Multiple mortality events in bats: a global review.

Despite conservation concerns for many species of bats, factors causing mortality in bats have not been reviewed since 1970. Here we review and qualitatively describe trends in the occurrence and apparent causes of multiple mortality events (MMEs) in bats around the world.We compiled a database of MMEs, defined as cases in which ≥ 10 dead bats were counted or estimated at a specific location within a maximum timescale of a year, and more typically within a few days or a season. We tabulated 1180 MMEs within nine categories.Prior to the year 2000, intentional killing by humans caused the greatest proportion of MMEs in bats. In North America and Europe, people typically killed bats because they were perceived as nuisances. Intentional killing occurred in South America for vampire bat control, in Asia and Australia for fruit depredation control, and in Africa and Asia for human food. Biotic factors, accidents, and natural abiotic factors were also important historically. Chemical contaminants were confirmed causes of MMEs in North America, Europe, and on islands. Viral and bacterial diseases ranked low as causes of MMEs in bats.Two factors led to a major shift in causes of MMEs in bats at around the year 2000: the global increase of industrial wind-power facilities and the outbreak of white-nose syndrome in North America. Collisions with wind turbines and white-nose syndrome are now the leading causes of reported MMEs in bats.Collectively, over half of all reported MMEs were of anthropogenic origin. The documented occurrence of MMEs in bats due to abiotic factors such as intense storms, flooding, heat waves, and drought is likely to increase in the future with climate change. Coupled with the chronic threats of roosting and foraging habitat loss, increasing mortality through MMEs is unlikely to be compensated for, given the need for high survival in the dynamics of bat populations.

Network analysis of host-virus communities in bats and rodents reveals determinants of cross-species transmission.

Bats are natural reservoirs of several important emerging viruses. Cross-species transmission appears to be quite common among bats, which may contribute to their unique reservoir potential. Therefore, understanding the importance of bats as reservoirs requires examining them in a community context rather than concentrating on individual species. Here, we use a network approach to identify ecological and biological correlates of cross-species virus transmission in bats and rodents, another important host group. We show that given our current knowledge the bat viral sharing network is more connected than the rodent network, suggesting viruses may pass more easily between bat species. We identify host traits associated with important reservoir species: gregarious bats are more likely to share more viruses and bats which migrate regionally are important for spreading viruses through the network. We identify multiple communities of viral sharing within bats and rodents and highlight potential species traits that can help guide studies of novel pathogen emergence.

Bat flight and zoonotic viruses.

Bats are sources of high viral diversity and high-profile zoonotic viruses worldwide. Although apparently not pathogenic in their reservoir hosts, some viruses from bats severely affect other mammals, including humans. Examples include severe acute respiratory syndrome coronaviruses, Ebola and Marburg viruses, and Nipah and Hendra viruses. Factors underlying high viral diversity in bats are the subject of speculation. We hypothesize that flight, a factor common to all bats but to no other mammals, provides an intensive selective force for coexistence with viral parasites through a daily cycle that elevates metabolism and body temperature analogous to the febrile response in other mammals. On an evolutionary scale, this host-virus interaction might have resulted in the large diversity of zoonotic viruses in bats, possibly through bat viruses adapting to be more tolerant of the fever response and less virulent to their natural hosts.

Variability in seroprevalence of rabies virus neutralizing antibodies and associated factors in a Colorado population of big brown bats (Eptesicus fuscus).

In 2001-2005 we sampled permanently marked big brown bats (Eptesicus fuscus) at summer roosts in buildings at Fort Collins, Colorado, for rabies virus neutralizing antibodies (RVNA). Seroprevalence was higher in adult females (17.9%, n = 2,332) than males (9.4%, n = 128; P = 0.007) or volant juveniles (10.2%, n = 738; P<0.0001). Seroprevalence was lowest in a drought year with local insecticide use and highest in the year with normal conditions, suggesting that environmental stress may suppress RVNA production in big brown bats. Seroprevalence also increased with age of bat, and varied from 6.2 to 26.7% among adult females at five roosts sampled each year for five years. Seroprevalence of adult females at 17 other roosts sampled for 1 to 4 years ranged from 0.0 to 47.1%. Using logistic regression, the only ranking model in our candidate set of explanatory variables for serological status at first sampling included year, day of season, and a year by day of season interaction that varied with relative drought conditions. The presence or absence of antibodies in individual bats showed temporal variability. Year alone provided the best model to explain the likelihood of adult female bats showing a transition to seronegative from a previously seropositive state. Day of the season was the only competitive model to explain the likelihood of a transition from seronegative to seropositive, which increased as the season progressed. We found no rabies viral RNA in oropharyngeal secretions of 261 seropositive bats or in organs of 13 euthanized seropositive bats. Survival of seropositive and seronegative bats did not differ. The presence of RVNA in serum of bats should not be interpreted as evidence for ongoing rabies infection.

