Ecological and anthropogenic drivers of rabies exposure in vampire bats: implications for transmission and control

Despite extensive culling of common vampire bats in Latin America, lethal human rabies outbreaks transmitted by this species are increasingly recognized, and livestock rabies occurs with striking frequency. To identify the individual and population-level factors driving rabies virus (RV) transmission in vampire bats, we conducted a longitudinal capture-recapture study in 20 vampire bat colonies spanning four regions of Peru. Serology demonstrated the circulation of RV in vampire bats from all regions in all years. Seroprevalence ranged from 3 to 28 per cent and was highest in juvenile and sub-adult bats. RV exposure was independent of bat colony size, consistent with an absence of population density thresholds for viral invasion and extinction. Culling campaigns implemented during our study failed to reduce seroprevalence and were perhaps counterproductive for disease control owing to the targeted removal of adults, but potentially greater importance of juvenile and sub-adult bats for transmission. These findings provide new insights into the mechanisms of RV maintenance in vampire bats and highlight the need for ecologically informed approaches to rabies prevention in Latin America.     

A comparative study of rabies virus isolates from hematophagous bats in Brazil

The Brazilian chiropteran fauna consists of 167 species; of which, three are hematophagous: the common vampire bat (Desmodus rotundus), the white-winged vampire bat (Diaemus youngi), and the hairy-legged vampire bat (Diphylla ecaudata). The aim of this study was to describe the isolation of Rabies virus from common and hairy-legged vampire bats and to report the first comparative antigenic and genetic studies of isolates from these bats. Antigenic and genetic typing of both isolates identified them as antigenic variant 3 (AgV3), the variant frequently isolated from common vampire bats. Phylogenetic analysis showed 99.3% identity between the isolates. This is the first time since 1934 that Rabies virus has been isolated from hairy-legged vampire bats in Brazil. Our analysis provides evidence that the existence of rabies-positive isolates from hairy-legged vampire bats may be the result of an interspecific rabies transmission event from common vampire bats and suggests that roost cohabitation may occur.     

Knowledge and Experience Predict Indiscriminate Bat‐Killing Intentions among Costa Rican Men

Human–wildlife conflicts create collateral damage when people attempt to control one problematic species and inadvertently kill others. I observed a collateral damage problem in southern Costa where people seeking to control common vampire bats (Desmodus rotundus) indiscriminately killed sympatric, non‐target bat species (e.g., by baiting bats with poisoned bananas). To learn about this phenomenon and its causes, I developed and implemented a questionnaire based on the theory of planned behavior. In a sample of 504 men, 14 percent had individually killed 1–115 bats within the past 5 yr, 68 percent had killed bats as children, and 27 percent said that they would kill bats that they found roosting on their farms, even if they could not identify the species. Men who intended to indiscriminately kill bats thought that it would reduce disease transmission to livestock, whereas men who did not intend to kill bats thought that killing bats would reduce ecosystem functioning and/or damage nature. Ultimately, men were more likely to intend to indiscriminately kill bats if they knew less about bat natural history and/or had previously suffered vampire bats attacking their livestock. Men knew more about bat natural history and were less likely to harbor indiscriminate bat‐killing intentions if they had experienced some form of environmental education. My results suggest that environmental education will be most effective for bat conservation when combined with farmer support to ameliorate perceived livelihood risks associated with vampire bats.     

吸血蝙蝠食性特化及其研究现状

翼手目动物（蝙蝠）的食性多样性丰富,其食物包括昆虫、鱼类、两栖动物、爬行动物、鸟类、哺乳动物、植物果实、花、花粉、花蜜、叶片和血液等。其中,大约70%的蝙蝠主要以昆虫为食,而以血液为食的吸血蝙蝠只有3种,它们是哺乳动物中唯一的仅以血液为食的动物类群。因此,吸血蝙蝠是研究动物食性演化的重要模式动物。本文综述了吸血蝙蝠在形态学、生理学、行为学、感觉系统和肠道微生物等方面的研究,指出了吸血蝙蝠食性特化的适应性特征。随着普通吸血蝠高质量基因组的公布,我们将有机会探究食性相关基因在吸血蝙蝠中的功能改变,阐明动物食性转变的分子机理。本文将为吸血蝙蝠和其它动物食性转变的研究提供有益的参考。     

