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Conventional nested PCR and Sanger sequencing methods are currently the gold standards for detecting trypanosomes in wildlife. However, these techniques are time-consuming and can often overlook mixed infections. True trypanosome prevalence can thus be underrepresented. Here, we designed an 18S rDNA-based real-time quantitative PCR (qPCR) assay coupled with High-Resolution Melting Analysis (HRMA) to detect and discriminate three Trypanosoma species (T. copemani, T. noyesi, and T. vegrandis) commonly infecting Australian marsupials. A total of 68 genetically characterised samples from blood and tissue were used to validate the High-Resolution Melting - Real Time Quantitative Polymerase Chain Reaction (HRM-qPCR) assay. A further 87 marsupial samples consisting of blood, tissue and in vitro cultures derived from wildlife blood samples, were screened for the first time using this assay, and species identity confirmed using conventional PCR and Sanger sequencing. All three Trypanosoma species were successfully detected in pure cultures using the HRM-qPCR assay, and in samples containing mixed trypanosome infections. Of the 87 marsupial samples screened using the HRM-qPCR assay, 93.1% were positive for trypanosomes, and 8.0% contained more than one trypanosome species. In addition to the three targeted Trypanosoma species, this assay was also able to detect and identify other native and exotic trypanosomes. The turnaround time for this assay, from sample preparation to obtaining results, was less than 2 h, with a detection limit of 10 copies of the amplicon in a reaction for each of the targeted trypanosome species. This more rapid and sensitive diagnostic tool provides a high throughput platform for the detection, identification and quantification of trypanosome infections. It will also improve understanding of host diversity and parasite relationships and facilitate conservation management decisions., Graphical abstract Image 1

Asiatic black bears (Ursus thibetanus) face chronic stress in bile farms. In this study, we investigated whether bile-farmed bears show significantly high levels of stress at rescue and whether stress levels reduce over time in a bear sanctuary where the bears are supported with environmental enrichment and veterinary care to improve animal welfare. We measured stress hormone levels using faecal cortisol metabolites (FCM) in 16 Asiatic black bears freshly rescued from bile farms in Vietnam. Fresh faeces were collected from each bear on the rescue truck and on a weekly basis for a 22-week study period at a bear sanctuary in Vietnam. Results showed that for all 16 rescued bears (with one exception) individual FCM levels from truck samples were above mean baseline FCMs of bears previously rehabilitated to a bear sanctuary. This suggested the majority of the rescued bears were still capable of showing a stress endocrine response during the rescue operation despite being exposed to conditions causing chronic stress in bears on bile farms. Results showed that mean FCM levels of the rescued bears differed significantly between time-periods (higher at the rescue [on truck samples] compared to week 22 samples) and mean FCM levels showed an overall decline over the first 22 weeks after they arrived at the bear sanctuary. The bears also demonstrated acute FCM stress responses to management interventions at the sanctuary, such as veterinary health checks and transportation. In conclusion, rescued bears tend to modulate their stress endocrine response after rehoming at the bear sanctuary. This is an important result, indicating that the rescue effort and rehabilitation of bile-farm bears is effective. Whether this also coincides with behavioural adjustments in rehabilitating bears (eg lessening of stereotypic behaviour) warrants further investigation.

A new synthetic route was developed to create an azo-based reactive disperse dye containing the vinyl sulphonyl reactive group. This dye was used to colour wool and cotton fibres using scCO2 as the dyeing medium. The optimum dyeing process was carried out with water pre-treatment at 90 °C and 140 bar during a 60 min dyeing time. Under optimal conditions, which are relatively moderate for supercritical dyeing processes, the dyed wool fabrics produced uniform dyeings with high colour strength and fastness properties. These wool dyeings provided good colour strength and fixation properties (fixation of 99.4%). Cotton also showed good colour strength results. Crucially, the process did not produce any dyeing effluent. Furthermore, there is a potential to modify a wide scope of disperse dyes using the thiol group via this synthetic method without the need for further purification.

