Unravelling the maternal evolutionary history of the African leopard (Panthera pardus pardus).

The African leopard (Panthera pardus pardus) has lost a significant proportion of its historical range, notably in north-western Africa and South Africa. Recent studies have explored the genetic diversity and population structure of African leopards across the continent. A notable genetic observation is the presence of two divergent mitochondrial lineages, PAR-I and PAR-II. Both lineages appeared to be distributed widely, with PAR-II frequently found in southern Africa. Until now, no study has attempted to date the emergence of either lineage, assess haplotype distribution, or explore their evolutionary histories in any detail. To investigate these underappreciated questions, we compiled the largest and most geographically representative leopard data set of the mitochondrial NADH-5 gene to date. We combined samples (n = 33) collected in an altitudinal transect across the Mpumalanga province of South Africa, where two populations of leopard are known to be in genetic contact, with previously published sequences of African leopard (n = 211). We estimate that the maternal PAR-I and PAR-II lineages diverged approximately 0.7051 (0.4477-0.9632) million years ago (Ma). Through spatial and demographic analyses, we show that while PAR-I underwent a mid-Pleistocene population expansion resulting in several closely related haplotypes with little geographic structure across much of its range, PAR-II remained at constant size and may even have declined slightly in the last 0.1 Ma. The higher genetic drift experienced within PAR-II drove a greater degree of structure with little haplotype sharing and unique haplotypes in central Africa, the Cape, KwaZulu-Natal and the South African Highveld. The phylogeographic structure of PAR-II, with its increasing frequency southward and its exclusive occurrence in south-eastern South Africa, suggests that this lineage may have been isolated in South Africa during the mid-Pleistocene. This hypothesis is supported by historical changes in paleoclimate that promoted intense aridification around the Limpopo Basin between 1.0-0.6 Ma, potentially reducing gene flow and promoting genetic drift. Interestingly, we ascertained that the two nuclear DNA populations identified by a previous study as East and West Mpumalanga correspond to PAR-I and PAR-II, respectively, and that they have come into secondary contact in the Lowveld region of South Africa. Our results suggest a subdivision of African leopard mtDNA into two clades, with one occurring almost exclusively in South Africa, and we identify the potential environmental drivers of this observed structure. We caution that our results are based on a single mtDNA locus, but it nevertheless provides a hypothesis that can be further tested with a dense sample of nuclear DNA data, preferably whole genomes. If our interpretation holds true, it would provide the first genetic explanation for the smaller observed size of leopards at the southernmost end of their range in Africa.

Dynamics of Antimicrobial Resistance Carriage in Koalas (Phascolarctos Cinereus) and Pteropid Bats (Pteropus Poliocephalus) Before, During and After Wildfires.

In the 2019-2020 summer, wildfires decimated the Australian bush environment and impacted wildlife species, including koalas (Phascolarctos cinereus) and grey headed flying fox pups (Pteropid bats, Pteropus poliocephalus). Consequently, hundreds of koalas and thousands of bat pups entered wildlife hospitals with fire-related injuries/illness, where some individuals received antimicrobial therapy. This study investigated the dynamics of antimicrobial resistance (AMR) in pre-fire, fire-affected and post-fire koalas and Pteropid bat pups. PCR and DNA sequencing were used to screen DNA samples extracted from faeces (koalas and bats) and cloacal swabs (koalas) for class 1 integrons, a genetic determinant of AMR, and to identify integron-associated antibiotic resistance genes. Class 1 integrons were detected in 25.5% of koalas (68 of 267) and 59.4% of bats (92 of 155). Integrons contained genes conferring resistance to aminoglycosides, trimethoprim and beta-lactams. Samples were also screened for blaTEM (beta-lactam) resistance genes, which were detected in 2.6% of koalas (7 of 267) and 25.2% of bats (39 of 155). Integron occurrence was significantly higher in fire-affected koalas in-care compared to wild pre-fire koalas (P < 0.0001). Integron and blaTEM occurrence were not significantly different in fire-affected bats compared to pre-fire bats (P > 0.05), however, their occurrence was significantly higher in fire-affected bats in-care compared to wild fire-affected bats (P < 0.0001 and P = 0.0488 respectively). The observed shifts of AMR dynamics in wildfire-impacted species flags the need for judicious antibiotic use when treating fire-affected wildlife to minimise unwanted selective pressure and negative treatment outcomes associated with carriage of resistance genes and antibiotic resistant bacteria.

