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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.

A total of 1,136 samples from 289 households in four provinces in northern Laos were subjected to Japanese encephalitis virus (JEV) and dengue virus hemagglutination inhibition (DENV HI). Overall, antibodies to JEV were detected by HI in 620 (54.6%) of 1,136 people; of which 217 (19.1%) had HI activity against JEV only. Antibodies to DENV4 were detected by HI in 526 (46.3%) of 1,136 people; of which 124 (10.9%) had HI activity against DENV4 only. Antibodies to DENV1-3 were detected by HI in 296 (26.1%), 274 (24.1%), and 283 (24.9) of 1,136 people, respectively; of which 7, 1, and 0, respectively, had HI activity against DENV1-3 only. JEV was the most prevalent Flavivirus in Oudomxay, Luangprabang, and Huaphan provinces and DENV4 was the most prevalent in Xiengkhouang province. Seroprevalence for JEV increased with increasing age and wealth and was higher in villages where rice was cultivated in paddy fields and highest for people of Lao-Tai ethnicity.

Automated extraction of DNA for testing of laboratory samples is an attractive alternative to labour-intensive manual methods when higher throughput is required. However, it is important to maintain the maximum detection sensitivity possible to reduce the occurrence of type II errors (false negatives; failure to detect the target when it is present), especially in the biomedical field, where PCR is used for diagnosis. We used blood infected with known concentrations of Trypanosoma copemani to test the impact of analysis techniques on trypanosome detection sensitivity by PCR. We compared combinations of a manual and an automated DNA extraction method and two different PCR primer sets to investigate the impact of each on detection levels. Both extraction techniques and specificity of primer sets had a significant impact on detection sensitivity. Samples extracted using the same DNA extraction technique performed substantially differently for each of the separate primer sets. Type I errors (false positives; detection of the target when it is not present), produced by contaminants, were avoided with both extraction methods. This study highlights the importance of testing laboratory techniques with known samples to optimise accuracy of test results.

The introduction of Echinococcus to Australia over 200 years ago and its establishment in sheep rearing areas of the country inflicted a serious medical and economic burden on the country. This resulted in an investment in both basic and applied research aimed at learning more about the biology and life cycle of Echinococcus. This research served to illustrate the uniqueness of the parasite in terms of developmental biology and ecology, and the value of Echinococcus as a model system in a broad range of research, from fundamental biology to theoretical control systems. These studies formed the foundation for an international, diverse and ongoing research effort on the hydatid organisms encompassing stem cell biology, gene regulation, strain variation, wildlife diseases and models of transmission dynamics. We describe the development, nature and diversity of this research, and how it was initiated in Australia but subsequently has stimulated much international and collaborative research on Echinococcus.

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.

We conducted cross-sectional surveys for taeniasis and cysticercosis in humans, pigs, and dogs in four northern provinces of Laos. Human cysticercosis and taeniasis prevalence was 2.2% (95% confidence interval [CI] = 1.4-3.0%) and 8.4% (95% CI = 6.9-9.9%), respectively. Eating uncooked beef, being male, province of residence, age, and ethnicity were significant risk factors for taeniasis and only province of residence was a significant risk factor for cystiercosis. Thirty-five human tapeworms were recovered during the survey and 33 (94.3%) and 2 (5.7%) were identified as Taenia saginata and T. solium, respectively. Maximum-likelihood adjusted prevalence of T. solium and T. hydatigena in pigs was 4.2% (95% CI = 0.5-7.9%) and 55.9% (95% CI = 47.5-64.3%), respectively, and T. hydatigena taeniasis in dogs was 4.8% (95% CI = 0.0-11.3%). Taenia hydatigena and T. saginata were the most prevalent taeniids in the respective pig and human populations and together may suppress T. solium transmission.