Host-parasite relationships are complex, and in wild animal populations individuals are commonly co-infected with various parasite species or intraspecific strains. While it is widely recognised that polyparasitism has the potential to reduce host fitness and increase susceptibility to predation or disease, the role of polyparasitism in influencing translocation success has never been investigated. Here we review the consequences of translocation for the host-parasite infracommunity and demonstrate how translocation-induced perturbations to within-host-parasite relationships may exacerbate the negative impacts of polyparasitism to the detriment of host health and translocation success. We also consider the ecological and immunological effects of altering host-parasite assemblages during translocation, and illustrate how the use of anti-parasitic drugs can further modify parasite infracommunity dynamics, with unintended impacts on target and non-target parasites. Importantly, as the evolutionary and ecological significance of the host-parasite relationship is increasingly recognised, we discuss the benefits of conserving parasites during fauna translocations.

Wildlife populations are increasingly subjected to perturbations that can impact their health. Yet, research on the health of endangered species in the face of disturbance is rare. This study investigated the impact of reserve expansion and wildfire on parasitism and body condition in a critically endangered marsupial, the woylie (Bettongia penicillata). We expected parasite prevalence to change in response to perturbation-induced changes in parasite abundance and host behaviour, depending on the transmission mode of the parasite. Similarly, we expected changes in resource availability associated with fire to impact body condition of hosts. Over 2 years, 999 captures were made of 136 woylies in Whiteman Park, a fenced reserve in Western Australia. Animals were assessed for body condition and the presence of ectoparasites, with blood and faeces collected for laboratory testing for haemo- and endoparasites. Generalized linear mixed models were used to determine what factors influenced parasitism and body condition. In the first 3 months post-expansion, the percentage of woylies shedding Strongyloides-like nematode eggs increased, while strongyle-like and oxyurid nematodes decreased, and ticks and trypanosomes were unaffected. Immediately after the fire, the proportion of woylies with ticks decreased and those with lice increased, though not significantly; low sample sizes limited our power. Body condition increased by 4 months post-fire. We suggest the variable response of parasites to expansion is likely multi-faceted and may relate to factors such as species-specificity and environmental dispersal. Conversely, the response to fire was more consistent with our expectations. As both fire and reserve expansion are highly relevant perturbations for remaining woylie populations, it is encouraging that neither appeared to have significant negative impacts on woylie health. To the best of our knowledge, this is the first study investigating changes in wildlife health parameters after reserve expansion.

A recent shift in conservation policy from the site scale to the ecosystem or landscape scale requires underpinning by large-scale species distribution data. This poses a significant challenge in conserving small/less charismatic species (SLCS's) whose often cryptic nature can result in spatially restricted sampling, thus preventing landscape scale conservation projects from being realised for these ecologically important groups. Species distribution models (SDMs) can provide a powerful tool to bridge this gap. However, in the case of SLCS's (here lichen epiphytes in temperate rainforests of western Scotland are used as a model system), direct predictor variables exist at micro-scales (millimetres to centimetres), which are not extensively available in landscape-scale datasets. Here we identify a group of well-mapped larger-scale ‘compound variables’ which capture the effect of multiple direct predictors (such as bark pH and topography), and test whether they can be successfully used to predict species distributions at the landscape scale, circumventing the need for direct (micro-scale) predictor data. By testing the SDMs more widely within western Scotland, accurate predictions of species presence/absence could be made throughout the region for 5 of the 9 lichen epiphytes, making these SDMs extremely valuable as a conservation planning tool. Species distribution models utilising compound variables as predictors offer a solution to the paucity of species distributional data for SLCS's, and present a valuable resource in conservation planning for such species. The importance of testing the SDMs outside of a training region to prevent prediction error is highlighted however.

Wildlife are increasingly subject to perturbations, which can impact pathogen transmission and lead to disease emergence. While a myriad of factors influence disease dynamics in wildlife, behaviour is emerging as a major influence. In this review, we examine how perturbations alter the behaviour of individuals and how, in turn, disease transmission may be impacted, with a focus on the use of network models as a powerful tool. There are emerging hypotheses as to how networks respond to different types of perturbations. The broad effects of perturbations make predicting potential outcomes and identifying mitigation opportunities for disease emergence critical; yet, the current paucity of data makes identification of underlying trends difficult. Social network analysis facilitates a mechanistic approach to how perturbation-induced behavioural changes result in shifts in pathogen transmission. However, the field is still developing, and future work should strive to address current deficits. There is particular need for empirical data to support modelling predictions and increased inclusion of pathogen monitoring in network studies.

