A rapid and inexpensive viability assay for zoospores and zoosporangia of Batrachochytrium dendrobatidis

The fungus Batrachochytrium dendrobatidis is causing global amphibian declines. Here we describe a simple, rapid and inexpensive methylene blue staining protocol to determine B. dendrobatidis viability, regardless of life-stage. The viability of cells in suspension or adherent monolayers can be determined using either manual microscopy counting or colorimetric assay

Nidoviruses in Reptiles: A Review

Since their discovery in 2014, reptile nidoviruses (also known as serpentoviruses) have emerged as significant pathogens worldwide. They are known for causing severe and often fatal respiratory disease in various captive snake species, especially pythons. Related viruses have been detected in other reptiles with and without respiratory disease, including captive and wild populations of lizards, and wild populations of freshwater turtles. There are many opportunities to better understand the viral diversity, species susceptibility, and clinical presentation in different species in this relatively new field of research. In captive snake collections, reptile nidoviruses can spread quickly and be associated with high morbidity and mortality, yet the potential disease risk to wild reptile populations remains largely unknown, despite reptile species declining on a global scale. Experimental studies or investigations of disease outbreaks in wild reptile populations are scarce, leaving the available literature limited mostly to exploring findings of naturally infected animals in captivity. Further studies into the pathogenesis of different reptile nidoviruses in a variety of reptile species is required to explore the complexity of disease and routes of transmission. This review focuses on the biology of these viruses, hosts and geographic distribution, clinical signs and pathology, laboratory diagnosis and management of reptile nidovirus infections to better understand nidovirus infections in reptiles

Glutathione is required for growth and cadmium tolerance in the amphibian chytrid fungus, Batrachochytrium dendrobatidis

Batrachochytrium dendrobatidis (Bd) is a lethal amphibian pathogen, partly due to its ability to evade the immune system of susceptible frog species. In many pathogenic fungi, the antioxidant glutathione is a virulence factor that helps neutralize oxidative stressors generated from host immune cells, as well as other environmental stressors such as heavy metals. The role of glutathione in stress tolerance in Bd has not been investigated. Here, we examine the changes in the glutathione pool after stress exposure and quantify the effect of glutathione depletion on cell growth and stress tolerance. Depletion of glutathione repressed growth and release of zoospores, indicating that glutathione is essential for life cycle completion in Bd. Supplementation with 2 mM were strongly inhibitory to Bd cells. While hydrogen peroxide exposure lowered the total cellular glutathione levels by 42 %, glutathione depletion did not increase the sensitivity to hydrogen peroxide. Exposure to cadmium increased total cellular glutathione levels by 93 %. Glutathione-depleted cells were more sensitive to cadmium, and this effect was attenuated by glutathione supplementation, suggesting that glutathione plays an important role in cadmium tolerance. The effects of heat and salt were exacerbated by the addition of exogenous glutathione. The impact of glutathione levels on Bd stress sensitivity may help explain differences in host susceptibility to chytridiomycosis and may provide opportunities for synergistic therapeutic

Chytridiomycosis causes catastrophic organism-wide metabolic dysregulation including profound failure of cellular energy pathways

Chytridiomycosis is among several recently emerged fungal diseases of wildlife that have caused decline or extinction of naive populations. Despite recent advances in understanding pathogenesis, host response to infection remains poorly understood. Here we modelled a total of 162 metabolites across skin and liver tissues of 61 frogs from four populations (three long-exposed and one naive to the fungus) of the Australian alpine tree frog (Litoria verreauxii alpina) throughout a longitudinal exposure experiment involving both infected and negative control individuals. We found that chytridiomycosis dramatically altered the organism-wide metabolism of clinically diseased frogs. Chytridiomycosis caused catastrophic failure of normal homeostatic mechanisms (interruption of biosynthetic and degradation metabolic pathways), and pronounced dysregulation of cellular energy metabolism. Key intermediates of the tricarboxylic acid cycle were markedly depleted, including in particular a-ketoglutarate and glutamate that together constitute a key nutrient pathway for immune processes. This study was the first to apply a non-targeted metabolomics approach to a fungal wildlife disease and specifically to dissect the host-pathogen interface of Bd-infected frogs. The patterns of metabolite accumulation we have identified reveal whole-body metabolic dysfunction induced by a fungal skin infection, and these findings have broad relevance for other fungal diseases

