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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

MOLECULAR DETECTION OF ANTIBIOTIC-RESISTANCE DETERMINANTS IN ESCHERICHIA COLI ISOLATED FROM THE ENDANGERED AUSTRALIAN SEA LION (NEOPHOCA CINEREA).

Greater interaction between humans and wildlife populations poses significant risks of anthropogenic impact to natural ecosystems, especially in the marine environment. Understanding the spread of microorganisms at the marine interface is therefore important if we are to mitigate adverse effects on marine wildlife. We investigated the establishment of Escherichia coli in the endangered Australian sea lion (Neophoca cinerea) by comparing fecal isolation from wild and captive sea lion populations. Fecal samples were collected from wild colonies March 2009-September 2010 and from captive individuals March 2011-May 2013. Using molecular screening, we assigned a phylotype to E. coli isolates and determined the presence of integrons, mobile genetic elements that capture gene cassettes conferring resistance to antimicrobial agents common in fecal coliforms. Group B2 was the most abundant phylotype in all E. coli isolates (n = 37), with groups A, B1, and D also identified. Integrons were not observed in E. coli (n = 21) isolated from wild sea lions, but were identified in E. coli from captive animals (n = 16), from which class I integrases were detected in eight isolates. Sequencing of gene cassette arrays identified genes conferring resistance to streptomycin-spectinomycin (aadA1) and trimethoprim (dfrA17, dfrB4). Class II integrases were not detected in the E. coli isolates. The frequent detection in captive sea lions of E. coli with resistance genes commonly identified in human clinical cases suggests that conditions experienced in captivity may contribute to establishment. Identification of antibiotic resistance in the microbiota of Australian sea lions provides crucial information for disease management. Our data will inform conservation management strategies and provide a mechanism to monitor microorganism dissemination to sensitive pinniped populations.

Diversity of Cryptosporidium in brush-tailed rock-wallabies (Petrogale penicillata) managed within a species recovery programme.

Host-parasite relationships are likely to be impacted by conservation management practices, potentially increasing the susceptibility of wildlife to emerging disease. Cryptosporidium, a parasitic protozoan genus comprising host-adapted and host-specific species, was used as an indicator of parasite movement between populations of a threatened marsupial, the brush-tailed rock-wallaby (Petrogale penicillata). PCR screening of faecal samples (n = 324) from seven wallaby populations across New South Wales, identified Cryptosporidium in 7.1% of samples. The sampled populations were characterised as captive, supplemented and wild populations. No significant difference was found in Cryptosporidium detection between each of the three population categories. The positive samples, detected using 18S rRNA screening, were amplified using the actin and gp60 loci. Multi-locus sequence analysis revealed the presence of Cryptosporidium fayeri, a marsupial-specific species, and C. meleagridis, which has a broad host range, in samples from the three population categories. Cryptosporidium meleagridis has not been previously reported in marsupials and hence the pathogenicity of this species to brush-tailed rock-wallabies is unknown. Based on these findings, we recommend further study into Cryptosporidium in animals undergoing conservation management, as well as surveying wild animals in release areas, to further understand the diversity and epidemiology of this parasite in threatened wildlife.

Distribution of Giardia duodenalis assemblages A and B among children living in a remote indigenous community of the Northern Territory, Australia.

Giardiasis is a communicable gastrointestinal disease caused by Giardia duodenalis and two genetic assemblages, A and B, cause human infection. In remote Indigenous communities of Australia, giardiasis is highly prevalent among children but disease transmission is poorly understood. This study investigated the prevalence of Giardia and genetic subtypes contributing to human disease in a remote Indigenous community, in the Northern Territory of Australia. Eighty-seven faecal samples were collected from 74 children (<15 years) over an 18 month period, and the distribution of positive cases relative to participant age and gender were examined. Screening by microscopy and 18S rRNA PCR amplification showed 66.7% (58/87) of faecal samples were positive for Giardia. Both males and females were equally affected and high detection rates were obtained for participants aged 0-<5 years and 5-<10 years (66.0 and 60.0% respectively). For 58.6% of the positive samples, Giardia was only detected by 18S rRNA PCR. Approximately 75% of cases were assemblage B, and subassemblage analyses using terminal restriction fragment length polymorphism of the glutamate dehydrogenase gene demonstrated that a variety of genetic variants were present. The high proportion of positive cases that were not detectable by microscopy, and dominance of assemblage B cases highlights the need for further research in this community, to assess the contribution of Giardia to chronic gastrointestinal disease among children, and to understand conditions conductive to assemblage B transmission.

Giardia duodenalis and Cryptosporidium occurrence in Australian sea lions (Neophoca cinerea) exposed to varied levels of human interaction.