Carriage of antibiotic resistance genes to treatments for chlamydial disease in koalas (Phascolarctos cinereus): A comparison of occurrence before and during catastrophic wildfires.

Growing reports of diverse antibiotic resistance genes in wildlife species around the world symbolises the extent of this global One Health issue. The health of wildlife is threatened by antimicrobial resistance in situations where wildlife species develop disease and require antibiotics. Chlamydial disease is a key threat for koalas in Australia, with infected koalas frequently entering wildlife hospitals and requiring antibiotic therapy, typically with chloramphenicol or doxycycline. This study investigated the occurrence and diversity of target chloramphenicol and doxycycline resistance genes (cat and tet respectively) in koala urogenital and faecal microbiomes. DNA was extracted from 394 urogenital swabs and 91 faecal swabs collected from koalas in mainland Australia and on Kangaroo Island (KI) located 14 km off the mainland, before (n = 145) and during (n = 340) the 2019-2020 wildfires. PCR screening and DNA sequencing determined 9.9% of samples (95%CI: 7.5% to 12.9%) carried cat and/or tet genes, with the highest frequency in fire-affected KI koalas (16.8%) and the lowest in wild KI koalas sampled prior to fires (6.5%). The diversity of cat and tet was greater in fire-affected koalas (seven variants detected), compared to pre-fire koalas (two variants detected). Fire-affected koalas in care that received antibiotics had a significantly higher proportion (p < 0.05) of cat and/or tet genes (37.5%) compared to koalas that did not receive antibiotics (9.8%). Of the cat and/or tet positive mainland koalas, 50.0% were Chlamydia-positive by qPCR test. Chloramphenicol and doxycycline resistance genes in koala microbiomes may contribute to negative treatment outcomes for koalas receiving anti-chlamydial antibiotics. Thus a secondary outcome of wildfires is increased risk of acquisition of cat and tet genes in fire-affected koalas that enter care, potentially exacerbating the already significant threat of chlamydial disease on Australia's koalas. This study highlights the importance of considering impacts to wildlife health within the One Health approach to AMR and identifies a need for greater understanding of AMR ecology in wildlife.

Characterisation of typical enteropathogenic Escherichia coli (tEPEC) lineages and novel bfpA variants detected in Australian fruit bats (Pteropus poliocephalus).

Enteropathogenic Escherichia coli (EPEC) is an important cause of diarrhoeal disease in human infants. EPEC strains are defined by the presence of specific virulence factors including intimin (encoded by the eae gene) and bundle forming pili (Bfp). Bfp is encoded by the bfp operon and includes the bfpA gene for the major pilus subunit. By definition, Bfp are only present in typical EPEC (tEPEC), for which, humans are considered to be the only known natural host. This study detected tEPEC in faecal samples from a wild Australian fruit bat species, the grey-headed flying-fox (Pteropus poliocephalus). Whole genome sequencing of 61 E. coli isolates from flying-foxes revealed that 21.3 % (95%CI: 13 %-33 %) were tEPEC. Phylogenetic analyses showed flying-fox tEPEC shared evolutionary lineages with human EPEC, but were predominantly novel sequence types (9 of 13) and typically harboured novel bfpA variants (11 of 13). HEp-2 cell adhesion assays showed adherence to human-derived epithelial cells by all 13 flying-fox tEPEC, indicating that they all carried functional Bfp. Using an EPEC-specific duplex PCR, it was determined that tEPEC comprised 17.4 % (95%CI: 13 %-22 %) of 270 flying-fox E. coli isolates. Furthermore, a tEPEC-specific multiplex PCR detected the eae and bfpA virulence genes in 18.0 % (95%CI: 8.0 %-33.7 %) of 506 flying-fox faecal DNA samples, with occurrences ranging from 1.3 % to 87.0 % across five geographic areas sampled over a four-year period. The identification of six novel tEPEC sequence types and five novel bfpA variants suggests flying-foxes carry bat-specific tEPEC lineages. However, their close relationship with human EPEC and functional Bfp, indicates that flying-fox tEPEC have zoonotic potential and that dissemination of flying-fox tEPEC into urban environments may pose a public health risk. The consistent detection of tEPEC in flying-foxes over extensive geographical and temporal scales indicates that both wild grey-headed flying-foxes and humans should be regarded as natural tEPEC hosts.

