Identifying historical and future global change drivers that place species recovery at risk.

Ecosystem management in the face of global change requires understanding how co-occurring threats affect species and communities. Such an understanding allows for effective management strategies to be identified and implemented. An important component of this is differentiating between factors that are within (e.g. invasive predators) or outside (e.g. drought, large wildfires) of a local manager's control. In the global biodiversity hotspot of south-western Australia, small- and medium-sized mammal species are severely affected by anthropogenic threats and environmental disturbances, including invasive predators, fire, and declining rainfall. However, the relative importance of different drivers has not been quantified. We used data from a long-term monitoring program to fit Bayesian state-space models that estimated spatial and temporal changes in the relative abundance of four threatened mammal species: the woylie (Bettongia penicillata), chuditch (Dasyurus geoffroii), koomal (Trichosurus vulpecula) and quenda (Isoodon fusciventor). We then use Bayesian structural equation modelling to identify the direct and indirect drivers of population changes, and scenario analysis to forecast population responses to future environmental change. We found that habitat loss or conversion and reduced primary productivity (caused by rainfall declines) had greater effects on species' spatial and temporal population change than the range of fire and invasive predator (the red fox Vulpes vulpes) management actions observed in the study area. Scenario analysis revealed that a greater extent of severe fire and further rainfall declines predicted under climate change, operating in concert are likely to further reduce the abundance of these species, but may be mitigated partially by invasive predator control. Considering both historical and future drivers of population change is necessary to identify the factors that risk species recovery. Given that both anthropogenic pressures and environmental disturbances can undermine conservation efforts, managers must consider how the relative benefit of conservation actions will be shaped by ongoing global change.

Eruptive dynamics are common in managed mammal populations.

Successful conservation management is often based on the principle that small or declining populations can recover if we identify and remove the factors that caused them to decline in the first place. But what form will that recovery take? Theory tells us that when a strong limiting factor is removed, a population should increase in size to where it becomes limited by some other factor. However, if the subsequent limitation involves feedbacks between the density of a consumer and its resource, there is potential for the consumer population to undergo substantial fluctuations in size that we would characterize as boom-bust or eruptive dynamics. We analysed long-term (7.6-29 yr) data documenting changes in the abundance of 169 populations of 20 mammal species released from a strong limiting factor (fox predation) in Australia. We show that many populations (44) exhibited eruptive dynamics, with exponential increase to a peak and subsequent population decline. Of 51 populations showing eruptive dynamics (the Australian populations plus seven translocated ungulate populations), the time taken for erupting populations to reach a peak before declining was related negatively to the intrinsic rate of population growth and positively to body mass, such that larger-bodied species with slow rates of population growth had a longer period of population increase before declining. Our results suggest that a substantial proportion of populations recovering after removal of a threatening process are likely to exhibit eruptive dynamics, and that managers of recovering or translocated populations should anticipate this outcome in conservation planning.

Identification of a novel species of Eimeria Schneider, 1875 from the woylie, Bettongia penicillata Gray (Diprotodontia: Potoroidae) and the genetic characterisation of three Eimeria spp. from other potoroid marsupials.

Faecal samples (n = 1,093) collected from the woylie Bettongia penicillata Gray, in south-western Australia were examined for the presence of coccidian parasites. Eimeria sp. oöcysts were detected in 15.2% of samples. Faecal samples obtained from the eastern bettong Bettongia gaimardi (Desmarest) (n = 4) and long-nosed potoroo Potorous tridactylus (Kerr) (n = 12) in Tasmania, were also screened for the presence of Eimeria spp. (prevalence 50% and 41.7%, respectively). Morphological and genetic comparison with other known species of Eimeria indicates that the material identified in woylies is novel. This study aimed to (i) morphologically describe and genetically characterise Eimeria woyliei n. sp. found in woylies; and (ii) genetically characterise Eimeria gaimardi Barker, O'Callaghan & Beveridge, 1988, Eimeria potoroi Barker, O'Callaghan & Beveridge, 1988, and Eimeria mundayi Barker, O'Callaghan & Beveridge, 1988, from other potoroid marsupials. Molecular phylogenetic analyses conducted at the 18S rDNA and mitochondrial cytochrome c oxidase subunit 1 (cox1) loci revealed that E. woyliei n. sp. was most closely related to Eimeria setonicis Barker, O'Callaghan & Beveridge, 1988, at the 18S rDNA locus, and Eimeria trichosuri O'Callaghan & O'Donoghue, 2001, at the cox1 locus. Eimeria woyliei n. sp. is the sixth species of Eimeria to be formally described from potoroid marsupials.