Australian Bat Lyssavirus: Analysis of National Bat Surveillance Data from 2010 to 2016

Australian bat lyssavirus (ABLV) was first described in 1996 and has been regularly detected in Australian bats since that time. While the virus does not cause population level impacts in bats and has minimal impacts on domestic animals, it does pose a public health risk. For this reason, bats are monitored for ABLV and a national dataset is collated and maintained by Wildlife Health Australia. The 2010–2016 dataset was analysed using logistic regression and time-series analysis to identify predictors of infection status in bats and the factors associated with human exposure to bats. In common with previous passive surveillance studies, we found that little red flying-foxes (Pteropus scapulatus) are more likely than other species to be infected with ABLV. In the four Australian mainland species of flying-fox, there are seasonal differences in infection risk that may be associated with reproductive cycles, with summer and autumn the seasons of greatest risk. The risk of human contact was also seasonal, with lower risk in winter. In line with other studies, we found that the circumstances in which the bat is encountered, such as exhibiting abnormal behaviour or being grounded, are risk factors for ABLV infection and human contact and should continue be key components of public health messaging. We also found evidence of biased recording of some types of information, which made interpretation of some findings more challenging. Strengthening of “One Health” linkages between public health and animal health services at the operational level could help overcome these biases in future, and greater harmonisation nationally would increase the value of the dataset

Ranaviruses and reptiles

Ranaviruses can infect many vertebrate classes including fish, amphibians and reptiles, but for the most part, research has been focused on non-reptilian hosts, amphibians in particular. More recently, reports of ranaviral infections of reptiles are increasing with over 12 families of reptiles currently susceptible to ranaviral infection. Reptiles are infected by ranaviruses that are genetically similar to, or the same as, the viruses that infect amphibians and fish; however, physiological and ecological differences result in differences in study designs. Although ranaviral disease in reptiles is often influenced by host species, viral strain and environmental differences, general trends in pathogenesis are emerging. More experimental studies using a variety of reptile species, life stages and routes of transmission are required to unravel the complexity of wild ranavirus transmission. Further, our understanding of the reptilian immune response to ranaviral infection is still lacking, although the considerable amount of work conducted in amphibians will serve as a useful guide for future studies in reptiles

Climate change could increase the geographic extent of Hendra virus spillover risk

Disease risk mapping is important for predicting and mitigating impacts of bat-borne viruses, including Hendra virus (Paramyxoviridae:Henipavirus), that can spillover to domestic animals and thence to humans. We produced two models to estimate areas at potential risk of HeV spillover explained by the climatic suitability for its flying fox reservoir hosts, Pteropus alecto and P. conspicillatus. We included additional climatic variables that might affect spillover risk through other biological processes (such as bat or horse behaviour, plant phenology and bat foraging habitat). Models were fit with a Poisson point process model and a log-Gaussian Cox process. In response to climate change, risk expanded southwards due to an expansion of P. alecto suitable habitat, which increased the number of horses at risk by 175–260% (110,000–165,000). In the northern limits of the current distribution, spillover risk was highly uncertain because of model extrapolation to novel climatic conditions. The extent of areas at risk of spillover from P. conspicillatus was predicted shrink. Due to a likely expansion of P. alecto into these areas, it could replace P. conspicillatus as the main HeV reservoir. We recommend: (1) HeV monitoring in bats, (2) enhancing HeV prevention in horses in areas predicted to be at risk, (3) investigate and develop mitigation strategies for areas that could experience reservoir host replacements

Review of the Amphibian Immune Response to Chytridiomycosis, and Future Directions