Faecal virome of the Australian grey-headed flying fox from urban/suburban environments contains novel coronaviruses, retroviruses and sapoviruses.

Bats are important reservoirs for viruses of public health and veterinary concern. Virus studies in Australian bats usually target the families Paramyxoviridae, Coronaviridae and Rhabdoviridae, with little known about their overall virome composition. We used metatranscriptomic sequencing to characterise the faecal virome of grey-headed flying foxes from three colonies in urban/suburban locations from two Australian states. We identified viruses from three mammalian-infecting (Coronaviridae, Caliciviridae, Retroviridae) and one possible mammalian-infecting (Birnaviridae) family. Of particular interest were a novel bat betacoronavirus (subgenus Nobecovirus) and a novel bat sapovirus (Caliciviridae), the first identified in Australian bats, as well as a potentially exogenous retrovirus. The novel betacoronavirus was detected in two sampling locations 1375 km apart and falls in a viral lineage likely with a long association with bats. This study highlights the utility of unbiased sequencing of faecal samples for identifying novel viruses and revealing broad-scale patterns of virus ecology and evolution.

Land use, season, and parasitism predict metal concentrations in Australian flying fox fur.

Urban-living wildlife can be exposed to metal contaminants dispersed into the environment through industrial, residential, and agricultural applications. Metal exposure carries lethal and sublethal consequences for animals; in particular, heavy metals (e.g. arsenic, lead, mercury) can damage organs and act as carcinogens. Many bat species reside and forage in human-modified habitats and could be exposed to contaminants in air, water, and food. We quantified metal concentrations in fur samples from three flying fox species (Pteropus fruit bats) captured at eight sites in eastern Australia. For subsets of bats, we assessed ectoparasite burden, haemoparasite infection, and viral infection, and performed white blood cell differential counts. We examined relationships among metal concentrations, environmental predictors (season, land use surrounding capture site), and individual predictors (species, sex, age, body condition, parasitism, neutrophil:lymphocyte ratio). As expected, bats captured at sites with greater human impact had higher metal loads. At one site with seasonal sampling, bats had higher metal concentrations in winter than in summer, possibly owing to changes in food availability and foraging. Relationships between ectoparasites and metal concentrations were mixed, suggesting multiple causal mechanisms. There was no association between overall metal load and neutrophil:lymphocyte ratio, but mercury concentrations were positively correlated with this ratio, which is associated with stress in other vertebrate taxa. Comparison of our findings to those of previous flying fox studies revealed potentially harmful levels of several metals; in particular, endangered spectacled flying foxes (P. conspicillatus) exhibited high concentrations of cadmium and lead. Because some bats harbor pathogens transmissible to humans and animals, future research should explore interactions between metal exposure, immunity, and infection to assess consequences for bat and human health.

Transmission of Klebsiella strains and plasmids within and between grey-headed flying fox colonies.

The grey-headed flying fox (Pteropus poliocephalus) is an endemic Australian fruit bat, known to carry zoonotic pathogens. We recently showed they harbour bacterial pathogen Klebsiella pneumoniae and closely related species in the K. pneumoniae species complex (KpSC); however, the dynamics of KpSC transmission and gene flow within flying fox colonies are poorly understood. High-resolution genome comparisons of 39 KpSC isolates from grey-headed flying foxes identified five putative strain transmission clusters (four intra- and one inter-colony). The instance of inter-colony strain transmission of K. africana was found between two flying fox populations within flying distance, indicating either direct or indirect transmission through a common food/water source. All 11 plasmids identified within the KpSC isolates showed 73% coverage (mean) and ≥95% identity to human-associated KpSC plasmids, indicating gene flow between human clinical and grey-headed flying fox isolates. Along with strain transmission, inter-species horizontal plasmid transmission between K. pneumoniae and Klebsiella africana was also identified within a flying fox colony. Finally, genome-scale metabolic models were generated to predict and compare substrate usage to previously published KpSC models, from human and environmental sources. These models indicated no distinction on the basis of metabolic capabilities. Instead, metabolic capabilities were consistent with population structure and ST/lineage.

