Pteropid Bats Research Articles

Hervey virus: Study on co-circulation with Henipaviruses in Pteropid bats within their distribution range from Australia to Africa.

In 2011, an unusually large number of independent Hendra virus outbreaks were recorded on horse properties in Queensland and New South Wales, Australia. Urine from bat colonies adjacent to the outbreak sites were sampled and screened for Hendra and other viruses. Several novel paramyxoviruses were also isolated at different locations. Here one of the novel viruses, named Hervey virus (HerPV), is fully characterized by genome sequencing, annotation, phylogeny and in vitro host range, and its serological cross-reactivity and neutralization patterns are examined. HerPV may have ecological and spatial and temporal patterns similar to Hendra virus and could serve as a sentinel virus for the surveillance of this highly pathogenic virus. The suitability of HerPV as potential sentinel virus is further assessed by determining the serological prevalence of HerPV antibodies in fruit-eating bats from Australia, Indonesia, Papua New Guinea, Tanzania and the Gulf of Guinea, indicating the presence of similar viruses in regions beyond the Australian border.

The impact of human population pressure on flying fox niches and the potential consequences for Hendra virus spillover

Hendra virus (HeV) is an emerging pathogen of concern in Australia given its ability to spillover from its reservoir host, pteropid bats, to horses and further on to humans, and the severe clinical presentation typical in these latter incidental hosts. Specific human pressures over recent decades, such as expanding human populations, urbanization, and forest fragmentation, may have altered the ecological niche of Pteropus species acting as natural HeV reservoirs and may modulate spillover risk. This study explored the influence of inter-decadal net human local migration between 1970 and 2000 on changes in the habitat suitability to P. alecto and P. conspicillatus from 1980 to 2015 in eastern Australia. These ecological niches were modeled using boosted regression trees and subsequently fitted, along with additional landscape factors, to HeV spillovers to explore the spatial dependency of this zoonosis. The spatial model showed that the ecological niche of these two flying fox species, the human footprint, and proximity to woody savanna were each strongly associated with HeV spillover and together explained most of the spatial dependency exhibited by this zoonosis. These findings reinforce the potential for anthropogenic pressures to shape the landscape epidemiology of HeV spillover.

Physiological stress and Hendra virus in flying-foxes (Pteropus spp.), Australia.

Pteropid bats (flying-foxes) are the natural reservoir of Hendra virus, an emergent paramyxovirus responsible for fatal infection in horses and humans in Australia. Pteropus alecto (the Black flying-fox) and the paraphyletic P. conspicillatus (the Spectacled flying-fox) appear to be the primary reservoir hosts. Previous studies have suggested that physiological and ecological factors may underpin infection dynamics in flying-foxes, and subsequent spillover to horses and in turn humans. We sought to examine temporal trends in urinary cortisol concentration in wild Australian flying-fox populations, to elucidate the putative relationship between Hendra virus infection and physiological stress. Pooled and individual urine samples were non-invasively collected from under roosting flying-foxes at two latitudinally disparate regions in the eastern Australian state of Queensland. Hendra virus detection, and (in individual urine samples) sex and species determination were PCR-based. Urinary cortisol measurement used a validated enzyme immunoassay. We found no direct correlation between increased urinary cortisol and Hendra virus excretion, but our findings do suggest a biologically plausible association between low winter temperatures and elevated cortisol levels in P. alecto in the lower latitude Southeast Queensland roosts. We hypothesize an indirect association between low winter temperatures and increased Hendra virus infection and excretion, mediated by the physiological cost of thermoregulation. Our findings and our approach are directly relevant to elaboration of the disease ecology of Nipah virus and other emerging henipaviruses in bats. More broadly, they inform investigation of emerging disease infection dynamics across the wildlife/livestock/human interface.