Molecular epidemiology, broadly defined, is the application of molecular genetic techniques to the dynamics of disease in a population. In this review, we briefly describe molecular and analytical tools available for molecular epidemiological studies and then provide an overview of how they can be applied to better understand parasitic disease. A range of new molecular tools have been developed in recent years, allowing for the direct examination of parasites from clinical or environmental samples, and providing access to relatively cheap, rapid, high throughput molecular assays. At the same time, new analytical approaches, in particular those derived from coalescent theory, have been developed to provide more robust estimates of evolutionary processes and demographic parameters from multilocus, genotypic data. To date, the primary application of molecular epidemiology has been to provide specific and sensitive identification of parasites and to resolve taxonomic issues, particularly at the species level and below. Population genetic studies have also been used to determine the extent of genetic diversity among populations of parasites and the degree to which this diversity is associated with different host cycles or epidemiologically important phenotypes. Many of these studies have also shed new light on transmission cycles of parasites, particularly the extent to which zoonotic transmission occurs, and on the prevalence and importance of mixed infections with different parasite species or intraspecific variants (polyparasitism). A major challenge, and one which is now being addressed by an increasing number of studies, is to find and utilize genetic markers for complex traits of epidemiological significance, such as drug resistance, zoonotic potential and virulence.

The Apicomplexan parasite Cryptosporidium parvum is responsible for the widespread disease cryptosporidiosis, in both humans and livestock. The nature of C. parvum infection is far from understood and many questions remain in regard to host-parasite interactions, limiting successful treatment of the disease. To definitively identify a range of C. parvum stages in cell culture and to begin to investigate host cell interactions in some of the lesser known life stages, we have utilized a combined scanning electron microscopy and immunolabeling approach, correlating high resolution microstructural information with definitive immunogold labeling of Cryptosporidium stages. Several life cycle stages, including oocysts, merozoites I, trophozoites, gamonts and microgametocytes, were successfully immunolabeled in an in vitro model system. Developing oocysts were clearly immunolabeled, but this did not persist once excystation had occurred. Immunolabeling visualized on the host cell surface adjacent to invasive merozoites is likely to be indicative of receptor shedding, with merozoites also initiating host responses that manifested as abnormal microvilli on the host cell surface. Small sub-micron stages such as microgametocytes, which were impossible to identify as single entities without immunolabeling, were readily visualized and observed to attach to host cells via novel membranous projections. Epicellular parasites also expressed Cryptosporidium-derived epitopes within their encapsulating membrane. These data have allowed us to confidently identify a variety of C. parvum stages in cell culture at high resolution. With this, we provide new insight into C. parvum - host cell interactions and highlight future opportunities for investigating and targeting receptor-mediated interactions between Cryptosporidium life cycle stages and host cells.

A growing number of enteric protozoan species are considered to have zoonotic potential. Their clinical impact varies and in many cases is poorly defined. Similarly, the epidemiology of infections, particularly the role of non-human hosts, requires further study. In this review, new information on the life cycles and transmission of Giardia, Cryptosporidium, Entamoeba, Blastocystis and Balantidium are examined in the context of zoonotic potential, as well as polyparasitism and clinical significance.

This is the first report of an intestinal Eimeria infection in Asian seabass (Lates calcarifer) at the histopathological and ultrastructural levels. The Eimeria infection was often associated with severe pathology and significant mortality in the absence of other pathogens. This showed that it is an important disease of juvenile L. calcarifer in small scale nurseries in Vietnam. Heavy infection and high prevalence levels of the Eimeria infection are suspected to be linked to the low daily water exchange rates practised in these nurseries. Although systemic iridovirus infection was concurrently observed in some of the fish examined, it was not as consistently present in diseased fish as the Eimeria infection.

The first large-scale urban survey of Giardia infections in dogs was undertaken in the USA. It involved several locations in the Western United States with Giardia isolates from microscopy-positive samples characterised by multi-locus PCR and sequencing. A high prevalence of Giardia was confirmed in asymptomatic domestic dogs, and for the first time, provides evidence that zoonotic assemblages/subgroups of Giardia occur frequently in domestic dogs living in urban environments, and more frequently than the dog specific assemblages.