An Analysis of Demographic and Triage Assessment Findings in Bushfire-Affected Koalas (Phascolarctos cinereus) on Kangaroo Island, South Australia, 2019-2020.

In the 2019-2020 Australian bushfires, Kangaroo Island, South Australia, experienced catastrophic bushfires that burnt approximately half the island, with an estimated 80% of the koala population lost. During and after the event, rescued koalas were triaged at a designated facility and a range of initial data were recorded including rescue location and date, sex, estimation of age, body condition and hydration, and assessment of burn severity (n = 304 records available). Koalas were presented to the triage facility over a span of 10 weeks, with 50.2% during the first 14 days of the bushfire response, the majority of which were rescued from regions of lower fire severity. Burns were observed in 67.4% of koalas, with the majority (60.9%) classified as superficial burns, primarily affecting the limbs and face. Poor body condition was recorded in 74.6% of burnt koalas and dehydration in 77.1%. Negative final outcomes (death or euthanasia, at triage or at a later date) occurred in 45.6% of koalas and were significantly associated with higher mean burn score, maximum burn severity, number of body regions burnt, poor body condition score, and dehydration severity. The findings of this retrospective study may assist clinicians in the field with decision making when triaging koalas in future fire rescue efforts.

Novel strains of Klebsiella africana and Klebsiella pneumoniae in Australian fruit bats (Pteropus poliocephalus).

Over the past decade human associated multidrug resistant (MDR) and hypervirulent Klebsiella pneumoniae lineages have been increasingly detected in wildlife. This study investigated the occurrence of K. pneumoniae species complex (KpSC) in grey-headed flying foxes (GHFF), an Australian fruit bat. Thirty-nine KpSC isolates were cultured from 275 GHFF faecal samples (14.2%), comprising K. pneumoniae (n = 30), Klebsiella africana (n = 8) and Klebsiella variicola subsp. variicola (n = 1). The majority (79.5%) of isolates belonged to novel sequence types (ST), including two novel K. africana STs. This is the first report of K. africana outside of Africa and in a non-human host. A minority (15.4%) of GHFF KpSC isolates shared STs with human clinical K. pneumoniae strains, of which, none belonged to MDR clonal lineages that cause frequent nosocomial outbreaks, and no isolates were characterised as hypervirulent. The occurrence of KpSC isolates carrying acquired antimicrobial resistance genes in GHFF was low (1.1%), with three K. pneumoniae isolates harbouring both fluoroquinolone and trimethoprim resistance genes. This study indicates that GHFF are not reservoirs for MDR and hypervirulent KpSC strains, but they do carry novel K. africana lineages. Health risks associated with KpSC carriage by GHFF are deemed low for the public and GHFF.

Porphyromonas spp., Fusobacterium spp., and Bacteroides spp. dominate microbiota in the course of macropod progressive periodontal disease.