Insights into the ancestral organisation of the mammalian MHC class II region from the genome of the pteropid bat, Pteropus alecto

BackgroundBats are an extremely successful group of mammals and possess a variety of unique characteristics, including their ability to co-exist with a diverse range of pathogens. The major histocompatibility complex (MHC) is the most gene dense and polymorphic region of the genome and MHC class II (MHC-II) molecules play a vital role in the presentation of antigens derived from extracellular pathogens and activation of the adaptive immune response. Characterisation of the MHC-II region of bats is crucial for understanding the evolution of the MHC and of the role of pathogens in shaping the immune system.ResultsHere we describe the relatively contracted MHC-II region of the Australian black flying-fox (Pteropus alecto), providing the first detailed insight into the MHC-II region of any species of bat. Twelve MHC-II genes, including one locus (DRB2) located outside the class II region, were identified on a single scaffold in the bat genome. The presence of a class II locus outside the MHC-II region is atypical and provides evidence for an ancient class II duplication block. Two non-classical loci, DO and DM and two classical, DQ and DR loci, were identified in P. alecto. A putative classical, DPB pseudogene was also identified. The bat’s antigen processing cluster, though contracted, remains highly conserved, thus supporting its importance in antigen presentation and disease resistance.ConclusionsThis detailed characterisation of the bat MHC-II region helps to fill a phylogenetic gap in the evolution of the mammalian class II region and is a stepping stone towards better understanding of the immune responses in bats to viral, bacterial, fungal and parasitic infections.

\u201cWhy won\u2019t they just vaccinate?\u201d Horse owner risk perception and uptake of the Hendra virus vaccine

BackgroundHendra virus is a paramyxovirus that causes periodic serious disease and fatalities in horses and humans in Australia first identified in 1994. Pteropid bats (commonly known as flying-foxes) are the natural host of the virus, and the putative route of infection in horses is by ingestion or inhalation of material contaminated by flying-fox urine or other bodily fluids. Humans become infected after close contact with infected horses. Horse owners in Australia are encouraged to vaccinate their horses against Hendra virus to reduce the risk of Hendra virus infection, and to prevent potential transmission to humans. After the vaccine was released in 2012, uptake by horse owners was slow, with some estimated 11-17% of horses in Australia vaccinated. This study was commissioned to examine barriers to vaccine uptake and potential drivers to future adoption of vaccination by horse owners.MethodsThis study examined qualitative comments from respondents to an on-line survey, reporting reasons for not vaccinating their horses. The study also investigated scenarios in which respondents felt they might consider vaccinating their horses.ResultsSelf-reported barriers to uptake of the Hendra virus vaccine by horse owners (N = 150) included concerns about vaccine safety, cost, and effectiveness. Reduction in vaccination costs and perception of immediacy of Hendra virus risk were reported as being likely to change future behaviour. However, the data also indicated that horse owners generally would not reconsider vaccinating their horses if advised by their veterinarian.ConclusionWhile changes to vaccine costs and the availability data supporting vaccine safety and efficacy may encourage more horse owners to vaccinate, this study highlights the importance of protecting the relationship between veterinarians and horse owners within the risk management strategies around Hendra virus. Interactions and trust between veterinarians and animal owners has important implications for management of and communication around Hendra virus and other zoonotic disease outbreaks.

Microclimates Might Limit Indirect Spillover of the Bat Borne Zoonotic Hendra Virus.

Infectious diseases are transmitted when susceptible hosts are exposed to pathogen particles that can replicate within them. Among factors that limit transmission, the environment is particularly important for indirectly transmitted parasites. To try and assess a pathogens' ability to be transmitted through the environment and mitigate risk, we need to quantify its decay where transmission occurs in space such as the microclimate harbouring the pathogen. Hendra virus, a Henipavirus from Australian Pteropid bats, spills-over to horses and humans, causing high mortality. While a vaccine is available, its limited uptake has reduced opportunities for adequate risk management to humans, hence the need to develop synergistic preventive measures, like disrupting its transmission pathways. Transmission likely occurs shortly after virus excretion in paddocks; however, no survival estimates to date have used real environmental conditions. Here, we recorded microclimate conditions and fitted models that predict temperatures and potential evaporation, which we used to simulate virus survival with a temperature-survival model and modification based on evaporation. Predicted survival was lower than previously estimated and likely to be even lower according to potential evaporation. Our results indicate that transmission should occur shortly after the virus is excreted, in a relatively direct way. When potential evaporation is low, and survival is more similar to temperature dependent estimates, transmission might be indirect because the virus can wait several hours until contact is made. We recommend restricting horses' access to trees during night time and reducing grass under trees to reduce virus survival.

Toxocara pteropodis in Free-Ranging Indian Flying Foxes (Pteropus medius) in Sri Lanka.

Toxocara pteropodis, an intestinal nematode, occurs in several captive and free-ranging pteropid bat species. We report infection in free-ranging Indian flying foxes (Pteropus medius) in Sri Lanka and contribute to our understanding of parasites in free-ranging P. medius .

