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.

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.

Colony Location and Captivity Influence the Gut Microbial Community Composition of the Australian Sea Lion (Neophoca cinerea).

UNLABELLED: Gut microbiota play an important role in maintenance of mammalian metabolism and immune system regulation, and disturbances to this community can have adverse impacts on animal health. To better understand the composition of gut microbiota in marine mammals, fecal bacterial communities of the Australian sea lion (Neophoca cinerea), an endangered pinniped with localized distribution, were examined. A comparison of samples from individuals across 11 wild colonies in South and Western Australia and three Australian captive populations showed five dominant bacterial phyla: Firmicutes, Proteobacteria, Bacteroidetes, Actinobacteria, and Fusobacteria The phylum Firmicutes was dominant in both wild (76.4% ± 4.73%) and captive animals (61.4% ± 10.8%), while Proteobacteria contributed more to captive (29.3% ± 11.5%) than to wild (10.6% ± 3.43%) fecal communities. Qualitative differences were observed between fecal communities from wild and captive animals based on principal-coordinate analysis. SIMPER (similarity percentage procedure) analyses indicated that operational taxonomic units (OTU) from the bacterial families Clostridiaceae and Ruminococcaceae were more abundant in wild than in captive animals and contributed most to the average dissimilarity between groups (SIMPER contributions of 19.1% and 10.9%, respectively). Differences in the biological environment, the foraging site fidelity, and anthropogenic impacts may provide various opportunities for unique microbial establishment in Australian sea lions. As anthropogenic disturbances to marine mammals are likely to increase, understanding the potential for such disturbances to impact microbial community compositions and subsequently affect animal health will be beneficial for management of these vulnerable species. IMPORTANCE: The Australian sea lion is an endangered species for which there is currently little information regarding disease and microbial ecology. In this work, we present an in-depth study of the fecal microbiota of a large number of Australian sea lions from geographically diverse wild and captive populations. Colony location and captivity were found to influence the gut microbial community compositions of these animals. Our findings significantly extend the baseline knowledge of marine mammal gut microbiome composition and variability.

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.

Evaluation of a PCR protocol for sensitive detection of Giardia intestinalis in human faeces.

Giardia intestinalis is a protozoan parasite and a human pathogen. It is a leading cause of human diarrheal disease and a significant cause of morbidity worldwide. At the molecular level, G. intestinalis is a species complex, consisting of genetic assemblages (A to G) and sub-assemblage strains. The genotypes that cause human disease have been characterised to assemblages A and B, and include strains AI, AII, BIII and BIV. PCR amplification of diagnostic loci is used to genotype samples and is required to understand different transmission cycles within communities. A multi-locus approach is required for validation of Giardia genotyping and molecular diagnostic techniques that are efficient across numerous loci have not been established. This study evaluated several published protocols for the 18S small subunit ribosomal RNA (18S rRNA) and glutamate dehydrogenase genes (gdh) genes. Assays were compared using spiked faecal samples and by measuring the concentration of DNA generated following DNA extraction and PCR amplification. An optimal molecular method for G. intestinalis identification was established from direct DNA extraction of faecal material and GC-rich PCR chemistry. The protocol was applied to 50 clinical samples and produced PCR success rates of 90% and 94% at the 18S rRNA and gdh loci. Cyst concentration prior to DNA extraction was not necessary, and the optimal protocol was highly sensitive and an efficient method for testing clinical samples.

Molecular epidemiology, spatiotemporal analysis, and ecology of sporadic human cryptosporidiosis in Australia.

Parasites from the Cryptosporidium genus are the most common cause of waterborne disease around the world. Successful management and prevention of this emerging disease requires knowledge of the diversity of species causing human disease and their zoonotic sources. This study employed a spatiotemporal approach to investigate sporadic human cryptosporidiosis in New South Wales, Australia, between January 2008 and December 2010. Analysis of 261 human fecal samples showed that sporadic human cryptosporidiosis is caused by four species; C. hominis, C. parvum, C. andersoni, and C. fayeri. Sequence analysis of the gp60 gene identified 5 subtype families and 31 subtypes. Cryptosporidium hominis IbA10G2 and C. parvum IIaA18G3R1 were the most frequent causes of human cryptosporidiosis in New South Wales, with 59% and 16% of infections, respectively, attributed to them. The results showed that infections were most prevalent in 0- to 4-year-olds. No gender bias or regional segregation was observed between the distribution of C. hominis and C. parvum infections. To determine the role of cattle in sporadic human infections in New South Wales, 205 cattle fecal samples were analyzed. Four Cryptosporidium species were identified, C. hominis, C. parvum, C. bovis, and C. ryanae. C. parvum subtype IIaA18G3R1 was the most common cause of cryptosporidiosis in cattle, with 47% of infections attributed to it. C. hominis subtype IbA10G2 was also identified in cattle isolates.

