Trends in national surveillance data for bat rabies in the United States: 2001-2009.

Rabies remains an important public health concern in the United States, with most human cases associated with bat rabies virus variants. Cases of rabies virus (RV) infection in bats are widely distributed across the continental United States and elsewhere in the Americas. In this retrospective study, data on bats submitted to state laboratories for RV diagnosis between 2001 and 2009 were analyzed to investigate epidemiological trends in the United States. Season, region, and roosting habits were the primary risk factors of interest. During the study interval, more than 205,439 bats were submitted for RV diagnosis, and 6.7% of these bats were rabid. Increased odds of a submitted bat being rabid were associated with species that exhibit inconspicuous roosting habits, bats originating in the Southwest, and bats submitted for diagnosis during the fall. Periodic analysis of zoonotic disease surveillance is recommended to detect changes in trends regarding geographic distribution, seasonal fluctuations, and host associations; this is particularly necessary, as existing trends may be influenced by climate change or other emerging factors.

A distinct lineage of influenza A virus from bats.

Influenza A virus reservoirs in animals have provided novel genetic elements leading to the emergence of global pandemics in humans. Most influenza A viruses circulate in waterfowl, but those that infect mammalian hosts are thought to pose the greatest risk for zoonotic spread to humans and the generation of pandemic or panzootic viruses. We have identified an influenza A virus from little yellow-shouldered bats captured at two locations in Guatemala. It is significantly divergent from known influenza A viruses. The HA of the bat virus was estimated to have diverged at roughly the same time as the known subtypes of HA and was designated as H17. The neuraminidase (NA) gene is highly divergent from all known influenza NAs, and the internal genes from the bat virus diverged from those of known influenza A viruses before the estimated divergence of the known influenza A internal gene lineages. Attempts to propagate this virus in cell cultures and chicken embryos were unsuccessful, suggesting distinct requirements compared with known influenza viruses. Despite its divergence from known influenza A viruses, the bat virus is compatible for genetic exchange with human influenza viruses in human cells, suggesting the potential capability for reassortment and contributions to new pandemic or panzootic influenza A viruses.

Commerson's leaf-nosed bat (Hipposideros commersoni) is the likely reservoir of Shimoni bat virus.

In this study we attempted to identify whether Commerson's leaf-nosed bat (Hipposideros commersoni) is the reservoir of Shimoni bat virus (SHIBV), which was isolated from a bat of this species in 2009. An alternative explanation is that the isolation of SHIBV from H. commersoni was a result of spill-over infection from other species, particularly from the Egyptian fruit bats (Rousettus aegyptiacus), which frequently sympatrically roost with H. commersoni and are known as the reservoir of the phylogenetically related Lagos bat virus (LBV). To evaluate these hypotheses, 769 bats of at least 17 species were sampled from 18 locations across Kenya during 2009?2010. Serum samples were subjected to virus neutralization tests against SHIBV and LBV. A limited amount of cross-neutralization between LBV and SHIBV was detected. However, H. commersoni bats demonstrated greater seroprevalence to SHIBV than to LBV, and greater virus-neutralizing titers to SHIBV than to LBV, with a mean difference of 1.16 log(10) (95% confidence intervals [CI]: 0.94-1.40; p<0.001). The opposite pattern was observed for sera of R. aegyptiacus bats, with a mean titer difference of 1.06 log(10) (95% CI: 0.83-1.30; p<0.001). Moreover, the seroprevalence in H. commersoni to SHIBV in the cave where these bats sympatrically roosted with R. aegyptiacus (and where SHIBV was isolated in 2009) was similar to their seroprevalence to SHIBV in a distant cave where no R. aegyptiacus were present (18.9% and 25.0%, respectively). These findings suggest that H. commersoni is the host species of SHIBV. Additional surveillance is needed to better understand the ecology of this virus and the potential risks of infection to humans and other mammalian species.

Bartonella spp. in Bats, Guatemala.

To better understand the role of bats as reservoirs of Bartonella spp., we estimated Bartonella spp. prevalence and genetic diversity in bats in Guatemala during 2009. We found prevalence of 33% and identified 21 genetic variants of 13 phylogroups. Vampire bat-associated Bartonella spp. may cause undiagnosed illnesses in humans.

Rabies prevalence in migratory tree-bats in Alberta and the influence of roosting ecology and sampling method on reported prevalence of rabies in bats.