Giardia and Cryptosporidium are amongst the most common protozoan parasites identified as causing enteric disease in pinnipeds. A number of Giardia assemblages and Cryptosporidium species and genotypes are common in humans and terrestrial mammals and have also been identified in marine mammals. To investigate the occurrence of these parasites in an endangered marine mammal, the Australian sea lion (Neophoca cinerea), genomic DNA was extracted from faecal samples collected from wild populations (n = 271) in Southern and Western Australia and three Australian captive populations (n = 19). These were screened using PCR targeting the 18S rRNA of Giardia and Cryptosporidium. Giardia duodenalis was detected in 28 wild sea lions and in seven captive individuals. Successful sequencing of the 18S rRNA gene assigned 27 Giardia isolates to assemblage B and one to assemblage A, both assemblages commonly found in humans. Subsequent screening at the gdh and ß-giardin loci resulted in amplification of only one of the 35 18S rRNA positive samples at the ß-giardin locus. Sequencing at the ß-giardin locus assigned the assemblage B 18S rRNA confirmed isolate to assemblage AI. The geographic distribution of sea lion populations sampled in relation to human settlements indicated that Giardia presence in sea lions was highest in populations less than 25 km from humans. Cryptosporidium was not detected by PCR screening in either wild colonies or captive sea lion populations. These data suggest that the presence of G. duodenalis in the endangered Australian sea lion is likely the result of dispersal from human sources. Multilocus molecular analyses are essential for the determination of G. duodenalis assemblages and subsequent inferences on transmission routes to endangered marine mammal populations.

Prevalence and Molecular Identification of Nematode and Dipteran Parasites in an Australian Alpine Grasshopper (Kosciuscola tristis).

In alpine Australia, Orthoptera are abundant, dominant herbivores, important prey species, and hosts for parasites and parasitoids. Despite the central role of orthopterans in alpine ecosystems, the impact of parasites on orthopteran populations is under-explored. In this study we describe the relationship between parasite prevalence and host sex, body size and year of collection. We accessed an existing, preserved collection of 640 Kosciuscola tristis collected from across its range between 2007 and 2011. Upon dissection we collected juvenile parasites and used molecular tools to identify them to three families (Nematoda; Mermithidae, and Arthropoda: Diptera: Tachinidae and Sarcophagidae). The prevalence of nematodes ranged from 3.5% to 25.0% and dipterans from 2.4% to 20.0%. Contrary to predictions, we found no associations between parasite prevalence and grasshopper sex or size. Although there was an association between prevalence of both nematodes and dipterans with year of collection, this is likely driven by a small sample size in the first year. Our results provide a foundation for future studies into parasite prevalence within the alpine environment and the abiotic factors that might influence these associations.

Cryptosporidium from a free-ranging marsupial host: bandicoots in urban Australia.

Expansion of human settlement has increased the interface between people and bandicoots with implications for the emergence and spread of zoonotic parasites. The host status of bandicoots inhabiting suburban areas and their potential role in Cryptosporidium transmission remains unresolved. Our study aimed to determine the prevalence and identity of Cryptosporidium in two sympatric bandicoot species. Cryptosporidium signatures were detected in twelve bandicoot faecal samples (n=98) through amplification of the 18S rRNA. Phylogenetic inference placed the isolates in a clade with Cryptosporidium parvum, a species with a broad host range and zoonotic potential, or loosely related to Cryptosporidium hominis. However, the identity of the bandicoot isolates was not fully resolved and whether they were infected or simply passively transmitting oocysts is unknown. This study revealed that free-ranging bandicoots of northern Sydney were shedding Cryptosporidium oocysts at a prevalence of 12.2% (95% CI [6.76, 20.8]), similar to marsupial species that act as reservoirs for Cryptosporidium. Our findings expand the range of hosts known to shed Cryptosporidium in urban areas.

Into the wild: dissemination of antibiotic resistance determinants via a species recovery program.

Management strategies associated with captive breeding of endangered species can establish opportunities for transfer of pathogens and genetic elements between human and animal microbiomes. The class 1 integron is a mobile genetic element associated with clinical antibiotic resistance in gram-negative bacteria. We examined the gut microbiota of endangered brush-tail rock wallabies Petrogale penicillata to determine if they carried class 1 integrons. No integrons were detected in 65 animals from five wild populations. In contrast, class 1 integrons were detected in 48% of fecal samples from captive wallabies. The integrons contained diverse cassette arrays that encoded resistance to streptomycin, spectinomycin, and trimethoprim. Evidence suggested that captive wallabies had acquired typical class 1 integrons on a number of independent occasions, and had done so in the absence of strong selection afforded by antibiotic therapy. Sufficient numbers of bacteria containing diverse class 1 integrons must have been present in the general environment occupied by the wallabies to account for this acquisition. The captive wallabies have now been released, in an attempt to bolster wild populations of the species. Consequently, they can potentially spread resistance integrons into wild wallabies and into new environments. This finding highlights the potential for genes and pathogens from human sources to be acquired during captive breeding and to be unwittingly spread to other populations.

Pinning down a polymorphic parasite: new genetic and morphological descriptions of Eimeria macropodis from the Tammar wallaby (Macropus eugenii).

Identification of the protozoan parasite, Eimeria has traditionally relied on oocyst morphology, host range and life-cycle attributes. However, it is increasingly recognized that Eimeria species can vary in size and shape across their host range, an attribute known as 'polymorphism' that presents a unique challenge for identification. Advances in molecular tools hold promise for characterising Eimeria that may otherwise be misclassified based on morphology. Our study used morphologic and molecular traits of the oocyst life stage to identify a polymorphic parasite, Eimeria macropodis in a captive Tammar wallaby (Macropus eugenii) population in Australia. Molecular characterization highlighted the need to use multiple genetic markers (18S SSU and cytochrome c oxidase subunit I) to accurately identify E. macropodis owing to heterozygous alleles at the 18S SSU locus. This study provided an opportunity to assess the utility and shortcomings of morphologic and molecular techniques for 'pinning down' a polymorphic species. Moreover, our study was able to place E. macropodis in an evolutionary context and enhance resolution of the under-studied marsupial clade.