Emerging amphibian diseases in Queensland and host immune response to disease

Emerging infectious diseases are a significant issue for amphibian conservation and the global spread of chytridiomycosis has decimated many frog populations. As well as developing effective disease management strategies, it is crucial to improve surveillance and health monitoring for the early detection and prevention of other emerging diseases. The aims of this research were to investigate emerging and endemic amphibian diseases in Queensland, to evaluate amphibian disease surveillance techniques, and to study the effect of chytridiomycosis on the host immune system.\ud \ud Significant diseases, including two new anuran pathogens, were discovered through the passive disease surveillance network I established at James Cook University in Cairns. Over 160 sick or dead amphibian specimens were received from various sources. Papovavirus particles were identified with electron microscopy in a dermal squamous papilloma from a white-lipped tree frog (Litoria infrafrenata). Subsequent PCR testing to further classify the virus was unsuccessful. This is the first known report describing a viral aetiology for dermal papillomas in anurans. Systemic microsporidiosis was diagnosed histologically in a common green tree frog (L. caerulea), and electron microscopic analysis identified the organism as belonging to the genus Anncaliia. This is the first known report of Anncaliia species infection, and only the third published report of a pathogenic microsporidial infection, in amphibians.\ud \ud A new endemic disease in L. infrafrenata, manifesting as irreversible emaciation, was detected by retrospective analysis of syndromic submission data from a community frog group. I identified the cestode Spirometra erinacei as a likely primary aetiologic agent of this fatal wasting syndrome. Over a six-year period, 877 L. infrafrenata submitted to the community group were classified according to origin, season and presenting category. Irreversible emaciation accounted for 9 % of submissions, but the lack of significant spatial or temporal patterns in case presentation suggests that this is not a currently emerging disease. I also found a high overall prevalence (27 %) of S. erinacei infection in L. infrafrenata populations in northern Queensland. This is the first known report of S. erinacei infection in this species. While community wildlife groups can play a valuable role in urban disease monitoring, passive syndromic surveillance has a number of limitations. All wildlife disease investigations must involve professionals to establish syndromic case definitions, and for diagnostic pathology, complementary active disease surveillance, and data analysis and interpretation.\ud \ud To improve diagnostic capabilities for amphibian disease investigations, I established blood reference intervals for two species of Australian tree frogs, described their leucocyte morphology, and analysed the effects of season, year and parasite status on blood values. This is currently the most comprehensive study that defines haematologic and biochemical reference intervals for amphibians. Blood was collected from reference sample populations of wild adult tree frogs (L. caerulea, n = 80, and L. infrafrenata, n = 66) for manual haematologic, automated plasma biochemical, and serum protein electrophoretic analysis. Intra-erythrocytic haemogregarine infections were found in 19 % of L. infrafrenata, and multiple haematologic and biochemical parameters varied in infected frogs. Wide inter-species and seasonal variations highlight the need to establish species- and season-specific reference intervals in amphibians.\ud \ud I also investigated host immune response to chytridiomycosis. Global spread of the amphibian chytrid fungus (Batrachochytrium dendrobatidis) has caused mass mortality leading to population declines and extinctions in many frog species. Wide variation in susceptibility to chytridiomycosis exists between species, populations and individuals, but the mechanisms of host immunity appear complex and much remains unknown.\ud \ud My first experimental infection trial is the first of its kind to show that B. dendrobatidis reduces systemic adaptive immune function in frogs. I used a range of haematologic and protein electrophoresis biomarkers, along with various functional tests, to assess immune competence in L. caerulea experimentally infected with B. dendrobatidis, and in experimentally exposed L. infrafrenata. Stimulation of infected L. caerulea resulted in a minimal immune system response. Compared with uninfected frogs, B. dendrobatidis infection reduced splenic, white blood cell, acute-phase protein and immunoglobulin responses, indicating a significantly impaired ability of infected frogs to respond adequately to antigenic stimulation. Although L. infrafrenata failed to maintain infection after exposure, sub-clinical immunologic effects occurred in recovered compared with unexposed frogs. This host immune suppression is likely a key factor enabling chytridiomycosis to be a formidable disease with unprecedented effects on biodiversity.