Statistical modeling based on structured surveys of Australian native possum excreta harboring Mycobacterium ulcerans predicts Buruli ulcer occurrence in humans.

Background: Buruli ulcer (BU) is a neglected tropical disease caused by infection of subcutaneous tissue with Mycobacterium ulcerans. BU is commonly reported across rural regions of Central and West Africa but has been increasing dramatically in temperate southeast Australia around the major metropolitan city of Melbourne, with most disease transmission occurring in the summer months. Previous research has shown that Australian native possums are reservoirs of M. ulcerans and that they shed the bacteria in their fecal material (excreta). Field surveys show that locales where possums harbor M. ulcerans overlap with human cases of BU, raising the possibility of using possum excreta surveys to predict the risk of disease occurrence in humans. Methods: We thus established a highly structured 12 month possum excreta surveillance program across an area of 350 km2 in the Mornington Peninsula area 70 km south of Melbourne, Australia. The primary objective of our study was to assess using statistical modeling if M. ulcerans surveillance of possum excreta provided useful information for predicting future human BU case locations. Results: Over two sampling campaigns in summer and winter, we collected 2,282 possum excreta specimens of which 11% were PCR positive for M. ulcerans-specific DNA. Using the spatial scanning statistical tool SaTScan, we observed non-random, co-correlated clustering of both M. ulcerans positive possum excreta and human BU cases. We next trained a statistical model with the Mornington Peninsula excreta survey data to predict the future likelihood of human BU cases occurring in the region. By observing where human BU cases subsequently occurred, we show that the excreta model performance was superior to a null model trained using the previous year's human BU case incidence data (AUC 0.66 vs 0.55). We then used data unseen by the excreta-informed model from a new survey of 661 possum excreta specimens in Geelong, a geographically separate BU endemic area to the southwest of Melbourne, to prospectively predict the location of human BU cases in that region. As for the Mornington Peninsula, the excreta-based BU prediction model outperformed the null model (AUC 0.75 vs 0.50) and pinpointed specific locations in Geelong where interventions could be deployed to interrupt disease spread. Conclusions: This study highlights the One Health nature of BU by confirming a quantitative relationship between possum excreta shedding of M. ulcerans and humans developing BU. The excreta survey-informed modeling we have described will be a powerful tool for the efficient targeting of public health responses to stop BU. Funding: This research was supported by the National Health and Medical Research Council of Australia and the Victorian Government Department of Health (GNT1152807 and GNT1196396).

The African ape-like foot of Ardipithecus ramidus and its implications for the origin of bipedalism.

The ancestral condition from which humans evolved is critical for understanding the adaptive origin of bipedal locomotion. The 4.4 million-year-old hominin partial skeleton attributed to Ardipithecus ramidus preserves a foot that purportedly shares morphometric affinities with monkeys, but this interpretation remains controversial. Here I show that the foot of Ar. ramidus is most similar to living chimpanzee and gorilla species among a large sample of anthropoid primates. The foot morphology of Ar. ramidus suggests that the evolutionary precursor of hominin bipedalism was African ape-like terrestrial quadrupedalism and climbing. The elongation of the midfoot and phalangeal reduction in Ar. ramidus relative to the African apes is consistent with hypotheses of increased propulsive capabilities associated with an early form of bipedalism. This study provides evidence that the modern human foot was derived from an ancestral form adapted to terrestrial plantigrade quadrupedalism.

The Effects of Indian Hedgehog Deletion on Mesenchyme Cells: Inducing Intermediate Cartilage Scaffold Ossification to Cause Growth Plate and Phalange Joint Absence, Short Limb, and Dwarfish Phenotypes.

