Transcriptomics reveal immune downregulation of newts overwhelmed by chytrid co-infection.

What happens when two emergent diseases infect the same host? In a From the Cover article in this issue of Molecular Ecology, McDonald et al. (2020) compare transcriptomic responses to co-infection by the two chytrid fungi in the skin, liver and spleen of Eastern newts (Notophthalmus viridescens). Novel molecular tools, such as high-throughput DNA sequencing for genome discovery and transcriptomics, have revolutionized our understanding of host-pathogen interactions and disease ecology (Güimil et al. 2005; Rosenblum et al. 2012). For example, epidemiologists are using genomic data to track the spread of the emergent SARS-CoV-2 in real time, both locally and globally. RNA sequencing (RNA-Seq) is routinely employed to study response to disease in humans, improving disease diagnostics, profiling and development of intervention strategies. Transcriptomic profiles may be particularly informative for emergent diseases, whose pathologies and effect on host phenotype are poorly known. Fungal pathogens increasingly threaten a variety of wild and domesticated organisms (Fisher et al. 2012), and two chytrid fungi attacking amphibians are causing one of the worst losses of vertebrate biodiversity ever recorded (Scheele et al. 2019).

Population-Level Resistance to Chytridiomycosis is Life-Stage Dependent in an Imperiled Anuran.

Amphibian declines caused by chytridiomycosis have been severe, but some susceptible populations have persisted or even recovered. Resistance to the causal agent Batrachochytrium dendrobatidis (Bd) could result from alleles of the adaptive immune system. During metamorphosis, however, immune systems may not be fully functional, implying that an effective immune response to Bd may be life-stage dependent. We evaluated the susceptibility of the relict leopard frog (Rana onca) sourced from two areas where Bd was present or absent, and where the populations appeared to show differences in pathogen resistance. We evaluated whether population-level resistance manifested across life stages using challenge experiments with late-stage tadpoles (Gosner stage 31-38), metamorphs (stage 45-46), and juvenile frogs. We used three different Bd isolates including one from wild R. onca to challenge juvenile frogs and focused on the isolate from R. onca to challenge tadpoles and resulting metamorphs. We found that juveniles from the Bd exposed population were 5.5 times more likely to survive Bd infection and 10 times more likely to clear infections than those from the area without Bd. In contrast, and regardless of the source area, we observed 98% survivorship of tadpoles, but only 19% survivorship of resulting metamorphs following re-exposure. Given the low survivorship of exposed metamorphs in the laboratory, we speculate on how resistance characteristics, whether adaptive or innate, that do not manifest at each life stage could develop in the wild. We suggest that seasonal high temperatures during times when metamorphosis appears common may modulate the effects of the pathogen during this most susceptible life stage.

Out in the cold and sick: low temperatures and fungal infections impair a frog's skin defenses.

Amphibians worldwide continue to battle an emerging infectious disease, chytridiomycosis, caused by Batrachochytrium dendrobatidis (Bd). Southern leopard frogs, Rana sphenocephala, are known to become infected with this pathogen, yet they are considered 'of least concern' for declines due to chytridiomycosis. Previous studies have shown that R. sphenocephala secretes four antimicrobial peptides (AMPs) onto their skin which may play an important role in limiting susceptibility to chytridiomycosis. Here, we examined (1) the effects of temperature and AMP depletion on infections with Bd and (2) the effects of temperature and Bd infection on the capacity to secrete AMPs in juvenile leopard frogs. Pathogen burden and mortality were greater in frogs exposed to Bd at low temperature but did not increase following monthly AMP depletion. Both low temperature and Bd exposure reduced the capacity of juvenile frogs to restore peptides after monthly depletions. Frogs held at 14°C were poorly able to restore peptides in comparison with those at 26°C. Frogs held at 26°C were better able to restore their peptides, but when exposed to Bd, this capacity was significantly reduced. These results strongly support the hypothesis that both colder temperatures and Bd infection impair the capacity of juvenile frogs to produce and secrete AMPs, an important component of their innate defense against chytrid fungi and other pathogens. Thus, in the face of unpredictable climate changes and enzootic pathogens, assessments of disease risk should consider the potential for effects of environmental variation and pathogen exposure on the quality of host defenses.

