Broad-scale pesticide screening finds anticoagulant rodenticide and legacy pesticides in Australian frogs.

Pesticide contamination poses a significant threat to non-target wildlife, including amphibians, many of which are already highly threatened. This study aims to assess the extent of pesticide exposure in dead frogs collected during a mass mortality event across eastern New South Wales, Australia between July 2021 and March 2022. Liver tissue from 77 individual frogs of six species were analysed for >600 legacy and contemporary pesticides, including rodenticides. More than a third (36 %) of the liver samples contained at least one pesticide, including brodifacoum, dieldrin, DDE, heptachlor/heptachlor epoxide, fipronil sulfone, and 2-methyl-4-chlorophenoxyacetic acid (MCPA). Brodifacoum, a second-generation anticoagulant rodenticide, was found in four of the six frog species analysed: the eastern banjo frog (Limnodynastes dumerilii), cane toad (Rhinella marina), green tree frog (Litoria caerulea) and Peron's tree frog (Litoria peronii). This is the first report of anticoagulant rodenticide detected in wild amphibians, raising concerns about potential impacts on frogs and extending the list of taxa shown to accumulate rodenticides. Dieldrin, a banned legacy pesticide, was also detected in two species: striped marsh frog (Limnodynastes peronii) and green tree frog (Litoria caerulea). The toxicological effects of these pesticides on frogs are difficult to infer due to limited comparable studies; however, due to the low frequency of detection the presence of these pesticides was not considered a major contributing factor to the mass mortality event. Additional research is needed to investigate the effects of pesticide exposure on amphibians, particularly regarding the impacts of second-generation anticoagulant rodenticides. There is also need for continued monitoring and improved conservation management strategies for the mitigation of the potential threat of pesticide exposure and accumulation in amphibian populations.

Close-kin mark-recapture informs critically endangered terrestrial mammal status.

Reliable information on population size is fundamental to the management of threatened species. For wild species, mark-recapture methods are a cornerstone of abundance estimation. Here, we show the first application of the close-kin mark-recapture (CKMR) method to a terrestrial species of high conservation value; the Christmas Island flying-fox (CIFF). The CIFF is the island's last remaining native terrestrial mammal and was recently listed as critically endangered. CKMR is a powerful tool for estimating the demographic parameters central to CIFF management and circumvents the complications arising from the species' cryptic nature, mobility, and difficult-to-survey habitat. To this end, we used genetic data from 450 CIFFs captured between 2015 and 2019 to detect kin pairs. We implemented a novel CKMR model that estimates sex-specific abundance, trend, and mortality and accommodates observations from the kin-pair distribution of male reproductive skew and mate persistence. CKMR estimated CIFF total adult female abundance to be approximately 2050 individuals (95% CI (950, 4300)). We showed that on average only 23% of the adult male population contributed to annual reproduction and strong evidence for between-year mate fidelity, an observation not previously quantified for a Pteropus species in the wild. Critically, our population estimates provide the most robust understanding of the status of this critically endangered population, informing immediate and future conservation initiatives.

Gammaretroviruses, novel viruses and pathogenic bacteria in Australian bats with neurological signs, pneumonia and skin lesions.

More than 70 bat species are found in mainland Australia. While most studies of bat viromes focus on sampling seemingly healthy individuals, little is known about the viruses and bacteria associated with diseased bats. We performed traditional diagnostic techniques and metatranscriptomic sequencing on tissue samples from 43 Australian bats, comprising three flying fox (Pteropodidae) and two microbat species experiencing a range of disease syndromes, including mass mortality, neurological signs, pneumonia and skin lesions. Of note, we identified the recently discovered Hervey pteropid gammaretrovirus in a bat with lymphoid leukemia, with evidence of replication consistent with an exogenous virus. The possible association of Hervey pteropid gammaretrovirus with lymphoid leukemia clearly merits additional investigation. One novel picornavirus and at least three new astroviruses and bat pegiviruses were also identified in a variety of tissue types, as well as a number of likely bacterial pathogens or opportunistic infections, most notably Pseudomonas aeruginosa.

