Endemic circulation of European bat lyssavirus type 1 in serotine bats, Spain.To determine the presence of European bat lyssavirus type 1 in southern Spain, we studied 19 colonies of serotine bats (Eptesicus isabellinus), its main reservoir, during 1998-2003. Viral genome and antibodies were detected in healthy bats, which suggests subclinical infection subclinical infection An infection in which Sx are mild or inapparent, and may not be diagnosed other than by positive confirmation of the ability to transmit the infection or serologically . The different temporal patterns of circulation found in each colony indicate independent endemic circulation.
The serotine bat (Eptesicus serotinus) is considered the main reservoir for European bat lyssavirus type 1 (EBLV EBLV European Bat Lyssa Virus 1). Recently, southern Iberian populations of this species have been classified as E. isabellinus (1), a bat species previously known to exist only in North Africa.
In 1989, 5 EBLV1-infected serotine bats were found dead during a survey of natural colonies in Huelva (Andalusia). The prevalence of EBLV1 antibodies was up to 20% among completely healthy bats that were recaptured 1 year later, providing the first direct evidence of the survival of serotine bats after EBLV1 infection (2). Furthermore, high seroprevalence seroprevalence Immunology The proportion of a population that is seropositive–ie, has been exposed to a particular pathogen or immunogen; the seropositivity of a population is calculated as the number of individuals who produce a particular antibody divided and presence of viral RNA RNA: see nucleic acid.
in full ribonucleic acid
One of the two main types of nucleic acid (the other being DNA), which functions in cellular protein synthesis in all living cells and replaces DNA as the carrier of genetic in the oral cavity and bloodstream of different lyssavirus species have been reported in healthy bats captured in natural colonies in which the numbers of deaths have not increased (3-5), showing direct evidence of subclinical subclinical /sub·clin·i·cal/ (sub-klin´i-k'l) without clinical manifestations.
Not manifesting characteristic clinical symptoms. Used of a disease or condition. or asymptomatic disease after viral infection. Even viral RNA and antigens have been detected in the brains of healthy captive bats (6). However, reports of experimental lyssavirus transmission have drawn discrepant dis·crep·ant
Marked by discrepancy; disagreeing.
[Middle English discrepaunt, from Latin discrep conclusions (7-9).
During 1998-2003, a total of 1,030 E. isabellinus from 19 colonies located in the provinces of Huelva, Seville, and Granada (Andalusia), in southern Spain, were sampled. We focused on this region because all EBLV1 cases reported in Spain were from southern Spain. Distances between colonies varied from 0.5 to 317 km.
Bats were captured mainly in so-called maternity colonies made up of adult females and young of both sexes. Each animal was banded and assessed for sex, age, size, and weight. A total of 150 (14.5%) were recaptured. The frequency of repeated captures reached 22.7% in individual bats from 3 colonies that were sampled in all 6 years of the study. All recaptured bats were always found in the same colony in which they were first marked.
Viral RNA was detected in 34 (2.8%) of 1,226 oropharyngeal oropharyngeal /oro·pha·ryn·ge·al/ (-fah-rin´je-al)
1. pertaining to the mouth and pharynx.
2. pertaining to the oropharynx. swab specimens from 33 bats of 8 colonies (1 bat was resampled after a 1-week interval). One bat that tested positive was recaptured and tested negative in a followup sampling effort. Positive results were identified as EBLV1 by direct sequencing as previously described (4,10). Samples for reverse transcription-PCR were stored in a buffer that was designed for RNA preservation but was not suitable for keeping the virus viable for isolation on cell culture.
A total of 626 plasma samples were tested for EBLV1-specific antibodies by using a modification of the rapid fluorescent focus inhibition test (11), but 77 (12.3%) of them were found to be toxic to cells. EBLV1 antibodies were found in 51 (9.3%) of the remaining 549 from 13 colonies. Only 2 of 22 samples showing viral RNA in the oral cavity were antibody positive (Table). Thirteen antibody-positive bats were recaptured in a healthy condition in the following campaigns.