Kapsulotaenia tidswelli is a proteocephalidean cestode that utilizes varanid lizards as definitive hosts. Fresh specimens of this cestode were observed with endogenous red pigmentation in the neck region that disappeared rapidly if specimens were not preserved in glutaraldehyde. The ultrastructural characteristics of the red pigment, which are described, suggest it is a carotenoid. Phylogenetic analysis confirmed a close relationship between K. tidswelli and other species of Kapsulotaenia for which sequence information is available. There is thus no reason to consider that the red pigmentation is because K. tidswelli is atypical, and it is proposed the carotenoids are likely to be associated with the diet of its varanid host.

Coatings were prepared by using NdFeB flake-like microparticles with aqueous polyurethane polyol dispersions and were coated onto the surface of textile fabrics in order to obtain textile fabrics with good magnetic properties. X-ray diffraction and energy dispersive spectroscopy analysis showed that there was no significant oxidation and magnetic decline in the samples after 6 months. The surface morphologies of the textiles were characterized using scanning electron microscopy and optical microscopy. The results showed that the crack density (the number of cracks per unit area) increased with increasing magnetic microparticle content, which can be attributed to two reasons: on one hand, the stress concentration at areas of high powder concentrations fractured more easily; on the other hand, the expansion coefficients of magnetic microparticles and polymer materials are different. Three kinds of textile fabrics with different microparticle concentrations were magnetized by means of a magnetizing machine. It was found that the cotton knitted fabrics had the highest average surface magnetic induction intensity, reaching 19 mT when the content of magnetic microparticle was 50 wt%. It was considered that this was related to the magnetic field distribution, due to the surface friction coefficient of the textile fabrics and the positions/angles of the flake-like microparticles. The hysteresis loops were measured using a vibrating sample magnetometer, and the results showed that the higher the concentration of magnetic microparticles, the higher the magnetization of the fabric. In short, this method can be used to prepare fabrics with high magnetization required under special conditions.

New information generated since 2016 from the application of molecular tools to infections with Giardia and Cryptosporidium is critically summarised. In the context of molecular epidemiology, nomenclature, taxonomy, in vitro culture, detection, zoonoses, population genetics and pathogenicity, are covered. Whole genome sequencing has had the greatest impact in the last three years. Future advances will provide a much better understanding of the zoonotic potential of both parasites, their diversity and how this is linked to pathogenesis in different hosts.

In the study reported in this paper, a series of reproducible conditions were employed to uniformly functionalize nylon 6 surfaces using a commercially available, low-frequency (40 kHz), low-pressure plasma system, utilizing oxygen plasma as a reactive gas. Initially, the plasma-treated samples were investigated using static contact angle measurements, showing a progressive increase in wettability with increasing plasma activation time between 10 and 40 s. Such an increase in wettability (and therefore increase in adhesive capabilities of the surfaces) was attributed to the creation of surface C-OH, C=O, and COOH groups. These surface-chemical modifications were characterized using x-ray photoelectron spectroscopy (XPS) and static secondary ion mass spectrometry (SSIMS). Surface radical densities were also shown to increase following plasma activation, having been quantified using a radical scavenging method based on the molecule 2,2-diphenyl-1-picrylhydrazyl (DPPH). The samples were imaged and analyzed using scanning electron microscopy (SEM) and atomic force microscopy (AFM), to confirm that there had been no detectable alteration to the surface roughness or morphology. Additionally, the “hydrophobic recovery” or “ageing” of the activated polymer samples, post-plasma treatment, was also investigated in terms of wettability and surface-chemistry, with the wettability of the sample surfaces decreasing over time due to a reduction in surface-oxygen concentration.

Faecal glucocorticoid metabolites are minimally invasive stress physiology indices that can be used to understand how animals respond to physical and/or psychological challenges (stressors) and inform how to optimize conservation management in view of these stressors. We investigated contextual biological, environmental and parasitological factors influencing variation in baseline faecal cortisol metabolite (FCM) concentration in a critically endangered marsupial, the woylie (syn. brush-tailed bettong, Bettongia penicillata). Woylies have undergone a rapid and significant population decline, with environmental stressors exacerbating disease suggested to contribute to these ongoing declines. We conducted a longitudinal field study of 15 adult woylies (9 females, 6 males) in a captive, naturalistic facility. FCM concentration in faecal samples (n = 269) collected monthly over 20 months was quantified by enzyme immunoassay in parallel with measures of body condition, sex, season, female reproductive status and the presence of endoparasites and ectoparasites. Linear mixed effect modelling revealed a significant effect of season, sex, body condition index and nematode parasite status on FCM. Overall, mean FCM was lowest in summer and highest in autumn and winter, and females had higher mean FCM than males. There was a significant but weak negative association between body condition and FCM. When woylies were shedding oxyurid nematode eggs they had higher mean FCM compared to when they were not shedding. In future, knowledge of factors that influence FCM fluctuations in woylies may be considered when carrying out potentially stressful conservation interventions that may influence the future survival of this unique and threatened species.