The migratory tree-roosting hoary bat (Lasiurus cinereus) and silver-haired bat (Lasionycteris noctivagans) are among the bat species with the highest reported prevalence of rabies in North America. However, bats submitted for rabies testing typically have been those that have come in contact with humans or pets. Given the roosting ecology of L. cinereus and L. noctivagans, contact with healthy individuals of these species is expected to be rare, with a bias in contact and submission of infected individuals and thus an overestimation of rabies prevalence. We tested 121 L. cinereus and 96 L. noctivagans specimens, collected during mortality surveys at wind energy facilities in Southern Alberta, Canada in 2007 and 2008, for rabies. None of the L. cinereus (0%) and one L. noctivagans (1%) tested positive for rabies. Prevalence of rabies was significantly lower than previously reported estimates, passive and active, for L. cinereus and L. noctivagans. In a review of the literature including multiple bat species, we found a significant difference in estimates of rabies prevalence based on passive versus active surveillance testing. Furthermore, roosting ecology influenced estimates of rabies prevalence, with significantly higher prevalence among passive surveillance submissions of nonsynanthropic species compared to synanthropic species, a trend not evident in active surveillance reports. We conclude that rabies prevalence in randomly collected L. cinereus and L. noctivagans is low and comparable to active surveillance estimates from other species (≤ 1%), and that roosting ecology influences estimates of rabies prevalence among bats submitted to public health laboratories in North America.

A perspective on lyssavirus emergence and perpetuation.

Rabies is propagated globally by viruses in the Family Rhabdoviridae, Genus Lyssavirus. These RNA viruses utilize the mammalian central nervous system as their ultimate niche, and exploit routine social mechanisms, as well as host behavioral alterations, to facilitate transmission by neural transport and innervations of the salivary glands, and ultimately excretion via the saliva, towards circulation thereafter in host populations. All mammals are susceptible to infection, but lyssavirus reservoirs are represented by several species of Carnivora, with viral global diversity and distribution in toto driven by a wide variety of the Chiroptera. Pathogen diversity is maintained by multiple faunas, and facilitated by pronounced host vagility, as exemplified by the ease of routine daily and seasonal movements by bats. Viral 'ensembles', or subpopulations associated with productive transmission events, emerge locally in vivo through a combination of naive host infections in some individuals versus acquired immunity by others, using complex metapopulation dynamics. Enhanced surveillance, improved diagnostics, increased pathogen detection, and an integrated One Health approach, targeting human, domestic animal and wildlife interfaces, provide modern insights to the ecology of bat lyssaviruses to augment future prevention and control.

A tale of two genomes: contrasting patterns of phylogeographic structure in a widely distributed bat.

One of the most widely distributed bats in the New World, the big brown bat (Eptesicus fuscus) exhibits well-documented geographic variation in morphology and life history traits, suggesting the potential for significant phylogeographic structure as well as adaptive differentiation among populations. In a pattern broadly consistent with morphologically defined subspecies, we found deeply divergent mitochondrial lineages restricted to different geographic regions. In contrast, sequence data from two nuclear loci suggest a general lack of regional genetic structure except for peripheral populations in the Caribbean and Mexico/South America. Coalescent analyses suggest that the striking difference in population structure between genomes cannot be attributed solely to different rates of lineage sorting, but is likely due to male-mediated gene flow homogenizing nuclear genetic diversity across most of the continental range. Despite this ongoing gene flow, selection has apparently been effective in producing and maintaining adaptive differentiation among populations, while strong female site fidelity, maintained over the course of millions of years, has produced remarkably deep divergence among geographically isolated matrilines. Our results highlight the importance of evaluating multiple genetic markers for a more complete understanding of population structure and history.

Variation in physiological stress between bridge- and cave-roosting Brazilian free-tailed bats.

Since the late 1980s, Brazilian free-tailed bats (Tadarida brasiliensis) have increasingly used bridges as roosts in the southern United States. We examined differences in blood cortisol levels, body condition, and parasite load, as measures of physiological stress in bats roosting in bridges and bats roosting in caves. We collected data during three periods, coinciding with female phases of reproduction. For all measures, bats were captured during the nightly emergence from the roost and immediately sampled. Cortisol levels were significantly higher during pregnancy and lactation and in individuals with lower body-condition scores (length of forearm to mass ratio) and significantly higher in bats roosting in caves than in those roosting in bridges. Thus, we concluded that individuals of this species that roost in bridges are not chronically stressed and seem to be unaffected by human activities present at bridges. This is a rare documented instance where a human-dominated environment does not appear to be adversely affecting the physiological health of a free-ranging animal.