\ud \ud In the second experimental infection trial, I found that prior infection with B. dendrobatidis had no significant effect on infection rate compared with naïve frogs, and also appeared to have a long-term adverse effect on host resistance. Litoria caerulea were experimentally infected with B. dendrobatidis, treated to clear infection, and then re-exposed to the pathogen. A greater proportion of exposed frogs (78%) became infected compared with naïve frogs (28%). Furthermore, infected re-exposed frogs had a higher infection intensity that increased at a greater rate compared with infected naïve frogs. A greater proportion of infected naïve frogs (59%) self-cured compared with the infected re-exposed group (0%), indicating a greater ability of naïve frogs to control and clear infection. These results indicate that vaccination-based control programmes for chytridiomycosis are unlikely to be successful.\ud \ud Collaborative research during my study identified fatal terminal pathophysiological changes in experimentally infected L. caerulea. While the virulence of B. dendrobatidis has been clearly demonstrated, the mechanism by which the pathogen kills its host has not been determined. We identified epidermal degeneration, inhibited epidermal electrolyte transport, systemic electrolyte disturbances and asystolic cardiac arrest as the pathogenic mechanisms of mortality in infected frogs. Amphibian skin exchanges respiratory gases, water and electrolytes to maintain homeostasis; this disruption to cutaneous function may be a key factor enabling the pathogen to be lethal to such a large range of host species, including phylogenetically distant amphibian taxa.\ud \ud I validated effective antifungal treatments and discovered a clinical protocol for curing terminally ill amphibians during B. dendrobatidis infection trials. There are few reports of successful treatment of chytridiomycosis, and none that include curing amphibians with severe disease. Three terminally ill L. caerulea with heavy B. dendrobatidis infections were cured using a combination of continuous shallow immersion in chloramphenicol solution, parenteral isotonic electrolyte fluid therapy, and increased ambient temperature. All terminally ill frogs recovered rapidly within five days of commencing treatment. In contrast, five untreated terminally ill L. caerulea with heavy B. dendrobatidis infections died within 24 to 48 hours of becoming moribund. Sub-clinical infections in 15 experimentally infected L. caerulea were cured within 28 days by continuous shallow immersion in chloramphenicol solution without adverse effects. This is the first known report of a clinical treatment protocol for curing terminally ill B. dendrobatidis-infected frogs.\ud \ud I identified key roles of zoological institutions to limit the impact of chytridiomycosis on wild amphibian populations during my review of captive disease management. Prevalence in the international amphibian trade is high and importation of infected frogs into zoos has caused disease epidemics in established amphibian collections. Control strategies for zoos to reduce the risk of pathogen spread must include strict quarantine, hygiene, disinfection and translocation protocols, increased public education, routine surveillance of captive and wild populations for rapid diagnosis of and response to outbreaks, and timely, well-planned captive-breeding and reintroduction programmes with extensive institutional collaboration worldwide.\ud \ud Outcomes from this research fill critical knowledge gaps about systemic adaptive immune function and host resistance in frogs with chytridiomycosis, and about successful clinical treatment of infected frogs. Validation of diagnostic tests and establishment of blood reference intervals, along with assessment of disease surveillance data and techniques, will contribute significantly to the future ability of researchers to detect and investigate emerging and endemic amphibian diseases. These are key results that contribute to global amphibian conservation efforts aimed firstly at managing chytridiomycosis, and secondly at improving disease surveillance and diagnosis.\ud \ud Future investigations are needed to better characterise the irreversible emaciation syndrome in L. infrafrenata, the role of S. erinacei infection in this disease, and the clinical significance of haemogregarines in Australian L. infrafrenata populations. Further work to understand mechanisms of B. dendrobatidis immune suppression should aim to identify and isolate pathogen-specific immunosuppressive factors. Researchers need to confirm whether susceptible amphibians can mount protective adaptive immune responses against B. dendrobatidis and if not, the focus of future research needs to shift beyond this to alternative methods of host immune modulation. Broader research to determine pathophysiology for chytridiomycosis among amphibian species should incorporate field studies with naturally infected susceptible host species, tolerant reservoir species and naturally resistant species. In the short-term, global resources must be dedicated to captive institutions for threatened species breeding programmes and for emergency response to species and population declines. While research is underway to improve in situ management of chytridiomycosis in wild amphibian populations, habitat protection and restoration also remain priorities for amphibian conservation