Validation of an Indirect Immunofluorescence Assay and Commercial Q Fever Enzyme-Linked Immunosorbent Assay for Use in Macropods.

Kangaroos are considered to be an important reservoir of Q fever in Australia, although there is limited knowledge on the true prevalence and distribution of coxiellosis in Australian macropod populations. Serological tests serve as useful surveillance tools, but formal test validation is needed to be able to estimate true seroprevalence rates, and few tests have been validated to screen wildlife species for Q fever. In this study, we modified and optimized a phase-specific indirect immunofluorescence assay (IFA) for the detection of IgG antibodies against Coxiella burnetii in macropod sera. The assay was validated against the commercially available ID Screen Q fever indirect multispecies enzyme-linked immunosorbent assay (ELISA) kit (IDVet, Grabels, France) to estimate the diagnostic sensitivity and specificity of each assay, using Bayesian latent class analysis. A direct comparison of the two tests was performed by testing 303 serum samples from 10 macropod populations from the east coast of Australia and New Zealand. The analysis indicated that the IFA had relatively high diagnostic sensitivity (97.6% [95% credible interval [CrI], 88.0 to 99.9]) and diagnostic specificity (98.5% [95% CrI, 94.4 to 99.9]). In comparison, the ELISA had relatively poor diagnostic sensitivity (42.1% [95% CrI, 33.7 to 50.8]) and similar diagnostic specificity (99.2% [95% CrI, 96.4 to 100]) using the cutoff values recommended by the manufacturer. The estimated true seroprevalence of C. burnetii exposure in the macropod populations included in this study ranged from 0% in New Zealand and Victoria, Australia, up to 94.2% in one population from New South Wales, Australia.

Contraceptive efficacy and dose-response effects of the gonadotrophin-releasing hormone (GnRH) agonist deslorelin in Tasmanian devils (Sarcophilus harrisii).

Contraception is increasingly used to manage breeding opportunities in conservation-dependent species. This study aimed to determine the efficacy, duration of effect, optimal dose and potential side effects of Suprelorin contraceptive implants in Tasmanian devils, for use in the conservation breeding program. In our pilot study, Suprelorin was found to effectively suppress oestrous cycles in female devils, yet caused a paradoxical increase in testosterone in males. Therefore, we focussed on females in further trials. Females received one (n=5), two (n=5) or no (n=5) Suprelorin implants, with quarterly gonadotrophin-releasing hormone (GnRH) challenges used to test pituitary responsiveness over two breeding seasons. Both Suprelorin doses suppressed pituitary responsiveness for at least one breeding season, with a reduced effect in the second. There was a dose-response effect on duration rather than magnitude of effect, with high-dose devils remaining suppressed for longer than low-dose animals. There were no apparent negative effects on general health, yet captivity and contraception together may cause weight gain. Suprelorin contraceptive implants are now routinely used in the Save the Tasmanian Devil Program insurance metapopulation to meet the aims of maintaining genetic and behavioural integrity by controlling individual reproductive contributions in group housing situations.

A role for selective contraception of individuals in conservation.

Contraception has an established role in managing overabundant populations and preventing undesirable breeding in zoos. We propose that it can also be used strategically and selectively in conservation to increase the genetic and behavioral quality of the animals. In captive breeding programs, it is becoming increasingly important to maximize the retention of genetic diversity by managing the reproductive contribution of each individual and preventing genetically suboptimal breeding through the use of selective contraception. Reproductive suppression of selected individuals in conservation programs has further benefits of allowing animals to be housed as a group in extensive enclosures without interfering with breeding recommendations, which reduces adaptation to captivity and facilitates the expression of wild behaviors and social structures. Before selective contraception can be incorporated into a breeding program, the most suitable method of fertility control must be selected, and this can be influenced by factors such as species life history, age, ease of treatment, potential for reversibility, and desired management outcome for the individual or population. Contraception should then be implemented in the population following a step-by-step process. In this way, it can provide crucial, flexible control over breeding to promote the physical and genetic health and sustainability of a conservation dependent species held in captivity. For Tasmanian devils (Sarcophilus harrisii), black-flanked rock wallabies (Petrogale lateralis), and burrowing bettongs (Bettongia lesueur), contraception can benefit their conservation by maximizing genetic diversity and behavioral integrity in the captive breeding program, or, in the case of the wallabies and bettongs, by reducing populations to a sustainable size when they become locally overabundant. In these examples, contraceptive duration relative to reproductive life, reversibility, and predictability of the contraceptive agent being used are important to ensure the potential for individuals to reproduce following cessation of contraception, as exemplified by the wallabies when their population crashed and needed females to resume breeding.

