Whether the Weather Drives Patterns of Endemic Amphibian Chytridiomycosis: A Pathogen Proliferation Approach

The pandemic amphibian disease chytridiomycosis often exhibits strong seasonality in both prevalence and disease-associated mortality once it becomes endemic. One hypothesis that could explain this temporal pattern is that simple weather-driven pathogen proliferation (population growth) is a major driver of chytridiomycosis disease dynamics. Despite various elaborations of this hypothesis in the literature for explaining amphibian declines (e.g., the chytrid thermal-optimum hypothesis) it has not been formally tested on infection patterns in the wild. In this study we developed a simple process-based model to simulate the growth of the pathogen Batrachochytrium dendrobatidis (Bd) under varying weather conditions to provide an a priori test of a weather-linked pathogen proliferation hypothesis for endemic chytridiomycosis. We found strong support for several predictions of the proliferation hypothesis when applied to our model species, Litoria pearsoniana, sampled across multiple sites and years: the weather-driven simulations of pathogen growth potential (represented as a growth index in the 30 days prior to sampling; GI₃₀) were positively related to both the prevalence and intensity of Bd infections, which were themselves strongly and positively correlated. In addition, a machine-learning classifier achieved ∼72% success in classifying positive qPCR results when utilising just three informative predictors 1) GI₃₀, 2) frog body size and 3) rain on the day of sampling. Hence, while intrinsic traits of the individuals sampled (species, size, sex) and nuisance sampling variables (rainfall when sampling) influenced infection patterns obtained when sampling via qPCR, our results also strongly suggest that weather-linked pathogen proliferation plays a key role in the infection dynamics of endemic chytridiomycosis in our study system. Predictive applications of the model include surveillance design, outbreak preparedness and response, climate change scenario modelling and the interpretation of historical patterns of amphibian decline.     

Experimental Infection and Repeat Survey Data Indicate the Amphibian Chytrid Batrachochytrium dendrobatidis May Not Occur on Freshwater Crustaceans in Northern Queensland, Australia

Chytridiomycosis is a fatal disease of amphibians, caused by the amphibian chytrid Batrachochytrium dendrobatidis. The disease is unusual in that it may drive many amphibian species to local extinction during outbreaks. These dramatic declines in host population numbers could be facilitated if the pathogen can grow as a saprobe or on alternative hosts, a feature common to other chytrid species. This is also supported by in vitro work that demonstrates B. dendrobatidis can grow and reproduce in the absence of amphibian cells. In a previous study, B. dendrobatidis was detected on freshwater shrimp from rain forest streams in northern Queensland, Australia, using diagnostic PCR. We set out to confirm and further investigate the presence of B. dendrobatidis on crustaceans by carrying out more extensive sampling of shrimp in the field, experimental B. dendrobatidis infection trials using shrimp and crayfish, and PCR verification of the presence of B. dendrobatidis from shrimp samples that previously tested positive. We could not confirm the presence of B. dendrobatidis on shrimp, and report that original positive tests in shrimp reported by Rowley et al. (2006) were likely false. Thus, we suggest that shrimp may not be an important reservoir host for B. dendrobatidis.     

Distribution of life cycle stages of Sarcoptes scabiei var wombati and effects of severe mange on common wombats in Victoria

Seven female and three male common wombats (Vombatus ursinus) collected from forested areas of Victoria (Australia) over a 10 mo period, 10 April 1997 to 22 February 1998 had at least 30% of their skin affected by severe hyperkeratotic sarcoptic mange. Mangy wombats were grazing during the day, could be readily approached, were in poor body condition, and lacked subcutaneous fat. The anterolateral surface of the body was most heavily parasitised with Sarcoptes scabiei var wombati followed by the posterolateral surface, the dorsal region between the ears, the ears, ventral abdomen, medial aspect of the legs, axillary and inguinal areas, and the dorsal midline. Larvae were the most prevalent life-cycle stage followed by eggs, nymphs, females, and males. Mite numbers and the severity of clinical signs, namely thickness of scale crust and the degree of alopecia, were correlated and were symmetrical on each side of the body. Fissuring of crust and skin only occurred when scale crust was present. Bacterial infections occurred in three of 10 wombats within lymph nodes or the pleural cavity. Lymphoid depletion did not occur in lymph nodes or spleens and prescapular lymph nodes contained a greater amount of nuclear debris in germinal centres than non-mangy wombats. Seven wombats had fatty change in their livers. Gonads of mature wombats were not active or had minimal activity. Significant histopathological changes were not seen in the gastrointestinal tract, kidney, brain, myocardium, spleen, thyroid, reproductive tract, and gonads. Hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, and concentrations of hemoglobin, lymphocytes, calcium, glucose, creatinine, total solids, total protein, albumin determined both colormetrically and electrophoretically, and globulins were significantly lower and concentrations of neutrophils, monocytes, phosphorus, urea, glutamate dehydrogenase, aspartate aminotransferase and creatine kinase were significantly higher in mangy versus captive wombats. Concentrations of erythrocytes, mean corpuscular hemoglobin, leucocytes, band neutrophils, eosinophils, nucleated erythrocytes, sodium, potassium, chloride, total bilirubin, alkaline phosphatase, and gamma glutamyltransferase for mangy wombats were not significantly different from that reported for captive wombats. Hematological and pathological changes in mangy wombats were consistent with anemia, inflammation, and changes seen with starvation.     