The Synchytrium endobioticum AvrSen1 Triggers a Hypersensitive Response in Sen1 Potatoes While Natural Variants Evade Detection.

Synchytrium endobioticum is an obligate biotrophic fungus of division Chytridiomycota. It causes potato wart disease, has a worldwide quarantine status and is included on the Health and Human Services and United States Department of Agriculture Select Agent list. S. endobioticum isolates are grouped in pathotypes based on their ability to evade host resistance in a set of differential potato varieties. Thus far, 39 pathotypes are reported. A single dominant gene (Sen1) governs pathotype 1 (D1) resistance and we anticipated that the underlying molecular model would involve a pathogen effector (AvrSen1) that is recognized by the host. The S. endobioticum-specific secretome of 14 isolates representing six different pathotypes was screened for effectors specifically present in pathotype 1 (D1) isolates but absent in others. We identified a single AvrSen1 candidate. Expression of this candidate in potato Sen1 plants showed a specific hypersensitive response (HR), which cosegregated with the Sen1 resistance in potato populations. No HR was obtained with truncated genes found in pathotypes that evaded recognition by Sen1. These findings established that our candidate gene was indeed Avrsen1. The S. endobioticum AvrSen1 is a single-copy gene and encodes a 376-amino-acid protein without predicted function or functional domains, and is the first effector gene identified in Chytridiomycota, an extremely diverse yet underrepresented basal lineage of fungi.

Metabolites Involved in Immune Evasion by Batrachochytrium dendrobatidis Include the Polyamine Spermidine.

Amphibians have been declining around the world for more than four decades. One recognized driver of these declines is the chytrid fungus Batrachochytrium dendrobatidis, which causes the disease chytridiomycosis. Amphibians have complex and varied immune defenses against B. dendrobatidis, but the fungus also has a number of counterdefenses. Previously, we identified two small molecules produced by the fungus that inhibit frog lymphocyte proliferation, methylthioadenosine (MTA) and kynurenine (KYN). Here, we report on the isolation and identification of the polyamine spermidine (SPD) as another significant immunomodulatory molecule produced by B. dendrobatidis SPD and its precursor, putrescine (PUT), are the major polyamines detected, and SPD is required for growth. The major pathway of biosynthesis is from ornithine through putrescine to spermidine. An alternative pathway from arginine to agmatine to putrescine appears to be absent. SPD is inhibitory at concentrations of ≥10 µM and is found at concentrations between 1 and 10 µM in active fungal supernatants. Although PUT is detected in the fungal supernatants, it is not inhibitory to lymphocytes even at concentrations as high as 100 µM. Two other related polyamines, norspermidine (NSP) and spermine (SPM), also inhibit amphibian lymphocyte proliferation, but a third polyamine, cadaverine (CAD), does not. A suboptimal (noninhibitory) concentration of MTA (10 µM), a by-product of spermidine synthesis, enhances the inhibition of SPD at 1 and 10 µM. We interpret these results to suggest that B. dendrobatidis produces an "armamentarium" of small molecules that, alone or in concert, may help it to evade clearance by the amphibian immune system.

Lymphocyte Deficiency Induced by Sublethal Irradiation in Xenopus.

In many studies of diseases affecting amphibians, it is important to determine to what extent lymphocyte-mediated defenses are involved. For example, in studies of the nature of the immune response of Xenopus laevis to the amphibian chytrid fungus, Batrachochytrium dendrobatidis, it was essential to determine if mucosal antimicrobial peptides or lymphocyte-mediated immunity was most important for resistance to this skin pathogen. In this protocol, we describe a method for sublethal irradiation to reduce lymphocyte numbers. Briefly, X. laevis adults or tadpoles are exposed to 9 Gy (900 rads) of irradiation applied by exposure to a cesium source or gamma irradiator to reduce lymphocyte populations in the spleen.

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.

Non-lethal isolation of the fungal pathogen Batrachochytrium dendrobatidis (Bd) from amphibians.