Trends in Rescue and Rehabilitation of Marsupials Surviving the Australian 2019-2020 Bushfires.

The 2019-2020 Australian bushfire season had a devastating impact on native wildlife. It was estimated that 3 billion native animals were impacted by the fires, yet there are few estimates of the number of animals that were rescued and rehabilitated post-fire. Focusing on the state of New South Wales (NSW) and Kangaroo Island, South Australia, we used a case study approach to determine the number of marsupials that were reported rescued due to the 2019-2020 bushfires in these areas and analysed species-specific trends in rescue and release success. In NSW, we found 889 reports of fire-affected marsupials in 2019-2020, mostly comprising kangaroos and wallabies (macropods; n = 458), koalas (n = 204), and possums (n = 162), with a smaller number of wombats (n = 43) and other marsupial species. Most reports of fire-affected marsupials occurred 6-8 weeks after fire ignition, and there was no difference in temporal frequency of rescues between marsupial groups. For the three main groups, the probability of survival and subsequent release differed, with macropods having the lowest probability of release after rescue (0.15 ± 0.04) compared to koalas (0.47 ± 0.04) and possums (0.55 ± 0.10). The type of injury was the main predictor of survival during rehabilitation for all three marsupial groups, with those malnourished/moribund or with traumatic injuries less likely to survive rehabilitation. Death or euthanasia occurred on the day of rescue for 77% of macropods, 48% of possums and 15% of koalas. Koalas most often died during rehabilitation rather than on the day of rescue, with 73% either dying or being euthanised between day 1 and 30 post-rescue, representing a potential welfare concern. On Kangaroo Island, koalas were the most frequently rescued marsupial species; most euthanasia cases and deaths occurred in a hospital, whereas other marsupials were mostly euthanised at triage. In both jurisdictions, koalas were over-represented while possums were under-represented relative to baseline population densities and wildlife rescue trends in the years before the 2019-2020 bushfires. These species differences in presentation post-fire warrant further investigation, as do the differences in triage, survival and release outcomes. It is hypothesised that the high intensity and large scale of the 2019-2020 fires impeded marsupial fire evasion tactics, as evidenced by the small number of animals found for rescue, and the differing rates of presentation relative to underlying population densities for the main marsupial groups. Based on our findings, there is a need for detailed record keeping and data sharing, development of consistent and evidence-based triage, treatment and euthanasia guidelines and deployment of trained wildlife emergency rescue teams with advanced search techniques to minimise animal suffering where safe to do so.

Development of the ethmoid in a wallaby and implications for the homology of turbinal elements in marsupials.

The homologies of the turbinals (scroll bones) of the ethmoid are not well understood, including the potential implication for understanding mammalian phylogeny. Here we examine the postnatal development of this anatomical system in a marsupial mammal because previous work has shown that the adult pattern of five endoturbinals (ethmoturbinals) and two ectoturbinals (frontoturbinals) is conserved. Furthermore, marsupial phylogeny is fairly well resolved and provides a solid evolutionary framework for examining turbinal homologies. In this study, we documented the development of the ethmoid of the tammar wallaby, Notamacropus eugenii, using histology and computed tomography imagery of a growth series of pouch young. The pattern of development of the turbinal elements in the wallaby was compared to that in didelphids, as described in previous work. We found that four ethmoturbinals initially develop, followed later in development by an interturbinal; these five elements then develop into the bony endoturbinals found in adults. These data support the idea that endoturbinal III, derived from an interturbinal, has a distinctive development pattern from the other endoturbinals. This is consistent with what is seen in the didelphid marsupials, Caluromys philander and Monodelphis domestica, suggesting this is a common developmental pattern for marsupials. This article is part of the theme issue 'The mammalian skull: development, structure and function'.

In Situ Provisioning Wildlife with Food, Water, or Shelter after Bushfires: Using a One Welfare Framework to Guide Responses.