Nipah Virus: A Public Health Concern

Nipah virus, a member of the genus Henipavirus, a new class of virus in the Paramyxoviridae family, has drawn attention as an emerging zoonotic virus in South-East and South Asian region. Case fatality rate of Nipah virus infection ranges from 40–70% although it has been as high as 100% in some outbreaks. Many of the outbreaks were attributed to pigs consuming fruits, partially eaten by fruit bats, and transmission of infection to humans. In Bangladesh, Nipah virus infection was associated with contact with a sick cow, consumption of fresh date palm sap (potentially contaminated with pteropid bat saliva), and person-to-person transmission. In 2014, 18 cases of Nipah virus infection have been reported in Bangladesh, of which 9 cases died. In the most recent epidemic at least 6 people died out of nine cases due to Nipah virus infection in the remote northern Bangladesh in 2015. Human infections range from asymptomatic infection to fatal encephalitis. Some people can also experience atypical pneumonia and severe respiratory problems. The virus is detected by ELISA, PCR, immunofluoroscence assay and isolation by cell culture. Treatment is mostly symptomatic and supportive as the effect of antiviral drugs is not satisfactory, and an effective vaccine is yet to be developed. So the very high case fatality addresses the need for adequate and strict control and preventive measures.J Enam Med Col 2016; 6(2): 101-105

Hendra Virus Infection Dynamics in the Grey-Headed Flying Fox (Pteropus poliocephalus) at the Southern-Most Extent of Its Range: Further Evidence This Species Does Not Readily Transmit the Virus to Horses.

Hendra virus (HeV) is an important emergent virus in Australia known to infect horses and humans in certain regions of the east coast. Whilst pteropid bats (“flying foxes”) are considered the natural reservoir of HeV, which of the four mainland species is the principal reservoir has been a source of ongoing debate, particularly as shared roosting is common. To help resolve this, we sampled a colony consisting of just one of these species, the grey-headed flying fox, (Pteropus poliocephalus), at the southernmost extent of its range. Using the pooled urine sampling technique at approximately weekly intervals over a two year period, we determined the prevalence of HeV and related paramyxoviruses using a novel multiplex (Luminex) platform. Whilst all the pooled urine samples were negative for HeV nucleic acid, we successfully identified four other paramyxoviruses, including Cedar virus; a henipavirus closely related to HeV. Collection of serum from individually caught bats from the colony showed that antibodies to HeV, as estimated by a serological Luminex assay, were present in between 14.6% and 44.5% of animals. The wide range of the estimate reflects uncertainties in interpreting intermediate results. Interpreting the study in the context of HeV studies from states to the north, we add support for an arising consensus that it is the black flying fox and not the grey-headed flying fox that is the principal source of HeV in spillover events to horses.

Spatiotemporal Aspects of Hendra Virus Infection in Pteropid Bats (Flying-Foxes) in Eastern Australia.

Hendra virus (HeV) causes highly lethal disease in horses and humans in the eastern Australian states of Queensland (QLD) and New South Wales (NSW), with multiple equine cases now reported on an annual basis. Infection and excretion dynamics in pteropid bats (flying-foxes), the recognised natural reservoir, are incompletely understood. We sought to identify key spatial and temporal factors associated with excretion in flying-foxes over a 2300 km latitudinal gradient from northern QLD to southern NSW which encompassed all known equine case locations. The aim was to strengthen knowledge of Hendra virus ecology in flying-foxes to improve spillover risk prediction and exposure risk mitigation strategies, and thus better protect horses and humans. Monthly pooled urine samples were collected from under roosting flying-foxes over a three-year period and screened for HeV RNA by quantitative RT-PCR. A generalised linear model was employed to investigate spatiotemporal associations with HeV detection in 13,968 samples from 27 roosts. There was a non-linear relationship between mean HeV excretion prevalence and five latitudinal regions, with excretion moderate in northern and central QLD, highest in southern QLD/northern NSW, moderate in central NSW, and negligible in southern NSW. Highest HeV positivity occurred where black or spectacled flying-foxes were present; nil or very low positivity rates occurred in exclusive grey-headed flying-fox roosts. Similarly, little red flying-foxes are evidently not a significant source of virus, as their periodic extreme increase in numbers at some roosts was not associated with any concurrent increase in HeV detection. There was a consistent, strong winter seasonality to excretion in the southern QLD/northern NSW and central NSW regions. This new information allows risk management strategies to be refined and targeted, mindful of the potential for spatial risk profiles to shift over time with changes in flying-fox species distribution.