PERMANENT GENETIC RESOURCES: Development of polymorphic microsatellite loci for the common vampire bat, Desmodus rotundus (Chiroptera: Phylostomidae)

The common vampire bat (Desmodus rotundus) is one of three haematophagous species of bats and the only species in this genus. These New World bats prey on mammals and create significant economic impacts through transmission of rabies in areas where livestock are prevalent. Furthermore, in some portions of their range, it is not uncommon for them to prey upon humans. It is critical to the management of this species and for understanding the spread of bat rabies that detailed studies of D. rotundus population structure be conducted. To further such studies, we have characterized 12 microsatellite loci for this species.     

Unravelling virus community ecology in bats through the integration of metagenomics and community ecology

The spillover of viruses from wildlife into agricultural animals or humans has profound socioeconomic and public health impact. Vampire bats, found throughout South America, feed directly on humans and other animals and are an important reservoir for zoonotic viruses, including rabies virus. This has resulted in considerable effort in understanding both the ecology of bat‐borne viruses and the composition and associated correlates of the structure of entire virus communities in wildlife, particularly in the context of disease control interventions. In a From the Cover article in this issue of Molecular Ecology, Bergner et al. (2019) set out to reveal virus community dynamics in vampire bats by interrogating factors that affect the structure, diversity and richness of these communities. Due to the linkage of metagenomic sequence data with community ecology, this study represents an important advance in the field of virus ecology.     

Phylodynamics of vampire bat‐transmitted rabies in Argentina

Common vampire bat populations distributed from Mexico to Argentina are important rabies reservoir hosts in Latin America. The aim of this work was to analyse the population structure of the rabies virus (RABV) variants associated with vampire bats in the Americas and to study their phylodynamic pattern within Argentina. The phylogenetic analysis based on all available vampire bat‐related N gene sequences showed both a geographical and a temporal structure. The two largest groups of RABV variants from Argentina were isolated from northwestern Argentina and from the central western zone of northeastern Argentina, corresponding to livestock areas with different climatic, topographic and biogeographical conditions, which determined their dissemination and evolutionary patterns. In addition, multiple introductions of the infection into Argentina, possibly from Brazil, were detected. The phylodynamic analysis suggests that RABV transmission dynamics is characterized by initial epizootic waves followed by local enzootic cycles with variable persistence. Anthropogenic interventions in the ecosystem should be assessed taking into account not only the environmental impact but also the potential risk of disease spreading through dissemination of current RABV lineages or the emergence of novel ones associated with vampire bats.     

Habitat fragmentation is associated with dietary shifts and microbiota variability in common vampire bats

Host ecological factors and external environmental factors are known to influence the structure of gut microbial communities, but few studies have examined the impacts of environmental changes on microbiotas in free‐ranging animals. Rapid land‐use change has the potential to shift gut microbial communities in wildlife through exposure to novel bacteria and/or by changing the availability or quality of local food resources. The consequences of such changes to host health and fitness remain unknown and may have important implications for pathogen spillover between humans and wildlife. To better understand the consequences of land‐use change on wildlife microbiotas, we analyzed long‐term dietary trends, gut microbiota composition, and innate immune function in common vampire bats (Desmodus rotundus) in two nearby sites in Belize that vary in landscape structure. We found that vampire bats living in a small forest fragment had more homogenous diets indicative of feeding on livestock and shifts in microbiota heterogeneity, but not overall composition, compared to those living in an intact forest reserve. We also found that irrespective of sampling site, vampire bats which consumed relatively more livestock showed shifts in some core bacteria compared with vampire bats which consumed relatively less livestock. The relative abundance of some core microbiota members was associated with innate immune function, suggesting that future research should consider the role of the host microbiota in immune defense and its relationship to zoonotic infection dynamics. We suggest that subsequent homogenization of diet and habitat loss through livestock rearing in the Neotropics may lead to disruption to the microbiota that could have downstream impacts on host immunity and cross‐species pathogen transmission.     