Australia's 2019-2020 bushfires had a devastating impact on animals, humans, and ecosystems. They also demonstrated the lack of evidence or guidance for wildlife provisioning in response to severe fire events when volunteers and wildlife organisations rose to respond. In addition, the unprecedented scale and intensity of the fires and an absence of institutional support for wildlife provisioning meant that well-intentioned interventions were largely uncoordinated and lacked clear short-term, mid-term, and long-term objectives. Fundamentally, a lack of consensus was revealed on whether any such interventions are advisable. Given the strong evidence indicating that future bushfire seasons will become longer and more intense in Australia and elsewhere, the welfare and survival of millions of wild animals are at risk every year. Understanding the impacts of supplementary resource interventions and contributing to the development of best practice information is crucial to inform the response to the next major fire event. Here, we contextualize the arguments for and against provisioning within a 'One Welfare' framework that recognizes that animal welfare, biodiversity, and the environment are intertwined with human welfare and community resilience. We propose that the One Welfare approach can facilitate appropriate consideration of the extant scientific and lay literature; local legislation; views of stakeholders; emerging data; and modelling from historic fire events. As a further step, we see merit in engaging with wildlife provisioners and the broader conservation community to build an evidence base for future wildlife provisioning activities. From an informed position, we can encourage beneficial interventions and reduce the risk of negative outcomes. Finally, we propose controlled experiments (e.g., using hazard reduction burns), ongoing data collection using emergent technology, and longitudinal analysis to address shifting research priorities as the climate changes. We conclude that the ordered collection of the necessary evidence relevant to each of the three stakeholder groups in the One Welfare framework has the greatest potential to support an informed policy platform on wildlife provisioning across Australia that is feasible, legal, and sustainable.

A systematic review of factors affecting wildlife survival during rehabilitation and release.

Millions of native animals around the world are rescued and rehabilitated each year by wildlife rehabilitators. Triage and rehabilitation protocols need to be robust and evidence-based, with outcomes consistently recorded, to promote animal welfare and better understand predictors of wildlife survival. We conducted a global systematic review and meta-analysis of 112 articles that reported survival rates of native mammals and birds during rehabilitation and after release to determine intrinsic and extrinsic factors associated with their survival. We assessed survival during rehabilitation and in the short- and long-term post-release, with the hypothesis that survival will vary as a function of species body size, diel activity pattern, trophic level and study location (region of the world). We aimed to determine the direction of effect of these factors on survival to assist in decision-making during triage and rehabilitation. Results showed that mammals and birds were equally likely to survive all stages of rehabilitation, and survival rates varied between locations. Birds in North America had the poorest survival rates post-release, particularly long-term, as did diurnal and carnivorous birds in the short-term post-release. Anthropogenic factors such as motor vehicle collisions and domestic or feral animal attack contributed to morbidity and post-release mortality in 45% (168 of 369) of instances. The reasons for rescue and associated severity of diagnosis were commonly reported to affect the likelihood of survival to release, but factors affecting survival were often species-specific, including bodyweight, age, and characteristics of the release location. Therefore, evidence-based, species-specific, and context-specific protocols need to be developed to ensure wildlife survival is maximised during rehabilitation and post-release. Such protocols are critical for enabling rapid, efficient rescue programs for wildlife following natural disasters and extreme weather events which are escalating globally, in part due to climate change.

Validation of an Enzyme Immunoassay to Measure Faecal Glucocorticoid Metabolites in Common Brushtail Possums (Trichosurus vulpecula) to Evaluate Responses to Rehabilitation.

Volunteer wildlife rehabilitators rescue and rehabilitate thousands of native animals every year in Australia. However, there is little known about how exposure to novel stimuli during rehabilitation could affect the physiology of wildlife. We investigated this question in a species that commonly enters rehabilitation, the common brushtail possum (Trichosurus vulpecula). We evaluated five enzyme immunoassays (EIA) to determine the most suitable for measuring faecal glucocorticoid metabolites (FGM) as a proxy for evaluating the response of brushtail possums to potential stressors during rehabilitation. An adrenocorticotrophin hormone (ACTH) challenge was conducted on wild-caught possums to determine the best-performing EIA based on the successful detection of FGM peaks in at least two of three possums. While a number of assays met these criteria, the 11-oxoaetiocholanolone (abbreviation: 72a) EIA was selected as it had the largest amplitude of change in response to the ACTH challenge. This assay was then used to measure FGM concentrations in 20 possums during rehabilitation. There was high variation in baseline FGM concentrations and response to captivity between possums. Significant changes in FGM levels were detected in most possums during captivity, but were not reliably associated with potentially stressful events that were identified by rehabilitators. The probability of an FGM peak occurring within five days of a potentially stressful event was about 50%, regardless of the type of event. Our study has demonstrated that injured and orphaned possums show changes in FGMs during captivity and rehabilitation and has identified events that can induce a physiological response in some individuals. We recommend that research now focus on the relationship between these responses during rehabilitation and pre- and post-release survival.