The temporal pattern of circulation was completely different in each colony (online Appendix Table, available from www.cdc.gov/EID/content/14/8/12653-appT.htm). Only 1 colony showed continuous circulation from 1998 through 2002.
Brains were obtained from 20 bats of the same colony that had been studied previously (4). Differences in body condition between noninfected bats, bats with positive oropharyngeal swabs only, and bats with positive oropharyngeal and brain specimens were tested by an analysis of variance; index of body condition was the dependent variable. Individual body condition was expressed as the residuals from an analysis of covariance Covariance
A measure of the degree to which returns on two risky assets move in tandem. A positive covariance means that asset returns move together. A negative covariance means returns vary inversely. with an optimized design, including body mass as dependent variable; forearm length as a covariate; and sex, age, and year as fixed factors. We found a significant negative association (Figure 1) between RNA presence and body condition (F = 11.78; degrees of freedom = 2,281; p<0.001, n = 292). Post hoc tests indicated that only bats whose brains tested positive had a significantly worse body condition (Tukey honestly significant differences [HSD HSD Human Services Department
HSD High Speed Data
HSD Hillsboro School District (Hillsboro, OR)
HSD Hybrid Synergy Drive (Toyota/Lexus)
HSD High School Diploma
HSD Historical Society of Delaware ] test -2.33, p<0.001, and HSD -2.27, p<0.001, respectively). Differences in body condition between oropharyngeal swab-negative and oropharyngeal swab-positive bats were not significant (Tukey HSD 0.57, p = 0.99).
Brain, cerebellum cerebellum (sĕr'əbĕl`əm), portion of the brain that coordinates movements of voluntary (skeletal) muscles. It contains about half of the brain's neurons, but these particular nerve cells are so small that the cerebellum accounts for , and spinal cord from 1 bat carcass with natural EBLV1 infection were studied by histopathologic and histochemical techniques. The bat was captured while flying but died during manipulation, which is an extremely rare event. The brain and oropharyngeal swab were positive for EBLV1 by reverse transcription-PCR, and the brain smear was also positive by immunofluorescence Immunofluorescence
A technique that uses a fluorochrome to indicate the occurrence of a specific antigen-antibody reaction. The fluorochrome labels either an antigen or an antibody. . Histopathologic studies showed moderate neuronal degeneration characterized by neuronal hyperchromatosis, chromatolysis chromatolysis /chro·ma·tol·y·sis/ (kro?mah-tol´i-sis) disintegration of Nissl bodies of a neuron as a result of injury, fatigue, or exhaustion.
n. , and satellitosis (Figure 2, panel A). The Sellers stain results were negative, proving the absence of Negri bodies (Figure 2, panel B). The occasional presence of basophilic basophilic /ba·so·phil·ic/ (-fil´ik)
1. pertaining to basophils.
2. staining readily with basic dyes.
staining readily with basic dyes. intracytoplasmic intracytoplasmic /in·tra·cy·to·plas·mic/ (-si?to-plaz´mik) within the cytoplasm of a cell. structures in neurons located in the cerebral cortex that underwent necrobiosis necrobiosis /nec·ro·bi·o·sis/ (-bi-o´sis) swelling, basophilia, and distortion of collagen bundles in the dermis, sometimes with obliteration of normal structure, but short of actual necrosis. was interpreted as a neurophagic process that was taking place in the glial cells because these inclusions were shown to be positive by the Feulgen reaction (Figure 2, panel C), whereas the Negri bodies have a strongly acidophilic acidophilic /ac·i·do·phil·ic/ (as?i-do-fil´ik)
1. easily stained with acid dyes.
2. growing best on acid media. nature. Moderate gliosis without perivascular perivascular /peri·vas·cu·lar/ (-vas´ku-lar) near or around a vessel.
around a vessel.
perivascular cellulitis infiltration was observed (Figure 2, panel 2D).