Altered parasite community structure in an endangered marsupial following translocation.

Fauna translocations play an integral role in the management of threatened wildlife, though we are limited by our understanding of how the host-parasite community changes during translocation. During this longitudinal field-based study, we monitored gastrointestinal, blood-borne and ectoparasite taxa infecting woylies (Bettongia penicillata) for up to 12 months following two fauna translocations to supplement existing wild woylie populations in three different sites (Dryandra, Walcott and Warrup East) within the south-west of Western Australia. We aimed to (a) identify changes in parasite community structure of both translocated and resident woylies following translocation; and (b) evaluate the efficacy of ivermectin treatment in translocated hosts. Destination site and time since translocation had the strongest effects on parasite prevalence and mean faecal egg counts following translocation. Ivermectin treatment did not significantly reduce parasite prevalence or mean faecal egg counts in treated hosts. Prior to translocation, parasite community composition differed significantly between woylies selected for translocation and resident woylies within each release site. Following translocation, the parasite communities of translocated and resident hosts converged to become more similar over time, with loss of parasite taxa and novel host-parasite associations emerging. This is the first study to examine changes to the broader parasite community in translocated and resident animals following translocation. The dominant site-specific response of parasites following translocation reinforces the importance of incorporating parasite studies to enhance our fundamental understanding of perturbations in host-parasite systems during translocation, in particular the site-level drivers of parasite dynamics.

Deconstructing compassionate conservation.

Compassionate conservation focuses on 4 tenets: first, do no harm; individuals matter; inclusivity of individual animals; and peaceful coexistence between humans and animals. Recently, compassionate conservation has been promoted as an alternative to conventional conservation philosophy. We believe examples presented by compassionate conservationists are deliberately or arbitrarily chosen to focus on mammals; inherently not compassionate; and offer ineffective conservation solutions. Compassionate conservation arbitrarily focuses on charismatic species, notably large predators and megaherbivores. The philosophy is not compassionate when it leaves invasive predators in the environment to cause harm to vastly more individuals of native species or uses the fear of harm by apex predators to terrorize mesopredators. Hindering the control of exotic species (megafauna, predators) in situ will not improve the conservation condition of the majority of biodiversity. The positions taken by so-called compassionate conservationists on particular species and on conservation actions could be extended to hinder other forms of conservation, including translocations, conservation fencing, and fertility control. Animal welfare is incredibly important to conservation, but ironically compassionate conservation does not offer the best welfare outcomes to animals and is often ineffective in achieving conservation goals. Consequently, compassionate conservation may threaten public and governmental support for conservation because of the limited understanding of conservation problems by the general public.

Deconstrucción de la Conservación Compasiva Resumen La conservación compasiva se enfoca en cuatro principios: no causar daño; los individuos importan; la integración de los animales individualmente; y la coexistencia pacífica entre los humanos u los animales. Recientemente, la conservación compasiva ha sido promovida como una alternativa a la filosofía convencional de la conservación. Creemos que los ejemplos presentados por los conservacionistas compasivos han sido elegidos arbitraria o deliberadamente por estar enfocados en los mamíferos; por ser inherentes y no compasivos; y por ofrecer soluciones de conservación poco efectivas. La conservación compasiva se enfoca arbitrariamente en las especies carismáticas, principalmente los grandes depredadores y los megaherbívoros. La filosofía no es compasiva cuando deja que los depredadores invasores dentro del ambiente causen daño a un vasto número de individuos nativos o usa el miedo al daño por superdepredadores para aterrorizar a los mesodepredadores. El entorpecimiento del control de especies exóticas (megafauna, depredadores) in situ no mejorará las condiciones de conservación de la mayoría de la biodiversidad, incluso si los conservacionistas compasivos no dañan a los individuos exóticos. Las posiciones que toman los llamados conservacionistas compasivos sobre especies particulares y sobre las acciones de conservación podrían extenderse para entorpecer otros tipos de conservación, incluyendo las reubicaciones, el encercado para la conservación y el control de la fertilidad. El bienestar animal es increíblemente importante para la conservación e irónicamente, la conservación compasiva no ofrece los mejores resultados de bienestar para los animales y comúnmente es poco efectiva en el logro de los objetivos de conservación. Como consecuencia, la conservación compasiva puede poner en peligro el apoyo público y del gobierno que tiene la conservación debido al entendimiento poco limitado que tiene el público general sobre los problemas de conservación.