Kangaroos at maximum capacity: health assessment of free-ranging eastern grey kangaroos on a coastal headland.

Sprawling urban development is fragmenting the landscape and native wildlife habitats on the Australian east coast. The impact of this rapid urbanization on wildlife health is largely unknown. This study surveyed the health of a high-density (5.4 individuals per ha) population of eastern grey kangaroos (Macropus giganteus) affected by urban encroachment and prolonged drought. Blood parameters (hematological and serum protein), trace element and heavy metal concentrations, and parasite counts (fecal worm egg counts, ticks, and mites) are reported for a sample of ≤ 54 kangaroos at Look at Me Now Headland, New South Wales, Australia. These parameters were compared to lower density kangaroo populations from other sites in New South Wales. We found the health and welfare of this population to be severely compromised, with nonregenerative anemia and nutritional deficiencies evident. Our results indicate that high-density kangaroo populations isolated by urban encroachment are at significant health risk. To prevent further decline in this population's health, we discuss management strategies that could be employed, concurrent with ongoing health and disease monitoring, to mitigate the poor health outcomes in this population. We conclude that it is essential to retain habitat connectivity when altering land use in areas with resident kangaroo populations if managers are to maintain healthy populations.

Dominance, body size and internal relatedness influence male reproductive success in eastern grey kangaroos (Macropus giganteus).

Knowledge of the determinants of reproductive success is essential for understanding the adaptive significance of particular traits. The present study examined whether particular behavioural, morphological, physiological or genetic traits were correlated with male dominance and reproductive success using three semi-free-ranging captive populations (n = 98) of the eastern grey kangaroo (Macropus giganteus). The morphological traits measured included bodyweight, head, forearm, tail, pes and leg length, forearm and bicep circumference, and testis size. Blood samples were collected to determine serum testosterone concentrations. All individuals were typed for 10 microsatellite loci and paternity determined for each pouch young. To determine the influence of relatedness and genetic diversity on male reproductive success, internal relatedness, standardised heterozygosity and mean d(2) were calculated. Dominant males sired a significantly higher proportion of offspring than smaller, lower-ranked males and had higher testosterone concentrations. Males that sired offspring were significantly heavier and had larger body size. Sires were significantly more heterozygous and genetically dissimilar to breeding females than non-sires. Despite the wealth of knowledge on the social organisation of kangaroos, this is the first study to assign parentage and male reproductive success using molecular evidence.

Effects of deslorelin implants on reproduction and feeding behavior in Tasmanian devils (Sarcophilus harrisii) housed in free-range enclosures.

In captive breeding programs, it is becoming increasingly important to maximize the retention of genetic diversity by managing the reproductive contribution of each individual, which can be facilitated through the use of selective contraception. This becomes critical when captive populations are held for several generations, and managers must prevent the confines of housing space and financial support from compromising genetic integrity. For example, the Tasmanian devil insurance population, established in 2006, is strategically managed to equalize founder representation. This becomes difficult when devils are housed in large groups in free-range enclosures (FREs). This study examined the efficacy, duration and potential side effects of Suprelorin® contraceptive implants (containing 4.7 mg of deslorelin) on Tasmanian devils housed in FREs. Females were monitored to assess post-treatment reproductive rates, feeding behavior and weight changes. Suprelorin® successfully prevented reproduction in all treated females (P < 0.001) for at least one breeding season. For one year after contraception, there was no difference in proportion of time spent feeding between contraception and control groups (P > 0.05) and there was no effect of contraception on order of arrival at food (P = 0.632), suggesting no alterations to social structure. Devils with pouch young spent more time feeding than those without (P < 0.001). Treatment and month had an interactive effect on weight (P < 0.001), yet contracepted females were only heavier than controls in one season, indicating no overall excessive weight gain. Suprelorin® implants inhibit reproduction for at least one breeding season, with no apparent negative effects on feeding behavior or social dynamic. Selective contraception has the potential to become an important tool for conservation managers, to meet multiple reproductive, genetic and behavioral goals for this species.