Despite the fact that serotine bats (E. serotinus and E. isabellinus) are the most frequently involved species in cases of bat lyssavirus exposure in humans in Europe, most published surveys of EBLV1 infection in natural bat colonies have focused on other bat species (5,12). Data from serotine bats have been obtained by either serologic se·rol·o·gy
n. pl. se·rol·o·gies
1. The science that deals with the properties and reactions of serums, especially blood serum.
2. or direct detection techniques but not from both (2,4), as in this report.
Our recapture data show an absence of switching among colonies, even between colonies located only a few kilometers apart. This result indicates highly philopatric behavior among female bats, as has been confirmed in a recent mtDNA-based study of these colonies (J. Juste, unpub. data). This could be the cause of the different temporal distribution of the positive results observed in each colony, which suggests a pattern of independent endemic viral circulation different from the model, based on periodic epidemic waves of fast viral spreading proposed for the mouse-eared bat (Myotis Myotis
genus of bats. Includes M. thysanodes (fringed myotis bat), M. myotis (European common mouse-eared bat), M. lucifugus (little brown bat). myotis) (12).
The analysis of body condition index as a measure of physiologic condition gives additional evidence for mild or subclinical infection in the previously described long-term survival of EBLV1 RNA or antibody-positive bats (2,4,5,12). Similar body condition values between bats with oropharyngeal swabs that were positive for the virus and those that were negative could be interpreted as a recent virus infection for which no symptoms have developed. However, 1 lyssavirus-positive bat showed no viral RNA 3 years later. One bat even showed RNA in the oral cavity in consecutive samples separated by 1 week. Our findings suggest that asymptomatic EBLV1 RNA carriage may be common in serotine bats (2-5,9,13,14). In only a small subset of them does symptomatic neurologic infection progress to more severe body condition. However, most of these bats were captured while they were flying, and only 1 of those involved in an episode of human exposure had lost the ability to fly. This bat had the poorest body condition. The absence of Negri bodies and the moderate percentage of nerve cells affected found in the bat, captured while flying, suggest a subclinical process, as has been proposed for EBLV1 in Rousettus aegyptiacus (6,14). We cannot predict whether severe encephalitis encephalitis (ĕnsĕf'əlī`təs), general term used to describe a diffuse inflammation of the brain and spinal cord, usually of viral origin, often transmitted by mosquitoes, in contrast to a bacterial infection of the meninges was about to develop in these bats. The lack of pathogenicity could have arisen through long-standing coevolution co·ev·o·lu·tion
The evolution of two or more interdependent species, each adapting to changes in the other. It occurs, for example, between predators and prey and between insects and the flowers that they pollinate. between bats and viruses, as the phylogenetic phy·lo·ge·net·ic
1. Of or relating to phylogeny or phylogenetics.
2. Relating to or based on evolutionary development or history. data suggest (15). Nevertheless, additional work is necessary to establish whether the presence of EBLV1 RNA in the oral cavity is associated with the excretion of live virus.
[FIGURE 1 OMITTED]
[FIGURE 2 OMITTED]
In a similar study of M. daubentonii in the United Kingdom, only EBLV2 antibodies, but not viral RNA in the oropharyngeal cavity, were reported (3). Another study of the experimental transmission of Aravan, Khujand, and Irkut viruses to E. fuscus found oral excretion only in bats whose conditions were progressing to neurologic infection and death (8). These facts suggest differences between models of pathogenesis of lyssavirus infections in bats.
In summary, our findings indicate that natural maternity colonies of E. isabellinus behave as close communities in which EBLV1 independently circulates, which suggests an endemic pattern associated with asymptomatic or mild disease. Further studies are needed to 1) investigate the circumstances under which neurologic manifestations develop in bats (which lead to the abnormal behavior usually associated with human exposures) and 2) to establish the epidemiologic implications for public health of asymptomatic oral RNA carriage. The anthropophilic behavior of this bat species makes active surveillance highly recommended to predict risk factors that allow taking political decisions relating to public health.
We gratefully acknowledge Juan L. Garcia-Mudarra, Jose A. Garrido, Elena Migens, Joaquin Munoz, Juan Quetglas, Jesus Nogueras, and Carlos Ruiz for providing technical assistance as bat handlers. We also thank the Genomic Unit of the National Center of Microbiology for analyzing the genomic sequences. We also gratefully acknowledge Thomas Muller who kindly donated the specific monoclonal antibody W239 used in this work.