Macropod progressive periodontal disease (MPPD) is a necrotizing, polymicrobial, inflammatory disease commonly diagnosed in captive macropods. MPPD is characterized by gingivitis associated with dental plaque formation, which progresses to periodontitis and then to osteomyelitis of the mandible or maxilla. However, the underlying microbial causes of this disease remain poorly understood. In this study, we collected 27 oral plaque samples and associated clinical records from 22 captive Macropodidae and Potoroidae individuals that were undergoing clinical examination at Adelaide and Monarto Zoos in South Australia (15 healthy, 7 gingivitis and 5 periodontitis-osteomyelitis samples). The V3-V4 region of the 16S ribosomal RNA gene was sequenced using an Illumina Miseq to explore links between MPPD and oral bacteria in these animals. Compositional differences were detected between the microbiota of periodontitis-osteomyelitis cases compared to healthy samples (p-value with Bonferroni correction < 0.01), as well as gingivitis cases compared to healthy samples (p-value with Bonferroni correction < 0.05) using Permutational Multivariate Analysis of Variance (PERMANOVA). An overabundance of Porphyromonas, Fusobacterium, and Bacteroides taxa was also identified in animals with MPPD compared to healthy individuals using linear discriminant analysis effect size (LEfSe; p = < 0.05). An increased abundance of Desulfomicrobium also was detected in MPPD samples (LEfSe; p < 0.05), which could potentially reflect differences in disease progression. This is the first microbiota analysis of MPPD in captive macropods, and these results support a polymicrobial pathogenesis of MPPD, suggesting that the microbial interactions underpinning MPPD may be more complex than previously documented.

INFECTION PRESSURE IS NECESSARY, BUT NOT SUFFICIENT BY ITSELF, TO EXPLAIN TOXOPLASMA GONDII SEROPREVALENCE IN INTERMEDIATE HOST SPECIES.

Parasite infection pressure is suggested to be a strong driver of transmission within ecosystems. We tested if infection pressure drives seroprevalence in intermediate host species for Toxoplasma gondii. We defined Toxoplasma infection pressure to intermediate host species as the combined influence of cat abundance, environmental conditions, and its prevalence in the cat population. We sampled and tested 2 species of rodent and collated information on Toxoplasma seroprevalence in koalas, wallabies, kangaroos, and sheep. All species were sampled using equivalent methods, within a 2-yr period, and from adjacent regions of low and high Toxoplasma infection pressure. The seroprevalence of Toxoplasma in kangaroos scaled with infection pressure, but we observed no statistical difference in seroprevalence for any other species between these 2 regions. Within the region of low infection pressure, Toxoplasma seroprevalence did not differ between species. However, within the region of high Toxoplasma infection pressure, we observed large variation in seroprevalence between species. Our results demonstrate that infection pressure is not sufficient by itself, but merely necessary, to drive Toxoplasma seroprevalence in intermediate host species. Where Toxoplasma seroprevalence in an intermediate host species is already low, further reducing infection pressure will not necessarily further decrease seroprevalence in those species. This has important ramifications for the mitigation of parasite infections and suggests that reductions in Toxoplasma infection pressure, intended to reduce infections, may be most effective and applicable to species that are known to experience high rates of infection.

Bat E-Commerce: Insights Into the Extent and Potential Implications of This Dark Trade.

Little is known about the global bat souvenir trade despite previous research efforts into bat harvest for bushmeat. We screened eBay listings of bats in Australia, Canada, Italy, Switzerland, United Kingdom and USA to assess the nature and extent of the online offers. A total of 237 listings were retrieved in between the 11th and 25th of May 2020 with a median price per item of US$38.50 (range: US$8.50-2,500.00). Items on offer were mostly taxidermy (61.2%) or skull (21.1%) specimens. Overall, 32 different species of bat were advertised, most of which (n = 28) are listed as "Least Concern" on the International Union for Conservation of Nature (IUCN) Red List. One species (Nycteris javanica) is classified as "Vulnerable" and one (Eidolon helvum) as "Near Threatened." Pteropus spp. specimens were the most expensive specimens on offer and the conservations status of these species may range from "Critically Endangered" to "Data Deficient" by IUCN and the entire genus is listed in the Appendix II by the Convention on the International Trade in Endangered Species of Wild Fauna and Flora (CITES). However, the exact species concerned, and their respective conservation status, could not be confirmed based on the listings' photos. The sourcing of bat was restricted to mostly South-East Asian countries (a third of items sourced from Indonesia) and to two African countries. Our survey revealed that the online offer of bat products is diverse, abundant, and facilitated by worldwide sellers although most offered bats species are from South-East Asia. With a few exceptions, the species on offer were of little present conservation concern, however, many unknowns remain on the potential animal welfare, biosecurity, legal implications, and most importantly public health risks associated with this dark trade.