Effects of deslorelin implants on reproduction in the common brushtail possum (Trichosurus vulpecula).

The present study investigated the effects of slow-release implants containing the gonadotrophin-releasing hormone (GnRH) agonist deslorelin on reproduction in the common brushtail possum (Trichosurus vulpecula). Captive female brushtail possums were assigned to control (placebo implant), low dose (4.7 mg deslorelin) or high dose (9.4 mg deslorelin) groups; males were assigned to control or high dose (9.4 mg deslorelin) groups. The acute effects of deslorelin treatment at the level of the pituitary gland were similar between the two sexes, where a transient rise in luteinising hormone concentration was induced over the first 24 h. In females, this was associated with the disruption of the normal oestrous cycle and mating within 2-10 days in some treated individuals, but no young were subsequently detected. By 3 weeks after treatment, treated females became anoestrus and remained infertile for at least one breeding season. The effects of treatment were reversible in a subset of females that had their implants removed, although the time taken to produce offspring was variable. Paradoxically, male brushtail possums remained fertile during chronic deslorelin exposure. Despite significant declines in basal follicle-stimulating hormone and testosterone concentrations, as well as an inability to respond to a GnRH challenge, treated males sired as many offspring as control males and there was no evidence of testicular regression. In conclusion, there is potential to control reproduction in female brushtail possums by using chronic GnRH agonist treatment.

Leptospirosis serology in the common brushtail possum (Trichosurus vulpecula) from urban Sydney, Australia.

The common brushtail possum (Trichosurus vulpecula) is indeed a common marsupial in major cities of Australia. This species is known to be susceptible to leptospirosis and often lives in close contact with humans, raising concerns about the potential for transmission of this disease in urban areas. A total of 192 brushtail possum blood samples were collected from 136 individuals in suburban areas of metropolitan Sydney from November 2002 to November 2004. Sera were screened against a reference panel of 21 Leptospira spp. using the microscopic agglutination test. Leptospiral antibodies were detected in 9.6% (13/136) of tested brushtail possums and represented two serovars; antibodies to Leptospira interrogans serovar Hardjo were most frequently identified (11/136). A representative of the exotic sero-group Ballum, most likely serovar Arborea, was found in two of 136 brushtail possums. Exposure to leptospirosis seemed to be associated with age, as older animals had a higher incidence, but there was no distinction in relation to gender. Antibody prevalence varied between the different sampling sites and seropositive animals were clustered and restricted to a few sites. These data support the possible role of brushtail possums as a maintenance host for Leptospira spp. in urban environments and also identified them as a previously unknown and potential source of serovar Arborea.

Extensive genetic differentiation detected within a model marsupial, the tammar wallaby (Notamacropus eugenii).

The tammar wallaby (Notamacropus eugenii) is one of the most intensively studied of all macropodids and was the first Australasian marsupial to have its genome sequenced. However, comparatively little is known about genetic diversity and differentiation amongst the morphologically distinct allopatric populations of tammar wallabies found in Western (WA) and South Australia (SA). Here we compare autosomal and Y-linked microsatellite genotypes, as well as sequence data (~600 bp) from the mitochondrial DNA (mtDNA) control region (CR) in tammar wallabies from across its distribution. Levels of diversity at autosomal microsatellite loci were typically high in the WA mainland and Kangaroo Island (SA) populations (A = 8.9-10.6; He = 0.77-0.78) but significantly reduced in other endemic island populations (A = 3.8-4.1; He = 0.41-0.48). Autosomal and Y-linked microsatellite loci revealed a pattern of significant differentiation amongst populations, especially between SA and WA. The Kangaroo Island and introduced New Zealand population showed limited differentiation. Multiple divergent mtDNA CR haplotypes were identified within both SA and WA populations. The CR haplotypes of tammar wallabies from SA and WA show reciprocal monophyly and are highly divergent (14.5%), with levels of sequence divergence more typical of different species. Within WA tammar wallabies, island populations each have unique clusters of highly related CR haplotypes and each is most closely related to different WA mainland haplotypes. Y-linked microsatellite haplotypes show a similar pattern of divergence although levels of diversity are lower. In light of these differences, we suggest that two subspecies of tammar wallaby be recognized; Notamacropus eugenii eugenii in SA and N. eugenii derbianus in WA. The extensive neutral genetic diversity and inter-population differentiation identified within tammar wallabies should further increase the species value and usefulness as a model organism.