Giardia and Cryptosporidium are protozoan parasites known to cause enteric disease in terrestrial wildlife species (mammals, reptiles and birds). Few surveys for Giardia and Cryptosporidium in marine wildlife species, such as pinnipeds, have been reported. The objective of this study was to determine the prevalence and genotype of Giardia and Cryptosporidium in two species of pinnipeds, harp seal (Phoca groenlandica) and hooded seal (Cystophora cristata), from the Gulf of St. Lawrence, Canada. Faecal samples were collected from pup and adult seals and examined for the presence of cysts of Giardia and oocysts of Cryptosporidium using microscopy and immunofluorescent staining. Tissues from the small intestine of adult seals were also collected and examined for infections using the polymerase chain reaction (PCR) technique. Giardia cysts were found in the faeces of 42% (16/38) of adult harp seals, but in none of the harp seal pups (0/20). Although Giardia cysts were not detected in faeces of adult hooded seals (0/10) using microscopy, 80% tested positive for Giardia using PCR of intestinal tissue indicative of a true replicating infection. Both harp and hooded seals harboured infections with the zoonotic strain, Giardia duodenalis Assemblage A, as determined using a nested-PCR technique to amplify a small subunit ribosomal (SSU-rRNA) gene of Giardia. Cryptosporidium was not detected by microscopy, nor using the PCR technique on intestinal tissues from any of the 68 seals examined.

In this review some emerging issues of parasite infections in wildlife, particularly in Australia, are considered. We discuss the importance of understanding parasite biodiversity in wildlife in terms of conservation, the role of wildlife as reservoirs of parasite infection, and the role of parasites within the broader context of the ecosystem. Using a number of parasite species, the value of undertaking longitudinal surveillance in natural systems using non-invasive sampling and molecular tools to characterise infectious agents is illustrated in terms of wildlife health, parasite biodiversity and ecology.

Meiotic sex evolved early in the history of eukaryotes. Giardia duodenalis (syn. Giardia lamblia, Giardia intestinalis), a parasitic protist belonging to an early diverging lineage of eukaryotes, shows no cytological or physiological evidence of meiotic or sexual processes. Recent molecular analyses challenge the idea that G. duodenalis is a strictly clonal organism by providing evidence of recombination between homologous chromosomes within one subgroup (Assemblage A) of this species as well as genetic transfer from one subgroup to another (Assemblage A-B). Because recombination is not well documented and because it is not known whether the observed inter-assemblage transfer represents true reciprocal genetic exchange or a non-sexual process, we analyzed genic sequences from all major subgroups (Assemblages A-G) of this species. For all assemblages, we detected molecular signatures consistent with meiotic sex or genetic exchange, including low levels of heterozygosity, as indicated by allelic sequence divergence within isolates, and intra- and inter-assemblage recombination. The identification of recombination between assemblages suggests a shared gene pool and calls into question whether it is appropriate to divide the genetically distinct assemblages of G. duodenalis into a species complex.

SUMMARYLittle is known regarding the diversity, distribution or host-parasite associations ofTrypanosomaspp. in Australian wildlife. Here we report on an investigation based on divergence of the 18S rRNA gene of trypanosomes isolated from a range of hosts and varied geographical locations. A total of 371 individuals representing 19 species of native animals from 14 different locations were screened. In total, 32 individuals from 9 different species tested positive for the parasite. Phylogenetic analysis revealed considerable parasite diversity with no clear geographical distribution and no evidence of host specificity. In general, it appears that AustralianTrypanosomaspp. are widespread, with several genotypes appearing in multiple host species and in varied locations including both mainland areas and offshore islands. Some host species were found to be susceptible to multiple genotypes, but no individuals were infected with more than a single isolate.