Prevalence of neutralizing antibodies to rabies virus in serum of seven species of insectivorous bats from Colorado and New Mexico, United States.

We determined the presence of rabies-virus-neutralizing antibodies (RVNA) in serum of 721 insectivorous bats of seven species captured, sampled, and released in Colorado and New Mexico, United States in 2003-2005. A subsample of 160 bats was tested for rabies-virus RNA in saliva. We sampled little brown bats (Myotis lucifugus) at two maternity roosts in Larimer County, Colorado; big brown bats (Eptesicus fuscus) at three maternity roosts in Morgan County, Colorado; and big brown bats at five maternity roosts in Larimer County. We also sampled hoary bats (Lasiurus cinereus) and silver-haired bats (Lasionycteris noctivagans) captured while drinking or foraging over water in Bernalillo County, New Mexico and at various locations in Larimer County. Big brown bats, little brown bats, long-legged myotis (Myotis volans), long-eared myotis (Myotis evotis), and fringed myotis (Myotis thysanodes) were also sampled over water in Larimer County. All species except long-eared myotis included individuals with RVNA, with prevalences ranging from 7% in adult female silver-haired bats to 32% in adult female hoary bats. None of the bats had detectable rabies-virus RNA in oropharyngeal swabs, including 51 bats of 5 species that had RVNA in serum. Antibody-positive bats were present in nine of the 10 maternity colonies sampled. These data suggest that wild bats are commonly exposed to rabies virus and develop a humoral immune response suggesting some degree of viral replication, but many infections fail to progress to clinical disease.

A comparison of bats and rodents as reservoirs of zoonotic viruses: are bats special?

Bats are the natural reservoirs of a number of high-impact viral zoonoses. We present a quantitative analysis to address the hypothesis that bats are unique in their propensity to host zoonotic viruses based on a comparison with rodents, another important host order. We found that bats indeed host more zoonotic viruses per species than rodents, and we identified life-history and ecological factors that promote zoonotic viral richness. More zoonotic viruses are hosted by species whose distributions overlap with a greater number of other species in the same taxonomic order (sympatry). Specifically in bats, there was evidence for increased zoonotic viral richness in species with smaller litters (one young), greater longevity and more litters per year. Furthermore, our results point to a new hypothesis to explain in part why bats host more zoonotic viruses per species: the stronger effect of sympatry in bats and more viruses shared between bat species suggests that interspecific transmission is more prevalent among bats than among rodents. Although bats host more zoonotic viruses per species, the total number of zoonotic viruses identified in bats (61) was lower than in rodents (68), a result of there being approximately twice the number of rodent species as bat species. Therefore, rodents should still be a serious concern as reservoirs of emerging viruses. These findings shed light on disease emergence and perpetuation mechanisms and may help lead to a predictive framework for identifying future emerging infectious virus reservoirs.

Model-guided fieldwork: practical guidelines for multidisciplinary research on wildlife ecological and epidemiological dynamics.

Infectious disease ecology has recently raised its public profile beyond the scientific community due to the major threats that wildlife infections pose to biological conservation, animal welfare, human health and food security. As we start unravelling the full extent of emerging infectious diseases, there is an urgent need to facilitate multidisciplinary research in this area. Even though research in ecology has always had a strong theoretical component, cultural and technical hurdles often hamper direct collaboration between theoreticians and empiricists. Building upon our collective experience of multidisciplinary research and teaching in this area, we propose practical guidelines to help with effective integration among mathematical modelling, fieldwork and laboratory work. Modelling tools can be used at all steps of a field-based research programme, from the formulation of working hypotheses to field study design and data analysis. We illustrate our model-guided fieldwork framework with two case studies we have been conducting on wildlife infectious diseases: plague transmission in prairie dogs and lyssavirus dynamics in American and African bats. These demonstrate that mechanistic models, if properly integrated in research programmes, can provide a framework for holistic approaches to complex biological systems.