Using noninvasive metagenomics to characterize viral communities from wildlife

Microbial communities play an important role in organismal and ecosystem health. While high‐throughput metabarcoding has revolutionized the study of bacterial communities, generating comparable viral communities has proven elusive, particularly in wildlife samples where the diversity of viruses and limited quantities of viral nucleic acid present distinctive challenges. Metagenomic sequencing is a promising solution for studying viral communities, but the lack of standardized methods currently precludes comparisons across host taxa or localities. Here, we developed an untargeted shotgun metagenomic sequencing protocol to generate comparable viral communities from noninvasively collected faecal and oropharyngeal swabs. Using samples from common vampire bats (Desmodus rotundus), a key species for virus transmission to humans and domestic animals, we tested how different storage media, nucleic acid extraction procedures and enrichment steps affect viral community detection. Based on finding viral contamination in foetal bovine serum, we recommend storing swabs in RNAlater or another nonbiological medium. We recommend extracting nucleic acid directly from swabs rather than from supernatant or pelleted material, which had undetectable levels of viral RNA. Results from a low‐input RNA library preparation protocol suggest that ribosomal RNA depletion and light DNase treatment reduce host and bacterial nucleic acid, and improve virus detection. Finally, applying our approach to twelve pooled samples from seven localities in Peru, we showed that detected viral communities saturated at the attained sequencing depth, allowing unbiased comparisons of viral community composition. Future studies using the methods outlined here will elucidate the determinants of viral communities across host species, environments and time.     

Bioecological Drivers of Rabies Virus Circulation in a Neotropical Bat Community

Introduction

In addition to the commonly accepted importance of the vampire bat in the maintenance and transmission of the rabies virus (RABV) in South America, RABV infection of other species is widely evidenced, challenging their role in the viral cycle.

Methodology / Principles findings

To identify the bioecological drivers of RABV circulation in neotropical bat communities, we conducted a molecular and serological survey on almost 1,000 bats from 30 species, and a 4-year longitudinal survey in two colonies of vampire bats in French Guiana. RABV was molecularly detected in a common vampire and in a frugivorous bat. The sequences corresponded to haematophagous bat-related strains and were close to viruses circulating in the Brazilian Amazon region. Species’ seroprevalence ranged from 0 to 20%, and the risk of seropositivity was higher in bats with a haematophagous diet, living in monospecific colonies and in dense forests. The longitudinal survey showed substantial temporal fluctuations, with individual waves of seroconversions and waning immunity. The high prevalences observed in bat communities, in most habitats and in species that do not share the same microhabitats and bioecological patterns, the temporal variations, and a rather short period of detectable antibodies as observed in recaptured vampires suggest (i) frequent exposure of animals, (ii) an ability of the infected host to control and eliminate the virus, (iii) more relaxed modes of exposure between bats than the commonly assumed infection via direct contact with saliva of infected animals, all of which should be further investigated.

Conclusions / significance

We hypothesize that RABV circulation in French Guiana is mainly maintained in the pristine forest habitats that may provide sufficient food resources to allow vampire bats, the main prevalent species, to survive and RABV to be propagated. However, on the forest edge and in disturbed areas, human activities may induce more insidious effects such as defaunation. One of the ecological consequences is the disappearance of resources for tertiary or secondary consumers. Populations of vampires may then shift to alternative resources such as cattle, domestic animals and humans. Therefore, a good forest status, allowing both a dilution effect in highly rich bat communities and the maintenance of large populations of medium-sized and large mammals used as prey by vampires, should prevent their migration to anthropized areas.     

Metabarcoding for the parallel identification of several hundred predators and their prey: Application to bat species diet analysis

Assessing diet variability is of main importance to better understand the biology of bats and design conservation strategies. Although the advent of metabarcoding has facilitated such analyses, this approach does not come without challenges. Biases may occur throughout the whole experiment, from fieldwork to biostatistics, resulting in the detection of false negatives, false positives or low taxonomic resolution. We detail a rigorous metabarcoding approach based on a short COI minibarcode and two‐step PCR protocol enabling the “all at once” taxonomic identification of bats and their arthropod prey for several hundreds of samples. Our study includes faecal pellets collected in France from 357 bats representing 16 species, as well as insect mock communities that mimic bat meals of known composition, negative and positive controls. All samples were analysed using three replicates. We compare the efficiency of DNA extraction methods, and we evaluate the effectiveness of our protocol using identification success, taxonomic resolution, sensitivity and amplification biases. Our parallel identification strategy of predators and prey reduces the risk of mis‐assigning prey to wrong predators and decreases the number of molecular steps. Controls and replicates enable to filter the data and limit the risk of false positives, hence guaranteeing high confidence results for both prey occurrence and bat species identification. We validate 551 COI variants from arthropod including 18 orders, 117 family, 282 genus and 290 species. Our method therefore provides a rapid, resolutive and cost‐effective screening tool for addressing evolutionary ecological issues or developing “chirosurveillance” and conservation strategies.     