SUMMARYTo date, little is known about the dynamics of vertical transmission of Toxoplasma gondii in Australian marsupials. Studies in mice demonstrate that vertical transmission of T. gondii is common and that chronically infected mice can transmit T. gondii to successive generations. In this study, PCR and immunohistochemistry were used to detect T. gondii in chronically infected marsupial dams and their offspring. T. gondii was detected in the unfurred pouch young of 2 out of 10 chronically infected western grey kangaroos (Macropus fuliginosus) and in the unfurred pouch young of a brush-tailed bettong (Bettongia penicillata). Results of the study suggest that vertical transmission of T. gondii can occur in chronically infected Australian marsupials.

Infection with Toxoplasma gondii is a significant problem in Australian marsupials, and can lead to devastating disease and predispose animals to predation. T. gondii infection in kangaroos is also of public health significance due to the kangaroo meat trade. A moderate seroprevalence of T. gondii was observed in a study of western grey kangaroos located in the Perth metropolitan area in Western Australia. Of 219 kangaroos tested, 15.5% (95%CI: 10.7-20.3) were positive for T. gondii antibodies using an ELISA developed to detect T. gondii IgG in macropod marsupials. When compared with the commercially available MAT (modified agglutination test), the ELISA developed was in absolute agreement and yielded a kappa coefficient of 1.00. Of 18 kangaroos tested for the presence of T. gondii DNA by PCR, the 9 ELISA positive kangaroos tested PCR positive and the 9 ELISA negative kangaroos tested PCR negative indicating the ELISA protocol was both highly specific and sensitive and correlated 100% with the more labour intensive PCR assay.

SUMMARYOf the 7 genetic assemblages of the parasiteGiardia duodenalisonly 2 (A and B) are known to cause infections in humans. These assemblages have been characterized in detail at the genomic level but few studies have examined differences in the proteins expressed. Employing one and two-dimensional PAGE we have identified an assemblage A-specific protein of human infectiveG. duodenalis; alpha 2 giardin. The protein difference was evident using both electrophoretic techniques. Alpha 2 giardin is known to be a structural protein and associates with the caudal flagella and the plasma membrane; however, its exact function is unknown. Although several proteins unique to assemblage B were also observed, we were unable to identify these proteins due to a lack of genomic data available for assemblage B isolates. Together, these proteins represent distinct phenotypic differences between the human infective assemblages ofG. duodenalisand support the need to revise the taxonomy of this parasite.

SUMMARYThe brush-tailed bettong (Bettongia penicillata), or woylie, is a medium-sized macropod marsupial that has undergone a rapid and substantial decline throughout its home range in the Upper Warren region of Western Australia over a period of approximately 5 years. As part of an investigation into possible causes of the decline a morphologically distinctTrypanosomasp. was discovered by light microscopy in the declining population but was absent in a stable population within the Karakamia Wildlife Sanctuary. Further investigations employing molecular methods targeting variations in the 18s rRNA gene determined that the trypanosome was novel and was also present within the Karakamia population albeit at a much lower overall prevalence and individual parasitaemia levels. Phylogenetic analysis suggests the novelTrypanosomasp. to be closely related to other trypanosomes isolated from native Australian wildlife species. Although it appears unlikely that the parasite is solely responsible for the decline in woylie population size, it may (singularly or in conjunction with other infectious agents) predispose woylies to increased mortality.

The application of molecular tools to the characterisation of the aetiological agents of echinococcosis has revealed a series of largely host-adapted species and genotypes that are maintained in distinct cycles of transmission. They can be defined on both genetic and phenotypic characteristics which complement previous observations made by descriptive parasitologists many years ago. A revised taxonomy for species in the genus Echinococcus has been proposed and widely accepted, particularly with respect to forms maintained in transmission cycles involving sheep, horses and cattle. However, molecular epidemiological studies are required in a number of endemic areas in order to determine cycles of transmission responsible for maintaining the parasite. The taxonomic status of forms in cervids, pigs and camels has still to be resolved, and the status and epidemiological significance of newly described species in China requires further research.