We aimed to validate the use of 1) the modified agglutination test and a polymerase chain reaction (PCR) protocol in detecting Toxoplasma gondii infection in quenda ( Isoodon obesulus) and brushtail possums ( Trichosurus vulpecula); 2) immunofluorescence microscopy of feces and a PCR and sequencing protocol in detecting Giardia spp. infection in quenda; and 3) a fecal flotation protocol in detecting gastrointestinal helminth infections of quenda. Quenda and brushtail possum carcasses, and samples from trapped quenda, were tested with 2 parasite detection tests per parasite, and results were modeled using Bayesian latent class analysis to estimate test sensitivity and specificity. The modified agglutination test and the PCR protocol were highly specific at detecting T. gondii infections in quenda and brushtail possums (≥93%); however, data were insufficient to assess sensitivity with adequate precision. Immunofluorescence microscopy and the PCR and sequencing protocol were both highly specific at detecting Giardia spp. in quenda (≥96%), but the PCR and sequencing protocol was relatively insensitive (69%, 95% credible interval [CrI]: 60–77%) compared to the highly sensitive immunofluorescence microscopy (98%, 95% CrI: 93–99%). The fecal flotation protocol was generally highly specific in the detection of gastrointestinal helminth infections (≥94%, with the exception of Trichuris spp. (88%, 95% CrI: 71–99%). The fecal flotation protocol was moderately to highly sensitive (≥74%) in the detection of strongyles, Labiobulura spp., Linstowinema spp., and Trichuris spp. Sensitivity was poor for detection of the cestode genus Potorolepis (36%, 95% CrI: 14–67%).

Giardia and Cryptosporidium are ubiquitous enteric protozoan pathogens of vertebrates. Although recognised as the aetiological agents of disease in humans and domestic animals for many years, fundamental questions concerning their ecology have been unresolved. Molecular tools have helped to better understand their genetic diversity and in so doing have helped to resolve questions about their transmission patterns and associated impacts on public health. However, the value of molecular tools is often complicated by questions concerning their applications, interpretation of results and terminology. Taxonomic issues have, until recently, made it difficult to determine the epidemiology of infections with both Giardia and Cryptosporidium. Similarly, improved understanding of their respective phylogenetic relationships has helped to resolve questions about zoonotic potential and distribution in wildlife. In the case of Cryptosporidium, imaging technologies have complemented phylogenetic studies in demonstrating the parasite's affinities with gregarine protozoa and have further supported its extracellular developmental capability and potential role as an environmental pathogen.

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.

Trypanosomes constitute a group of flagellate protozoan parasites responsible for a number of important, yet neglected, diseases in both humans and livestock. The most significantly studied include the causative agents of African sleeping sickness (Trypanosoma brucei) and Chagas disease (Trypanosoma cruzi) in humans. Much of our knowledge about trypanosome host–parasite relationships and life histories has come from these two human pathogens. Recent investigations into the diversity and life histories of wildlife trypanosomes in Australia highlight that there exists a great degree of biological and behavioural variation within and between trypanosomes. In addition, the genetic relationships between some Australian trypanosomes show that they are unexpectedly more closely related to species outside Australia than within it. These findings have led to a growing focus on the importance of understanding parasites occurring naturally in wildlife to (1) better document parasite biodiversity, (2) determine evolutionary relationships and degree of host specificity, (3) understand host–parasite interactions and the role of parasites in the natural ecosystem and (4) identify biosecurity issues of emerging disease in both wildlife and human populations. Here we review what is known about the diversity, life histories, host–parasite interactions and evolutionary relationships of trypanosomes in Australian wildlife. In this context, we focus upon the genetic proximity of key Australian species to the pathogenic T. cruzi and discuss similarities in their biology and behaviour that present a potential risk of human disease transmission by Australian vectors and wildlife.