Molecular epidemiology and spatial distribution of a waterborne cryptosporidiosis outbreak in Australia.

Cryptosporidiosis is one of the most common waterborne diseases reported worldwide. Outbreaks of this gastrointestinal disease, which is caused by the Cryptosporidium parasite, are often attributed to public swimming pools and municipal water supplies. Between the months of January and April in 2009, New South Wales, Australia, experienced the largest waterborne cryptosporidiosis outbreak reported in Australia to date. Through the course of the contamination event, 1,141 individuals became infected with Cryptosporidium. Health authorities in New South Wales indicated that public swimming pool use was a contributing factor in the outbreak. To identify the Cryptosporidium species responsible for the outbreak, fecal samples from infected patients were collected from hospitals and pathology companies throughout New South Wales for genetic analyses. Genetic characterization of Cryptosporidium oocysts from the fecal samples identified the anthroponotic Cryptosporidium hominis IbA10G2 subtype as the causative parasite. Equal proportions of infections were found in males and females, and an increased susceptibility was observed in the 0- to 4-year age group. Spatiotemporal analysis indicated that the outbreak was primarily confined to the densely populated coastal cities of Sydney and Newcastle.

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.

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.

Into the sea: Antimicrobial resistance determinants in the microbiota of little penguins (Eudyptula minor).

Terrestrial and aquatic birds have been proposed as sentinels for the spread of antimicrobial resistant bacteria, but few species have been investigated specifically in the context of AMR in the marine ecosystem. This study contrasts the occurrence of class 1 integrons and associated antimicrobial resistance genes in wild and captive little penguins (Eudyptula minor), an Australian seabird with local population declines. PCR screening of faecal samples (n = 448) revealed a significant difference in the prevalence of class 1 integrons in wild and captive groups, 3.2% and 44.7% respectively, with genes that confer resistance to streptomycin, spectinomycin, trimethoprim and multidrug efflux pumps detected. Class 1 integrons were not detected in two clinically relevant bacterial species, Klebsiella pneumoniae or Escherichia coli, isolated from penguin faeces. The presence of class 1 integrons in the little penguin supports the use of marine birds as sentinels of AMR in marine environments.

Biology of Cryptosporidium from marsupial hosts.

The majority of biological data on Cryptosporidium has been collected from humans and domestic animal hosts which creates a bias in knowledge on the biodiversity and evolution of this parasite genus. Further to understanding Cryptosporidium biology are studies encompassing broad hosts that represent diverse taxa sampled across wide geographic ranges. Marsupials represent a group of wildlife hosts from which limited information on Cryptosporidium is available. As marsupial hosts are an ancient mammalian lineage they represent an important group for studying parasite evolution. This review summarises information of the biology, epidemiology and evolution of Cryptosporidium in marsupial hosts, and discusses the importance of further understanding interactions in this parasite-host system.

Cryptosporidium fayeri: diversity within the GP60 locus of isolates from different marsupial hosts.

The highly polymorphic 60 kDa glycoprotein (GP60) of Cryptosporidium is an important tool for investigating the epidemiology of this parasite. Characterization of the GP60 gene has only been performed for 3 of the 20 known Cryptosporidium species, and has already enabled sub-typing and source tracking of species with human significance. We have characterised a fourth species, Cryptosporidium fayeri, at the GP60 locus using isolates (n=26) from different marsupial hosts to assess the diversity of GP60 within this species. The analysis demonstrated that C. fayeri isolates could be assigned to 6 subtypes which were associated with host species and locality. The intra-species diversity for the host-adapted C. fayeri was less than the diversity for human pathogenic species suggesting that the GP60 locus is under less selective pressure in these than host-adapted species.

Prevalence and genetic characterization of Cryptosporidium isolates from common brushtail possums (Trichosurus vulpecula) adapted to urban settings.

The common brushtail possum (Trichosurus vulpecula) is one of the most abundant native marsupials in urban Australia, having successfully adapted to utilize anthropogenic resources. The habituation of possums to food and shelter available in human settlements has facilitated interaction with people, pets, and zoo animals, increasing the potential for transmission of zoonotic Cryptosporidium pathogens. This study sought to examine the identity and prevalence of Cryptosporidium species occurring in possums adapted to urban settings compared to possums inhabiting remote woodlands far from urban areas and to characterize the health of the host in response to oocyst shedding. Findings indicated that both populations were shedding oocysts of the same genotype (brushtail possum 1 [BTP1]) that were genetically and morphologically distinct from zoonotic species and genotypes and most closely related to Cryptosporidium species from marsupials. The urban population was shedding an additional five Cryptosporidium isolates that were genetically distinct from BTP1 and formed a sister clade with Cryptosporidium parvum and Cryptosporidium hominis. Possums that were shedding oocysts showed no evidence of pathogenic changes, including elevated levels of white blood cells, diminished body condition (body mass divided by skeletal body length), or reduced nutritional state, suggesting a stable host-parasite relationship typical of Cryptosporidium species that are adapted to the host. Overall, Cryptosporidium occurred with a higher prevalence in possums from urban habitat (11.3%) than in possums from woodland habitat (5.6%); however, the host-specific nature of the genotypes may limit spillover infection in the urban setting. This study determined that the coexistence of possums with sympatric populations of humans, pets, and zoo animals in the urban Australian environment is unlikely to present a threat to public health safety.