Serological evidence of a pararubulavirus and a betacoronavirus in the geographically isolated Christmas Island flying-fox (Pteropus natalis).

Due to their geographical isolation and small populations, insular bats may not be able to maintain acute immunizing viruses that rely on a large population for viral maintenance. Instead, endemic transmission may rely on viruses establishing persistent infections within hosts or inducing only short-lived neutralizing immunity. Therefore, studies on insular populations are valuable for developing broader understanding of viral maintenance in bats. The Christmas Island flying-fox (CIFF; Pteropus natalis) is endemic on Christmas Island, a remote Australian territory, and is an ideal model species to understand viral maintenance in small, geographically isolated bat populations. Serum or plasma (n = 190), oral swabs (n = 199), faeces (n = 31), urine (n = 32) and urine swabs (n = 25) were collected from 228 CIFFs. Samples were tested using multiplex serological and molecular assays, and attempts at virus isolation to determine the presence of paramyxoviruses, betacoronaviruses and Australian bat lyssavirus. Analysis of serological data provides evidence that the species is maintaining a pararubulavirus and a betacoronavirus. There was little serological evidence supporting the presence of active circulation of the other viruses assessed in the present study. No viral nucleic acid was detected and no viruses were isolated. Age-seropositivity results support the hypothesis that geographically isolated bat populations can maintain some paramyxoviruses and coronaviruses. Further studies are required to elucidate infection dynamics and characterize viruses in the CIFF. Lastly, apparent absence of some pathogens could have implications for the conservation of the CIFF if a novel disease were introduced into the population through human carriage or an invasive species. Adopting increased biosecurity protocols for ships porting on Christmas Island and for researchers and bat carers working with flying-foxes are recommended to decrease the risk of pathogen introduction and contribute to the health and conservation of the species.

Morphological and quantitative analysis of leukocytes in free-living Australian black flying foxes (Pteropus alecto).

The black flying fox (Pteropus alecto) is a natural reservoir for Hendra virus, a paramyxovirus that causes fatal infections in humans and horses in Australia. Increased excretion of Hendra virus by flying foxes has been hypothesized to be associated with physiological or energetic stress in the reservoir hosts. The objective of this study was to explore the leukocyte profiles of wild-caught P. alecto, with a focus on describing the morphology of each cell type to facilitate identification for clinical purposes and future virus spillover research. To this end, we have created an atlas of images displaying the commonly observed morphological variations across each cell type. We provide quantitative and morphological information regarding the leukocyte profiles in bats captured at two roost sites located in Redcliffe and Toowoomba, Queensland, Australia, over the course of two years. We examined the morphology of leukocytes, platelets, and erythrocytes of P. alecto using cytochemical staining and characterization of blood films through light microscopy. Leukocyte profiles were broadly consistent with previous studies of P. alecto and other Pteropus species. A small proportion of individual samples presented evidence of hemoparasitic infection or leukocyte morphological traits that are relevant for future research on bat health, including unique large granular lymphocytes. Considering hematology is done by visual inspection of blood smears, examples of the varied cell morphologies are included as a visual guide. To the best of our knowledge, this study provides the first qualitative assessment of P. alecto leukocytes, as well as the first set of published hematology reference images for this species.

Body-size dependent foraging strategies in the Christmas Island flying-fox: implications for seed and pollen dispersal within a threatened island ecosystem.