Serologic survey for Toxoplasma gondii and Neospora caninum in the common brushtail possum (Trichosurus vulpecula) from urban Sydney, Australia.

The common brushtail possum (Trichosurus vulpecula) has well adapted to increasing urbanization, resulting in greater interaction with humans and their domestic pets. Wildlife species in urban areas face a higher risk of exposure to zoonotic pathogens and may be affected by parasites hosted by cats (Toxoplasma gondii) or dogs (Neospora caninum), yet it is unknown to what extent urban T. vulpecula are exposed to these parasites. Antibodies to T. gondii and N. caninum were assayed in sera of 142 adult possums from the city of Sydney, Australia. Using the modified agglutination test, antibodies to T. gondii were found in 9 (6.3%) of the 142 animals in titers of 1:25 (4), 1:50 (1), 1:100 (1), 1:800 (1), 1:3,200 (1), 1:6,400 (1), and 1:12,800 (1). Of some T. vulpecula multiple sera samples within a 2-yr frame could be collected, but seropositive animals in general were not recaptured after initial seroconversion. One possum had a high T. gondii titer on 2 consecutive bleedings, 14 mo apart, and seropositive possums appeared normal when captured. Sex seemed not to have an affect on antibody prevalence, but age and location may play a role. Antibodies to N. caninum were not detected in 1:25 dilution of sera in the N. caninum agglutination test, indicating that T. vulpecula may not have been exposed to this parasite. This is the first serological survey for T. gondii and N. caninum infections in urban T. vulpecula.

Evaluation of the management of rectal cancers before and after the consensus conference in France.

OBJECTIVES: Population-based registries are the best way to draw a picture of the management of a disease. The purpose of this study was to analyse therapeutic modalities for rectal cancers in seven French areas in 1990 and 1995, before and just after a consensus conference devoted to this topic. METHODS: A community-based series of 945 patients (402 in 1990, 543 in 1995) with rectal cancer was used to assess therapeutic modalities and stage at diagnosis. RESULTS: Colonoscopy was performed in most of the cases (90% in 1990 and 1995). There was significant change between 1990 and 1995 in stage at diagnosis and cancer resection. The rate of continence-preserving operations was similar in 1990 and in 1995, as was the rate of adjuvant radiotherapy. There was a shift between 1990 and 1995 from postoperative radiotherapy to preoperative radiotherapy. There was an increase in the use of adjuvant chemotherapy. CONCLUSION: Changes in the management of rectal cancer in France over the past few years have concerned mainly resection rate, stage at diagnosis and adjuvant therapy. The recommendations of the consensus conference were followed only partly, in particular for adjuvant preoperative radiotherapy, which has not reached its full development, and adjuvant chemotherapy, which tends to be overprescribed, considering how little is known about its effectiveness.

Ultrasonography of wallaby prenatal development shows that the climb to the pouch begins in utero.

Marsupials have a functional placenta for a shorter period of time compared to that of eutherian species, and their altricial young reach the teats without any help from the mother. We have monitored the short intrauterine development of one marsupial, the tammar wallaby, with high-resolution ultrasound from reactivation of the 100-cell diapausing blastocyst to birth. The expanding blastocyst could be visualized when it had reached a diameter of 1.5 mm. From at least halfway through pregnancy, there are strong undulating movements of the endometrium that massage the expanding vesicle against the highly secretory endometrial surface. These unique movements possibly enhance exchange of uterine secretions and gases between the mother and embryo. There was a constant rate of development measured ultrasonographically from mid-gestation, regardless of when the blastocyst reactivated. Interestingly climbing movements by the fetus began in utero about 3 days before birth, mimicking those required to climb to the pouch.