This project was financially supported by grants from the Instituto de Salud Carlos III (MPY MPY Mils Per Year
MPY Multiply 1364/99, MPY 1271/01, and EXP 03/0020), by an agreement between the Public Health Department of the Spanish Ministry of Health and the Instituto de Salud Carlos III for the development of "Improvement rabies surveillance in Spain through the development of molecular tools and the establishment of a new system for active rabies surveillance," by the Spanish program of Research in Biomedicine biomedicine /bio·med·i·cine/ (bi?o-med´i-sin) clinical medicine based on the principles of the natural sciences (biology, biochemistry, etc.).biomed´ical
1. (FIS FIS n abbr (BRIT) (= Family Income Supplement) → ayuda estatal familiar 98/0945), by the WP5 of the European Med-Vet-Net network of excellence, by the project SAF SAF Safety
SAF Society of American Foresters
SAF Society of American Florists
SAF Secretary of the Air Force
SAF Second Amendment Foundation
SAF Singapore Armed Forces
SAF Students for Academic Freedom
SAF Store And Forward 2006-12784-C02-01/ of the General Research Program of the Spanish Ministry of Science and Education, and by the Environmental Delegation of Seville. This research has been approved by the appropriate animal review board of the Spanish Society for Conservation and Study of Bats.
DOI (Digital Object Identifier) A method of applying a persistent name to documents, publications and other resources on the Internet rather than using a URL, which can change over time. : 10.3201/1408.080068
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Sonia Vazquez-Moron, * ([dagger]) Javier Juste, ([double dagger]) Carlos Ibanez, ([double dagger]) Eduardo Ruiz-Villamor, ([section]) Ana Avellon, * Manuel Vera, * and Juan E. Echevarria * ([dagger])
* Instituto de Salud Carlos III, Majadahonda, Madrid, Spain; ([dagger]) Centro de Investigacion Biomedica en Red de Epidemiologia y Salud Publica (CIBERESP), Spain; ([double dagger]) Estacion Biologica de Dofana CSIC (Customer Specific Integrated Circuit) Pronounced "c-sick." Another term for ASIC, which was coined by Motorola. Some feel this is a more accurate description of an ASIC chip. See ASIC. , Sevilla. Spain; and ([section]) Laboratorio Central de Veterinaria de Santa Fe, Granada Santa Fe is a Andalucian municipality in the province of Granada, Spain, situated in the Vega de Granada, irrigated by the river Genil. External links
Ms Vazquez-Moron is a doctoral candidate in science at the Complutense
University of Madrid and a microbiologist who is developing a new system for active rabies surveillance at the Instituto de Salud Carlos III, Madrid. Her research interests include emergent viruses in bats and their public health implications.
Address for correspondence: Sonia Vazquez-Moron, Diagnostic Microbiology Service, National Center for Microbiology, Instituto de Salud Carlos III, Carretera de Majadahonda-Pozuelo km 2 Majadahonda 28220, Madrid, Spain; email: firstname.lastname@example.org
Table. European bat lyssavirus type 1 in Eptesicus isabellinus bats, Spain, 1999-2003 * Type of testing RT-PCR RFFIT Total captures 1,226 626 No. colonies 19 13 Total no. (%) positive 34 (2.8) 51 (9.3) 1998 No. captures 164 151 No. (%) positive 4 (2.4) 10 (6.6) 1999 No. (%) captures 161 90 No. positive 4 (2.5) 5 (5.6) 2000 No. captures 204 128 No. (%) positive 16 (7.8) 12 (9.4) 2001 No. captures 209 96 No. positive 0 4 (4.2%) 2002 No. captures 287 100 No. (%) positive 10 (3.4) 5 (5.0) 2003 No. captures 201 54 No. positive 0 0 * Results obtained during active surveillance. RT-PCR, reverse transcription-PCR, RFFIT, rapid fluorescent focus inhibition test.