BACKGROUND: Animals are important vectors for the dispersal of a wide variety of plant species, and thus play a key role in maintaining the health and biodiversity of natural ecosystems. On oceanic islands, flying-foxes are often the only seed dispersers or pollinators. However, many flying-fox populations are currently in decline, particularly those of insular species, and this has consequences for the ecological services they provide. Knowledge of the drivers and the scale of flying-fox movements is important in determining the ecological roles that flying-foxes play on islands. This information is also useful for understanding the potential long-term consequences for forest dynamics resulting from population declines or extinction, and so can aid in the development of evidence-based ecological management strategies. To these ends, we examined the foraging movements, floral resource use, and social interactions of the Critically Endangered Christmas Island flying-fox (Pteropus natalis). METHODS: Utilization distributions, using movement-based kernel estimates (MBKE) were generated to determine nightly foraging movements of GPS-tracked P. natalis (n = 24). Generalized linear models (GLMs), linear mixed-effect models (LMMs), and Generalized linear mixed-effects model (GLMMs) were constructed to explain how intrinsic factors (body mass, skeletal size, and sex) affected the extent of foraging movements. In addition, we identified pollen collected from facial and body swabs of P. natalis (n = 216) to determine foraging resource use. Direct observations (n = 272) of foraging P. natalis enabled us to assess the various behaviors used to defend foraging resources. RESULTS: Larger P. natalis individuals spent more time foraging and less time traveling between foraging patches, traveled shorter nightly distances, and had smaller overall foraging ranges than smaller conspecifics. Additionally, larger individuals visited a lower diversity of floral resources. CONCLUSIONS: Our findings suggest that smaller P. natalis individuals are the primary vectors of long-distance dispersal of pollen and digested seeds in this species, providing a vital mechanism for maintaining the flow of plant genetic diversity across Christmas Island. Overall, our study highlights the need for more holistic research approaches that incorporate population demographics when assessing a species' ecological services.

Meta-transcriptomic analysis of the virome and microbiome of the invasive Indian myna (Acridotheres tristis) in Australia.

Invasive species exert a serious impact on native fauna and flora and have become the target of eradication and management efforts worldwide. Invasive avian species can also be important pathogen reservoirs, although their viromes and microbiomes have rarely been studied. As one of the top 100 invasive pest species globally, the expansion of Indian mynas (Acridotheres tristis) into peri-urban and rural environments, in conjunction with increasing free-ranging avian agricultural practices, may increase the risk of microbial pathogens jumping species boundaries. Herein, we used a meta-transcriptomic approach to explore the microbes present in brain, liver and large intestine of 16 invasive Indian myna birds in Sydney, Australia. From this, we discovered seven novel viruses from the families Adenoviridae, Caliciviridae, Flaviviridae, Parvoviridae and Picornaviridae. Interestingly, each of the novel viruses identified shared less than 80% genomic similarity with their closest relatives from other avian species, indicative of a lack of detectable virus transmission between invasive mynas to native or domestic species. Of note, we also identified two coccidian protozoa, Isospora superbusi and Isospora greineri, from the liver and gut tissues of mynas. Overall, these data demonstrate that invasive mynas can harbor a diversity of viruses and other microorganisms such that ongoing pathogen surveillance in this species is warranted.

Experimental infection of Asian house geckos with Enterococcus lacertideformus demonstrates multiple disease transmission routes and the in-vivo efficacy of antibiotics.

The disease caused by Enterococcus lacertideformus is multisystemic and ultimately fatal. Since its emergence, the bacterium has significantly impacted the captive breeding programs of the extinct in the wild Christmas Island Lister's gecko (Lepidodactylus listeri) and blue-tailed skink (Cryptoblepharus egeriae). The bacterium's pathogenicity, inability to grow in-vitro, and occurrence beyond the confines of Christmas Island necessitated the development of an experimental infection and treatment model. Asian house geckos (Hemidactylus frenatus) were challenged with a single dose of E. lacertideformus inoculum either by mouth, application to mucosal abrasion or skin laceration, subcutaneous injection, coelomic injection, or via co-housing with an infected gecko. Five healthy geckos acted as controls. Each transmission route resulted in disease in at least 40% (n = 2) geckos, expanding to 100% (n = 5) when E. lacertideformus was applied to skin laceration and mucosal abrasion groups. Incubation periods post-infection ranged between 54 and 102 days. To determine the efficacy of antibiotic treatment, infected geckos were divided into six groups (enrofloxacin 10 mg/kg, per os (PO), every 24 h (q24), amoxicillin-clavulanic acid 10 mg/kg, PO, q24, enrofloxacin 10 mg/kg combined with amoxicillin-clavulanic acid 10 mg/kg, PO, q24, rifampicin 15 mg/kg, PO, q24, clarithromycin 15 mg/kg, PO, q24, and untreated controls) for 21 days. Response to treatment was assessed by the change in lesion size, bacterial dissemination, and histological evidence of a host immune response. Irrespective of the antibiotic given, histology revealed that geckos inoculated by skin laceration were observed to have more extensive disease spread throughout the animal's body compared to other inoculation routes. The reduction in the average surface area of gross lesions was 83.6% for geckos treated with enrofloxacin, followed by the combination therapy amoxicillin-clavulanic acid and enrofloxacin (62.4%), amoxicillin-clavulanic acid (58.2%), rifampicin (45.5%), and clarithromycin (26.5%). Lesions in geckos untreated with antibiotics increased in size between 100 and 300%. In summary, enrofloxacin and amoxicillin-clavulanic acid show promising properties for the treatment of E. lacertideformus infection in geckos. The Asian house gecko E. lacertideformus infection model therefore provides foundational findings for the development of effective therapeutic treatment protocols aimed at conserving the health of infected and at-risk reptiles.

