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

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

The High aerobic capacity of a small, marsupial rat-kangaroo (Bettongia penicillata) is matched by the mitochondrial and capillary morphology of its skeletal muscles

We examined the structure-function relationships that underlie the aerobic capacities of marsupial mammals that hop. Marsupials have relatively low basal metabolic rates (BMR) and historically were seen as 'low energy' mammals. However, the red kangaroo, Macropus rufus (family Macropodidae), has aerobic capacities equivalent to athletic placentals. It has an extreme aerobic scope (fAS) and its large locomotor muscles feature high mitochondrial and capillary volumes. M. rufus belongs to a modern group of kangaroos and its high fAS is not general for marsupials. However, other hopping marsupials may have elevated aerobic capacities. Bettongia penicillata, a rat-kangaroo (family Potoroidae), is a small (1 kg), active hopper whose fAS is somewhat elevated. We examined the oxygen delivery system in its muscles to ascertain links with hopping. An elevated fAS of 23 provided a relatively high maximal aerobic oxygen consumption (VO₂,max) in B. penicillata; associated with this is a skeletal muscle mass of 44% of body mass. Ten muscles were sampled to estimate the total mitochondrial and capillary volume of the locomotor muscles. Values in B. penicillata were similar to those in M. rufus and in athletic placentals. This small hopper had high muscle mitochondrial volume densities (7.1-11.9%) and both a large total capillary volume (6 ml kg⁻¹ body mass) and total capillary erythrocyte volume (3.2 ml kg⁻¹). Apparently, a considerable aerobic capacity is required to achieve the benefits of the extended stride in fast hopping. Of note, the ratio of VO₂,max to total muscle mitochondrial volume in B. penicillata was 4.9 ml O₂ min⁻¹ ml⁻¹. Similar values occur in M. rufus and also placental mammals generally, not only athletic species. If such relationships occur in other marsupials, a fundamental structure-function relationship for oxygen delivery to muscles likely originated with or before the earliest mammals.8 page(s

Faecal glucocorticoid metabolite concentrations in the free-ranging bandicoots (Perameles nasuta and Isoodon obesulus) of northern Sydney

Knowledge of how animals cope with their environment is fundamental to the management of free-ranging populations. Urban animals face increased competition for resources, habitat fragmentation and predation. These pressures may impact an individual's welfare by releasing glucocorticoid hormones in the blood through a response from the hypothalamic-pituitary-adrenal axis, resulting in altered energy storage and utilisation. This study aimed to determine the applicability of measuring faecal glucocorticoid metabolites in free-ranging bandicoots by using a simple enzyme immunoassay. We used long-nosed and southern brown bandicoots in northern Sydney to investigate whether environmental and demographic variables can influence faecal glucocorticoid metabolites. Long-nosed bandicoots showed similar faecal glucocorticoid metabolite concentrations between suburban backyards and National Park populations. Higher faecal glucocorticoid metabolites were recorded in female southern brown bandicoots than in males, whilst female and male long-nosed bandicoots had similar glucocorticoid metabolite levels. Ectoparasite load, body condition and season did not influence faecal glucocorticoid metabolites. This non-invasive method has a broad application and can be used to provide biological information to guide management of populations within a conservation context.7 page(s

The fur of mammals in exposed environments; do crypsis and thermal needs necessarily conflict? The polar bear and marsupial koala compared

The furs of mammals have varied and complex functions. Other than for thermoregulation, fur is involved in physical protection, sensory input, waterproofing and colouration, the latter being important for crypsis or camouflage. Some of these diverse functions potentially conflict. We have investigated how variation in cryptic colouration and thermal features may interact in the coats of mammals and influence potential heat inflows from solar radiation, much of which is outside the visible spectral range. The coats of the polar bear (Ursus maritimus) and the marsupial koala (Phascolarctus cinereus) have insulative similarities but, while they feature cryptic colouration, they are of contrasting colour, i.e. whitish and dark grey. The reflectance of solar radiation by coats was measured across the full solar spectrum using a spectroradiometer. The modulation of incident solar radiation and resultant heat flows in these coats were determined at a range of wind speeds by mounting them on a heat flux transducer/temperature-controlled plate apparatus in a wind tunnel. A lamp with a spectral distribution of radiation similar to the solar spectrum was used as a proxy for the sun. Crypsis by colour matching was apparent within the visible spectrum for the two species, U. maritimus being matched against snow and P. cinereus against Eucalyptus forest foliage. While reflectances across the full solar spectrum differed markedly, that of U. maritimus being 66 % as opposed to 10 % for P. cinereus, the heat influxes from solar radiation reaching the skin were similar. For both coats at low wind speed (1 m s⁻¹), 19 % of incident solar radiation impacted as heat at the skin surface; at higher wind speed (10 m s⁻¹) this decreased to approximately 10 %. Ursus maritimus and P. cinereus have high and comparable levels of fur insulation and although the patterns of reflectance and depths of penetrance of solar radiation differ for the coats, the considerable insulation limited the radiant heat reaching the skin. These data suggest that generally, if mammal coats have high insulation then heat flow from solar radiation into an animal is much restricted and the impact of coat colour is negligible. However, comparisons with published data from other species suggest that as fur insulation decreases, colour increasingly influences the heat inflow associated with solar radiation.12 page(s

Ectoparasite infestation patterns, haematology and serum biochemistry of urban-dwelling common brushtail possums