Increased Trypanosoma spp. richness and prevalence of haemoparasite co-infection following translocation.

BACKGROUND: Understanding how fauna translocation and antiparasitic drug treatment impact parasite community structure within a host is vital for optimising translocation outcomes. Trypanosoma spp. and piroplasms (Babesia and Theileria spp.) are known to infect Australian marsupials, including the woylie (Bettongia penicillata). However relatively little is known about these haemoparasites, or how they respond to management practices such as translocation. We monitored haemoparasites infecting woylies for up to 12 months during two fauna translocations to supplement existing woylie populations in three different sites (Dryandra, Walcott and Warrup East) within south-western Australia between 2014 and 2016, with the aim of investigating (i) how haemoparasite prevalence, Trypanosoma spp. richness and Trypanosoma spp. community composition varied over time and between different sites following translocation; and (ii) whether ivermectin treatment indirectly impacts haemoparasite prevalence. Using molecular methods, 1211 blood samples were screened for the presence of trypanosomes, and a subset of these samples (n = 264) were also tested for piroplasms. RESULTS: Trypanosomes and piroplasms were identified in 55% and 94% of blood samples, respectively. We identified five Trypanosoma species, two Theileria species, a single species of Babesia and a novel Bodo species. Trypanosoma spp. richness and the prevalence of haemoparasite co-infection increased after translocation. Prior to translocation, Trypanosoma spp. community composition differed significantly between translocated and resident woylies within Walcott and Warrup East, but not Dryandra. Six months later, there was a significant difference between translocated and resident woylies within Dryandra, but not Walcott or Warrup East. The response of haemoparasites to translocation was highly site-specific, with predominant changes to the haemoparasite community in translocated woylies occurring within the first few months following translocation. Ivermectin treatment had no significant effect on haemoparasite prevalence. CONCLUSIONS: This study contributes to our understanding of haemoparasite dynamics in woylies following translocation. The highly site-specific and rapid response of haemoparasites to translocation highlights the need to better understand what drives these effects. Given that haemoparasite prevalence and composition of translocated and resident animals changed significantly following translocation, we propose that parasite monitoring should form an essential component of translocation protocols, and such protocols should endeavour to monitor translocated hosts and cohabiting species.

Debilitating disease in a polyparasitised woylie (Bettongia penicillata): A diagnostic investigation.

During monitoring of critically endangered woylie (Bettongia penicillata) populations within the south-west of Western Australia, an adult female woylie was euthanased after being found in extremely poor body condition with diffuse alopecia, debilitating skin lesions and severe ectoparasite infestation. Trypanosoma copemani G2 and Sarcocystis sp. were detected molecularly within tissue samples collected post-mortem. Potorostrongylus woyliei and Paraustrostrongylus sp. nematodes were present within the stomach and small intestine, respectively. Blood collected ante-mortem revealed the presence of moderate hypomagnesaemia, mild hypokalaemia, mild hyperglobulinaemia and mild hypoalbuminaemia. Diffuse megakaryocytic hypoplasia was evident within the bone marrow. We propose various hypotheses that may explain the presence of severe ectoparasite infection, skin disease and poor body condition in this woylie. Given the potential deleterious effects of parasite infection, the importance of monitoring parasites cannot be over-emphasised.

Trypanosome co-infections increase in a declining marsupial population.

Understanding the impacts of parasites on wildlife is growing in importance as diseases pose a threat to wildlife populations. Woylie (syn. brush-tailed bettong, Bettongia penicillata) populations have undergone enigmatic declines in south-western Western Australia over the past decade. Trypanosomes have been suggested as a possible factor contributing towards these declines because of their high prevalence in the declining population. We asked whether temporal patterns of infection with Trypanosoma spp. were associated with the decline patterns of the host, or if other factors (host sex, body condition, co-infection or rainfall) were more influential in predicting infection patterns. Species-specific nested PCRs were used to detect the two most common trypanosomes (T. copemani and T. vegrandis) from 444 woylie blood samples collected between 2006 and 2012. Time relative to the decline (year) and an interaction with co-infection by the other trypanosome best explained patterns of infection for both trypanosomes. The prevalence of single species infections for both T. copemani and T. vegrandis was lower after the population crash, however, the occurrence of co-infections increased after the crash compared to before the crash. Our results suggest an interaction between the two parasites with the decline of their host, leading to a higher level of co-infection after the decline. We discuss the possible mechanisms that may have led to a higher level of co-infection after the population crash, and highlight the importance of considering co-infection when investigating the role of parasites in species declines.