A comparison of nutritional value of native and alien food plants for a critically endangered island flying-fox.

Habitat loss and alteration are two of the biggest threats facing insular flying-foxes. Altered habitats are often re-vegetated with introduced or domestic plant species on which flying-foxes may forage. However, these alien food plants may not meet the nutritional requirements of flying-foxes. The critically endangered Christmas Island flying-fox (CIFF; Pteropus natalis) is subject to habitat alteration and the introduction of alien food plants, and therefore is a good model species to evaluate the potential impact of alien plant species on insular flying-foxes. In this study, we evaluated nutritional content of native food plants to determine how flying-foxes historically met their nutritional requirements. Furthermore, we compared the nutritional content of native and alien fruits to predict possible impacts of alien plants on insular flying-foxes. Native and alien fruits and flowers, and native foliage (leaves, petals, and petioles) commonly consumed by the CIFF were collected and evaluated for soluble sugars, crude protein, non-fiber carbohydrates, and nine minerals. Evaluation of native food plants suggests that flying-foxes meet energy requirements by consuming fruit and nectar. However, fruit and nectar are low in protein and essential minerals required for demanding life periods; therefore, flying-foxes likely supplement their diets with pollen and foliage. Thus, flying-foxes require a diverse array of plants to meet their nutritional requirements. Compared to native fruits, alien fruits contained significantly higher non-fiber carbohydrates, and this may provide an important energy source, particularly from species that bear fruit year-round. Median mineral concentrations in alien fruit species, however, were deficient compared to native fruits, suggesting major (or even seasonal) shifts in the proportion of alien species in the CIFF diet could lead to nutritional imbalances. This study confirms the need to quantify nutritional parameters in addition to feeding ecology when evaluating habitat quality to inform conservation actions that can be applied both locally and globally.

BASELINE HEALTH PARAMETERS FOR A NEWLY ESTABLISHED POPULATION OF LONG-NOSED POTOROO (POTOROUS TRIDACTYLUS) AT BOODEREE NATIONAL PARK, AUSTRALIA.

Over two field seasons during 2014-15, 35 long-nosed potoroos (Potorous tridactylus) were captured in state forests in South Eastern New South Wales for translocation to Booderee National Park, Jervis Bay Territory, Australia. Animals were anesthetized for physical examination and collection of samples to assess general health and screen for select diseases identified during a disease risk assessment. Morphologic, hematologic, and biochemical parameters were determined, and parasites were identified where possible. Trypanosoma gilletti, Trypanosoma vegrandis, and novel genotypes most similar to a Trypanosoma wallaby-derived isolate (ABF) were identified from blood samples by PCR; the first time Trypanosoma has been described in this species. Also reported is the first confirmation of the Australian paralysis tick, Ixodes holocyclus, from the long-nosed potoroo. Surveillance showed that Cryptococcus sp. may form part of the normal nasal flora for long-nosed potoroo. Salmonella enterica serotype Dublin and Salmonella enterica subsp. enterica was identified from rectal swabs of otherwise healthy animals. The data provide baseline health and disease parameters for this newly established population and the source population and will inform future translocation and conservation management activities. These data expand current knowledge on aspects of the biology and microbiology of the long-nosed potoroo, both locally and nationally.