Characterization of beta-lactam-resistant Escherichia coli from Australian fruit bats indicates anthropogenic origins.

Antimicrobial-resistant Escherichia coli, particularly those resistant to critically important antimicrobials, are increasingly reported in wildlife. The dissemination of antimicrobial-resistant bacteria to wildlife indicates the far-reaching impact of selective pressures imposed by humans on bacteria through misuse of antimicrobials. The grey-headed flying fox (GHFF; Pteropus poliocephalus), a fruit bat endemic to eastern Australia, commonly inhabits urban environments and encounters human microbial pollution. To determine if GHFF have acquired human-associated bacteria, faecal samples from wild GHFF (n=287) and captive GHFF undergoing rehabilitation following illness or injury (n=31) were cultured to detect beta-lactam-resistant E. coli. Antimicrobial susceptibility testing, PCR and whole genome sequencing were used to determine phenotypic and genotypic antimicrobial resistance profiles, strain type and virulence factor profiles. Overall, 3.8 % of GHFF carried amoxicillin-resistant E. coli (wild 3.5 % and captive 6.5 %), with 38.5 % of the 13 GHFF E. coli isolates exhibiting multidrug resistance. Carbapenem (blaNDM-5) and fluoroquinolone resistance were detected in one E. coli isolate, and two isolates were resistant to third-generation cephalosporins (blaCTX-M-27 and ampC). Resistance to tetracycline and trimethoprim plus sulfamethoxazole were detected in 69.2% and 30.8 % of isolates respectively. Class 1 integrons, a genetic determinant of resistance, were detected in 38.5 % of isolates. Nine of the GHFF isolates (69.2 %) harboured extraintestinal virulence factors. Phylogenetic analysis placed the 13 GHFF isolates in lineages associated with humans and/or domestic animals. Three isolates were human-associated extraintestinal pathogenic E. coli (ST10 O89:H9, ST73 and ST394) and seven isolates belonged to lineages associated with extraintestinal disease in both humans and domestic animals (ST88, ST117, ST131, ST155 complex, ST398 and ST1850). This study provides evidence of anthropogenic multidrug-resistant and pathogenic E. coli transmission to wildlife, further demonstrating the necessity for incorporating wildlife surveillance within the One Health approach to managing antimicrobial resistance.

Serological evidence of exposure to a coronavirus antigenically related to severe acute respiratory syndrome virus (SARS-CoV-1) in the Grey-headed flying fox (Pteropus poliocephalus).

Many infectious pathogens can be transmitted by highly mobile species, like bats that can act as reservoir hosts for viruses such as henipaviruses, lyssaviruses and coronaviruses. In this study, we investigated the seroepidemiology of protein antigens to Severe acute respiratory syndrome virus (SARS-CoV-1) and Middle eastern respiratory syndrome virus (MERS-CoV) in Grey-headed flying foxes (Pteropus poliocephalus) in Adelaide, Australia sampled between September 2015 and February 2018. A total of 301 serum samples were collected and evaluated using a multiplex Luminex binding assay, and median fluorescence intensity thresholds were determined using finite-mixture modelling. We found evidence of antibodies reactive to SARS-CoV-1 or a related antigen with 42.5% (CI: 34.3%-51.2%) seroprevalence but insufficient evidence of reactivity to MERS-CoV antigen. This study provides evidence that the Grey-headed flying foxes sampled in Adelaide have been exposed to a SARS-like coronavirus.

Reply to Wolf et al.: Why Trap-Neuter-Return (TNR) Is Not an Ethical Solution for Stray Cat Management.

We critique the recent article by Wolf et al. (2019) that claims scientific merit for reducing the number of stray cats in Australia through Trap-Neuter-Return (TNR) programs, and then we provide an inventory of biological, welfare, and economic reasons why TNR is less successful than adoption and euthanasia for managing unowned cats. Like Crawford et al. (2019) and multiple other comprehensive and unbiased Australian and international scientific reviews, we refute the idea that returning neutered unowned cats to stray populations has any valid role in responsible, ethical, affordable, and effective cat management, or in wildlife conservation. The main purported objective of TNR proponents along with animal welfare, human health, and wildlife advocacy stakeholders is to reduce the number of unhomed cats. We contend that cessation of provisioning unowned cats with food is the most effective approach to achieve this objective. We also present evidence from the Brisbane City Council that informed cat management policy, advocacy, and laws, backed up by responsible rehoming or prompt ethical euthanasia, are together effective at reducing the stray cat problem.