The endochondral ossification plays a critical role in vertebrate limb development and skeletal homeostasis, where limb mesenchyme cells form an intermediate cartilage scaffold that develops into growth plates and then replaced by bone. Although Indian hedgehog (Ihh) is known to control the hypertrophic differentiation process of chondrocytes, its role from the mesenchyme cells to the early stages of chondrogenesis is unclear. To define the function of Ihh in the mesenchymal cell's early stages of chondrogenesis, we specifically delete Ihh in Prx1-expressed mesenchyme cells at E9.5 using Prx1-Cre;Ihhfl/fl;Rosa26-ZsGreen1 mice. We found that deleting Ihh in the mesenchyme cells results in an early and quick ossification of the intermediate cartilage scaffold, causing the growth plate and phalange joint absence, short limbs, and dwarfishness. The green fluorescent protein (GFP)-positive cells derived from deleted Ihh mesenchyme cells overlap with von Kossa- and osteocalcin-positive staining area. These deleted Ihh/GFP-positive cells isolated from Prx1-Cre;Ihhfl/fl;Rosa26-ZsGreen1 newborn mice had osteogenic differentiation by showing a positive Alizarin red and von Kossa staining, as well as an enhanced Col1a1, osteocalcin, and Runx2 expression. Our findings demonstrate that deleting Ihh in mesenchyme cells during early limb development promotes intermediate cartilage scaffold ossification, which prevents growth plate formation that causes phalange joint absence, short limb, and dwarfish phenotype.

Uterine molecular changes for non-invasive embryonic attachment in the marsupials Macropus eugenii (Macropodidae) and Trichosurus vulpecula (Phalangeridae).

Pregnancy in mammals requires remodeling of the uterus to become receptive to the implanting embryo. Remarkably similar morphological changes to the uterine epithelium occur in both eutherian and marsupial mammals, irrespective of placental type. Nevertheless, molecular differences in uterine remodeling indicate that the marsupial uterus employs maternal defences, including molecular reinforcement of the uterine epithelium, to regulate embryonic invasion. Non-invasive (epitheliochorial) embryonic attachment in marsupials likely evolved secondarily from invasive attachment, so uterine defences in these species may prevent embryonic invasion. We tested this hypothesis by identifying localization patterns of Talin, a key basal anchoring molecule, in the uterine epithelium during pregnancy in the tammar wallaby (Macropus eugenii; Macropodidae) and the brush tail possum (Trichosurus vulpecula; Phalangeridae). Embryonic attachment is non-invasive in both species, yet Talin undergoes a clear distributional change during pregnancy in M. eugenii, including recruitment to the base of the uterine epithelium just before attachment, that closely resembles that of invasive implantation in the marsupial species Sminthopsis crassicaudata. Basal localization occurs throughout pregnancy in T. vulpecula, although, as for M. eugenii, this pattern is most specific prior to attachment. Such molecular reinforcement of the uterine epithelium for non-invasive embryonic attachment in marsupials supports the hypothesis that less-invasive and non-invasive embryonic attachment in marsupials may have evolved via accrual of maternal defences. Recruitment of basal molecules, including Talin, to the uterine epithelium may have played a key role in this transition.

Plasmodium infection in a Leadbeater's possum (Gymnobelideus leadbeateri).

CASE REPORT: A wild-caught, adult female Leadbeater's possum (Gymnobelideus leadbeateri) died while in captivity after suffering from chronic ill-thrift that progressed to acute respiratory distress. On histopathological examination of tissues, the cause of death was determined to be severe acute pneumonia with pulmonary oedema associated with an intracellular protozoan parasite present within erythrocytes. Transmission electron microscopy was performed on lung tissues and organisms consistent for Plasmodium sp. were identified within numerous erythrocytes. Molecular characterisation of the parasite from DNA extracted from tissue blocks of fixed lung determined the organism to belong to the genus Plasmodium (100% similarity to Plasmodium species when a BLAST analysis was performed); however, speciation of the organism was not possible. CONCLUSION: This is the first report of Plasmodium sp. infection and subsequent disease in a native Australian mammal. The lifecycle of this parasite remains unknown. It is also unknown what effects haemoparasitism may have on the population dynamics of this endangered possum species.

Concurrent progressive-ratio and fixed-ratio schedule performance under geometric and arithmetic progressions by brushtail possums.