Genomic Insights Into the Pathogenicity of a Novel Biofilm-Forming Enterococcus sp. Bacteria (Enterococcus lacertideformus) Identified in Reptiles.

Whole genome analysis of a novel species of enterococci, Enterococcus lacertideformus, causing multi-systemic and invariably fatal disease in critically endangered Christmas Island reptiles was undertaken to determine the genetic elements and potential mechanisms conferring its pathogenic nature, biofilm-forming capabilities, immune recognition avoidance, and inability to grow in vitro. Comparative genomic analyses with related and clinically significant enterococci were further undertaken to infer the evolutionary history of the bacterium and identify genes both novel and absent. The genome had a G + C content of 35.1%, consisted of a circular chromosome, no plasmids, and was 2,419,934 bp in length (2,321 genes, 47 tRNAs, and 13 rRNAs). Multi-locus sequence typing (MLST), and single nucleotide polymorphism (SNP) analysis of multiple E. lacertideformus samples revealed they were effectively indistinguishable from one another and highly clonal. E. lacertideformus was found to be located within the Enterococcus faecium species clade and was closely related to Enterococcus villorum F1129D based on 16S rDNA and MLST house-keeping gene analysis. Antimicrobial resistance (DfreE, EfrB, tetM, bcrRABD, and sat4) and virulence genes (Fss3 and ClpP), and genes conferring tolerance to metals and biocides (n = 9) were identified. The detection of relatively few genes encoding antimicrobial resistance and virulence indicates that this bacterium may have had no exposure to recently developed and clinically significant antibiotics. Genes potentially imparting beneficial functional properties were identified, including prophages, insertion elements, integrative conjugative elements, and genomic islands. Functional CRISPR-Cas arrays, and a defective prophage region were identified in the genome. The study also revealed many genomic loci unique to E. lacertideformus which contained genes enriched in cell wall/membrane/envelop biogenesis, and carbohydrate metabolism and transport functionality. This finding and the detection of putative enterococcal biofilm determinants (EfaAfs, srtC, and scm) may underpin the novel biofilm phenotype observed for this bacterium. Comparative analysis of E. lacertideformus with phylogenetically related and clinically significant enterococci (E. villorum F1129D, Enterococcus hirae R17, E. faecium AUS0085, and Enterococcus faecalis OG1RF) revealed an absence of genes (n = 54) in E. lacertideformus, that encode metabolic functionality, which potentially hinders nutrient acquisition and/or utilization by the bacterium and precludes growth in vitro. These data provide genetic insights into the previously determined phenotype and pathogenic nature of the bacterium.

Evidence of chronic cadmium exposure identified in the critically endangered Christmas Island flying-fox (Pteropus natalis).

The Christmas Island flying-fox (Pteropus natalis) is the last native mammal on Christmas Island and its population is in decline. Phosphate mining occurs across much of the eastern side of Christmas Island. The phosphate deposits are naturally rich in cadmium, and potentially other metals, which may be threatening the Christmas Island flying-fox population. To test this, concentrations of metals (cadmium, copper, iron, mercury, lead, and zinc) were measured in fur and urine collected from Christmas Island flying-foxes and interpreted concurrently with urinalysis and serum biochemistry data. In addition, metal concentrations in liver and kidney samples from two Christmas Island flying-foxes and associated histological findings from one of these individuals are reported. Fur cadmium concentrations were significantly higher in the Christmas Island flying-fox compared to concentrations found in flying-foxes in mainland Australia. Additionally, 30% of Christmas Island flying-foxes had urine cadmium concentrations exceeding maximum concentrations previously reported in flying-foxes in mainland Australia. Glucosuria and proteinuria were identified in two Christmas Island flying-foxes, suggestive of renal dysfunction. In one aged flying-fox, kidney cadmium concentrations were four-fold higher than toxic thresholds reported for domestic mammals. Microscopic evaluation of this individual identified bone lesions consistent with those described in laboratory animals with chronic cadmium poisoning. These results suggest that Christmas Island flying-foxes are being exposed to cadmium and identification of these sources is recommended as a focus of future research. Unexpectedly, urine iron concentrations in Christmas Island flying-foxes were higher compared to previous studies of Australian mainland flying-foxes, which suggests that urinary excretion of iron may be an important aspect of iron homeostasis in this species whose diet is iron rich.