Discovery of bacterial NAD⁺-dependent DNA ligase inhibitors: improvements in clearance of adenosine series.

Optimization of clearance of adenosine inhibitors of bacterial NAD(+)-dependent DNA ligase is discussed. To reduce Cytochrome P-450-mediated metabolic clearance, many strategies were explored; however, most modifications resulted in compounds with reduced antibacterial activity and/or unchanged total clearance. The alkyl side chains of the 2-cycloalkoxyadenosines were fluorinated, and compounds with moderate antibacterial activity and favorable pharmacokinetic properties in rat and dog were identified.

Host and viral ecology determine bat rabies seasonality and maintenance.

Rabies is an acute viral infection that is typically fatal. Most rabies modeling has focused on disease dynamics and control within terrestrial mammals (e.g., raccoons and foxes). As such, rabies in bats has been largely neglected until recently. Because bats have been implicated as natural reservoirs for several emerging zoonotic viruses, including SARS-like corona viruses, henipaviruses, and lyssaviruses, understanding how pathogens are maintained within a population becomes vital. Unfortunately, little is known about maintenance mechanisms for any pathogen in bat populations. We present a mathematical model parameterized with unique data from an extensive study of rabies in a Colorado population of big brown bats (Eptesicus fuscus) to elucidate general maintenance mechanisms. We propose that life history patterns of many species of temperate-zone bats, coupled with sufficiently long incubation periods, allows for rabies virus maintenance. Seasonal variability in bat mortality rates, specifically low mortality during hibernation, allows long-term bat population viability. Within viable bat populations, sufficiently long incubation periods allow enough infected individuals to enter hibernation and survive until the following year, and hence avoid an epizootic fadeout of rabies virus. We hypothesize that the slowing effects of hibernation on metabolic and viral activity maintains infected individuals and their pathogens until susceptibles from the annual birth pulse become infected and continue the cycle. This research provides a context to explore similar host ecology and viral dynamics that may explain seasonal patterns and maintenance of other bat-borne diseases.

Alphacoronaviruses in New World bats: prevalence, persistence, phylogeny, and potential for interaction with humans.

Bats are reservoirs for many different coronaviruses (CoVs) as well as many other important zoonotic viruses. We sampled feces and/or anal swabs of 1,044 insectivorous bats of 2 families and 17 species from 21 different locations within Colorado from 2007 to 2009. We detected alphacoronavirus RNA in bats of 4 species: big brown bats (Eptesicus fuscus), 10% prevalence; long-legged bats (Myotis volans), 8% prevalence; little brown bats (Myotis lucifugus), 3% prevalence; and western long-eared bats (Myotis evotis), 2% prevalence. Overall, juvenile bats were twice as likely to be positive for CoV RNA as adult bats. At two of the rural sampling sites, CoV RNAs were detected in big brown and long-legged bats during the three sequential summers of this study. CoV RNA was detected in big brown bats in all five of the urban maternity roosts sampled throughout each of the periods tested. Individually tagged big brown bats that were positive for CoV RNA and later sampled again all became CoV RNA negative. Nucleotide sequences in the RdRp gene fell into 3 main clusters, all distinct from those of Old World bats. Similar nucleotide sequences were found in amplicons from gene 1b and the spike gene in both a big-brown and a long-legged bat, indicating that a CoV may be capable of infecting bats of different genera. These data suggest that ongoing evolution of CoVs in bats creates the possibility of a continued threat for emergence into hosts of other species. Alphacoronavirus RNA was detected at a high prevalence in big brown bats in roosts in close proximity to human habitations (10%) and known to have direct contact with people (19%), suggesting that significant potential opportunities exist for cross-species transmission of these viruses. Further CoV surveillance studies in bats throughout the Americas are warranted.

Optimization of Potent Inhibitors of P. falciparum Dihydroorotate Dehydrogenase for the Treatment of Malaria.