The aim of this study was to compare the demand for food under concurrent progressive- and fixed-ratio schedules. Twelve brushtail possums participated in 16 conditions where schedule, progression and food type were varied. An incrementing schedule increased the fixed-ratio requirement within and across sessions and was arranged as either a geometric sequence (base 2), or an arithmetic sequence (step 5). Two foods were tested: a flaked barley and coco-pop(®) mix versus rolled oats. Overall, performance was similar for most possums in the within- and across-session incrementing schedules. An analysis of the estimates of essential value and break point produced the same account of demand for foods under the geometric or arithmetic progressions and within- and across-session procedures for 8 of 12 possums. Six possums showed higher demand for rolled oats compared to flaked barley, and two possums showed higher demand for flaked barley compared to rolled oats. Incrementing ratios within, rather than between sessions using an arithmetic progression was demonstrated to be a time efficient procedure for investigating demand for different food types without affecting conclusions about the relative demand for those foods.

An Endangered Arboreal Specialist, the Western Ringtail Possum (Pseudocheirus occidentalis), Shows a Greater Genetic Divergence across a Narrow Artificial Waterway than a Major Road.

The fragmentation of habitats by roads and other artificial linear structures can have a profound effect on the movement of arboreal species due to their strong fidelity to canopies. Here, we used 12 microsatellite DNA loci to investigate the fine-scale spatial genetic structure and the effects of a major road and a narrow artificial waterway on a population of the endangered western ringtail possum (Pseudocheirus occidentalis) in Busselton, Western Australia. Using spatial autocorrelation analysis, we found positive genetic structure in continuous habitat over distances up to 600 m. These patterns are consistent with the sedentary nature of P. occidentalis and highlight their vulnerability to the effects of habitat fragmentation. Pairwise relatedness values and Bayesian cluster analysis also revealed significant genetic divergences across an artificial waterway, suggesting that it was a barrier to gene flow. By contrast, no genetic divergences were detected across the major road. While studies often focus on roads when assessing the effects of artificial linear structures on wildlife, this study provides an example of an often overlooked artificial linear structure other than a road that has a significant impact on wildlife dispersal leading to genetic subdivision.

Retrospective analysis of causes of death in mountain pygmy-possums (Burramys parvus) at Healesville Sanctuary, Victoria, Australia.

BACKGROUND: Identification and characterisation of deaths is important for the veterinary management of both wild and captive animals. It is especially important as a tool for monitoring health and disease within populations of endangered species for which little information on morbidity and mortality is known. Investigations into the causes of death and other important necropsy findings were made in a captive population of the critically endangered mountain pygmy-possum (Burramys parvus). METHODS: Necropsy records from January 2000-December 2013 were reviewed for all possums that had lived and died at Healesville Sanctuary (n = 48). RESULTS: The average age of death of possums in this population was 4.7 years. The most common histological change in mountain pygmy-possums was varying degrees of chronic progressive kidney disease (n = 17). Of these cases, eight animals (47%) had histological changes suggesting the kidney disease was the likely cause of death. Other causes of death included neoplasia (n = 5), necrotising pancreatitis (n = 4), pneumonia (n = 2), reproductive disease (n = 2) and trauma (n = 2). No cause of death was able to be identified in 33.3% (n = 16) of cases. Hepatic lipidosis (n = 5), pneumonia (n = 2) and degenerative joint disease (n = 2) were the most common comorbidities found. CONCLUSION: Progressive renal disease, often with secondary metastatic mineralisation, appears to be a significant cause of mortality in captive mountain pygmy-possums and further investigation into its pathophysiology, antemortem diagnosis and treatment is warranted.

Pharmacokinetics of enrofloxacin following oral and subcutaneous administration in the common ringtail possum (Pseudocheirus peregrinus).