Cross-continental emergence of Nannizziopsis barbatae disease may threaten wild Australian lizards.

Members of the genus Nannizziopsis are emerging fungal pathogens of reptiles that have been documented as the cause of fatal mycoses in a wide range of reptiles in captivity. Cases of severe, proliferative dermatitis, debility and death have been detected in multiple free-living lizard species from locations across Australia, including a substantial outbreak among Eastern water dragons (Intellagama lesueurii) in Brisbane, Queensland. We investigated this disease in a subset of severely affected lizards and identified a clinically consistent syndrome characterized by hyperkeratosis, epidermal hyperplasia, dermal inflammation, necrosis, ulceration, and emaciation. Using a novel fungal isolation method, histopathology, and molecular techniques, we identified the etiologic agent as Nannizziopsis barbatae, a species reported only once previously from captive lizards in Australia. Here we report severe dermatomycosis caused by N. barbatae in five species of Australian lizard, representing the first cases of Nannizziopsis infection among free-living reptiles, globally. Further, we evaluate key pathogen and host characteristics that indicate N. barbatae-associated dermatomycosis may pose a concerning threat to Australian lizards.

Novel hepaci- and pegi-like viruses in native Australian wildlife and non-human primates.

The Flaviviridae family of positive-sense RNA viruses contains important pathogens of humans and other animals, including Zika virus, dengue virus, and hepatitis C virus. The Flaviviridae are currently divided into four genera-Hepacivirus, Pegivirus, Pestivirus, and Flavivirus-each with a diverse host range. Members of the genus Hepacivirus are associated with an array of animal species, including humans, non-human primates, other mammalian species, as well as birds and fish, while the closely related pegiviruses have been identified in a variety of mammalian taxa, also including humans. Using a combination of total RNA and whole-genome sequencing we identified four novel hepaci-like viruses and one novel variant of a known hepacivirus in five species of Australian wildlife. The hosts infected comprised native Australian marsupials and birds, as well as a native gecko (Gehyra lauta). From these data we identified a distinct marsupial clade of hepaci-like viruses that also included an engorged Ixodes holocyclus tick collected while feeding on Australian long-nosed bandicoots (Perameles nasuta). Distinct lineages of hepaci-like viruses associated with geckos and birds were also identified. By mining the SRA database we similarly identified three new hepaci-like viruses from avian and primate hosts, as well as two novel pegi-like viruses associated with primates. The phylogenetic history of the hepaci- and pegi-like viruses as a whole, combined with co-phylogenetic analysis, provided support for virus-host co-divergence over the course of vertebrate evolution, although with frequent cross-species virus transmission. Overall, our work highlights the diversity of the Hepacivirus and Pegivirus genera as well as the uncertain phylogenetic distinction between.

Meta-Transcriptomic Discovery of a Divergent Circovirus and a Chaphamaparvovirus in Captive Reptiles with Proliferative Respiratory Syndrome.

Viral pathogens are being increasingly described in association with mass morbidity and mortality events in reptiles. However, our knowledge of reptile viruses remains limited. Herein, we describe the meta-transcriptomic investigation of a mass morbidity and mortality event in a colony of central bearded dragons (Pogona vitticeps) in 2014. Severe, extensive proliferation of the respiratory epithelium was consistently found in affected dragons. Similar proliferative lung lesions were identified in bearded dragons from the same colony in 2020 in association with increased intermittent mortality. Total RNA sequencing identified two divergent DNA viruses: a reptile-infecting circovirus, denoted bearded dragon circovirus (BDCV), and the first exogeneous reptilian chaphamaparvovirus-bearded dragon chaphamaparvovirus (BDchPV). Phylogenetic analysis revealed that BDCV was most closely related to bat-associated circoviruses, exhibiting 70% amino acid sequence identity in the Replicase (Rep) protein. In contrast, in the nonstructural (NS) protein, the newly discovered BDchPV showed approximately 31%-35% identity to parvoviruses obtained from tilapia fish and crocodiles in China. Subsequent specific PCR assays revealed BDCV and BDchPV in both diseased and apparently normal captive reptiles, although only BDCV was found in those animals with proliferative pulmonary lesions and respiratory disease. This study expands our understanding of viral diversity in captive reptiles.