Do predators influence the behaviour of bats?

Many aspects of animal behaviour are affected by real‐time changes in the risk of predation. This conclusion holds for virtually all taxa and ecological systems studied, but does it hold for bats? Bats are poorly represented in the literature on anti‐predator behaviour, which may reflect a lack of nocturnal predators specialized on bats. If bats actually experience a world with minimal anti‐predator concerns, then they will provide a unique contrast within the realm of vertebrate ecology. Alternatively, such predator‐driven behaviour in bats may not yet be fully understood, given the difficulties in working with these highly mobile and nocturnal animals. We provide a wide‐ranging exploration of these issues in bat behaviour. We first cover the basic predator‐prey information available on bats, both on potential predators and the ways in which bats might perceive predators and respond to attacks. We then cover work relevant to key aspects of bat behaviour, such as choice of daytime roosts, the nature of sleep and torpor, evening roost departures, moonlight avoidance, landscape‐related movement patterns, and habitat selection. Overall, the evidence in favour of a strong influence of predators on bat behaviour is equivocal, with the picture clouded by contradictory results and a lack of information on potential predators and the perception of risk by bats. It seems clear that day‐active bats run a considerable risk of being killed by diurnal raptors, which are able to capture bats with relative ease. Thus, bats taking advantage of a pulse of insects just prior to sunset are likely taking risks to gain much‐needed energy. Further, the choice of daytime roosts by bats is probably strongly influenced by roost safety. Few studies, however, have directly addressed either of these topics. As a group, insectivorous temperate‐zone bats show no clear tendency to avoid apparently risky situations, such as activity on moonlit nights. However, some observations are consistent with the idea that predation risk affects choice of movement paths and feeding areas by temperate‐zone bats, as well as the timing of roost departures. The behaviour of tropical bats, on the other hand, seems more generally influenced by predators; this is especially true for tropical nectarivores and frugivores, but also for insectivorous bats. Presumably there are more serious predators on bats in the tropics (e.g. specialized raptors or carnivorous bats), but the identity of these predators is unclear. More information is needed to assess fully the influence of predators on bat behaviour. There is much need for work on the ways in which bats perceive predators via auditory, visual, and olfactory cues, and whether bats have some knowledge of the risks posed by different predators. Also needed is information on how predators attack bats and how bats react to attacking predators. Difficult to obtain, but of critical value, will be information on the nature of the predation risk experienced by bats while away from roosts and during the full darkness of night.     

Colour vision variation in leaf‐nosed bats (Phyllostomidae): Links to cave roosting and dietary specialization

Bats are a diverse radiation of mammals of enduring interest for understanding the evolution of sensory specialization. Colour vision variation among species has previously been linked to roosting preferences and echolocation form in the suborder Yinpterochiroptera, yet questions remain about the roles of diet and habitat in shaping bat visual ecology. We sequenced OPN1SW and OPN1LW opsin genes for 20 species of leaf‐nosed bats (family Phyllostomidae; suborder Yangochiroptera) with diverse roosting and dietary ecologies, along with one vespertilionid species (Myotis lavali). OPN1LW genes appear intact for all species, and predicted spectral tuning of long‐wavelength opsins varied among lineages. OPN1SW genes appear intact and under purifying selection for Myotis lavali and most phyllostomid bats, with two exceptions: (a) We found evidence of ancient OPN1SW pseudogenization in the vampire bat lineage, and loss‐of‐function mutations in all three species of extant vampire bats; (b) we additionally found a recent, independently derived OPN1SW pseudogene in Lonchophylla mordax, a cave‐roosting species. These mutations in leaf‐nosed bats are independent of the OPN1SW pseudogenization events previously reported in Yinpterochiropterans. Therefore, the evolution of monochromacy (complete colour blindness) has occurred in both suborders of bats and under various evolutionary drivers; we find independent support for the hypothesis that obligate cave roosting drives colour vision loss. We additionally suggest that haematophagous dietary specialization and corresponding selection on nonvisual senses led to loss of colour vision through evolutionary sensory trade‐off. Our results underscore the evolutionary plasticity of opsins among nocturnal mammals.     