Infectious Disease Surveillance in the Woylie (Bettongia penicillata).

Wild populations of the critically endangered woylie (Bettongia penicillata) recently declined by 90% in southwest Western Australia. Increased predation is the leading hypothesis for decline, but disease may be playing a role increasing susceptibility to predation. To explore this possibility, we surveyed woylie populations in the wild, in captivity and in a predator-free sanctuary for exposure to, and infection with, four known pathogens of macropods: herpesviruses, Wallal and Warrego orbiviruses, and Toxoplasma gondii. Our study found two of 68 individuals positive for neutralizing antibodies against known macropodid alphaherpesviruses. Three of 45 individuals were PCR positive for a herpesvirus that was shown to be a novel gammaherpesvirus or a new strain/variant of Potoroid Herpesvirus 1. Further sequence information is required to definitively determine its correct classification. There was no evidence of antibodies to orbivirus Wallal and Warrego serogroups, and all serological samples tested for T. gondii were negative. This is the first report of PCR and serological detection of herpesviruses in the woylie. Positive individuals did not demonstrate clinical signs of herpesviral diseases; therefore, the clinical significance of herpesviruses to wild woylie populations remains unclear. Further monitoring for herpesvirus infections will be important to inform disease risk analysis for this virus and determine temporal trends in herpesvirus activity that may relate to population health and conservation outcomes.

Evaluating Stress Physiology and Parasite Infection Parameters in the Translocation of Critically Endangered Woylies (Bettongia penicillata).

Translocation can be stressful for wildlife. Stress may be important in fauna translocation because it has been suggested that it can exacerbate the impact of infectious disease on translocated wildlife. However, few studies explore this hypothesis by measuring stress physiology and infection indices in parallel during wildlife translocations. We analysed faecal cortisol metabolite (FCM) concentration and endoparasite parameters (nematodes, coccidians and haemoparasites) in a critically endangered marsupial, the woylie (Bettongia penicillata), 1-3 months prior to translocation, at translocation, and 6 months later. FCM for both translocated and resident woylies was significantly higher after translocation compared to before or at translocation. In addition, body condition decreased with increasing FCM after translocation. These patterns in host condition and physiology may be indicative of translocation stress or stress associated with factors independent of the translocation. Parasite factors also influenced FCM in translocated woylies. When haemoparasites were detected, there was a significant negative relationship between strongyle egg count and FCM. This may reflect the influence of glucocorticoids on the immune response to micro- and macro-parasites. Our results indicate that host physiology and infection patterns can change significantly during translocation, but further investigation is required to determine how these patterns influence translocation success.

Morphological and molecular description of Ixodes woyliei n. sp. (Ixodidae) with consideration for co-extinction with its critically endangered marsupial host.

BACKGROUND: Taxonomic identification of ticks obtained during a longitudinal survey of the critically endangered marsupial, Bettongia penicillata Gray, 1837 (woylie, brush-tailed bettong) revealed a new species of Ixodes Latrielle, 1795. Here we provide morphological data for the female and nymphal life stages of this novel species (Ixodes woyliei n. sp.), in combination with molecular characterisation using the mitochondrial cytochrome c oxidase subunit 1 gene (cox1). In addition, molecular characterisation was conducted on several described Ixodes species and used to provide phylogenetic context. RESULTS: Ixodes spp. ticks were collected from the two remaining indigenous B. penicillata populations in south-western Australia. Of 624 individual B. penicillata sampled, 290 (47%) were host to ticks of the genus Ixodes; specifically I. woyliei n. sp., I. australiensis Neumann, 1904, I. myrmecobii Roberts, 1962, I. tasmani Neumann, 1899 and I. fecialis Warburton & Nuttall, 1909. Of these, 123 (42%) were host to the newly described I. woyliei n. sp. In addition, 268 individuals from sympatric marsupial species (166 Trichosurus vulpecula hypoleucus Wagner, 1855 (brushtail possum), 89 Dasyurus geoffroii Gould, 1841 (Western quoll) and 13 Isoodon obesulus fusciventer Gray, 1841 (southern brown bandicoot)) were sampled for ectoparasites and of these, I. woyliei n. sp. was only found on two I. o. fusciventer. CONCLUSIONS: Morphological and molecular data have confirmed the first new Australian Ixodes tick species described in over 50 years, Ixodes woyliei n. sp. Based on the long-term data collected, it appears this tick has a strong predilection for B. penicillata, with 42% of Ixodes infections on this host identified as I. woyliei n. sp. The implications for this host-parasite relationship are unclear but there may be potential for a future co-extinction event. In addition, new molecular data have been generated for collected specimens of I. australiensis, I. tasmani and museum specimens of I. victoriensis Nuttall, 1916, which for the first time provides molecular support for the subgenus Endopalpiger Schulze, 1935 as initially defined. These genetic data provide essential information for future studies relying on genotyping for species identification or for those tackling the phylogenetic relationships of Australian Ixodes species.