Urban environments support high concentrations of humans, domestic pets and introduced animals, creating conditions conducive to the transmission of parasites. This study compared patterns of ectoparasite infestation of the common brushtail possum Trichosurus vulpecula in urbanised Sydney (n = 161) to those from a remote woodland site (n = 18) from February 2005 - November 2006. We found differences in ectoparasite species prevalence between the two groups: the flea Echidnophaga myrmecobii was only found on urban possums and the tick Ixodes trichosuri was much more prevalent in the urban habitat, while the mite Atellana papilio was more prevalent on woodland possums. E. myrmecobii and I. trichosuri differed from other ectoparasites by showing an association with host sex and host age. Potential physiological costs of ectoparasitism to urban-dwelling possums were determined using multivariate analysis of haematology, serum biochemistry and body condition. Changes in serum iron levels were seen in the presence of both the tick Ixodes trichosuri and the flea E. myrmecobii, and E. myrmecobii was associated with elevated serum levels of the liver enzyme ALT. However, ectoparasite-related changes in haematology and serum biochemistry were not indicative of long-term pathology. In this urban possum population, the costs of ectoparasitism appear to be limited and unlikely to pose a major threat to the health of the population.11 page(s

Molecular detection of antibiotic-resistance determinants in Escherichia coli isolated from the endangered Australian sea lion (Neophoca cinerea)

Greater interaction between humans and wildlife populations poses significant risks of anthropogenic impact to natural ecosystems, especially in the marine environment. Understanding the spread of microorganisms at the marine interface is therefore important if we are to mitigate adverse effects on marine wildlife. We investigated the establishment of Escherichia coli in the endangered Australian sea lion (Neophoca cinerea) by comparing fecal isolation from wild and captive sea lion populations. Fecal samples were collected from wild colonies March 2009-September 2010 and from captive individuals March 2011-May 2013. Using molecular screening, we assigned a phylotype to E. coli isolates and determined the presence of integrons, mobile genetic elements that capture gene cassettes conferring resistance to antimicrobial agents common in fecal coliforms. Group B2 was the most abundant phylotype in all E. coli isolates (n = 37), with groups A, B1, and D also identified. Integrons were not observed in E. coli (n = 21) isolated from wild sea lions, but were identified in E. coli from captive animals (n = 16), from which class I integrases were detected in eight isolates. Sequencing of gene cassette arrays identified genes conferring resistance to streptomycin-spectinomycin (aadA1) and trimethoprim (dfrA17, dfrB4). Class II integrases were not detected in the E. coli isolates. The frequent detection in captive sea lions of E. coli with resistance genes commonly identified in human clinical cases suggests that conditions experienced in captivity may contribute to establishment. Identification of antibiotic resistance in the microbiota of Australian sea lions provides crucial information for disease management. Our data will inform conservation management strategies and provide a mechanism to monitor microorganism dissemination to sensitive pinniped populations.9 page(s

Colony location and captivity influence the gut microbial community composition of the Australian sea lion (Neophoca cinerea)

Gut microbiota play an important role in maintenance of mammalian metabolism and immune system regulation, and disturbances to this community can have adverse impacts on animal health. To better understand the composition of gut microbiota in marine mammals, fecal bacterial communities of the Australian sea lion (Neophoca cinerea), an endangered pinniped with localized distribution, were examined. A comparison of samples from individuals across 11 wild colonies in South and Western Australia and three Australian captive populations showed five dominant bacterial phyla: Firmicutes, Proteobacteria, Bacteroidetes, Actinobacteria, and Fusobacteria. The phylum Firmicutes was dominant in both wild (76.4%±4.73%) and captive animals (61.4%±10.8%), while Proteobacteria contributed more to captive (29.3%±11.5%) than to wild (10.6%±3.43%) fecal communities. Qualitative differences were observed between fecal communities from wild and captive animals based on principal-coordinate analysis. SIMPER (similarity percentage procedure) analyses indicated that operational taxonomic units (OTU) from the bacterial families Clostridiaceae and Ruminococcaceae were more abundant in wild than in captive animals and contributed most to the average dissimilarity between groups (SIMPER contributions of 19.1% and 10.9%, respectively). Differences in the biological environment, the foraging site fidelity, and anthropogenic impacts may provide various opportunities for unique microbial establishment in Australian sea lions. As anthropogenic disturbances to marine mammals are likely to increase, understanding the potential for such disturbances to impact microbial community compositions and subsequently affect animal health will be beneficial for management of these vulnerable species.10 page(s

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

One size does not fit all : monitoring faecal glucocorticoid metabolites in marsupials

Marsupial research, conservation, and management can benefit greatly from knowledge about glucocorticoid (GC) secretion patterns because GCs influence numerous aspects of physiology and play a crucial role in regulating an animal's response to stressors. Faecal glucocorticoid metabolites (FGM) offer a non-invasive tool for tracking changes in GCs over time. To date, there are relatively few validated assays for marsupials compared with other taxa, and those that have been published generally test only one assay. However, different assays can yield very different signals of adrenal activity. The goal of this study was to compare the performance of five different enzyme immunoassays (EIAs) for monitoring adrenocortical activity via FGM in 13 marsupial species. We monitored FGM response to two types of events: biological stressors (e.g., transport, novel environment) and pharmacological stimulation (ACTH injection). For each individual animal and assay, FGM peaks were identified using the iterative baseline approach. Performance of the EIAs for each species was evaluated by determining (1) the percent of individuals with a detectable peak 0.125–4.5 days post-event, and (2) the biological sensitivity of the assay as measured by strength of the post-event response relative to baseline variability (Z-score). Assays were defined as successful if they detected a peak in at least 50% of the individuals and the mean species response had a Z ⩾ 2. By this criterion, at least one assay was successful in 10 of the 13 species, but the best-performing assay varied among species, even those species that were closely related. Furthermore, the ability to confidently assess assay performance was influenced by the experimental protocols used. We discuss the implications of our findings for biological validation studies