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ABSTRACT |
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Information about bats and WNV is meager. Antibodies to WNV in Chiroptera were first identified in healthy Rousettus aegyptiacus fruit bats collected in Uganda and Israel.12 WNV was isolated in India from the nearly indistinguishable Rousettus leschenaulti.13 Subsequent interest in WNV as a Chiropterian pathogen appeared limited until its arrival into the New World. During the year 2000, 150 dead bats were submitted to the New York State Department of Health after initial testing for rabies, and two bats (one Eptesicus fuscus and one Myotis lucifugus) were found to be positive for WNV by immunofluorescence.14 Subsequently, attempts were made to demonstrate WNV in an additional 306 bats submitted during 2000 to 2002 from the WNV-endemic zones of New York State. These animals represented several species, most of which were E. fuscus, and none were found to be positive by virus isolation and polymerase chain reaction testing (Baryl J, Bernard K, personal communication).
Like the numerous species of birds in the class of Aves, diversity within the order Chiroptera is extraordinary, with more than 1,100 species found throughout the world.15 The current study focused on two species that are most numerous within the United States, E. fuscus (big brown bat) and Tadarida brasiliensis (Mexican free-tailed bat). Individuals from both of these species are likely to live within or near urban populations and frequently come into contact with humans.16 E. fuscus bats are considered to be nonmigratory and hibernate during the winter months. Previous demonstrations of persistence of JE17,18 and SLE10 viruses through a period of experimental hibernation implied the possibility that hibernating E. fuscus might play a role in overwintering of WNV. The majority of T. brasiliensis are migratory, and millions of these animals fly between the southern United States and Latin America every year.19 If T. brasiliensis were found to be significant hosts for WNV, this yearly event could promote the spread of WNV into countries currently free of the disease, as well as reintroduce the agent back into the southern United States. The purpose of this study was to elucidate the role these two species of bats might play as hosts in the WNV transmission cycle and to determine their clinical response to WNV infection. We also assessed the prevalence of WNV infection in T. brasiliensis bats living within a focus of endemic infection in Louisiana.
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MATERIALS AND METHODS |
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Bats of both species were inoculated subcutaneously with 0.1 mL containing between 6,000 and 7,000 plaque-forming units (PFU) of New York 99 strain of WNV (4132), isolated initially from an infected crow and passaged once in Vero cells, once in C6/36 mosquito cells, and once in BHK-21 cells. All bats were examined for clinical signs of disease twice daily for the duration of the project by rousing for feeding and observing their movement in their cages. Three E. fuscus bats were euthanized via pentobarbital overdose daily on Days 1 through 6 and on Days 14 and 40 after inoculation. Four T. brasiliensis bats were euthanized daily from Days 1 to 6 after inoculation; within each group of four bats, two were among those that had antibodies that reacted in the flavivirus group-specific immunoassay, and two were serologically negative in that assay. At the time of euthanasia, blood was collected by heart puncture, and a cotton swab wet with BA-1 (medium M199-Hanks’ salts, 0.05 M Tris pH 7.5, 1% bovine serum albumin, 0.35 g/L sodium bicarbonate, 50 mg gentamicin/L, 2.5 mg amphotericin B/L) was rotated within the oral cavity, then swirled in 0.5 mL of BA-1. Duplicate 0.1 mL samples of undiluted sera and oral swab samples were assayed for WNV using a double overlay plaque assay on Vero cells, with plaques counted on Days 3, 4, and 5. For E. fuscus bats, samples of brain, heart, lung, liver, kidney, and spleen collected at necropsy were homogenized in BA-1 to yield 10% suspensions, and those were assayed for virus by plaque assay on Vero cells. Sera from the six E. fuscus bats euthanized 14 and 40 days after virus inoculation were tested for neutralizing antibody using a plaque-reduction neutralization test (PRNT) as previously described.22 Maintenance and care of all bats complied with the National Institutes of Health guidelines for the humane use of laboratory animals.
Field studies. As part of a larger field study in St. Tammany Parish, Louisiana, in late summer 2002, 149 T. brasiliensis bats were bled by cardiac puncture under isoflurane anesthesia. An 0.2-mL sample of blood was added to 0.9 mL BA-1, allowed to clot, and centrifuged to recover the equivalent of a 10–1 dilution of serum. These sera were tested for neutralizing antibodies to WNV by PRNT.
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RESULTS |
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Seven of the 24 E. fuscus bats were viremic at the time of euthanasia (Table 1
). Viremia was detected between 2 and 6 days after inoculation, with titers ranging from 10 (limit of detection) to 180 PFU/mL. In contrast, WNV was not isolated from any of the sera collected between Days 1 and 6 from the 24 T. brasiliensis bats. Virus was not isolated from any of the oral swabs collected from either species of bat, nor from any of the tissues collected at necropsy from E. fuscus bats. None of the sera collected from the E. fuscus bats euthanized 14 and 40 days after inoculation with WNV had neutralizing antibody to WNV when tested at a dilution of 1:10 (90% reduction).