Evaluating the Effects of Ivermectin Treatment on Communities of Gastrointestinal Parasites in Translocated Woylies (Bettongia penicillata).

Wildlife species are often treated with anti-parasitic drugs prior to translocation, despite the effects of this treatment being relatively unknown. Disruption of normal host-parasite relationships is inevitable during translocation, and targeted anti-parasitic drug treatment may exacerbate this phenomenon with inadvertent impacts on both target and non-target parasite species. Here, we investigate the effects of ivermectin treatment on communities of gastrointestinal parasites in translocated woylies (Bettongia penicillata). Faecal samples were collected at three time points (at the time of translocation, and 1 and 3 months post-translocation) and examined for nematode eggs and coccidian oocysts. Parasite prevalence and (for nematodes) abundance were estimated in both treated and untreated hosts. In our study, a single subcutaneous injection of ivermectin significantly reduced Strongyloides-like egg counts 1 month post-translocation. Strongyle egg counts and coccidia prevalence were not reduced by ivermectin treatment, but were strongly influenced by site. Likewise, month of sampling rather than ivermectin treatment positively influenced body condition in woylies post-translocation. Our results demonstrate the efficacy of ivermectin in temporarily reducing Strongyloides-like nematode abundance in woylies. We also highlight the possibility that translocation-induced changes to host density may influence coinfecting parasite abundance and host body condition post-translocation.

Temporal Patterns in the Abundance of a Critically Endangered Marsupial Relates to Disturbance by Roads and Agriculture.

The aim of this study was to investigate how landscape disturbance associated with roads, agriculture and forestry influenced temporal patterns in woylie (Bettongia penicillata) abundance before, during and after periods of rapid population change. Data were collected from an area of approximately 140,000 ha of forest within the Upper Warren region in south-western Australia. Woylie abundance was measured using cage trapping at 22 grid and five transect locations with varying degrees of landscape disturbance between 1994 and 2012. We found evidence that the distribution and abundance of woylies over time appears to be related to the degree of fragmentation by roads and proximity to agriculture. Sites furthest from agriculture supported a greater abundance of woylies and had slower rates of population decline. Sites with fewer roads had a greater abundance of woylies generally and a greater rate of increase in abundance after the implementation of invasive predator control. The results of this study suggest that landscape disturbance is less important at peak population densities, but during times of environmental and population change, sites less dissected by roads and agriculture better support woylie populations. This may be due to the role these factors play in increasing the vulnerability of woylies to introduced predators, population fragmentation, weed species invasion, mortality from road collisions or a reduction in available habitat. Strategies that reduce the impact of disturbance on woylie populations could include the rationalisation of forest tracks and consolidation of contiguous habitat through the acquisition of private property. Reducing the impact of disturbance in the Upper Warren region could improve the resilience of this critically important woylie population during future environmental change.

Genetic diversity loss in a biodiversity hotspot: ancient DNA quantifies genetic decline and former connectivity in a critically endangered marsupial.