Routes of Hendra Virus Excretion in Naturally-Infected Flying-Foxes: Implications for Viral Transmission and Spillover Risk.

Pteropid bats or flying-foxes (Chiroptera: Pteropodidae) are the natural host of Hendra virus (HeV) which sporadically causes fatal disease in horses and humans in eastern Australia. While there is strong evidence that urine is an important infectious medium that likely drives bat to bat transmission and bat to horse transmission, there is uncertainty about the relative importance of alternative routes of excretion such as nasal and oral secretions, and faeces. Identifying the potential routes of HeV excretion in flying-foxes is important to effectively mitigate equine exposure risk at the bat-horse interface, and in determining transmission rates in host-pathogen models. The aim of this study was to identify the major routes of HeV excretion in naturally infected flying-foxes, and secondarily, to identify between-species variation in excretion prevalence. A total of 2840 flying-foxes from three of the four Australian mainland species (Pteropus alecto, P. poliocephalus and P. scapulatus) were captured and sampled at multiple roost locations in the eastern states of Queensland and New South Wales between 2012 and 2014. A range of biological samples (urine and serum, and urogenital, nasal, oral and rectal swabs) were collected from anaesthetized bats, and tested for HeV RNA using a qRT-PCR assay targeting the M gene. Forty-two P. alecto (n = 1410) had HeV RNA detected in at least one sample, and yielded a total of 78 positive samples, at an overall detection rate of 1.76% across all samples tested in this species (78/4436). The rate of detection, and the amount of viral RNA, was highest in urine samples (>serum, packed haemocytes >faecal >nasal >oral), identifying urine as the most plausible source of infection for flying-foxes and for horses. Detection in a urine sample was more efficient than detection in urogenital swabs, identifying the former as the preferred diagnostic sample. The detection of HeV RNA in serum is consistent with haematogenous spread, and with hypothesised latency and recrudesence in flying-foxes. There were no detections in P. poliocephalus (n = 1168 animals; n = 2958 samples) or P. scapulatus (n = 262 animals; n = 985 samples), suggesting (consistent with other recent studies) that these species are epidemiologically less important than P. alecto in HeV infection dynamics. The study is unprecedented in terms of the individual animal approach, the large sample size, and the use of a molecular assay to directly determine infection status. These features provide a high level of confidence in the veracity of our findings, and a sound basis from which to more precisely target equine risk mitigation strategies.

Landscape Utilisation, Animal Behaviour and Hendra Virus Risk.

Hendra virus causes sporadic fatal disease in horses and humans in eastern Australia. Pteropid bats (flying-foxes) are the natural host of the virus. The mode of flying-fox to horse transmission remains unclear, but oro-nasal contact with flying-fox urine, faeces or saliva is the most plausible. We used GPS data logger technology to explore the landscape utilisation of black flying-foxes and horses to gain new insight into equine exposure risk. Flying-fox foraging was repetitious, with individuals returning night after night to the same location. There was a preference for fragmented arboreal landscape and non-native plant species, resulting in increased flying-fox activity around rural infrastructure. Our preliminary equine data logger study identified significant variation between diurnal and nocturnal grazing behaviour that, combined with the observed flying-fox foraging behaviour, could contribute to Hendra virus exposure risk. While we found no significant risk-exposing difference in individual horse movement behaviour in this study, the prospect warrants further investigation, as does the broader role of animal behaviour and landscape utilisation on the transmission dynamics of Hendra virus.

Flying-foxes in the Australian urban environment—community attitudes and opinions

The urban presence of flying-foxes (pteropid bats) in eastern Australia has increased in the last 20years, putatively reflecting broader landscape change. The influx of large numbers often precipitates community angst, typically stemming from concerns about loss of social amenity, economic loss or negative health impacts from recently emerged bat-mediated zoonotic diseases such as Hendra virus and Australian bat lyssavirus. Local authorities and state wildlife authorities are increasingly asked to approve the dispersal or modification of flying-fox roosts to address expressed concerns, yet the scale of this concern within the community, and the veracity of the basis for concern are often unclear. We conducted an on-line survey to capture community attitudes and opinions on flying-foxes in the urban environment to inform management policy and decision-making. Analysis focused on awareness, concerns, and management options, and primarily compared responses from communities where flying-fox management was and was not topical at the time of the survey. While a majority of respondents indicated a moderate to high level of knowledge of both flying-foxes and Hendra virus, a substantial minority mistakenly believed that flying-foxes pose a direct infection risk to humans, suggesting miscommunication or misinformation, and the need for additional risk communication strategies. Secondly, a minority of community members indicated they were directly impacted by urban roosts, most plausibly those living in close proximity to the roost, suggesting that targeted management options are warranted. Thirdly, neither dispersal nor culling was seen as an appropriate management strategy by the majority of respondents, including those from postcodes where flying-fox management was topical. These findings usefully inform community debate and policy development and demonstrate the value of social analysis in defining the issues and options in this complex human–wildlife interaction. The mobile nature of flying-foxes underlines the need for a management strategy at a regional or larger scale, and independent of state borders.

