cor-rectly predicted for 95% of the birds. At the species level,prediction error was 11%: 89% of the species were correctlyclassified either as low prevalence (�10%) species or as highprevalence (�10%) species.

When considering the 19 species for which �5 birds weretrapped (Table 1), the predicted status (low versus highprevalence) was correct in all species. High prevalence sta-tus was predicted (and observed) for four resident speciesand none of the migratory species. Low prevalence statuswas predicted for 11 migratory species and four residentspecies.

Model predictions were correct for two-thirds of the 30weakly represented species (�5 tested birds; Table 2). Ahigh-prevalence status was predicted for 10 resident species,among which, Urocolius macrourus and Laniarius barbaruswere found positive with prevalence figures of 1/3 and 1/1,respectively. Conversely, the model predicted a low preva-lence level for nine resident species and for 11 migratoryspecies, positive results being observed in one residentspecies (Ploceus velatus: 1/1) and in three migratory species(Anthus trivialis: 1/2, Lanius senator: 1/3, and Jynx torquilla:2/3).

Discussion

The overall serological prevalence rate (5.5%) confirmedthat WNV was transmitted to wild birds in Senegal either inthe Ferlo or in the Senegal River basin. Senegal, where the

WNV circulation is endemic, is also a major area of winter-ing for Palearctic migrating birds. To explain the WNV pres-ence in Palearctic areas, current epidemiological knowledgeassumes that the virus may be regularly transported fromAfrica to Europe by migrating birds (Malkinson and Banet2002). The higher prevalence rate observed in our study inresident birds (8.4%)—compared with migratory species(3%)—is consistent with this assumption: the exposure to thevirus was longer in the former than in the latter species.

In migratory birds, the prevalence rate of 2.7% was closeto observations made in birds sampled in southern Francein 2004 possibly coming from sub-Saharan Africa (Jourdain2006). Birds’ serological status is not related to their epi-demiological role in WNV transmission. A large number ofbirds should be ringed in Senegal and trapped and tested forviral presence when arriving in Europe to accurately assessthe risk of introduction of the virus in Europe by these birds.

Large variations of the observed point prevalence are re-ported in the literature. In the West Indies in 2002, it rangedfrom 3.3% in Jamaica (n � 542; Dupuis et al. 2003) to 15%(n � 33) in Dominican Republic (Komar et al. 2003). In theAfrican continent, point serological prevalence rates was40% in the Nile delta, Egypt, in 1950 (n � 420; Taylor et al.1956) and 12.5% in South Africa in 1962–1965 (Jupp 2001). Inthe Middle East, seroprevalence rate was 11.5% and 11.3%on resident birds in Israel in 1965–1966 (Nir et al. 1969, 1972).However, comparisons are difficult because the ecosystemsand epidemiological situations differ. Within the same area,large differences were also observed between species: inEgypt, the point prevalence was 13% in domestic chicken(n � 15) and 65% in hooded crows (n � 163; Taylor et al.1956). However, our results corroborate some results of pre-vious studies: the high point prevalence observed in theColumbidae family was reported in Israel (Nir et al. 1969,1972); Woodchat Strike (Lanius senator) was identified as can-didate species for virus introduction from sub-SaharanAfrica to Europe (Jourdain 2006).

More information is needed on the biology of the sampledspecies. As a matter of fact, seroprevalence is an indirect in-dicator of the exposure since other factors may influence itsvalue. The high prevalence rate in Streptopelia species maybe explained by the fact that they live in, or close to, urbanareas, which are preferential habitats for Culex mosquitoes,but also by a greater longevity compared with birds of otherfamilies. Bird age may be a confounding factor to study theexposure level using serological data. Juvenile and adultbirds can be easily distinguished. However, this distinctionis not accurate enough to take into account the age as a con-founding factor. Due to a lack of field method to precisely

CHEVALIER ET AL.594

Table 3. Results of the Logistic Model Selected by the Stepwise Regression Procedure

Variable Reference Value OR (95% CI) p valuea

Migration status Migrating Resident 1.4 (0.2–29.6) 0.8Nest type: migrating birds Bulky or soil nest Medium nest 1.9 (0.3–37.9) 0.6Nest type: resident birds Bulky or soil nest Medium nest 11.0 (3.6–49.8) 0.0003Herd instinct Low High 0.3 (0.1–08) 0.01Intercept 0.006 (0.0003–0.03) �0.0001

OR, odds ratio; CI, confidence interval.aWald test.

estimate bird ages, this factor could not be considered in thisstudy.