The extent of genetic diversity loss and former connectivity between fragmented populations are often unknown factors when studying endangered species. While genetic techniques are commonly applied in extant populations to assess temporal and spatial demographic changes, it is no substitute for directly measuring past diversity using ancient DNA (aDNA). We analysed both mitochondrial DNA (mtDNA) and nuclear microsatellite loci from 64 historical fossil and skin samples of the critically endangered Western Australian woylie (Bettongia penicillata ogilbyi), and compared them with 231 (n = 152 for mtDNA) modern samples. In modern woylie populations 15 mitochondrial control region (CR) haplotypes were identified. Interestingly, mtDNA CR data from only 29 historical samples demonstrated 15 previously unknown haplotypes and detected an extinct divergent clade. Through modelling, we estimated the loss of CR mtDNA diversity to be between 46% and 91% and estimated this to have occurred in the past 2000-4000 years in association with a dramatic population decline. In addition, we obtained near-complete 11-loci microsatellite profiles from 21 historical samples. In agreement with the mtDNA data, a number of 'new' microsatellite alleles was only detected in the historical populations despite extensive modern sampling, indicating a nuclear genetic diversity loss >20%. Calculations of genetic diversity (heterozygosity and allelic rarefaction) showed that these were significantly higher in the past and that there was a high degree of gene flow across the woylie's historical range. These findings have an immediate impact on how the extant populations are managed and we recommend the implementation of an assisted migration programme to prevent further loss of genetic diversity. Our study demonstrates the value of integrating aDNA data into current-day conservation strategies.

Temporal and spatial dynamics of trypanosomes infecting the brush-tailed bettong (Bettongia penicillata): a cautionary note of disease-induced population decline.

BACKGROUND: The brush-tailed bettong or woylie (Bettongia penicillata) is on the brink of extinction. Its numbers have declined by 90% since 1999, with their current distribution occupying less than 1% of their former Australian range. Woylies are known to be infected with three different trypanosomes (Trypanosoma vegrandis, Trypanosoma copemani and Trypanosoma sp. H25) and two different strains of T. copemani that vary in virulence. However, the role that these haemoparasites have played during the recent decline of their host is unclear and is part of ongoing investigation. METHODS: Woylies were sampled from five locations in southern Western Australia, including two neighbouring indigenous populations, two enclosed (fenced) populations and a captive colony. PCR was used to individually identify the three different trypanosomes from blood and tissues of the host, and to investigate the temporal and spatial dynamics of trypanosome infections. RESULTS: The spatial pattern of trypanosome infection varied among the five study sites, with a greater proportion of woylies from the Perup indigenous population being infected with T. copemani than from the neighbouring Kingston indigenous population. For an established infection, T. copemani detection was temporally inconsistent. The more virulent strain of T. copemani appeared to regress at a faster rate than the less virulent strain, with the infection possibly transitioning from the acute to chronic phase. Interspecific competition may also exist between T. copemani and T. vegrandis, where an existing T. vegrandis infection may moderate the sequential establishment of the more virulent T. copemani. CONCLUSION: In this study, we provide a possible temporal connection implicating T. copemani as the disease agent linked with the recent decline of the Kingston indigenous woylie population within the Upper Warren region of Western Australia. The chronic association of trypanosomes with the internal organs of its host may be potentially pathogenic and adversely affect their long term fitness and coordination, making the woylie more susceptible to predation.

Morphological polymorphism of Trypanosoma copemani and description of the genetically diverse T. vegrandis sp. nov. from the critically endangered Australian potoroid, the brush-tailed bettong (Bettongia penicillata (Gray, 1837)).

BACKGROUND: The trypanosome diversity of the Brush-tailed Bettong (Bettongia penicillata), known locally as the woylie, has been further investigated. At a species level, woylies are critically endangered and have declined by 90% since 1999. The predation of individuals made more vulnerable by disease is thought to be the primary cause of this decline, but remains to be proven. METHODS: Woylies were sampled from three locations in southern Western Australia. Blood samples were collected and analysed using fluorescence in situ hybridization, conventional staining techniques and microscopy. Molecular techniques were also used to confirm morphological observations. RESULTS: The trypanosomes in the blood of woylies were grouped into three morphologically distinct trypomastigote forms, encompassing two separate species. The larger of the two species, Trypanosoma copemani exhibited polymorphic trypomastigote forms, with morphological phenotypes being distinguishable, primarily by the distance between the kinetoplast and nucleus. The second trypanosome species was only 20% of the length of T. copemani and is believed to be one of the smallest recorded trypanosome species from mammals. No morphological polymorphism was identified for this genetically diverse second species. We described the trypomastigote morphology of this new, smaller species from the peripheral blood of the woylie and proposed the name T. vegrandis sp. nov. Temporal results indicate that during T. copemani Phenotype 1 infections, the blood forms remain numerous and are continuously detectable by molecular methodology. In contrast, the trypomastigote forms of T. copemani Phenotype 2 appear to decrease in prevalence in the blood to below molecular detectable levels. CONCLUSIONS: Here we report for the first time on the morphological diversity of trypanosomes infecting the woylie and provide the first visual evidence of a mixed infection of both T. vegrandis sp. nov and T. copemani. We also provide supporting evidence that over time, the intracellular T. copemani Phenotype 2 may become localised in the tissues of woylies as the infection progresses from the active acute to chronic phase. As evidence grows, further research will be necessary to investigate whether the morphologically diverse trypanosomes of woylies have impacted on the health of their hosts during recent population declines.