Lamblien (Giardia) sind die beim Menschen sowie bei domestizierten Tieren und Wildtieren am haufigsten vorkommenden Darmprotozoen. Am gefahrlichsten sind die klinischen Auswirkungen der Giardiasis (auch Giardiose und Lamblienruhr genannt) bei Kindern, insbesondere Kindern in Entwicklungslandern und Kindern, die in einem sozial benachteiligten Umfeld aufwachsen. Molekularepidemiologische Studien haben zur Aufklarung der Infektionsquellen und der Bedeutung von Tierreservoiren fur die offentliche Gesundheit beigetragen. Obwohl wir inzwischen einige Aspekte der Pathogenese der Giardiasis kennen, wissen wir noch lange nicht, welche Faktoren eine Pradisposition fur diese klinische Erkrankung auslosen. Zwar verfugen wir inzwischen uber wirksame Arzneimittel zur Behandlung von Infektionen mit Lamblien, aber diese Arzneimittel konnen in endemischen Umgebungen, in denen Kinder haufig mit Lamblien infiziert sind, lediglich als Begleittherapie zur Erganzung der traditionellen Ansatze des offentlichen Gesundheitswesens eingesetzt werden.

Giardia is the most common enteric protozoan pathogen of humans, domestic animals and wildlife. Children are at the most risk from the clinical consequences of Giardia infection, particularly those in developing countries and living in disadvantaged community settings. Molecular epidemiological studies have helped to elucidate sources of infection and the public health significance of animal reservoirs. Although aspects of the pathogenesis of Giardia infection are now understood, we are still a long way from understanding the factors that predispose to clinical disease. Effective drugs are available to treat giardial infections but can serve only as an adjunct to traditional public health approaches in endemic settings where children are commonly infected.

Faecal samples were obtained randomly from asymptomatic dogs visiting veterinary clinics (kept individually) and sanctuaries (kept in groups) in southern Germany. Of 60 Giardia positive samples, 55 samples successfully amplified at the 18S rDNA locus. In both dogs kept individually and dogs kept in groups, assemblage A was most prevalent (overall, n = 33, 60%), followed by mixed infections with assemblages A&C (overall, n = 15, 27.3%), while assemblages C&D alone were less often detected (overall, n = 5, 9.1% and n = 2, 3.6%). The occurrence of zoonotic genotypes was more prevalent in individual than in group dogs. Genotyping at the gdh locus confirmed the 18S results and clustered the assemblage A isolates into A-I. This is the first large-scale urban survey in Central Europe, which has not only confirmed the high prevalence of Giardia in asymptomatic domestic dogs, but has also shown that the zoonotic assemblage A occurs commonly in domestic dogs living in urban environments, and more frequently than the dog-specific assemblage. Although it was not possible to obtain samples from pet owners in this study, the results demonstrate that a significant proportion of dogs in urban areas of southern Germany harbour zoonotic Giardia, and should thus be considered a potential reservoir for infection in humans.

Recent studies have shown that dogs in Germany harbour different genotypic assemblages, predominantly C and D, but also the assemblage A, which has been confirmed as a pathogenic human genotype. To determine the prevalence of assemblage A, a first study was conducted in dogs which showed clinical signs of diarrhoea or other symptoms indicating a possible infection with Giardia. A total of 92 dogs were identified positive for Giardia by a coproantigen test, of which 65 samples were positive for cysts of Giardia using theMIFC technique.These samples were genotyped, 50 Giardia isolates were identified as assemblage D, 33 as assemblage C and eight as a mixture of both. One dog harboured a mixture of assemblage D and A. It was concluded that the predominant dog-specific assemblages C and D in this first study might be more commonly associated with GI disorders than the zoonotic genotype A. Alternatively the coproantigen test used in this study might select assemblages C + D in dogs. Therefore, in a second study randomly selected dogs presented at local veterinary clinics were examined for cysts of Giardia using theMIFC technique. A total of 58 samples of Giardia-positive dogs were genotyped: 57% belonged to assemblage D, 36% to assemblage C and 7% could be identified as the zoonotic genotype assemblage A. Comparing the 65 MIFC-positive samples from the first study with the 58 samples of the second study a significantly higher prevalence of assemblage A (P = 0.0467) was found in randomly selected dogs.