Inhibition of dihydroorotate dehydrogenase (DHODH) for P. falciparum potentially represents a new treatment option for malaria, since DHODH catalyzes the rate-limiting step in the pyrimidine biosynthetic pathway and P. falciparum is unable to salvage pyrimidines and must rely on de novo biosynthesis for survival. We report herein the synthesis and structure-activity relationship of a series of 5-(2-methylbenzimidazol-1-yl)-N-alkylthiophene-2-carboxamides that are potent inhibitors against PfDHODH but do not inhibit the human enzyme. On the basis of efficacy observed in three mouse models of malaria, acceptable safety pharmacology risk assessment and safety toxicology profile in rodents, lack of potential drug-drug interactions, acceptable ADME/pharmacokinetic profile, and projected human dose, 5-(4-cyano-2-methyl-1H-benzo[d]imidazol-1-yl)-N-cyclopropylthiophene-2-carboxamide 2q was identified as a potential drug development candidate.

Synthesis and antiplasmodial activity of novel 2,4-diaminopyrimidines.

Two sets of diaminopyrimidines, totalling 45 compounds, were synthesized and assayed against Plasmodium falciparum. The SAR was relatively shallow, with only the presence of a 2-(pyrrolidin-1-yl)ethyl group at R(2) significantly affecting activity. A subsequent series addressed high LogD values by introducing more polar side groups, with the most active compounds possessing diazepine and N-benzyl-4-aminopiperidyl groups at R(1)/R(2). A final series attempted to address high in vitro microsomal clearance by replacing the C6-Me group with CF(3), however antiplasmodial activity decreased without any improvement in clearance. The C6-CF(3) group decreased hERG inhibition, probably as a result of decreased amine basicity at C2/C4.

Synthesis and in vitro DMPK profiling of a 1,2-dioxolane-based library with activity against Plasmodium falciparum.

A 43-member 1,2-dioxolane library was synthesized by coupling a 1,2-dioxolane-3-acetic acid derivative to a range of amines. Ten compounds had EC(50)s30nM against Plasmodium falciparum 3D7 and Dd2 strains, and another 15 compounds had EC(50)s50nM against both 3D7 and Dd2. The library was then subjected to a range of in vitro DMPK assays, which revealed that side chains with a heteroatom were required for favorable solubility, LogD and membrane permeability. CYP450 inhibition was isoform dependent, with 2C19 and 3A4 particularly susceptible, and the majority of compounds tested against rat and human microsomes were metabolized rapidly.

Lack of association between ectoparasite intensities and rabies virus neutralizing antibody seroprevalence in wild big brown bats (Eptesicus fuscus), Fort Collins, Colorado.

Recently, bat ectoparasites have been demonstrated to harbor pathogens of potential importance to humans. We evaluated antirabies antibody seroprevalence and the presence of ectoparasites in big brown bats (Eptesicus fuscus) sampled in 2002 and 2003 in Colorado to investigate if an association existed between ectoparasite intensity and exposure to rabies virus (RV). We used logistic regression and Akaike's Information Criteria adjusted for sample size (AICc) in a post-hoc analysis to investigate the relative importance of three ectoparasite species, as well as bat colony size, year sampled, age class, colony size, and year interaction on the presence of rabies virus neutralizing antibodies (VNA) in serum of wild E. fuscus. We obtained serum samples and ectoparasite counts from big brown bats simultaneously in 2002 and 2003. Although the presence of ectoparasites (Steatonyssus occidentalis and Spinturnix bakeri) were important in elucidating VNA seroprevalence, their intensities were higher in seronegative bats than in seropositive bats, and the presence of a third ectoparasite (Cimex pilosellus) was inconsequential. Colony size and year sampled were the most important variables in these AICc models. These findings suggest that these ectoparasites do not enhance exposure of big brown bats to RV.

Factors influencing movement probabilities of big brown bats (Eptesicus fuscus) in buildings.