The fungal skin disease, chytridiomycosis (caused by Batrachochytrium dendrobatidis and B. salamandrivorans), has caused amphibian declines and extinctions globally since its emergence. Characterizing the host immune response to chytridiomycosis has been a focus of study with the aim of disease mitigation. However, many aspects of the innate and adaptive arms of this response are still poorly understood, likely due to the wide range of species' responses to infection. In this paper we provide an overview of expected immunological responses (with inference based on amphibian and mammalian immunology), together with a synthesis of current knowledge about these responses for the amphibian-chytridiomycosis system. We structure our review around four key immune stages: (1) the naïve immunocompetent state, (2) immune defenses that are always present (constitutive defenses), (3) mechanisms for recognition of a pathogen threat and innate immune defenses, and (4) adaptive immune responses. We also evaluate the current hot topics of immunosuppression and immunopathology in chytridiomycosis, and discuss their respective roles in pathogenesis. Our synthesis reveals that susceptibility to chytridiomycosis is likely to be multifactorial. Susceptible amphibians appear to have ineffective constitutive and innate defenses, and a late-stage response characterized by immunopathology and Bd-induced suppression of lymphocyte responses. Overall, we identify substantial gaps in current knowledge, particularly concerning the entire innate immune response (mechanisms of initial pathogen detection and possible immunoevasion by Bd, degree of activation and efficacy of the innate immune response, the unexpected absence of innate leukocyte infiltration, and the cause and role of late-stage immunopathology in pathogenesis). There are also gaps concerning most of the adaptive immune system (the relative importance of B and T cell responses for pathogen clearance, the capacity and extent of immunological memory, and specific mechanisms of pathogen-induced immunosuppression). Improving our capacity for amphibian immunological research will require selection of an appropriate Bd-susceptible model species, the development of taxon-specific affinity reagents and cell lines for functional assays, and the application of a suite of conventional and emerging immunological methods. Despite current knowledge gaps, immunological research remains a promising avenue for amphibian conservation management

The hookworm Ancylostoma ceylanicum: an emerging public health risk in Australian tropical rainforests and Indigenous communities

Ancylostoma ceylanicum is the common hookworm of domestic dogs and cats throughout Asia, and is an emerging but little understood public health risk in tropical northern Australia. We investigated the prevalence of A. ceylanicum in soil and free-ranging domestic dogs at six rainforest locations in Far North Queensland that are Indigenous Australian communities and popular tourist attractions within the Wet Tropics World Heritage Area. By combining PCR-based techniques with traditional methods of hookworm species identification, we found the prevalence of hookworm in Indigenous community dogs was high (96.3% and 91.9% from necropsy and faecal samples, respectively). The majority of these infections were A. caninum. We also observed, for the first time, the presence of A. ceylanicum infection in domestic dogs (21.7%) and soil (55.6%) in an Indigenous community. A. ceylanicum was present in soil samples from two out of the three popular tourist locations sampled. Our results contribute to the understanding of dogs as a public health risk to Indigenous communities and tourists in the Wet Tropics. Dog health needs to be more fully addressed as part of the Australian Government's commitments to "closing the gap" in chronic disease between Indigenous and other Australians, and encouraging tourism in similar locations

Climatic suitability influences species specific abundance patterns of Australian flying foxes and risk of Hendra virus spillover

Hendra virus is a paramyxovirus of Australian flying fox bats. It was first detected in August 1994, after the death of 20 horses and one human. Since then it has occurred regularly within a portion of the geographical distribution of all Australian flying fox (fruit bat) species. There is, however, little understanding about which species are most likely responsible for spillover, or why spillover does not occur in other areas occupied by reservoir and spillover hosts. Using ecological niche models of the four flying fox species we were able to identify which species are most likely linked to spillover events using the concept of distance to the niche centroid of each species. With this novel approach we found that 20 out of 27 events occur disproportionately closer to the niche centroid of two species (P. alecto and P. conspicillatus). With linear regressions we found a negative relationship between distance to the niche centroid and abundance of these two species. Thus, we suggest that the bioclimatic niche of these two species is likely driving the spatial pattern of spillover of Hendra virus into horses and ultimately humans