Seroprevalence of three paramyxoviruses; Hendra virus, Tioman virus, Cedar virus and a rhabdovirus, Australian bat lyssavirus, in a range expanding fruit bat, the Grey-headed flying fox (Pteropus poliocephalus).

Habitat-mediated global change is driving shifts in species' distributions which can alter the spatial risks associated with emerging zoonotic pathogens. Many emerging infectious pathogens are transmitted by highly mobile species, including bats, which can act as spill-over hosts for pathogenic viruses. Over three years, we investigated the seroepidemiology of paramyxoviruses and Australian bat lyssavirus in a range-expanding fruit bat, the Grey-headed flying fox (Pteropus poliocephalus), in a new camp in Adelaide, South Australia. Over six, biannual, sampling sessions, we quantified median florescent intensity (MFI) antibody levels for four viruses for a total of 297 individual bats using a multiplex Luminex binding assay. Where appropriate, florescence thresholds were determined using finite mixture modelling to classify bats' serological status. Overall, apparent seroprevalence of antibodies directed at Hendra, Cedar and Tioman virus antigens was 43.2%, 26.6% and 95.7%, respectively. We used hurdle models to explore correlates of seropositivity and antibody levels when seropositive. Increased body condition was significantly associated with Hendra seropositivity (Odds ratio = 3.67; p = 0.002) and Hendra virus levels were significantly higher in pregnant females (p = 0.002). While most bats were seropositive for Tioman virus, antibody levels for this virus were significantly higher in adults (p < 0.001). Unexpectedly, all sera were negative for Australian bat lyssavirus. Temporal variation in antibody levels suggests that antibodies to Hendra virus and Tioman virus may wax and wane on a seasonal basis. These findings suggest a common exposure to Hendra virus and other paramyxoviruses in this flying fox camp in South Australia.

Field immobilization using alfaxalone and alfaxalone-medetomidine in free-ranging koalas (Phascolarctos cinereus): a randomized comparative study.

OBJECTIVE: To characterize and compare two intramuscular drug protocols using alfaxalone and alfaxalone-medetomidine combination for the field immobilization of free-ranging koalas. STUDY DESIGN: Blinded, randomized, comparative field study. ANIMALS: A total of 66 free-ranging koalas from the Mount Lofty Ranges, South Australia. METHODS: Koalas were randomly allocated into two groups. Group A animals were given alfaxalone alone at 3.5 mg kg-1. Group AM animals were given alfaxalone 2 mg kg-1 and medetomidine 40 µg kg-1, reversed with atipamezole at 0.16 mg kg-1. Blinded operators recorded heart rate (HR), respiratory rate (fR), cloacal temperature, depth of sedation and times to: first effect, sedation suitable for clinical interventions, first arousal and full recovery. Data were analysed using independent t test, Mann-Whitney U test, chi-square analysis and log-rank test at 5% level of significance. RESULTS: Suitable immobilization for clinical examination and sample collection was achieved in all animals. In groups A and AM, median time to working depth was 6.5 minutes (range: 3.4-15) and 8.1 minutes (range: 4.3-24) and time to complete recovery was 66 minutes (range: 12-138) and 34 minutes (range: 4-84), respectively, following reversal. Time to first effect was significantly shorter in group A (p = 0.013), whereas time to full arousal was significantly shorter in group AM (p = 0.007) probably due to the administration of atipamezole. Maximum HR was 117 ± 28 beats minute-1 in group A, which was a significant increase from baseline values (p < 0.0001), whereas group AM showed a significant tachypnoea of 67 ± 25 (normal fR 10-15; p < 0.0001). CONCLUSIONS AND CLINICAL RELEVANCE: Both the protocols produced immobilization, enabling clinical examination and sample collection; however, protocol AM was more suitable for field work due to shorter recovery times.