Meta-transcriptomic identification of Trypanosoma spp. in native wildlife species from Australia.

BACKGROUND: Wildlife species carry a remarkable diversity of trypanosomes. The detection of trypanosome infection in native Australian fauna is central to understanding their diversity and host-parasite associations. The implementation of total RNA sequencing (meta-transcriptomics) in trypanosome surveillance and diagnosis provides a powerful methodological approach to better understand the host species distribution of this important group of parasites. METHODS: We implemented a meta-transcriptomic approach to detect trypanosomes in a variety of tissues (brain, liver, lung, skin, gonads) sampled from native Australian wildlife, comprising four marsupials (koala, Phascolarctos cinereus; southern brown bandicoot, Isoodon obesulus; swamp wallaby, Wallabia bicolor; bare-nosed wombat, Vombatus ursinus), one bird (regent honeyeater, Anthochaera phrygia) and one amphibian (eastern dwarf tree frog, Litoria fallax). Samples corresponded to both clinically healthy and diseased individuals. Sequencing reads were de novo assembled into contigs and annotated. The evolutionary relationships among the trypanosomatid sequences identified were determined through phylogenetic analysis of 18S rRNA sequences. RESULTS: We detected trypanosome sequences in all six species of vertebrates sampled, with positive samples in multiple organs and tissues confirmed by PCR. Phylogenetic analysis indicated that the trypanosomes infecting marsupials were related to those previously detected in placental and marsupial mammals, while the trypanosome in the regent honeyeater grouped with avian trypanosomes. In contrast, we provide the first evidence for a trypanosome in the eastern dwarf tree frog that was phylogenetically distinct from those described in other amphibians. CONCLUSIONS: To our knowledge, this is the first meta-transcriptomic analysis of trypanosomes in native Australian wildlife, expanding the known genetic diversity of these important parasites. We demonstrated that RNA sequencing is sufficiently sensitive to detect low numbers of Trypanosoma transcripts and from diverse hosts and tissues types, thereby representing an effective means to detect trypanosomes that are divergent in genome sequence.

Pharmacokinetic profile of enrofloxacin and its metabolite ciprofloxacin in Asian house geckos (Hemidactylus frenatus) after single-dose oral administration of enrofloxacin.

The pharmacokinetics of enrofloxacin and its active metabolite ciprofloxacin were determined following oral administration in 21 Asian house geckos (Hemidactylus frenatus) at a dose of 10 mg/kg. Changes in enrofloxacin and ciprofloxacin plasma concentrations were quantified at regular intervals over 72 h (1, 2, 6, 12, 24, 48, and 72 h). Samples were analysed by high-pressure liquid chromatography (HPLC) and the enrofloxacin pharmacokinetic data underwent a two-compartment analysis. Due to the limited ciprofloxacin plasma concentrations above the lower limit of quantification (LLOQ), the ciprofloxacin data underwent non-compartment analysis and the half-life was determined by the Lineweaver-Burke plot and analysis. The enrofloxacin and ciprofloxacin mean half-lives (t ½) were 0.95 h (α) / 24.36 h (ß), and 11.06 h respectively, area under the curve (AUC0-24h) were 60.56 and 3.14 µg/mL*h, respectively, maximum concentrations (C max) were 12.31 and 0.24 µg/mL, respectively, and time required to reach the C max (T max) were 1 and 2 h respectively. Enrofloxacin was minimally converted to the active metabolite ciprofloxacin, with ciprofloxacin concentrations contributing only 4.91% of the total fluoroquinolone concentrations (AUC0-24h). Based on the pharmacokinetic indices when using susceptibility breakpoints when determined at mammalian body temperature it is predicted that single oral administration of enrofloxacin (10 mg/kg) would result in plasma concentrations effective against susceptible bacterial species inhibited by an enrofloxacin MIC ≤ 0.5 µg/mL in vitro, but additional studies will be required to determine its efficacy in vivo.