SUMMARYA review of theGiardia duodenalissequences currently available on the GenBank database was completed to compare the different genotyping loci (small subunit ribosomal DNA, glutamate dehydrogenase, triose-phosphate isomerase and beta giardin) for their ability to discern assemblage and subassemblage groups and infer phylogenetic relationships. In total, 405Giardia duodenalissequences were sorted and aligned to examine the substitutions within and between the assemblages – A and B (zoonotic), C and D (dogs), E (livestock), F (cats) and G (rodents). It was found that all of the genes could reproducibly group isolates into their assemblages and that the AI/AII subassemblage groups were robust and identifiable at all loci. However, the assemblage B subgroups were not reproducible at half of the loci (small subunit ribosomal DNA and beta giardin), not due to their conserved nature, but because there was insufficient sequence data of reference isolates available for comparison. It is anticipated that further investigation of these loci may reveal the core subgroups of this medically important and zoonotic assemblage and also those of others. The closer, more recent, phylogenetic relationships amongst the assemblages appear to be resolved; however, more sequence data from the current loci, and possibly new loci, will be required to establish the remaining relationships.

Studies on genetic variability in Trypanosoma evansi have been limited by a lack of high-resolution techniques. In this study, we have investigated the use of inter-simple sequence repeats (ISSR) and microsatellites in revealing polymorphism among T. evansi isolates. Twelve ISSR primers and five microsatellite loci were used to generate polymorphic bands and alleles, respectively, to investigate the genetic variability among T. evansi isolates from Africa and Asia. Seven of the twelve ISSR primers showed variability between isolates with a total of 71 fragments of which 49(69%) were polymorphic. Microsatellite analysis revealed a total of 60 alleles. On average the ISSR markers revealed a higher genetic diversity (23%) than microsatellites (21.1%). The two techniques showed a strong agreement of r = 0.95 for Dice and r = 0.91 for Jaccard indices in estimating the genetic distances between isolates. The distance UPGMA tree revealed two major clusters of T. evansi which correlate with the minicircle classification of subtype A and B. The cophenetic correlation coefficient between Dice and Jaccard based matrices were r = 0.79 for microsatellites and r = 0.73 for ISSR indicating a strong agreement between dendrograms. The results suggest that both ISSR and microsatellites markers are useful in detecting genetic variability within T. evansi.

A total of 15 red foxes, 15 raccoon dogs, 15 domestic dogs and 15 domestic cats were each infected with 20,000 protoscolices of Echinococcus multilocularis. At 35, 63, and 90 days post inoculation (dpi), five animals from each group were necropsied and the worm burdens determined. The highest worm burdens in foxes (mean of 16,792) and raccoon dogs (mean of 7930) were found at 35 dpi. These declined to a mean of just 331 worms in foxes and 3213 worms in raccoon dogs by day 63 with a further decline to 134 worms in foxes and 67 worms in raccoon dogs by day 90. In dogs, there was no significant difference between worm burdens recovered at days 35 (mean of 2466) and day 90 (mean of 1563), although reduced numbers were recovered on day 63 (mean of 899). In cats, worms were found in four animals 35 dpi (mean of 642), in three at 63 dpi (mean of 28) and in two at 90 dpi (mean of 57). Faecal egg counts were determined at 3 day intervals from 25 dpi. A mathematical model of egg excretion dynamics suggested that the mean biotic potential per infected animal was high in foxes (346,473 eggs); raccoon dogs (335,361 eggs) and dogs (279,910 eggs) but very low for cats (573 eggs). It also indicated that approximately 114, 42 and 27 eggs per worm were excreted in the faeces of dogs, raccoon dogs and foxes, respectively. The fecundity of worms in cats was low with an average of less than one egg per worm. The peak levels of coproantigen were detected earlier in foxes and raccoon dogs than in dogs. Eggs recovered from foxes, raccoon dogs and dogs resulted in massive infections in experimental mice. However, metacestodes did not develop from eggs originating from infected cats. It is concluded that foxes, raccoon dogs and dogs are good hosts of E. multilocularis. In contrast, the low worm establishment, the very few excreted eggs and the lack of infectivity of eggs strongly indicate that cats play an insignificant role in parasite transmission.