Host phylogeny constrains cross-species emergence and establishment of rabies virus in bats.

For RNA viruses, rapid viral evolution and the biological similarity of closely related host species have been proposed as key determinants of the occurrence and long-term outcome of cross-species transmission. Using a data set of hundreds of rabies viruses sampled from 23 North American bat species, we present a general framework to quantify per capita rates of cross-species transmission and reconstruct historical patterns of viral establishment in new host species using molecular sequence data. These estimates demonstrate diminishing frequencies of both cross-species transmission and host shifts with increasing phylogenetic distance between bat species. Evolutionary constraints on viral host range indicate that host species barriers may trump the intrinsic mutability of RNA viruses in determining the fate of emerging host-virus interactions.

Response to vaccination with a commercial inactivated rabies vaccine in a captive colony of Brazilian free-tailed bats (Tadarida brasiliensis).

A captive colony of Brazilian free-tailed bats (Tadarida brasiliensis) was vaccinated with a commercial monovalent inactivated rabies virus (RABV) vaccine (RABVAC 1). Baseline rabies virus neutralizing antibodies (VNA) and the response to vaccination were measured in 50 bats. Rabies VNA was detected in the plasma of 64% (27/42) of bats that had been vaccinated 1 yr prior, but only 19% (8/42) had levels considered adequate. Rabies VNA was detected in the plasma of 63% (5/8) of bats with no record of previous vaccination, suggesting natural RABV exposure before captivity. All bats demonstrated a VNA response by 10 days postvaccination, and baseline titer significantly predicted humoral response to vaccination. No adverse reactions to vaccination or clinical signs of RABV infection were observed in the bats during a 6-mo observation period. Annual vaccination may maintain immunity against RABV infection in captive colonies of bats. Bat, rabies virus, Tadarida brasiliensis, vaccination, virus neutralizing antibodies.

Histological assessment of cellular immune response to the phytohemagglutinin skin test in Brazilian free-tailed bats (Tadarida brasiliensis).

Bats are known reservoirs for numerous emerging infectious diseases, occupy unique ecological niches, and occur globally except for Antarctica. Given their impact on human and agricultural health, it is critical to understand the mechanisms underlying immunocompetence in this reservoir host. To date, few studies have examined immune function in the Order Chiroptera, particularly among natural colonies of bats. The phytohemagglutinin (PHA) skin test has been widely used to measure delayed-type cellular immune response in a wide variety of vertebrates, and has been routinely employed in immunoecological studies. Although this test is frequently described as a measure of T cell proliferation, recent studies indicate it may represent a combination of immune responses. In mammals, the immune response is differentially, temporally and spatially regulated, therefore, we characterized the infiltrating leukocyte response to the PHA skin test in bats by examining a time-series of histological sections from PHA and saline injection areas in 41 Brazilian free-tailed bats (Tadarida brasiliensis). Results suggest that bats exhibit diverse leukocyte traffic within 6 h, and up to 24 h following subcutaneous PHA injection. There was a significant presence of lymphocytes and neutrophils, as well as eosinophils, basophils, and macrophages observed in the PHA-injected tissues, compared with saline-injected control tissues. We observed a highly significant negative correlation between the number of lymphocytes and neutrophils in PHA-injected tissue, with peak lymphocyte response at 12 h, and peak neutrophil response at 24 h post-injection. These results indicate substantial variation in the immune response of individuals, and may aid our understanding of disease emergence in natural populations of bats.

Ecology of rabies virus exposure in colonies of Brazilian free-tailed bats (Tadarida brasiliensis) at natural and man-made roosts in Texas.

Previous studies have investigated rabies virus (RABV) epizootiology in Brazilian free-tailed bats (Tadarida brasiliensis) in natural cave roosts. However, little is known about geographic variation in RABV exposure, or if the use of man-made roosts by this species affects enzootic RABV infection dynamics within colonies. We sampled rabies viral neutralizing antibodies in bats at three bridge and three cave roosts at multiple time points during the reproductive season to investigate temporal and roost variation in RABV exposure. We report seropositive bats in all age and sex classes with minimal geographic variation in RABV seroprevalence among Brazilian free-tailed bat colonies in south-central Texas. While roost type was not a significant predictor of RABV seroprevalence, it was significantly associated with seasonal fluctuations, suggesting patterns of exposure that differ between roosts. Temporal patterns suggest increased RABV seroprevalence after parturition in cave colonies, potentially related to an influx of susceptible young, in contrast to more uniform seroprevalence in bridge colonies. This study highlights the importance of life history and roost ecology in understanding patterns of RABV seroprevalence in colonies of the Brazilian free-tailed bat.