Metatranscriptomic Analysis of Virus Diversity in Urban Wild Birds with Paretic Disease.

Wild birds are major natural reservoirs and potential dispersers of a variety of infectious diseases. As such, it is important to determine the diversity of viruses they carry and use this information to help understand the potential risks of spillover to humans, domestic animals, and other wildlife. We investigated the potential viral causes of paresis in long-standing, but undiagnosed, disease syndromes in wild Australian birds. RNA from diseased birds was extracted and pooled based on tissue type, host species, and clinical manifestation for metagenomic sequencing. Using a bulk and unbiased metatranscriptomic approach, combined with clinical investigation and histopathology, we identified a number of novel viruses from the families Astroviridae, Adenoviridae, Picornaviridae, Polyomaviridae, Paramyxoviridae, Parvoviridae, and Circoviridae in common urban wild birds, including Australian magpies, magpie larks, pied currawongs, Australian ravens, and rainbow lorikeets. In each case, the presence of the virus was confirmed by reverse transcription (RT)-PCR. These data revealed a number of candidate viral pathogens that may contribute to coronary, skeletal muscle, vascular, and neuropathology in birds of the Corvidae and Artamidae families and neuropathology in members of the Psittaculidae The existence of such a diverse virome in urban avian species highlights the importance and challenges in elucidating the etiology and ecology of wildlife pathogens in urban environments. This information will be increasingly important for managing disease risks and conducting surveillance for potential viral threats to wildlife, livestock, and human health.IMPORTANCE Wildlife naturally harbor a diverse array of infectious microorganisms and can be a source of novel diseases in domestic animals and human populations. Using unbiased RNA sequencing, we identified highly diverse viruses in native birds from Australian urban environments presenting with paresis. This research included the clinical investigation and description of poorly understood recurring syndromes of unknown etiology: clenched claw syndrome and black and white bird disease. As well as identifying a range of potentially disease-causing viral pathogens, this study describes methods that can effectively and efficiently characterize emergent disease syndromes in free-ranging wildlife and promotes further surveillance for specific pathogens of potential conservation and zoonotic concern.

Investigation into the utility of flying foxes as bioindicators for environmental metal pollution reveals evidence of diminished lead but significant cadmium exposure.

Due to their large range across diverse habitats, flying-foxes are potential bioindicator species for environmental metal exposure. To test this hypothesis, blood spots, urine, fur, liver and kidney samples were collected from grey-headed flying-foxes (Pteropus poliocephalus) and black flying-foxes (P. alecto) from the Sydney basin, Australia. Concentrations of arsenic, cadmium, copper, lead, mercury and zinc and 11 other trace metals were determined using inductively coupled plasma mass spectrometry. As predicted, kidney and fur lead concentrations were lower compared to concentrations found in flying-foxes in the early 1990's, due to reduced environmental lead emissions. Tissue cadmium concentrations in flying-foxes were higher compared to previous studies of flying-foxes and other bat species, suggesting that flying-foxes were exposed to unrecognized cadmium sources. Identification of these sources should be a focus of future research. Urine concentrations of arsenic, cadmium, mercury, and lead were proportional to kidney concentrations. Given that urine can be collected from flying-foxes without handling, this demonstrates that many flying-foxes can be assessed for metal exposure with relative ease. The analysis of blood spots was not viable because of variable metal concentrations in the filter paper used. Fur concentrations of metals correlated poorly with tissue concentrations at the low levels of metals found in this study, but fur could still be a useful sample if flying-foxes are exposed to high levels of metals. Lastly, heat inactivation had minimal impact on metal concentrations in kidney and liver samples and should be considered as a tool to protect personnel working with biohazardous samples.