We investigated movements of female big brown bats (Eptesicus fuscus) roosting in maternity colonies in buildings in Fort Collins, Colorado (USA), during the summers of 2002, 2003, and 2005. This behavior can be of public health concern where bats that may carry diseases (e.g., rabies) move among buildings occupied by people. We used passive integrated transponders (PIT tags) to mark individual bats and hoop PIT readers at emergence points to passively monitor the use of building roosts by marked adult females on a daily basis during the lactation phase of reproduction. Multi-strata models were used to examine movements among roosts in relation to ambient temperatures and ectoparasite loads. Our results suggest that high ambient temperatures influence movements. Numbers of mites (Steatonyssus occidentalis) did not appear to influence movements of female bats among building roosts. In an urban landscape, periods with unusually hot conditions are accompanied by shifting of bats to different buildings or segments of buildings, and this behavior may increase the potential for contact with people in settings where, in comparison to their more regularly used buildings, the bats may be more likely to be of public concern as nuisances or health risks.

Evaluation of 3-O-methylfluorescein as a selective fluorometric substrate for CYP2C19 in human liver microsomes.

Cytochrome P450 (P450) fluorometric high-throughput inhibition assays have been widely used for drug-drug interaction screening particularly at the preclinical drug discovery stages. Many fluorometric substrates have been investigated for their selectivity, but most are found to be catalyzed by multiple P450 isozymes, limiting their utility. In this study, 3-O-methylfluorescein (OMF) was examined as a selective fluorescence substrate for CYP2C19 in human liver microsomes (HLMs). The kinetic studies of OMF O-demethylation in HLMs using a liquid chromatography/mass spectrometry method exhibited two-enzyme kinetics with apparent K(m) and V(max) values of 1.14 +/- 0.90 microM and 11.3 +/- 4.6 pmol/mg/min, respectively, for the high affinity component(s) and 57.0 +/- 6.4 microM and 258 +/- 6 pmol/mg/min, respectively, for the low affinity component(s). Studies utilizing cDNA-expressed individual P450 isoforms and P450-selective chemical inhibitors showed that OMF O-demethylation to fluorescein was selective for CYP2C19 at substrate concentrations < or =1 microM. At substrate concentrations > or =10 microM, other P450 isozymes were found to catalyze OMF O-demethylation. In HLMs, analysis of the two-enzyme kinetics in the presence of P450 isozyme-selective chemical inhibitors (ticlopidine for CYP2C19, sulfaphenazole for CYP2C9, and furafylline for CYP1A2) indicated that CYP2C19 was the high affinity component and CYP2C9 was the low affinity component. Based on these findings, a fluorometric assay was developed using 1 microM OMF and 2 microM sulfaphenazole for probing CYP2C19-mediated inhibition in HLMs. The IC(50) data of 13 substrates obtained from the fluorometric assay developed in this study correlated well with that reported in the literature using nonfluorescence assays.

Detection of group 1 coronaviruses in bats in North America.

The epidemic of severe acute respiratory syndrome (SARS) was caused by a newly emerged coronavirus (SARS-CoV). Bats of several species in southern People's Republic of China harbor SARS-like CoVs and may be reservoir hosts for them. To determine whether bats in North America also harbor coronaviruses, we used reverse transcription-PCR to detect coronavirus RNA in bats. We found coronavirus RNA in 6 of 28 fecal specimens from bats of 2 of 7 species tested. The prevalence of viral RNA shedding was high: 17% in Eptesicus fuscus and 50% in Myotis occultus. Sequence analysis of a 440-bp amplicon in gene 1b showed that these Rocky Mountain bat coronaviruses formed 3 clusters in phylogenetic group 1 that were distinct from group 1 coronaviruses of Asian bats. Because of the potential for bat coronaviruses to cause disease in humans and animals, further surveillance and characterization of bat coronaviruses in North America are needed.

Sampling blood from big brown bats (Eptesicus fuscus) in the field with and without anesthesia: impacts on survival.

Blood was collected from wild big brown bats (Eptesicus fuscus) with and without anesthesia in Fort Collins, Colorado in 2004 to assess the impacts of these procedures on short-term survival and 1-yr return rates. Short-term survival and 1-yr return rates after release were passively monitored using PIT tag detection hoops placed at selected buildings. Comparison of 14-day maximum likelihood survival estimates from bats not bled (142 adult females, 62 volant juveniles), and bats sampled for blood with anesthesia (96 adult females, 23 volant juveniles) and without anesthesia (112 adult females, 22 volant juveniles) indicated no adverse effects of either treatment (juveniles: chi(2) = 53.38, df = 41, P = 0.09; adults: chi(2) = 39.09, df = 44, P = 0.68). Return rates of bats one year after sampling were similar among adult female controls (75.4%, n = 142, 95% CI = 67.4-82.2%), females sampled for blood with anesthesia (83.0%, n = 112, 95% CI = 74.8-89.5%), and females sampled without anesthesia (87.5%, n = 96, 95% CI = 79.2-93.4%). Lack of an effect was also noted in 1-yr return rates of juvenile females. These data suggest that the use of anesthesia during sampling of blood has no advantages in terms of enhancement of survival in big brown bats.