A new species of Cryptosporidium (Apicomplexa: Cryptosporidiidae) from eastern grey kangaroos (Macropus giganteus).

Cryptosporidium macropodum n. sp is described. Oocysts of C. macropodum from the feces of kangaroos (Macropus spp.) are morphologically indistinguishable from other mammalian Cryptosporidium species, including C. parvum, C. hominis, C. suis, and C. canis. The oocysts are fully sporulated on excretion, lack sporocysts, and have an average width of 4.9 microm (4.5-6.0), a length of 5.4 microm (5.0-6.0), and a length:width ratio of 1.1. Phylogenetic analyses of the 18S ribosomal RNA, actin, and heat shock protein 70 (HSP70) loci demonstrate that C. macropodum is genetically distinct from all described Cryptosporidium species, including others found in marsupials. The parasite seems to be highly host-specific, because it has been found only in marsupials to date. Therefore, based on biological and molecular data, we consider C. macropodum a new species.

Molecular characterization of Cryptosporidium and Giardia from the Tasmanian devil (Sarcophilus harrisii).

The Tasmanian devil (Sarcophilus harrisii) is a carnivorous marsupial found only in the wild in Tasmania, Australia. Tasmanian devils are classified as endangered and are currently threatened by devil facial tumour disease, a lethal transmissible cancer that has decimated the wild population in Tasmania. To prevent extinction of Tasmanian devils, conservation management was implemented in 2003 under the Save the Tasmanian Devil Program. This study aimed to assess if conservation management was altering the interactions between Tasmanian devils and their parasites. Molecular tools were used to investigate the prevalence and diversity of two protozoan parasites, Cryptosporidium and Giardia, in Tasmanian devils. A comparison of parasite prevalence between wild and captive Tasmanian devils showed that both Cryptosporidium and Giardia were significantly more prevalent in wild devils (p < 0.05); Cryptosporidium was identified in 37.9% of wild devils but only 10.7% of captive devils, while Giardia was identified in 24.1% of wild devils but only 0.82% of captive devils. Molecular analysis identified the presence of novel genotypes of both Cryptosporidium and Giardia. The novel Cryptosporidium genotype was 98.1% similar at the 18S rDNA to Cryptosporidium varanii (syn. C. saurophilum) with additional samples identified as C. fayeri, C. muris, and C. galli. Two novel Giardia genotypes, TD genotype 1 and TD genotype 2, were similar to G. duodenalis from dogs (94.4%) and a Giardia assemblage A isolate from humans (86.9%). Giardia duodenalis BIV, a zoonotic genotype of Giardia, was also identified in a single captive Tasmanian devil. These findings suggest that conservation management may be altering host-parasite interactions in the Tasmanian devil, and the presence of G. duodenalis BIV in a captive devil points to possible human-devil parasite transmission.

Evaluation of next generation sequencing for the analysis of Eimeria communities in wildlife.

Next-generation sequencing (NGS) techniques are well-established for studying bacterial communities but not yet for microbial eukaryotes. Parasite communities remain poorly studied, due in part to the lack of reliable and accessible molecular methods to analyse eukaryotic communities. We aimed to develop and evaluate a methodology to analyse communities of the protozoan parasite Eimeria from populations of the Australian marsupial Petrogale penicillata (brush-tailed rock-wallaby) using NGS. An oocyst purification method for small sample sizes and polymerase chain reaction (PCR) protocol for the 18S rRNA locus targeting Eimeria was developed and optimised prior to sequencing on the Illumina MiSeq platform. A data analysis approach was developed by modifying methods from bacterial metagenomics and utilising existing Eimeria sequences in GenBank. Operational taxonomic unit (OTU) assignment at a high similarity threshold (97%) was more accurate at assigning Eimeria contigs into Eimeria OTUs but at a lower threshold (95%) there was greater resolution between OTU consensus sequences. The assessment of two amplification PCR methods prior to Illumina MiSeq, single and nested PCR, determined that single PCR was more sensitive to Eimeria as more Eimeria OTUs were detected in single amplicons. We have developed a simple and cost-effective approach to a data analysis pipeline for community analysis of eukaryotic organisms using Eimeria communities as a model. The pipeline provides a basis for evaluation using other eukaryotic organisms and potential for diverse community analysis studies.