Echinococcosis is a zoonosis whose history dates back to antiquity. This article provides an overview on the general history of echinococcosis, including the elucidation of Echinococcus life cycles and the long controversy on the aetiology of the cystic and alveolar forms of echinococcosis (CE and AE), lasting about 100 years since the middle of the 19th century. Furthermore, selected historical aspects of some fields of echinococcosis research are discussed and compared with our current knowledge, such as geographic distribution and epidemiology of CE ( Echinococcus granulosus ) and AE ( Echinococcus multilocularis ), clinical aspects and pathology, diagnosis in humans and animals, treatment (with focus on chemotherapy), control and basic research. A short paragraph is devoted to the neotropical forms of echinococcosis, caused by Echinococcus vogeli and Echinococcus oligarthrus . In this context the achievements of some ancestral pioneers of echinococcosis research are particularly highlighted and appreciated. Finally, the role of associations, international organizations (World Health Organization and others) and international working groups in echinococcosis research and control is briefly outlined. The retrospective reveals both the admirable achievements of our ancestors and the scientific progress of more recent times. But, it also shows the gaps in our knowledge, skills and resources that we need to control or even eradicate echinococcosis.

Alveolar echinococcosis (AE) and cystic echinococcosis (CE) are severe helminthic zoonoses. Echinococcus multilocularis (causative agent of AE) is widely distributed in the northern hemisphere where it is typically maintained in a wild animal cycle including canids as definitive hosts and rodents as intermediate hosts. The species Echinococcus granulosus, Echinococcus ortleppi, Echinococcus canadensis and Echinococcus intermedius are the causative agents of CE with a worldwide distribution and a highly variable human disease burden in the different endemic areas depending upon human behavioural risk factors, the diversity and ecology of animal host assemblages and the genetic diversity within Echinococcus species which differ in their zoonotic potential and pathogenicity. Both AE and CE are regarded as neglected zoonoses, with a higher overall burden of disease for CE due to its global distribution and high regional prevalence, but a higher pathogenicity and case fatality rate for AE, especially in Asia. Over the past two decades, numerous studies have addressed the epidemiology and distribution of these Echinococcus species worldwide, resulting in better-defined boundaries of the endemic areas. This chapter presents the global distribution of Echinococcus species and human AE and CE in maps and summarizes the global data on host assemblages, transmission, prevalence in animal definitive hosts, incidence in people and molecular epidemiology.

We conducted a serologic survey of four high-priority pig-associated viral zoonoses, Japanese encephalitis virus (JEV), hepatitis E virus (HEV), Nipah virus (NiV), and swine influenza virus (SIV), in Laos. We collected blood from pigs at slaughter during May 2008-January 2009 in four northern provinces. Japanese encephalitis virus hemagglutination inhibition seroprevalence was 74.7% (95% confidence interval [CI] = 71.5-77.9%), JEV IgM seroprevalence was 2.3% (95% CI = 1.2-3.2%), and HEV seroprevalence was 21.1% (95% CI = 18.1-24.0%). Antibodies to SIV were detected in 1.8% (95% CI = 0.8-2.8%) of pigs by screening enzyme-linked immunosorbent assay, and only subtype H3N2 was detected by hemagglutination inhibition in two animals with an inconclusive enzyme-linked immunosorbent assay result. No NiV antibody-positive pigs were detected. Our evidence indicates that peak JEV and HEV transmission coincides with the start of the monsoonal wet season and poses the greatest risk for human infection.

The hyperfine transitions in lithium-like and hydrogen-like bismuth were remeasured by direct laser spectroscopy at the experimental storage ring. For this we have now employed a voltage divider which enabled us to monitor the electron cooler voltage in situ. This will improve the experimental accuracy by about one order of magnitude with respect to our previous measurement using the same technique.

We report an improved measurement of the hyperfine splitting in hydrogen-like bismuth (209Bi82+) at the experimental storage ring ESR at GSI by laser spectroscopy on a coasting beam. Accuracy was improved by about an order of magnitude compared to the first observation in 1994. The most important improvement is an in situ high voltage measurement at the electron cooler (EC) platform with an accuracy at the 10 ppm level. Furthermore, the space charge effect of the EC current on the ion velocity was determined with two independent techniques that provided consistent results. The result of nm provides an important reference value for experiments testing bound-state quantum electrodynamics in the strong magnetic field regime by evaluating the specific difference between the splittings in the hydrogen-like and lithium-like ions.