Anesthesia and blood sampling of wild big brown bats (eptesicus fuscus) with an assessment of impacts on survival.

We anesthetized and blood sampled wild big brown bats (Eptesicus fuscus) in Fort Collins, Colorado (USA) in 2001 and 2002 and assessed effects on survival. Inhalant anesthesia was delivered into a specially designed restraint and inhalation capsule that minimized handling and bite exposures. Bats were immobilized an average of 9.1+/-5.1 (SD) min (range 1-71, n=876); blood sample volumes averaged 58+/-12 microl (range 13-126, n=718). We randomly selected control (subject to multiple procedures before release) and treatment (control procedures plus inhalant anesthesia and 1% of body weight blood sampling) groups in 2002 to assess treatment effects on daily survival over a 14-day period for adult female and volant juvenile bats captured at maternity roosts in buildings. We monitored survival after release using passive integrated transponder tag detection hoops placed at openings to selected roosts. Annual return rates of bats sampled in 2001 were used to assess long-term outcomes. Comparison of 14-day maximum-likelihood daily survival estimates from control (86 adult females, 92 volant juveniles) and treated bats (187 adult females, 87 volant juveniles) indicated no adverse effect from anesthesia and blood sampling (juveniles: chi2=22.22, df=27, P>0.05; adults: chi2=9.72, df=18, P>0.05). One-year return rates were similar among adult female controls (81%, n=72, 95% confidence interval [CI]=70-91%), females treated once (82%, n=276, 95% CI=81-84%), and females treated twice (84%, n=50, 95% CI=74-94%). Lack of an effect was also noted in 1-yr return rates of juvenile female controls (55%, n=29, 95% CI=37-73%), juveniles treated once (66%, n=113, 95% CI=58-75%), and juveniles treated twice (71%, n=17, 95% CI=49-92%). These data suggest that anesthesia and blood sampling for health monitoring did not measurably affect survival of adult female and volant juvenile big brown bats.

Genetic divergence of rabies viruses from bat species of Colorado, USA.

Molecular epidemiological studies have linked many cryptic human rabies cases in the United States with exposure to rabies virus (RV) variants associated with insectivorous bats. In Colorado, bats accounted for 98% of all reported animal rabies cases between 1977 and 1996. The genetic divergence of RV was investigated in bat and terrestrial animal specimens that were submitted for rabies diagnosis to the Colorado Department of Public Health and Environment (CDPHE), Colorado, USA. RV isolates from animal specimens across the United States were also included in the analysis. Phylogenetic analyses were performed on partial nucleoprotein (N) gene sequences, which revealed seven principal clades. RV associated with the colonial big brown bat, Eptesicus fuscus, an bats of the genus Myotis were found to segregate into two distinct clades (I and IV). Clade I was harbored by E. fuscus and Myotis species, but was also identified in terrestrial animals such as domestic cats and striped skunks (Mephitis mephitis). Clade IV was divided into subclades IVA, IVB, and IVC; IVA was identified in E. fuscus, and Myotis species bats, and also in a fox; subclades IVB and IVC circulated predominantly in E. fuscus. Clade II was formed by big free-tailed bat (Nyctinomops macrotis) and striped skunk (Mephitis mephitis) samples. Clade III included RVs that are maintained by generally solitary, migratory bats such as the silver-haired bat (Lasionycteris noctivagans) and bats of the genus Lasiurus. Big brown bats were found to harbor this RV variant. None of the Colorado specimens segregated with clades V and VII that harbor RVs associated with terrestrial animals. Different species of bats had the same RV variant, indicating active inter-species rabies transmission. In Colorado, animal rabies occurs principally in bats, and the identification of bat RVs in cat, gray fox Urocyon cinereoargenteus), and striped skunks demonstrated the importance of rabies spillover from bats to domestic and terrestrial wildlife species.