Clinical response of captive common wombats (Vombatus ursinus) infected with Sarcoptes scabiei var. wombati

Seven common wombats (Vombatus ursinus) were exposed and two of these were re-exposed to Sarcoptes scabiei var. wombati (Acari: Sarcoptidae). For wombats exposed for the first time, five exposed to 5,000 mites on their shoulder developed moderate to severe parakeratotic mange after 11 wk compared with two given 1,000 mites that developed mild clinical signs of mange after 11 wk. For re-exposed wombats, one of two given 5,000 mites developed mild parakeratotic mange and the other developed severe parakeratotic mange. Initial signs of mange were erythema followed by parakeratosis, alopecia, excoriation and fissuring of parakeratotic crust and skin. Erythema usually became apparent within 14 days after exposure (DAE) or within 24 hrs of re-exposure. Parakeratosis was visible 14-21 DAE and alopecia first occurred 35-77 DAE. Clinical signs increased in severity over time and lesions spread slowly from the site of exposure. Mangy wombats scratched excessively, lost weight, and exhibited a significant neutrophilia compared with control wombats. Treatment of mange with three injections of ivermectin, 300 micrograms/kg, 10 days apart led to complete resolution of clinical signs. However mites were not entirely eliminated until wombats received a second regime of treatment.     

Treatment of chytridiomycosis requires urgent clinical trials

Effective and safe treatments of amphibian chytridiomycosis, caused by Batrachochytrium dendrobatidis (Bd), are needed to prevent mortality in captive programs, reduce the risk of disease spread, and better manage the disease in threatened wild populations. Bd is susceptible to a range of antifungal agents and low levels of heat (>30 degrees C) when tested in vitro, but there are few proven methods for clearing adult amphibians of Bd, and acute drug toxicity is a problem for tadpoles and juveniles. In postmetamorphic animals, heat (32 and 37 degrees C) is the only well-supported treatment. Antifungal drugs have not undergone rigorous testing--for example, trials were small or lacked controls and thorough post-treatment testing. In addition, pharmacokinetic studies have not been performed so there are no data on blood or tissue levels of antifungal agents. However, itraconazole baths have been widely used in amphibian rescue and conservation programs and anecdotal evidence suggests that they are effective for adults and subadults. In an experimental trial with tadpoles, a low dose of itraconazole cleared Bd but may have been associated with cutaneous depigmentation. Fluconazole appeared safe for tadpoles as it did not cause mortality, and future attempts to find an effective dose may be worthwhile. Palliative restoration of blood sodium and potassium levels by administration of electrolyte solutions appears useful in frogs with clinical chytridiomycosis. Randomised and blinded clinical trials, which include basic pharmacological studies, are urgently needed to provide comparable evidence for the safety and efficacy of treatment options which are likely to vary with amphibian species. Priorities are to validate and optimize the use of heat and itraconazole regimes.     

Batrachochytrium dendrobatidis: requirement for further isolate collection and archiving

The fungal pathogen Batrachochytrium dendrobatidis (Bd) causes the disease chytridiomycosis, which is lethal to many species of amphibians worldwide. Many studies have investigated the epidemiology of chytridiomycosis in amphibian populations, but few have considered possible host-pathogen coevolution. More specifically, investigations focused on the evolution of Bd, and the link with Bd virulence, are needed. Such studies, which may be important for conservation management of amphibians, depend on access to Bd isolates. Here we provide a summary of known Bd isolates that have been collected and archived in various locations around the world. Of 257 Bd isolates, we found that 53% originate from ranids in the United States. In many cases, detailed information on isolate origin is unavailable, and it is unknown how many isolates are cryo-archived. We suggest the creation of a centralized database of isolate information, and we urge researchers and managers to isolate and archive Bd to facilitate future research on chytridiomycosis.     