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DISCUSSION |
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Seven of 24 serum samples collected from E. fuscus bats over the first 6 days after inoculation with WNV contained virus. However, the level of viremia observed was low, the highest titer being 180 PFU/mL, and unlikely to serve as a source of virus for feeding mosquitoes.22–24 In the case of T. brasiliensis bats, we were unable to isolate WNV from any of the sera collected during the 6 days after inoculation. Prior to challenge, half of these bats did have antibodies that cross-reacted with WNV antigen. However, these antibodies recognized a flavivirus group-specific epitope, did not react with a WNV-specific epitope, and may thus have represented antibody to SLE or Rio Bravo viruses.
It was surprising that we failed to detect a neutralizing antibody response on Days 14 or 40 after inoculation of WNV into E. fuscus bats. This lack of immune responsiveness may be due to poor viral replication in these animals. Lack of neutralizing antibody was also observed in E. fuscus bats sampled 17 days after inoculation with SLE virus.11
Insectivorous bats have been experimentally infected with several flaviviruses. Needle inoculation of SLE virus resulted in development of viremia in some E. fuscus bats, but the magnitude of viremia was very low and not considered infectious to mosquitoes, and clinical signs of disease were not observed.11 Inoculation of E. fuscus and Pipistrellus subflavus bats with JE virus also resulted in development of viremia, but the titers achieved were not reported.25 In another study, it was found that bats experimentally infected with JE virus were capable of producing a viremia sufficient to infect mosquitoes.17
Insectivorous bats feed on numerous types of insects, including occasional mosquitoes, and may possibly be infected with arboviruses through oral exposure. Indeed, it has been demonstrated that bats could become infected with YF virus through feeding on a single infected mosquito,26 and that feeding of three JE virus-infected mosquitoes to a P. subflavus led to development of viremia.17
We found 2 of 149 Mexican free-tailed bats from an endemic focus in Louisiana to have neutralizing antibody to WNV. Free-ranging birds sampled from this region during the same period of time had an overall WNV seroprevalence rate of 28% (Komar and others, submitted).
Our objective was to assess the potential of bats to serve as amplifying hosts and thereby contribute to WNV transmission in the Americas. Although it is possible that some bat species may react to WNV infection in a substantively different manner than the two species we investigated, E. fuscus and T. brasiliensis bats together probably represent the most prevalent bats in North America. The major finding of this study is that neither species of bat investigated developed more than a low-titer viremia after inoculation with a low-passage isolate of WNV. It was also found that the prevalence of antibodies to WNV in bats collected from an endemic focus was very low. Collectively, these observations suggest that E. fuscus and T. brasiliensis bats are unlikely to serve as amplifying hosts for WNV. Additional surveillance to characterize seroprevalence and presence of virus in dead bats is indicated to more fully understand possible interactions of WNV and bats.
Received September 30, 2004. Accepted for publication April 2, 2005.
Acknowledgments: Appreciation is extended to Dr. Tom O’Shea and student assistants for providing the Eptesicus bats, to personnel from the Louisiana Department of Health for assistance in bleeding bats in Louisiana and to Dr. Nick Komar for reviewing the manuscript.
This study was supported in part by NIH contract NO1-AI25489. Dr. Blitvich was supported in part by grant U50 CCU820510 from the CDC and grant AI45430 from NIH.
* Address correspondence to Richard Bowen, Department of Biomedical Sciences, Colorado State University, Fort Collins, CO 80523. E-mail: rbowen{at}colostate.edu 
Authors’ addresses: April Davis, Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO 80523, E-mail: April.Davis{at}colostate.edu. Michel Bunning, Division of Vector-Borne Infectious Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, P.O. Box 2087, Fort Collins, CO 80522, E-mail: zyd7{at}cdc.gov. Paul Gordy, Department of Biomedical Sciences, Colorado State University, Fort Collins, CO 80523, E-mail: pgordy{at}colostate.edu. Nicholas Panella, Division of Vector-Borne Infectious Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, P.O. Box 2087, Fort Collins, CO 80522, E-mail: nap4{at}cdc.gov. Bradley Blitvich, Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO 80523, E-mail: blitvich{at}colostate.edu. Richard Bowen, Department of Biomedical Sciences, Colorado State University, Fort Collins, CO 80523, Telephone: (970) 491-5768, Fax: (970) 491-3557, E-mail: rbowen{at}colostate.edu.
Reprint requests: Richard Bowen, Department of Biomedical Sciences, Colorado State University, Fort Collins, CO 80523, E-mail: rbowen{at}colostate.edu.
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