bats were sampled in january and may 2007 at 6 sites in ghana : the center of accra ( urban habitat ) ; the wooded outskirts of accra ( savannah habitat ) ; and forested habitats at pra , kibi , adoagyiri , and oyibi ( a plantation with woodland / forest border ) . bats were captured by using 618-m mist nets ; roosting eidolon helvum were captured by using nets on poles . a sample size of 59 would provide 95% confidence of finding at least 1 lbv - seropositive bat in a large population ( > 5,000 ) , given a seroprevalence of 5% and assuming random sampling ( 3 ) . captured bats were manually restrained and anesthetized by intravenous injection ; 0.21.0 ml of blood was collected from the propatagial vein before the bat was released . blood was centrifuged in the field at ambient temperature at 3,000 rpm for 15 min . two species , epomophorus gambianus and e. helvum , were caught in sufficient numbers ( 117 and 66 , respectively ) for reasonable inferences to be made about lbv seroprevalence rates ( table ) . a standard approach was used to calculate 95% confidence intervals ( cis ) for seroprevalence ( 3 ) . because of the relatively short distances between study sites and the likelihood of bats mixing between these sites , bats of each species were considered to belong to single populations . all but 3 e. helvum were derived from a colony in accra , whereas e. gambianus were derived from all habitat types . * phylogroup 1 contains challenge virus standard ( cvs ) , genotype 1 ; phylogroup 2 contains lagos bat virus ( lbv ) , genotype 2 ; ci , confidence interval ; nt , not tested . gambianus was caught in all habitats , including at the city colony and in plantations . bat serum samples were tested for virus neutralizing antibody against classical rabies virus ( challenge virus standard ) by using a standard fluorescent antibody virus neutralization ( favn ) test ( 5 ) . antibodies to lbv were measured by using a modified favn test ( 6 ) . because positive bat antiserum from naturally infected bats was not available , for positive controls we used human rabies immunoglobulin , lbv - positive rabbit serum , and serum from mice vaccinated with human diploid cell vaccine . all samples were analyzed in duplicate and serially diluted by using a 3-fold series ( representing reciprocal titers of 9 , 27 , 81 , and 24319,683 ) ( 6 ) . the modified favn test requires a cut - off threshold , which in prior bat lyssavirus studies has been a titer of 27 , to avoid false - positive results ( 6,7 ) . the first 121 samples collected were tested against the challenge virus standard ; no tested bat was seropositive at 1:3 dilutions . a mean antibody titers to lagos bat virus ( lbv ) in 6 species of fruit bat in ghana . an lbv - specific modified fluorescent antibody neutralization test was used to determine the level of antibody in bats ; it used two 3-fold serial dilutions and derived a mean dilution at which the bats neutralized lbv . 1 , epomops franqueti ; 2 , epomophorus gambianus ; 3 , epomops buettikoferi ; 4 , eidolon helvum ; 5 , hypsignathus monstrosus ; 6 , nanonycteris veldkampii . levels of specific virus neutralizing antibodies against lbv were higher in e. helvum ( seroprevalence 37% , 95% ci 24%49% ) than in e. gambianus ( 3% , 95% ci 0%7% ) . of 6 epomops buettikoferi , no sex differences in e. helvum seroprevalence were evident (   1.0 , p>0.9 ) . because of the high level of seropositivity in e. helvum , we attempted to determine a possible case reproduction rate ( r0 ) for lbv infection in this species by using the equation r0  =  1/x * , where x *  =  proportion of susceptible hosts in a population ( 9 ) . we assumed that infection with each virus within the bat populations is endemic , stable , and randomly dispersed ; that all seropositive animals have lifelong immunity that is detectable serologically ; and that seropositivity is to 1 virus . on the basis of these assumptions , r0  =  1.6 ( 95% ci 1.32.0 ) . previous studies have suggested that healthy bats develop antibodies to other lyssavirus infections ( 7,10,11 ) , which may reflect past exposure , rather than survival from clinical disease . lbv likely co - evolved with its natural megachiropteran host until a genetic stasis had been reached in which the virus this situation would result in high seroprevalence rates after a wave of virus circulation in a colony . nine seropositive bats ( 8 e. helvum , 1 e. buettikoferi ) were apparently healthy pregnant females . these results support theories that lyssaviruses are endemic within specific bat populations , that they may not cause high mortality rates , that exposure rates of lbv between megachiroptera in old world african bats are high , and that bats may breed successfully after lbv exposure ( 7,8 ) . the number of high reciprocal titers against lbv ( figure 1 ) and the history of lbv isolation in e. helvum suggest that lbv circulates in megachiroptera in ghana . however , further work is needed to determine the specific phylogroup 2 virus and its prevalence within specific bat populations . no previous estimate of r0 for genotype 2 lyssavirus has been calculated , and although anamnesis may lead to no detectable antibodies in bats with immunity and a consequent underestimate of r0 , this value indicates the potential r0 and is comparable to values previously estimated for lyssavirus infections in bats ( 7,11 ) . more detailed analysis relating to age - specific infection and survival rates or mode of transmission was precluded by the difficulty in determining the age of adult bats , the lack of juveniles in the sample , and the cross - sectional sample used . the underlying cause of the difference in seroprevalence between e. gambianus and e. helvum with respect to lbv infection is unclear . possible explanations include differential susceptibilities to infection ; virus  host adaptation ; different contact with the virus , including a recent epidemic in the e. helvum colony ; or different population ecology . e. helvum resides in high - density populations ( hundreds of thousands ) ( figure 2 , panel a ) and migrates annually , compared with e. gambianus , which resides in less dense colonies of tens or hundreds ( 4 ) . e. helvum commonly forms large colonies in african cities in close proximity to humans and domestic animals and is a food source in west africa ( figure 2 , panel b ) . no investigations into infections of humans were made during these investigations , but lyssavirus infections in humans in africa are underdiagnosed ( 12 ) . despite reduced pathogenicity of lbv in the laboratory , it has been isolated from dogs , cats , and a mongoose ( 2 ) . conversely , mokv has caused a fatal case of encephalitis in a human ( 1 ) . lbv and mokv each have a substitution in the r333 glycoprotein residue ( 1 ) . although it is not the only protein to determine the pathogenicity of lbv , the r333 substitution still remains an important marker of rabies pathogenicity . in conclusion , the high seroprevalence to lbv in this population may pose a substantial public health risk because e. helvum is widely distributed in africa and a food source in west africa .
to investigate the presence of lagos bat virus ( lbv)specific antibodies in megachiroptera from west africa , we conducted fluorescent antibody virus neutralization tests . neutralizing antibodies were detected in eidolon helvum ( 37% ) , epomophorus gambianus ( 3% ) , and epomops buettikoferi ( 33% , 2/6 ) from ghana . 
 these findings confirm the presence of lbv in west africa .