This could be the start of something big—20 years since the identification of bats as the natural host of Hendra virus

Hendra virus was first described in 1994 in Australia, causally associated with a cluster of fatal equine and human cases at a thoroughbred racing stable in the Brisbane suburb of Hendra. This year marks the twentieth anniversary of the identification of pteropid bats (flying-foxes) as the natural host of the virus, and it is timely to reflect on a pivotal meeting of an eclectic group of scientists in that process. They included animal and public health experts, environmental scientists, veterinary and horse industry representatives, and wildlife experts. The task was to review and prioritise wildlife surveillance seeking the origin of the previously unknown virus. The group determined that the likely reservoir must occur in disparate locations, and be capable of moving between locations, or exist in continuous, overlapping populations spanning multiple locations. Flying-foxes were considered to be a more probable source of the novel virus than birds. Within weeks, antibodies were detected in several species of flying-fox, and the virus was subsequently isolated. While the identification of the natural host of Hendra virus within 18months of its description was remarkable in itself, a broader legacy followed. In the subsequent years, a suite of zoonotic viruses including Australian bat lyssavirus, Nipah virus, SARS coronavirus, and Ebola and Marburg viruses have been detected in bats. Bats are now the “go to” taxa for novel viruses. History has repeatedly demonstrated that knowledge begets knowledge. This simple notion of bringing a diverse group of people together in an environment of mutual respect reinforced this principle and proves that the sum is often so much more powerful than the parts.

Structural characterization by transmission electron microscopy and immunoreactivity of recombinant Hendra virus nucleocapsid protein expressed and purified from Escherichia coli

Hendra virus (family Paramyxoviridae) is a negative sense single-stranded RNA virus (NSRV) which has been found to cause disease in humans, horses, and experimentally in other animals, e.g. pigs and cats. Pteropid bats commonly known as flying foxes have been identified as the natural host reservoir. The Hendra virus nucleocapsid protein (HeV N) represents the most abundant viral protein produced by the host cell, and is highly immunogenic with naturally infected humans and horses producing specific antibodies towards this protein.The purpose of this study was to express and purify soluble, functionally active recombinant HeV N, suitable for use as an immunodiagnostic reagent to detect antibodies against HeV. We expressed both full-length HeV N, (HeV NFL), and a C-terminal truncated form, (HeV NCORE), using a bacterial heterologous expression system. Both HeV N constructs were engineered with an N-terminal Hisx6 tag, and purified using a combination of immobilized metal affinity chromatography (IMAC) and size exclusion chromatography (SEC). Purified recombinant HeV N proteins self-assembled into soluble higher order oligomers as determined by SEC and negative-stain transmission electron microscopy. Both HeV N proteins were highly immuno-reactive with sera from animals and humans infected with either HeV or the closely related Nipah virus (NiV), but displayed no immuno-reactivity towards sera from animals infected with a non-pathogenic paramyxovirus (CedPV), or animals receiving Equivac® (HeV G glycoprotein subunit vaccine), using a Luminex-based multiplexed microsphere assay.

Natural Hendra Virus Infection in Flying-Foxes - Tissue Tropism and Risk Factors.