The most important risk factor observed in this study wasthe interaction between migration status and nest type. Aspreviously suggested, nesting could be a predominant pe-riod for WNV infection: the limited mobility of adults andfledglings, and the bare skin of the latter, presumably makethem easy prey for mosquitoes (Marra et al. 2004). The nest-ing type would then be an important factor to explain birdexposure to mosquito bites. In resident birds, high preva-lence was associated with medium nests that include plat-form, cupped, and cavity nest. Platform nests are rudimen-tary, flat structures located on the ground, in a tree, or onthe tops of rooted vegetation or debris in shallow water.Cupped nests are an arrangement of hard and soft materialfrom floor and walls shaped in a cup. Cavity nest is a hol-lowed-up opening in the trunk of a tree. These three kindsof nests are located in the midstory and provide a large ac-cess to mosquitoes. Ground nests are usually just scrapes onthe ground forming a depression. Many mosquito’ speciesexhibit vertical height specialization for host seeking(Balenghien et al. 2006): birds building nests on the groundmay be less exposed than birds building medium nests. Thestructure of bulky nests is complex and compact. They aremade of twigs or grass weave or not with a grass-lined cham-ber inside. Access to outside is rather reduced; thus, the ex-posure to mosquito bites of birds building these kinds of nestis probably weak.

The increased point prevalence in nongregarious birdsmight be explained by a dilution effect previously suggestedin horses (Durand et al. 2002).

Results of model internal validation were satisfactory at theindividual and species level. Considering two prevalenceclasses (below or above 10%), the model adequately fitted thestatus of each species with �5 trapped birds, thus giving someconfidence in model predictions. However, an external vali-dation remains to be performed using independent data. Thehigher predicted prevalence in migratory birds was 7%, inparticular for White Wagtail (Motacilla alba), the Common Red-start (Phoenicurus phoenicurus), and the Rufous Scrub-Robin(Cercotrichas galactotes). Previous studies in 1965–1967 in Israelsuggested that the White Wagtail was exposed to the WNVand might be a relevant indicator of WNV circulation for sur-veillance (Nir et al. 1969, 1972). The Common Redstart wasreported as having WNV antibodies in another study in south-ern France in 2004 (Jourdain 2006). To our knowledge, no in-fection of Rufous Scrub-Robin by WNV was reported in theliterature. However, our analysis provides evidence that thesemigratory species are possibly exposed to WNV and mightplay a role in the dissemination of WNV from Africa to Eu-rope. Finally, high prevalence rates were predicted for threeresident species: the Black Shrub-Robin (Cercotrichas podobe),the Common Gonolek (Laniarius barbarus), and a woodpeckerspecies (Mesopicos goertae). Nothing is known about the sus-ceptibility to the WNV and the reservoir competence of thesetwo species. More field and biological data are needed to con-firm these preliminary results.

Acknowledgments

This publication is catalogued by the EDEN Steering Com-mittee as EDEN00116 (www.edenfp6project. net). The con-

tents of this publication are the sole responsibility of the au-thors and do not necessarily reflect the views of the Euro-pean Commission.

Disclosure Statement

This publication was partially funded by the CORUS proj-ect (French Ministry of Foreign Affairs) and GOCE-2003-010284 EDEN grants.

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Address correspondence to:Dr. Veronique Chevalier

CIRADCampus International de Baillarguet

Montpellier 34398France

E-mail: chevalier@cirad.fr

CHEVALIER ET AL.596