Correlates of viral richness in bats (order Chiroptera).

Historic and contemporary host ecology and evolutionary dynamics have profound impacts on viral diversity, virulence, and associated disease emergence. Bats have been recognized as reservoirs for several emerging viral pathogens, and are unique among mammals in their vagility, potential for long-distance dispersal, and often very large, colonial populations. We investigate the relative influences of host ecology and population genetic structure for predictions of viral richness in relevant reservoir species. We test the hypothesis that host geographic range area, distribution, population genetic structure, migratory behavior, International Union for Conservation of Nature and Natural Resources (IUCN) threat status, body mass, and colony size, are associated with known viral richness in bats. We analyze host traits and viral richness in a generalized linear regression model framework, and include a correction for sampling effort and phylogeny. We find evidence that sampling effort, IUCN status, and population genetic structure correlate with observed viral species richness in bats, and that these associations are independent of phylogeny. This study is an important first step in understanding the mechanisms that promote viral richness in reservoir species, and may aid in predicting the emergence of viral zoonoses from bats.

Roosting ecology and variation in adaptive and innate immune system function in the Brazilian free-tailed bat (Tadarida brasiliensis).

Bats have recently been implicated as reservoirs of important emerging diseases. However, few studies have examined immune responses in bats, and even fewer have evaluated these responses in an ecological context. We examined aspects of both innate and adaptive immune response in adult female Brazilian free-tailed bats (Tadarida brasiliensis) at four maternity roosts (two natural caves and two human-made bridges) in south-central Texas. Immune measurements included in vitro bactericidal ability of whole blood and in vivo T cell mediated response to mitogenic challenge. Bactericidal activity in T. brasiliensis varied with roosting ecology, but appears to be sensitive to colony-level effects. Blood from females living at one cave had significantly lower bactericidal ability than blood from females at three other sites. T cell mediated response in this species was associated with variation in roost ecology, with females from two caves having greater responses than females from two bridges. T cell mediated response and bactericidal activity were negatively correlated with one another within individuals that were tested for both. Variation in immunological response of T. brasiliensis is important for understanding the influence of the environment on the frequency and distribution of immunologically competent individuals and for understanding disease-host dynamics in this and other colonial species.

Experimental rabies virus infection of big brown bats (Eptesicus fuscus).

A captive colony of adult Big Brown Bats (Eptesicus fuscus) was experimentally infected with a rabies virus (RABV) variant isolated from the salivary glands of a naturally infected Big Brown Bat and passaged once through murine neuroblastoma cell culture. Bats were divided into 11 groups, which were composed of one to three noninfected and one to three infected individuals each. Twenty of 38 animals were infected intramuscularly into both left and right masseter muscles; they received a total of 10(3.2) median mouse intracerebral lethal dose (MICLD50) of Big Brown Bat RABV variant. Experimental outcome after viral exposure was followed in the bats for 140 days postinoculation (PI). Of 20 infected bats, 16 developed clinical rabies, and the mean incubation period was 24 days (range: 13-52 days). Three infected bats never seroconverted and succumbed early to infection (13 days). Four infected bats that survived until the end of the experiment without any signs of disease maintained detectable antibody titers until the third month PI, peaking between days 13 and 43, and consequent drop-off below the threshold for detection occurred by day 140. Limited excretion of virus in saliva of infected bats during the clinical course of disease was observed in two individuals on days 13 and 15 PI (<24 hr prior to onset of clinical illness). No bat-to-bat transmission of RABV to noninfected bats was detected.

Integrative models of bat rabies immunology, epizootiology and disease demography.

Bats are natural reservoirs of rabies. We address the maintenance of the disease in bat colonies by developing individual and population models that generate indicators of risk of rabies to bats, that provide dynamic estimates of effects of rabies on population densities, and that suggest consequences of viral exposures and infections in bats relative to physiological and ecological characteristics of bats in different habitats. We present individual models (within host) for the immune responses to a rabies virus challenge, an immunotypic disease model that describes the evolution of the disease and a disease demographics model, which is structured by immunotypic response governed by immune system efficiency. Model simulations are consistent with available data, characterized by relatively low prevalence of the virus in colonies and much higher prevalence of rabies virus-neutralizing antibodies. Under model conditions, there is a robust non-clinical state that can be attained by the exposed individual that allows persistence of the disease in the population.