Ecological dynamics of emerging bat virus spillover

Viruses that originate in bats may be the most notorious emerging zoonoses that spill over from wildlife into domestic animals and humans. Understanding how these infections filter through ecological systems to cause disease in humans is of profound importance to public health. Transmission of viruses from bats to humans requires a hierarchy of enabling conditions that connect the distribution of reservoir hosts, viral infection within these hosts, and exposure and susceptibility of recipient hosts. For many emerging bat viruses, spillover also requires viral shedding from bats, and survival of the virus in the environment. Focusing on Hendra virus, but also addressing Nipah virus, Ebola virus, Marburg virus and coronaviruses, we delineate this cross-species spillover dynamic from the within-host processes that drive virus excretion to land-use changes that increase interaction among species. We describe how land-use changes may affect co-occurrence and contact between bats and recipient hosts. Two hypotheses may explain temporal and spatial pulses of virus shedding in bat populations: episodic shedding from persistently infected bats or transient epidemics that occur as virus is transmitted among bat populations. Management of livestock also may affect the probability of exposure and disease. Interventions to decrease the probability of virus spillover can be implemented at multiple levels from targeting the reservoir host to managing recipient host exposure and susceptibility.     

Survey protocol for detecting chytridiomycosis in all Australian frog populations

Spread of the amphibian chytrid fungus Batrachochytrium dendrobatidis (Bd) has caused the decline and extinction of frogs, but the distribution of Bd is not completely known. This information is crucial to implementing appropriate quarantine strategies, preparing for outbreaks of chytridiomycosis due to introduction of Bd, and for directing conservation actions towards affected species. This survey protocol provides a simple and standard method for sampling all frog populations in Australia to maximise the chances of detecting Bd. In order to structure and prioritise the protocol, areas are divided by bioregion and frog species are allocated depending on the water bodies they utilize into 3 groups representing different levels of risk of exposure to Bd. Sixty individuals per population need to be tested to achieve 95% certainty of detecting 1 positive frog, based on the minimum apparent prevalence of > or =5% in infected Australian frog populations and using a quantitative real-time TaqMan PCR test. The appropriate season to sample varies among bioregions and will ideally incorporate temperatures favourable for chytridiomycosis (e.g. maximum air temperatures generally <27 degrees C). Opportunistic collection and testing of sick frogs and tadpoles with abnormal mouth-parts should also be done to increase the probability of detecting Bd. The survey priorities in order are (1) threatened species that may have been exposed to Bd, (2) bioregions surrounding infected bioregions/ecological groups, and (3) species of frogs of unknown infection status in infected bioregions. Within these priority groups, sampling should first target ecological groups and species likely to be exposed to Bd, such as those associated with permanent water, and areas within bioregions that have high risk for Bd as indicated by climatic modelling. This protocol can be adapted for use in other countries and a standard protocol will enable comparison among amphibian populations globally.     

Storage of samples at high temperatures reduces the amount of amphibian chytrid fungus Batrachochytrium dendrobatidis DNA detectable by PCR assay

Chytridiomycosis, caused by the amphibian chytrid fungus Batrachochytrium dendrobatidis (Bd), is an emerging infectious disease responsible for amphibian declines on several continents. In laboratory conditions, optimal temperatures for Bd growth and survivorship are between 17 and 25 degrees C. We investigated the effect of different storage temperatures, both in field and laboratory conditions, on detection of Bd from swabs stored for 7 d. We sampled 52 wild Litoria wilcoxii males for Bd by simultaneously running 2 cotton swabs along the skin of the frog. One group of swabs was stored in a freezer within 2 h of sampling and the other was kept in a car in an exposed environment for 7 d before being stored in the freezer. In the laboratory experiment, swabs were inoculated with zoospores of Bd and underwent one of 4 treatments: immediate DNA extraction, or storage at 27, 38 or 45 degrees C for 7 d prior to DNA extraction. Swabs from all treatments were analyzed by quantitative (real-time) PCR test. Though prevalence of Bd did not differ significantly between swabs that were frozen and those that remained in a car for 7 d (19.2 vs. 17.3%, respectively), the number of Bd zoospores detected on car swabs taken from infected frogs was, on average, 67% less than that detected on the corresponding frozen swab. In the laboratory experiment, the number of zoospore equivalents varied significantly with treatment (F(3,35) = 4.769, p = 0.007), indicating that there was reduced recovery of Bd DNA from swabs stored at higher temperatures compared with those stored at lower temperatures or processed immediately. We conclude that failure to store swabs in cool conditions can result in a significant reduction in the amount of Bd DNA detected using the PCR assay. Our results have important implications for researchers conducting field sampling of amphibians for Bd.