Viral Communities Among Sympatric Vampire Bats and Cattle

Vampire bats are the only mammals known to feed exclusively on blood from other animals, often from domestic cattle. We tested the hypothesis that the adaptation of vampire bats to hematophagy would have resulted in shared viral communities among vampire bats and cattle, as a direct result of historic spillover events occurring due to hematophagy. We analyzed the presence of different viruses in sample populations of sympatric bat and prey populations and searched for shared viruses between taxa. A limited number of DNA viral groups were detected within each species. However, there was no evidence for a shared viral community among the vampire bat and cattle populations tested.     

In silico and empirical evaluation of twelve metabarcoding primer sets for insectivorous diet analyses

During the most recent decade, environmental DNA metabarcoding approaches have been both developed and improved to minimize the biological and technical biases in these protocols. However, challenges remain, notably those relating to primer design. In the current study, we comprehensively assessed the performance of ten COI and two 16S primer pairs for eDNA metabarcoding, including novel and previously published primers. We used a combined approach of in silico, in vivo‐mock community (33 arthropod taxa from 16 orders), and guano‐based analyses to identify primer sets that would maximize arthropod detection and taxonomic identification, successfully identify the predator (bat) species, and minimize the time and financial costs of the experiment. We focused on two insectivorous bat species that live together in mixed colonies: the greater horseshoe bat (Rhinolophus ferrumequinum) and Geoffroy's bat (Myotis emarginatus). We found that primer degeneracy is the main factor that influences arthropod detection in silico and mock community analyses, while amplicon length is critical for the detection of arthropods from degraded DNA samples. Our guano‐based results highlight the importance of detecting and identifying both predator and prey, as guano samples can be contaminated by other insectivorous species. Moreover, we demonstrate that amplifying bat DNA does not reduce the primers' capacity to detect arthropods. We therefore recommend the simultaneous identification of predator and prey. Finally, our results suggest that up to one‐third of prey occurrences may be unreliable and are probably not of primary interest in diet studies, which may decrease the relevance of combining several primer sets instead of using a single efficient one. In conclusion, this study provides a pragmatic framework for eDNA primer selection with respect to scientific and methodological constraints.     

Evolutionary insights into the ecology of coronaviruses

Although many novel members of the Coronaviridae have recently been recognized in different species, the ecology of coronaviruses has not been established. Our study indicates that bats harbor a much wider diversity of coronaviruses than any other animal species. Dating of different coronavirus lineages suggests that bat coronaviruses are older than those recognized in other animals and that the human severe acute respiratory syndrome (SARS) coronavirus was directly derived from viruses from wild animals in wet markets of southern China. Furthermore, the most closely related bat and SARS coronaviruses diverged in 1986, an estimated divergence time of 17 years prior to the outbreak, suggesting that there may have been transmission via an unknown intermediate host. Analysis of lineage-specific selection pressure also indicated that only SARS coronaviruses in civets and humans were under significant positive selection, also demonstrating a recent interspecies transmission. Analysis of population dynamics revealed that coronavirus populations in bats have constant population growth, while viruses from all other hosts show epidemic-like increases in population. These results indicate that diverse coronaviruses are endemic in different bat species, with repeated introductions to other animals and occasional establishment in other species. Our findings suggest that bats are likely the natural hosts for all presently known coronavirus lineages and that all coronaviruses recognized in other species were derived from viruses residing in bats. Further surveillance of bat and other animal populations is needed to fully describe the ecology and evolution of this virus family.     

