Rapid decline and extinction of a montane frog population in southern Australia follows detection of the amphibian pathogen Batrachochytrium dendrobatidis

The amphibian pathogen Batrachochytrium dendrobatidis (Bd) has been implicated as a leading agent of amphibian declines. However, few studies have documented the real-time host/pathogen dynamics during the arrival of Bd and the decline of an amphibian population. Diagnosing population declines is difficult because multiple factors may be involved and data are often inadequate. However, identifying the relative impact among different threatening processes is critical to understanding population declines. We report the decline and extinction of a population of a threatened temperate montane frog species Litoria spenceri in south-eastern Australia. Unlike most threatened species at the time, its ecology and population demography were well understood, and a monitoring programme was in place, enabling prompt detection of the decline and evaluation of its cause. Histology for Bd on a unique series of phalange samples showed that the decline was strongly linked to the arrival and increased prevalence of Bd, estimated to have emerged in the population within 39 days of first detection. Our study provides the first real-time observation of a mass die-off and decline in a temperate Australian species and the first tight estimate of Bd arrival in a declining population in Australia. These findings not only provide further evidence that Bd has contributed to amphibian declines but also demonstrates how Bd may work in concert with other threatening processes, resulting in extinction

Resistance to chytridiomycosis varies among amphibian species and is correlated with skin peptide defenses

Innate immune mechanisms of defense are especially important to ectothermic vertebrates in which adaptive immune responses may be slow to develop. One innate defense in amphibian skin is the release of abundant quantities of antimicrobial peptides. Chytridiomycosis is an emerging infectious disease of amphibians caused by the skin fungus, Batrachochytrium dendrobatidis. Susceptibility to chytridiomycosis varies among species, and mechanisms of disease resistance are not well understood. Previously, we have shown that Australian and Panamanian amphibian species that possess skin peptides that effectively inhibit the growth of B. dendrobatidis in vitro tend to survive better in the wild or are predicted to survive the first encounter with this lethal pathogen. For most species, it has been difficult to experimentally infect individuals with B. dendrobatidis and directly evaluate both survival and antimicrobial peptide defenses. Here, we demonstrate differences in susceptibility to chytridiomycosis among four Australian species (Litoria caerulea, Litoria chloris, Mixophyes fasciolatus and Limnodynastes tasmaniensis) after experimental infection with B. dendrobatidis, and show that the survival rate increases with the in vitro effectiveness of the skin peptides. We also observed that circulating granulocyte, but not lymphocyte, counts differed between infected and uninfected Lit. chloris. This suggests that innate granulocyte defenses may be activated by pathogen exposure. Taken together, our data suggest that multiple innate defense mechanisms are involved in resistance to chytridiomycosis, and the efficacy of these defenses varies by amphibian species

Genetic potential for disease resistance in critically endangered amphibians decimated by chytridiomycosis

Hundreds of amphibian species have declined worldwide after the emergence of the amphibian fungal pathogen Batrachochytrium dendrobatidis (Bd). Despite captive breeding efforts, it is unlikely that wild populations for many species will be reestablished unless Bd resistance increases. We performed a Bd‐challenge study in the functionally extinct southern corroboree frog Pseudophryne corroboree to investigate differences in Bd susceptibility among individuals and populations, identify genetic [major histocompatibility complex (MHC) class I] and genome‐wide variants associated with Bd resistance, and measure genetic diversity and population genetic structure. We found three MHC variants and one MHC supertype associated with Bd infection load and survival along with a suggestively associated single nucleotide polymorphism. We also showed that genome‐wide heterozygosity is associated with increased survival. Additionally, we found evidence of significant population structure among the four P. corroboree populations studied and high MHC genetic diversity. Our results indicate that there are immunogenetic differences among captive southern corroboree frogs; such differences could be manipulated to increase disease resistance and mitigate the significant threat of chytridiomycosis. These results demonstrate a potential long‐term solution to chytridiomycosis that could include breeding more resistant individuals and returning them to the wild

Production of polyclonal antibodies to Batrachochytrium dendrobatidis and their use in an immunoperoxidase test for chytridiomycosis in amphibians

Polyclonal antibodies were produced for diagnosing chytridiomycosis in amphibians. Two sheep and 4 rabbits were inoculated with homogenized whole culture of Batrachochytrium dendrobatidis in Freund¹s complete adjuvant or triple adjuvant. Antisera from all animals reacted strongly with all stages of B. dendrobatidis and stained the walls, cytoplasm, rhizoids and zoospores in an indirect immunoperoxidase test. Significant cross-reactivity occurred only with some fungi in the Chytridiomycota, and there are no members of this phylum besides B. dendrobatidis that infect frogs. The immunoperoxidase stain is a useful screening test when combined with recognition of the morphology and infection site of B. dendrobatidis

Diagnostic assays and sampling protocols for the detection of Batrachochytrium dendrobatidis

Batrachochytrium dendrobatidis is a fungus belonging to the Phylum Chytridiomycota, Class Chytridiomycetes, Order Chytridiales, and is the highly infectious aetiological agent responsible for a potentially fatal disease, chytridiomycosis, which is currently decimating many of the world’s amphibian populations. The fungus infects 2 amphibian orders (Anura and Caudata), 14 families and at least 200 species and is responsible for at least 1 species extinction. Whilst the origin of the agent and routes of transmission are being debated, it has been recognised that successful management of the disease will require effective sampling regimes and detection assays. We have developed a range of unique sampling protocols together with diagnostic assays for the detection of B. dendrobatidis in both living and deceased tadpoles and adults. Here, we formally present our data and discuss them in respect to assay sensitivity, specificity, repeatability and reproducibility. We suggest that compliance with the recommended protocols will avoid the generation of spurious results, thereby providing the international scientific and regulatory community with a set of validated procedures which will assist in the successful management of chytridiomycosis i

Chytridiomycosis causes amphibian mortality associated with population declines in the rain forests of Australia and Central America

Epidermal changes caused by a chytridiomycete fungus (Chytridiomycota; Chytridiales) were found in sick and dead adult anurans collected from montane rain forests in Queensland (Australia) and Panama during mass mortality events associated with significant population declines. We also have found this new disease associated with morbidity and mortality in wild and captive anurans from additional locations in Australia and Central America. This is the first report of parasitism of a vertebrate by a member of the phylum Chytridiomycota. Experimental data support the conclusion that cutaneous chytridiomycosis is a fatal disease of anurans, and we hypothesize that it is the proximate cause of these recent amphibian declines

Experimental exposures of boreal toads (Bufo boreas) to a pathogenic Chytrid fungus (Batrachochytrium dendrobatidis)

One of the major causes of worldwide amphibian declines is a skin infection caused by a pathogenic chytrid fungus (Batrachochytrium dendrobatidis). This study documents the interactions between this pathogen and a susceptible amphibian host, the boreal toad (Bufo boreas). The amount of time following exposure until death is influenced by the dosage of infectious zoospores, duration of exposure, and body size of the toad. The significant relation between dosage and the number of days survived (dose-response curve) supports the hypothesis that the degree of infection must reach a particular threshold of about 107–108 zoosporangia before death results. Variation in air temperature between 12°C and 23°C had no significant effect on survival time. The infection can be transmitted from infected to healthy animals by contact with water containing zoospores; no physical contact between animals is required. These results are correlated with observations on the population biology of boreal toads in which mortalities associated with B. dendrobatidis have been identified