[Correction added on 23 March 2015, after first online publication: Terminal half-life values of enrofloxacin is corrected in the fourth sentence of the abstract] Clinically healthy common ringtail possums (n = 5) received single doses of 10 mg/kg enrofloxacin orally and then 2 weeks later subcutaneously. Serial plasma samples were collected over 24 h for each treatment phase, and enrofloxacin concentrations were determined using a validated HPLC assay. Pharmacokinetic parameters were determined by noncompartmental analysis. Following oral administration, plasma concentrations were of therapeutic relevance (Cmax median 5.45 µg/mL, range 2.98-6.9 µg/mL), with terminal-phase half-life (t½ ) shorter than in other species (median 3.09 h, range 1.79-5.30 h). In contrast, subcutaneous administration of enrofloxacin did not achieve effective plasma concentrations, with plasma concentrations too erratic to fit the noncompartmental model except in one animal. On the basis of the AUC:MIC, enrofloxacin administered at 10 mg/kg orally, but not subcutaneously, is likely to be effective against a range of bacterial species that have been reported in common ringtail possums.

Stress triangle: do introduced predators exert indirect costs on native predators and prey?

Non-consumptive effects of predators on each other and on prey populations often exceed the effects of direct predation. These effects can arise from fear responses elevating glucocorticoid (GC) hormone levels (predator stress hypothesis) or from increased vigilance that reduces foraging efficiency and body condition (predator sensitive foraging hypothesis); both responses can lead to immunosuppression and increased parasite loads. Non-consumptive effects of invasive predators have been little studied, even though their direct impacts on local species are usually greater than those of their native counterparts. To address this issue, we explored the non-consumptive effects of the invasive red fox Vulpes vulpes on two native species in eastern Australia: a reptilian predator, the lace monitor Varanus varius and a marsupial, the ringtail possum Pseudocheirus peregrinus. In particular, we tested predictions derived from the above two hypotheses by comparing the basal glucocorticoid levels, foraging behaviour, body condition and haemoparasite loads of both native species in areas with and without fox suppression. Lace monitors showed no GC response or differences in haemoparasite loads but were more likely to trade safety for higher food rewards, and had higher body condition, in areas of fox suppression than in areas where foxes remained abundant. In contrast, ringtails showed no physiological or behavioural differences between fox-suppressed and control areas. Predator sensitive foraging is a non-consumptive cost for lace monitors in the presence of the fox and most likely represents a response to competition. The ringtail's lack of response to the fox potentially represents complete naiveté or strong and rapid selection to the invasive predator. We suggest evolutionary responses are often overlooked in interactions between native and introduced species, but must be incorporated if we are to understand the suite of forces that shape community assembly and function in the wake of biological invasions.

Fecal indicators and zoonotic pathogens in household drinking water taps fed from rainwater tanks in Southeast Queensland, Australia.

In this study, the microbiological quality of household tap water samples fed from rainwater tanks was assessed by monitoring the numbers of Escherichia coli bacteria and enterococci from 24 households in Southeast Queensland (SEQ), Australia. Quantitative PCR (qPCR) was also used for the quantitative detection of zoonotic pathogens in water samples from rainwater tanks and connected household taps. The numbers of zoonotic pathogens were also estimated in fecal samples from possums and various species of birds by using qPCR, as possums and birds are considered to be the potential sources of fecal contamination in roof-harvested rainwater (RHRW). Among the 24 households, 63% of rainwater tank and 58% of connected household tap water (CHTW) samples contained E. coli and exceeded Australian drinking water guidelines of <1 CFU E. coli per 100 ml water. Similarly, 92% of rainwater tanks and 83% of CHTW samples also contained enterococci. In all, 21%, 4%, and 13% of rainwater tank samples contained Campylobacter spp., Salmonella spp., and Giardia lamblia, respectively. Similarly, 21% of rainwater tank and 13% of CHTW samples contained Campylobacter spp. and G. lamblia, respectively. The number of E. coli (P = 0.78), Enterococcus (P = 0.64), Campylobacter (P = 0.44), and G. lamblia (P = 0.50) cells in rainwater tanks did not differ significantly from the numbers observed in the CHTW samples. Among the 40 possum fecal samples tested, Campylobacter spp., Cryptosporidium parvum, and G. lamblia were detected in 60%, 13%, and 30% of samples, respectively. Among the 38 bird fecal samples tested, Campylobacter spp., Salmonella spp., C. parvum, and G. lamblia were detected in 24%, 11%, 5%, and 13% of the samples, respectively. Household tap water samples fed from rainwater tanks tested in the study appeared to be highly variable. Regular cleaning of roofs and gutters, along with pruning of overhanging tree branches, might also prove effective in reducing animal fecal contamination of rainwater tanks.