Spatial expansions and travelling waves of rabies in vampire bats

A major obstacle to anticipating the cross-species transmission of zoonotic diseases and developing novel strategies for their control is the scarcity of data informing how these pathogens circulate within natural reservoir populations. Vampire bats are the primary reservoir of rabies in Latin America, where the disease remains among the most important viral zoonoses affecting humans and livestock. Unpredictable spatiotemporal dynamics of rabies within bat populations have precluded anticipation of outbreaks and undermined widespread bat culling programs. By analysing 1146 vampire bat-transmitted rabies (VBR) outbreaks in livestock across 12 years in Peru, we demonstrate that viral expansions into historically uninfected zones have doubled the recent burden of VBR. Viral expansions are geographically widespread, but severely constrained by high elevation peaks in the Andes mountains. Within Andean valleys, invasions form wavefronts that are advancing towards large, unvaccinated livestock populations that are heavily bitten by bats, which together will fuel high transmission and mortality. Using spatial models, we forecast the pathways of ongoing VBR epizootics across heterogeneous landscapes. These results directly inform vaccination strategies to mitigate impending viral emergence, reveal VBR as an emerging rather than an enzootic disease and create opportunities to test novel interventions to manage viruses in bat reservoirs.     

Longitudinal deep sequencing informs vector selection and future deployment strategies for transmissible vaccines

Vaccination is a powerful tool in combating infectious diseases of humans and companion animals. In most wildlife, including reservoirs of emerging human diseases, achieving sufficient vaccine coverage to mitigate disease burdens remains logistically unattainable. Virally vectored “transmissible” vaccines that deliberately spread among hosts are a potentially transformative, but still theoretical, solution to the challenge of immunising inaccessible wildlife. Progress towards real-world application is frustrated by the absence of frameworks to guide vector selection and vaccine deployment prior to major in vitro and in vivo investments in vaccine engineering and testing. Here, we performed deep sequencing on field-collected samples of Desmodus rotundus betaherpesvirus (DrBHV), a candidate vector for a transmissible vaccine targeting vampire bat–transmitted rabies. We discovered 11 strains of DrBHV that varied in prevalence and geographic distribution across Peru. The phylogeographic structure of DrBHV strains was predictable from both host genetics and landscape topology, informing long-term DrBHV-vectored vaccine deployment strategies and identifying geographic areas for field trials where vaccine spread would be naturally contained. Multistrain infections were observed in 79% of infected bats. Resampling of marked individuals over 4 years showed within-host persistence kinetics characteristic of latency and reactivation, properties that might boost individual immunity and lead to sporadic vaccine transmission over the lifetime of the host. Further, strain acquisitions by already infected individuals implied that preexisting immunity and strain competition are unlikely to inhibit vaccine spread. Our results support the development of a transmissible vaccine targeting a major source of human and animal rabies in Latin America and show how genomics can enlighten vector selection and deployment strategies for transmissible vaccines.

Deep sequencing of vampire bat betaherpesviruses reveals prevalent multi-strain, multi-year infections with predictable patterns of spread and no evidence of cross immunity, supporting the herpesvirus as a promising transmissible vaccine candidate for bat rabies.     

Vampire bats exhibit evolutionary reduction of bitter taste receptor genes common to other bats

The bitter taste serves as an important natural defence against the ingestion of poisonous foods and is thus believed to be indispensable in animals. However, vampire bats are obligate blood feeders that show a reduced behavioural response towards bitter-tasting compounds. To test whether bitter taste receptor genes (T2Rs) have been relaxed from selective constraint in vampire bats, we sampled all three vampire bat species and 11 non-vampire bats, and sequenced nine one-to-one orthologous T2Rs that are assumed to be functionally conserved in all bats. We generated 85 T2R sequences and found that vampire bats have a significantly greater percentage of pseudogenes than other bats. These results strongly suggest a relaxation of selective constraint and a reduction of bitter taste function in vampire bats. We also found that vampire bats retain many intact T2Rs, and that the taste signalling pathway gene Calhm1 remains complete and intact with strong functional constraint. These results suggest the presence of some bitter taste function in vampire bats, although it is not likely to play a major role in food selection. Together, our study suggests that the evolutionary reduction of bitter taste function in animals is more pervasive than previously believed, and highlights the importance of extra-oral functions of taste receptor genes.