Escherichia coli out in the cold: Dissemination of human-derived bacteria into the Antarctic microbiome.

Discharge of untreated sewage into Antarctic environments presents a risk of introducing non-native microorganisms, but until now, adverse consequences have not been conclusively identified. Here we show that sewage disposal introduces human derived Escherichia coli carrying mobile genetic elements and virulence traits with the potential to affect the diversity and evolution of native Antarctic microbial communities. We compared E. coli recovered from environmental and animal sources in Antarctica to a reference collection of E. coli from humans and non-Antarctic animals. The distribution of phylogenetic groups and frequency of 11 virulence factors amongst the Antarctic isolates were characteristic of E. coli strains more commonly associated with humans. The rapidly emerging E. coli ST131 and ST95 clones were found amongst the Antarctic isolates, and ST95 was the predominant E. coli recovered from Weddell seals. Class 1 integrons were found in 15% of the Antarctic E. coli with 4 of 5 identified gene cassette arrays containing antibiotic resistance genes matching those common in clinical contexts. Disposing untreated sewage into the Antarctic environment does disseminate non-native microorganisms, but the extent of this impact and implications for Antarctic ecosystem health are, as yet, poorly understood.

The environmental impact of sewage and wastewater outfalls in Antarctica: An example from Davis station, East Antarctica.

We present a comprehensive scientific assessment of the environmental impacts of an Antarctic wastewater ocean outfall, at Davis station in East Antarctica. We assessed the effectiveness of current wastewater treatment and disposal requirements under the Protocol on Environmental Protection to the Antarctic Treaty. Macerated wastewater has been discharged from an outfall at Davis since the failure of the secondary treatment plant in 2005. Water, sediment and wildlife were tested for presence of human enteric bacteria and antibiotic resistance mechanisms. Epibiotic and sediment macrofaunal communities were tested for differences between sites near the outfall and controls. Local fish were examined for evidence of histopathological abnormalities. Sediments, fish and gastropods were tested for uptake of sewage as measured by stable isotopes of N and C. Escherichia coli carrying antibiotic resistance determinants were found in water, sediments and wildlife (the filter feeding bivalve Laternula eliptica). Fish (Trematomus bernacchii) within close proximity to the outfall had significantly more severe and greater occurrences of histopathological abnormalities than at controls, consistent with exposure to sewage. There was significant enrichment of 15N in T. bernacchii and the predatory gastropod Neobuccinum eatoni around the outfall, providing evidence of uptake of sewage. There were significant differences between epibiotic and sediment macrofaunal communities at control and outfall sites (<1.5 km), when sites were separated into groups of similar habitat types. Benthic community composition was also strongly related to habitat and environmental drivers such as sea ice. The combined evidence indicated that the discharge of wastewater from the Davis outfall is causing environmental impacts. These findings suggest that conditions in Antarctic coastal locations, such as Davis, are unlikely to be conducive to initial dilution and rapid dispersal of wastewater as required under the Protocol on Environmental Protection to the Antarctic Treaty. Current minimum requirements for wastewater treatment and disposal in Antarctica are insufficient to ameliorate these risks and are likely to lead to accumulation of contaminants and introduction of non-native microbes and associated genetic elements. This new understanding suggests that modernised approaches to the treatment and disposal of wastewater are required in Antarctica. The most effective solution is advanced levels of wastewater treatment, which are now possible, feasible and a high priority for installation. As a direct outcome of the study, a new advanced treatment system is being installed at Davis, effectively avoiding environmental risks.

Investigation into potential transmission sources of Giardia duodenalis in a threatened marsupial (Petrogale penicillata).

Assemblages of the protozoan parasite Giardia duodenalis common in humans and domestic species are increasingly identified in wildlife species, raising concern about the spill-over of pathogens from humans and domestic animals into wildlife. Here, the identity and prevalence of G. duodenalis in populations of a threatened marsupial, the brush-tailed rock-wallaby (Petrogale penicillata), was investigated. Identification of G. duodenalis isolates, across three loci (18S rRNA, ß-giardin and gdh), from rock-wallaby fecal samples (n = 318) identified an overall detection rate of 6.3%. No significant difference in G. duodenalis detection was found among captive, wild and supplemented populations. Isolates were assigned to the zoonotic assemblages A and B at 18S rRNA, with sub-assemblages AI and BIV identified at the ß-giardin and gdh loci, respectively. Assemblages AI and BIV have previously been identified in human clinical cases, but also in domestic animals and wildlife. The identification of these assemblages in brush-tailed rock-wallabies suggests there are transmission routes of G. duodenalis from humans or other animals to Australian wildlife, both in captivity and in the wild.