Hendra virus (HeV) is a lethal zoonotic agent that emerged in 1994 in Australia. Pteropid bats (flying-foxes) are the natural reservoir. To date, HeV has spilled over from flying-foxes to horses on 51 known occasions, and from infected horses to close-contact humans on seven occasions. We undertook screening of archived bat tissues for HeV by reverse transcription quantitative polymerase chain reaction (RT-qPCR). Tissues were tested from 310 bats including 295 Pteropodiformes and 15 Vespertilioniformes. HeV was detected in 20 individual flying-foxes (6.4%) from various tissues including spleen, kidney, liver, lung, placenta and blood components. Detection was significantly higher in Pteropus Alecto and P. conspicillatus, identifying species as a risk factor for infection. Further, our findings indicate that HeV has a predilection for the spleen, suggesting this organ plays an important role in HeV infection. The lack of detections in the foetal tissues of HeV-positive females suggests that vertical transmission is not a regular mode of transmission in naturally infected flying-foxes, and that placental and foetal tissues are not a major source of infection for horses. A better understanding of HeV tissue tropism will strengthen management of the risk of spillover from flying-foxes to horses and ultimately humans.

Complete genome sequence of teviot paramyxovirus, a novel rubulavirus isolated from fruit bats in australia.

The causative agents of a number of emerging zoonotic diseases have been identified as paramyxoviruses originating in bats. We report here the complete genome sequence of two Teviot paramyxoviruses, novel rubulaviruses isolated from urine samples collected from pteropid bats in Australia. The zoonotic potential of Teviot paramyxovirus is undetermined.

Isolation of multiple novel paramyxoviruses from pteropid bat urine.

Bats have been found to harbour a number of new emerging viruses with zoonotic potential, and there has been a great deal of interest in identifying novel bat pathogens to determine the risk to human and animal health. Many groups have identified novel viruses in bats by detection of viral nucleic acid; however, virus isolation is still a challenge, and there are few reports of viral isolates from bats. In recent years, our group has developed optimized procedures for virus isolation from bat urine, including the use of primary bat cells. In previous reports, we have described the isolation of Hendra virus, Menangle virus and Cedar virus in Queensland, Australia. Here, we report the isolation of four additional novel bat paramyxoviruses from urine collected from beneath pteropid bat (flying fox) colonies in Queensland and New South Wales during 2009-2011.

IRF7 in the Australian black flying fox, Pteropus alecto: evidence for a unique expression pattern and functional conservation.

As the only flying mammal, bats harbor a number of emerging and re-emerging viruses, many of which cause severe diseases in humans and other mammals yet result in no clinical symptoms in bats. As the master regulator of the interferon (IFN)-dependent immune response, IFN regulatory factor 7 (IRF7) plays a central role in innate antiviral immunity. To explore the role of bat IRF7 in the regulation of the IFN response, we performed sequence and functional analysis of IRF7 from the pteropid bat, Pteropus alecto. Our results demonstrate that bat IRF7 retains the ability to bind to MyD88 and activate the IFN response despite unique changes in the MyD88 binding domain. We also demonstrate that bat IRF7 has a unique expression pattern across both immune and non-immune related tissues and is inducible by double-strand RNA. The broad tissue distribution of IRF7 may provide bats with an enhanced ability to rapidly activate the IFN response in a wider range of tissues compared to other mammals. The importance of IRF7 in antiviral activity against the bat reovirus, Pulau virus was confirmed by siRNA knockdown of IRF7 in bat cells resulting in enhanced viral replication. Our results highlight the importance of IRF7 in innate antiviral immunity in bats.

Measuring physiological stress in Australian flying-fox populations.

Flying-foxes (pteropid bats) are the natural host of Hendra virus, a recently emerged zoonotic virus responsible for mortality or morbidity in horses and humans in Australia since 1994. Previous studies have suggested physiological and ecological risk factors for infection in flying-foxes, including physiological stress. However, little work has been done measuring and interpreting stress hormones in flying-foxes. Over a 12-month period, we collected pooled urine samples from underneath roosting flying-foxes, and urine and blood samples from captured individuals. Urine and plasma samples were assayed for cortisol using a commercially available enzyme immunoassay. We demonstrated a typical post-capture stress response in flying-foxes, established urine specific gravity as an attractive alternative to creatinine to correct urine concentration, and established population-level urinary cortisol ranges (and geometric means) for the four Australian species: Pteropus alecto 0.5–305.1 ng/mL (20.1 ng/mL); Pteropus conspicillatus 0.3–370.9 ng/mL (18.9 ng/mL); Pteropus poliocephalus 0.3–311.3 ng/mL (10.1 ng/mL); Pteropus scapulatus 5.2–205.4 ng/mL (40.7 ng/mL). Geometric means differed significantly except for P. alecto and P. conspicillatus. Our approach is methodologically robust, and has application both as a research or clinical tool for flying-foxes, and for other free-living colonial wildlife species.