Advanced diagnostic approaches and current management of internal disorders of select species (rodents, sugar gliders, hedgehogs).

African pygmy and European hedgehogs, sugar gliders, and rodents such as rats, mice, gerbils, hamsters, guinea pigs, and chinchillas are becoming increasingly popular as pets in the United States, and more practitioners are being asked to examine, diagnose, and treat these animals for a bevy of disorders and diseases. Many procedures and techniques used in traditional small and large animal medicine are used for these species, with minor adaptations or considerations. This article examines available diagnostic tools and treatment methodologies for use in hedgehogs, sugar gliders, and selected rodents.

Population genetic analysis reveals a long-term decline of a threatened endemic Australian marsupial.

Since European colonization, Leadbeater's possum (Gymnobelideus leadbeateri) has declined across its range to the point where it is now only patchily distributed within the montane ash forests of the Central Highlands of Victoria. The loss of large hollow-bearing trees coupled with inadequate recruitment of mature ash forest has been predicted to result in a reduction in population size of up to 90% by 2020. Furthermore, bioclimatic analyses have suggested additional reductions in the species' distribution under a variety of climate change scenarios. Using a panel of 15 highly resolving microsatellite markers and mitochondrial control region sequence data, we infer past and present gene flow. Populations in the northern part of the core range were highly admixed, and showed no signs of either current or historical barriers to gene flow. A marginal, isolated and inbred population at Yellingbo was highly genetically differentiated, both in terms of current and historic genetic structure. Sequence data confirmed the conclusions from earlier genetic simulation studies that the Yellingbo population has been isolated from the rest of the species range since before European-induced changes to the montane landscape, and formed part of a larger genetic unit that is now otherwise extinct. Historic loss of maternal lineages in the Central Highlands of Victoria was detected despite signals of immigration, indicating population declines that most probably coincided with changes in climate at the end of the Pleistocene. Given ongoing habitat loss and the recent (February 2009) wildfire in the Central Highlands, we forecast (potentially extensive) demographic declines, in line with predicted range reductions under climate change scenarios.

Looks can deceive: molecular identity of an intraerythrocytic apicomplexan parasite in Australian gliders.

Two yellow-bellied gliders (Petaurus australis) had an intraerythrocytic parasite closely related to the cyst-forming coccidia (Apicomplexa: Sarcocystidae). The parasitaemia persisted for 3 months or more but was observed to clear within 3 years in captivity. The parasite appears not to significantly debilitate its infected host. Traditionally, using morphological identification, the intraerythrocytic parasite would have been classified within the Hepatozoon species typically found in red blood cells. However, molecular diagnostic techniques targeting the parasite's SSU rDNA and LSU rDNA demonstrated the unusual identity of this blood parasite and disputed its identity as a haemogregarine parasite of the genus Hepatozoon. The sequence was compared with available sequences from diverse mammalian and non-mammalian blood parasites (malaria, piroplasms, hemosporidia and sarcosporidia). The intraerythrocytic blood parasite was found to be most closely related to the cyst-forming coccidia including Besnoitia spp., Cystoisospora spp., Hammondia spp., Hyaloklossia lieberkuehni, Neospora caninum, Sarcocystis spp. and Toxoplasma gondii. The life cycle of this intraerythrocytic parasite remains unknown. The presented DNA identification demonstrates its suitability for an improved identification of blood parasites.

Chemotherapy and radiotherapy for treatment of cutaneous lymphoma in a ground cuscus (Phalanger gymnotis).