Many issues concerning the taxonomy ofEchinococcushave been resolved in recent years with the application of molecular tools. However, the status ofEchinococcusmaintained in transmission cycles involving cervid intermediate hosts remains to be determined. The recent characterization of the parasite from cervids in Finland has highlighted the paucity of data available, particularly that from North America. In this study, we have characterized a large number ofEchinococcusisolates from cervids from Western Canada on the basis of morphology and molecular genetic techniques. Our results support earlier studies suggesting thatEchinococcusof cervid origin is phenotypically and genetically distinct toEchinococcusmaintained in domestic host assemblages, and also confirms thatEchinococcusof cervid origin does not constitute a genetically homogeneous group. However, our data do not support the existence of 2 distinct genotypes (strains/subspecies) with separate geographical distributions. Our data appear to support the existence of only 1 species in cervids, but additional isolates from cervids and wolves in other endemic regions should be characterized before a final decision is made on the taxonomic status ofEchinococcusin cervids.

A small survey was undertaken of commercially reared free-range chickens in Western Australia using serology and molecular detection. Eighteen out of 20 serum samples showed antibody responses with titers of 1:64 in 5 chickens and ≥ 1:128 in 13 chickens. DNA extracted from 22 out of 50 tissue samples, 10 brains and 12 spleens, were positive by nested PCR, and sequencing at the B1 locus on DNA from 3 brain and 3 spleen samples confirmed that 2 isolates were Toxoplasma gondii, Type I, and 4 Type II/III. The high prevalence of Toxoplasma infection found in commercial, free-range chickens raises public health issues with respect to both exposure in the workplace, during carcass processing, and subsequent transmission during food handling and/or consumption as food. The results of this study emphasize the need for more data on the incidence of Toxoplasma infection in domestic animals and humans in Australia.

There are 11 different pathogenic trypanosomes in trypanosomiasis endemic regions of Africa. Their detection and characterisation by molecular methods relies on species-specific primers; consequently several PCR tests have to be made on each sample. Primers ITS1 CF and ITS1 BR, previously designed to amplify the internal transcribed spacer (ITS1) of rDNA, have been evaluated for use in a universal diagnostic test for all pathogenic trypanosomes. Blood was collected from 373 cattle and 185 camels. The primers gave constant PCR products with the stocks of each taxon tested. Members of subgenus Trypanozoon (T. brucei brucei, T. evansi, T. b. rhodesiense and T. b. gambiense) gave a constant product of approximately 480 bp; T. congolense, savannah 700 bp, T. congolense kilifi 620 bp and T. congolense forest 710 bp: T. simiae 400 bp, T. simiae tsavo 370 bp, T. godfreyi 300 bp and T. vivax 250 bp. The sensitivity of the test ranged from 10 pg for Trypanozoon, T. congolense clade and T. vivax to 100 pg for T. simiae and T. godfreyi. The primers detected cases of multi-taxa samples, although the sensitivity was reduced with an increase in the combinations. A better detection rate of trypanosome DNA was recorded with buffy coats than from direct blood. With the field samples, the diagnostic sensitivity was close to the sensitivity obtained using single reactions with species-specific primers for Trypanozoon 38/40 (95%) and T. congolense savannah 30/33 (90.9%) but was lower with T. vivax 25/31 (77.4%). The primers offer promise as a routine diagnostic tool through the use of a single PCR; however, further evaluation is recommended.