Trypanosomes genetic diversity, polyparasitism and the population decline of the critically endangered Australian marsupial, the brush tailed bettong or woylie (Bettongia penicillata).

While much is known of the impact of trypanosomes on human and livestock health, trypanosomes in wildlife, although ubiquitous, have largely been considered to be non-pathogenic. We describe the genetic diversity, tissue tropism and potential pathogenicity of trypanosomes naturally infecting Western Australian marsupials. Blood samples collected from 554 live-animals and 250 tissue samples extracted from 50 carcasses of sick-euthanized or road-killed animals, belonging to 10 species of marsupials, were screened for the presence of trypanosomes using a PCR of the 18S rDNA gene. PCR results revealed a rate of infection of 67% in blood and 60% in tissues. Inferred phylogenetic trees using 18S rDNA and glycosomal glyceraldehyde phosphate dehydrogenase (gGAPDH) sequences showed the presence of eight genotypes that clustered into three clades: a clade including Trypanosoma copemani, a new clade closely related to Trypanosoma gilletti, and a clade including Trypanosoma H25 from an Australian kangaroo. Trypanosome infections were compared in a declining and in a stable population of the endangered Australian marsupial, the brush tailed bettong or woylie (Bettongia penicillata). This marsupial showed high rates of infection with Clade A genotypes (96%) in the declining population, whereas in the stable population, Clade B genotypes were predominant (89%). Mixed infections were common in woylies from the declining but not from the stable population. Histopathological findings associated with either mixed or single infections involving Clade A genotypes, showed a strong inflammatory process and tissue degeneration predominantly in heart, oesophagus and tongue. Trypanosomes were successfully grown in culture and for the first time we demonstrate that a genotype within Clade A has the capacity to not only colonize different tissues in the host but also to invade cells in vitro. These results provide evidence for the potential role of trypanosomes in the decline of a formerly abundant marsupial that is now critically endangered.

Hematologic characteristics of the woylie (Bettongia penicillata ogilbyi).

An accurate assessment of animal health is fundamental to disease investigation in wildlife. Blood samples (n = 609) from several populations of the endangered woylie or brush-tailed bettong (Bettongia penicillata ogilbyi), collected between March 2006 and April 2010 in Western Australia and South Australia, were used to establish hematologic reference ranges. Differences between populations, sexes, and seasons were also investigated. Significant sex differences in hematocrit, red blood cell, total white blood cell, neutrophil, lymphocyte, and eosinophil counts were evident in at least one population. Generally, males had higher hematocrit and blood cell concentrations than did females. A positive association of the erythron parameters with rainfall was also detected. The hematologic characteristics of woylie populations described in this study greatly increase knowledge of the health status in these populations. The data also represent a baseline to enable monitoring and detection of changes in the health status in these populations as well as representing a valid dataset for comparison with hematologic investigations in other macropods and marsupials.

A high prevalence of Theileria penicillata in woylies (Bettongia penicillata).

The woylie or brush-tailed bettong (Bettongia penicillata) is a medium-sized native Australian marsupial that has undergone a dramatic decline in numbers in recent years. Trypanosome parasites have been identified in the woylie but little is known about the prevalence and clinical impact of other haemoprotozoan parasites in these marsupials. In the present study, the occurrence and molecular phylogeny of a piroplasm was studied in woylies from six different sites in Western Australia (WA). Blood samples were screened by PCR at the 18S rRNA locus and 80.4% (123/153) of the blood samples were positive for piroplasm DNA. Sequence and phylogenetic analysis of 12 of these positives identified them as Theileria penicillata, and sequencing of cloned PCR products indicated that no other species of Theileria were present. Infected woylies had a lower body weight but microscopic evaluation of the blood films indicated that T. penicillata did not appear to cause red cell injury or anaemia. Further studies are required to determine the clinical significance of T. penicillata in woylies.