A 15-year-old female ground cuscus (Phalanger gymnotis) was presented with an isolated ulcerated, nonhealing lesion on the lateral thorax. Histopathology and immunohistochemistry were diagnostic for cutaneous T-cell lymphoma with incomplete excision. Oral chemotherapy with CCNU (lomustine) resulted in clinical remission that lasted 255 days, with no appreciable toxicity. Tumor recurrence was treated with radiation therapy, which resulted in 120 additional days of clinical remission. Subsequently, the tumor developed at a distant site and the cuscus was humanely euthanized. A slight decrease in appetite early in disease progression was the only adverse effect noted throughout the treatment period. Oral, minimally invasive chemotherapy, and adjunct radiation therapy were viable treatment options for this ground cuscus and should be considered for treatment of neoplasia in other nontraditional species.

A common tendency for phylogenetic overdispersion in mammalian assemblages.

Competition has long been proposed as an important force in structuring mammalian communities. Although early work recognized that competition has a phylogenetic dimension, only with recent increases in the availability of phylogenies have true phylogenetic investigations of mammalian community structure become possible. We test whether the phylogenetic structure of 142 assemblages from three mammalian clades (New World monkeys, North American ground squirrels and Australasian possums) shows the imprint of competition. The full set of assemblages display a highly significant tendency for members to be more distantly related than expected by chance (phylogenetic overdispersion). The overdispersion is also significant within two of the clades (monkeys and squirrels) separately. This is the first demonstration of widespread overdispersion in mammal assemblages and implies an important role for either competition between close relatives where traits are conserved, habitat filtering where distant relatives share convergent traits, or both.

Testosterone secretion, testicular histology and the cryopreservation of cauda epididymidal spermatozoa in the common ringtail possum (Pseudocheirus peregrinus).

The present study reports novel aspects of the reproductive biology of the male common ringtail possum (Pseudocheirus peregrinus). Plasma testosterone was measured through a stimulation test using the gonadotrophin-releasing hormone agonist, buserelin. Following intra-muscular administration of buserelin, there was an increase (P<0.05) in testosterone concentration in the peripheral circulation 4 h later. Quantitative testicular histology of this species was described for the first time. Eight stages of the seminiferous epithelium cycle were identified in 10 possums and their relative frequency determined. Spermatozoa were recovered from the cauda epididymides of hemi-castrated possums and cryopreservation conducted in straws (6 degrees C min(-1)) using final glycerol concentrations ranging between 2 and 20% in Tris-citrate egg yolk extender (v/v). Frozen straws were thawed and post-thaw motility, rate of motility, the percentage of live-dead spermatozoa and the percentage of sperm with swollen decondensed nuclei recorded. Similar to other marsupial sperm, common ringtail possum cauda epididymidal spermatozoa required high levels of glycerol (10-16%) in order to maintain post-thaw viability.

Family-level relationships among the Australasian marsupial "herbivores" (Diprotodontia: Koala, wombats, kangaroos and possums).

The marsupial order Diprotodontia includes 10 extant families, which occupy all terrestrial habitats across Australia and New Guinea and have evolved remarkable dietary and locomotory diversity. Despite considerable attention, the interrelations of these families have for the most part remained elusive. In this study, we separately model mitochondrial RNA and protein-coding sequences in addition to nuclear protein-coding sequences to provide near-complete resolution of diprotodontian family-level phylogeny. We show that alternative topologies inferred in some previous studies are likely to be artifactual, resulting from branch-length and compositional biases. Subordinal groupings resolved herein include Vombatiformes (wombats and koala) and Phalangerida, which in turn comprises Petauroidea (petaurid gliders and striped, feathertail, ringtail and honey possums) and a clade whose plesiomorphic members possess blade-like premolars (phalangerid possums, kangaroos and their allies and most likely, pygmy possums). The topology resolved reveals ecological niche structuring among diprotodontians that has likely been maintained for more than 40 million years.

Rapid loss of genetic variation in an endangered possum.

The endangered mountain pygmy possum is the only Australian marsupial that hibernates under snow cover. Most of its alpine habitat was burnt by a rare fire in 2003, and habitat loss and disturbance have also occurred owing to ski resort development. Here we show that there has been a rapid loss of genetic variation following habitat loss associated with resort development, but no detectable loss of alleles or decrease in heterozygosity following the fire.