Dual-locus DNA metabarcoding reveals southern hairy-nosed wombats (Lasiorhinus latifrons Owen) have a summer diet dominated by toxic invasive plants.

Habitat degradation and summer droughts severely restrict feeding options for the endangered southern hairy-nosed wombat (SHNW; Lasiorhinus latifrons). We reconstructed SHNW summer diets by DNA metabarcoding from feces. We initially validated rbcL and ndhJ diet reconstructions using autopsied and captive animals. Subsequent diet reconstructions of wild wombats broadly reflected vegetative ground cover, implying local rather than long-range foraging. Diets were all dominated by alien invasives. Chemical analysis of alien food revealed Carrichtera annua contains high levels of glucosinolates. Clinical examination (7 animals) and autopsy (12 animals) revealed that the most degraded site also contained most individuals showing signs of glucosinolate poisoning. We infer that dietary poisoning through the ingestion of alien invasives may have contributed to the recent population crashes in the region. In floristically diverse sites, individuals appear to be able to manage glucosinolate intake by avoidance or episodic feeding but this strategy is less tractable in the most degraded sites. We conclude that recovery of the most affected populations may require effective Carrichtera management and interim supplementary feeding. More generally, we argue that protection against population decline by poisoning in territorial herbivores requires knowledge of their diet and of those food plants containing toxic principles.

SERUM BIOCHEMISTRY OF FREE-RANGING SOUTHERN HAIRY-NOSED WOMBATS (LASIORHINUS LATIFRONS).

Listed as near-threatened by the International Union for Conservation of Nature (IUCN), the southern hairy-nosed wombat (SHNW, Lasiorhinus latifrons) faces threats such as drought, habitat degradation and loss, disease, and persecution because of competition with agriculture. To assist with evaluation of wombat health, this study reports serum biochemical reference intervals (RIs) for wild-caught SHNW from South Australia established from 126 apparently healthy SHNW using a Beckman Coulter AU480® Automated Chemistry Analyzer using RefVal Advisor. Partitioning of RIs for male and female wombats and for the two methods of sampling was performed as appropriate, and additional significant differences (P < 0.05) in biochemical profiles were identified across age class and season examined. A number of differences were observed between male and female wombats, most notably higher creatinine, urea, and sodium in females. Subadult and juvenile wombats had significantly lower total protein, globulin, and ALT activity, and significantly higher ALP activity than adults. Wombats sampled in winter and spring had significantly higher total protein, albumin, potassium, bicarbonate, and enzyme activities (ALP, ALT, AST, GGT, GLDH, lipase), and significantly lower glucose and creatinine when compared to individuals sampled in summer and autumn. Differences in CK activity and anion gap observed between the two methods of sampling likely reflect delay and handling of animals between capture and blood collection. The serum biochemical RIs documented here are considered representative of a population of healthy SHNW, providing a tool for health assessment and monitoring of SHNW health in South Australia and elsewhere.

Molecular investigation of Hepatocystis parasites in the Australian flying fox Pteropus poliocephalus across its distribution range.

Phylogenetic inference of Hepatocystis, a haemosporidian parasite of diverse primate and bat hosts, revealed that the parasites from Australasian Pteropus bat species form a distinct clade to all other Hepatocystis parasites from Africa and Asia. Here, we investigate the phylogenetic placement of Hepatocystis in the Australian bat Pteropus poliocephalus for the first time and examine parasite morphology and prevalence from selected points across its range. Hepatocystis infections were detected in low prevalences in P. poliocephalus in contrast to high numbers in P. alecto and P. scapulatus. The prevalence in P. poliocephalus varied across its distribution range with 15% in the central biogeographic areas (central Queensland and New South Wales) and 1% in the southern-most edge (South Australia) of its range. Sequencing of five genes revealed high genetic similarity in Hepatocystis of P. poliocephalus independent of sampling location. Phylogenetic analysis placed these parasites with Hepatocystis from other Pteropus species from Australia and Asia. While numerous haplotypes were identified among sequences from the Pteropus hosts, no patterns of host specificity were recovered within the Pteropus-specific parasite group.