The ability to isolate and purify pathogens is important for the study of infectious disease. A protocol for isolating Batrachochytrium dendrobatidis (Bd), a lethal pathogen of amphibians, has been available for over a decade, but the method relies on sacrificing infected animals. We validated a non-lethal protocol for Bd isolation that uses biopsy punches from toe webbing to collect skin samples from live amphibians in remote field locations. We successfully isolated Bd from the Cascades frog Rana cascadae and found a positive association between Bd infection and probability of Bd growth in culture. Recapture rates of sampled animals suggest that our isolation protocol did not affect frog survival. The ability to collect isolates from live animals will facilitate investigations of the biology of Bd and enhance amphibian conservation efforts.

Opening the file drawer: Unexpected insights from a chytrid infection experiment.

Infection experiments are critical for understanding wildlife disease dynamics. Although infection experiments are typically designed to reduce complexity, disease outcomes still result from complex interactions between host, pathogen, and environmental factors. Cryptic variation across factors can lead to decreased repeatability of infection experiments within and between research groups and hinder research progress. Furthermore, studies with unexpected results are often relegated to the "file drawer" and potential insights gained from these experimental outcomes are lost. Here, we report unexpected results from an infection experiment studying the response of two differentially-susceptible but related frogs (American Bullfrog Rana catesbeiana and the Mountain yellow-legged frog Rana muscosa) to the amphibian-killing chytrid fungus (Batrachochytrium dendrobatidis, Bd). Despite well-documented differences in susceptibility between species, we found no evidence for antibody-mediated immune response and no Bd-related mortality in either species. Additionally, during the study, the sham-inoculated R. catesbeiana control group became unexpectedly Bd-positive. We used a custom genotyping assay to demonstrate that the aberrantly-infected R. catesbeiana carried a Bd genotype distinct from the inoculation genotype. Thus R. catesbeiana individuals were acquired with low-intensity infections that could not be detected with qPCR. In the Bd-inoculated R. catesbeiana treatment group, the inoculated genotype appeared to out-compete the cryptic infection. Thus, our results provide insight into Bd coinfection dynamics, a phenomenon that is increasingly relevant as different pathogen strains are moved around the globe. Our experiment highlights how unexpected experimental outcomes can serve as both cautionary tales and opportunities to explore unanswered research questions. We use our results as a case study to highlight common sources of anomalous results for infection experiments. We argue that understanding these factors will aid researchers in the design, execution, and interpretation of experiments to understand wildlife disease processes.

Recommendations on diagnostic tools for Batrachochytrium salamandrivorans.

Batrachochytrium salamandrivorans (Bsal) poses a major threat to amphibian, and more specifically caudata, diversity. Bsal is currently spreading through Europe, and mitigation measures aimed at stopping its spread and preventing its introduction into naïve environments are urgently needed. Screening for presence of Bsal and diagnosis of Bsal-induced disease in amphibians are essential core components of effective mitigation plans. Therefore, the aim of this study was to present an overview of all Bsal diagnostic tools together with their limitations and to suggest guidelines to allow uniform interpretation. Here, we investigate the use of different diagnostic tools in post-mortem detection of Bsal and whether competition between Bd and Bsal occurs in the species-specific Bd and Bsal duplex real-time PCR. We also investigate the diagnostic sensitivity, diagnostic specificity and reproducibility of the Bsal real-time PCR and show the use of immunohistochemistry in diagnosis of Bsal-induced chytridiomycosis in amphibian samples stored in formaldehyde. Additionally, we have drawn up guidelines for the use and interpretation of the different diagnostic tools for Bsal currently available, to facilitate standardization of execution and interpretation.

White blood cell profiles in amphibians help to explain disease susceptibility following temperature shifts.

Temperature variability, and in particular temperature decreases, can increase susceptibility of amphibians to infections by the fungus Batrachochytrium dendrobatidis (Bd). However, the effects of temperature shifts on the immune systems of Bd-infected amphibians are unresolved. We acclimated frogs to 16 °C and 26 °C (baseline), simultaneously transferred them to an intermediate temperature (21 °C) and inoculated them with Bd (treatment), and tracked their infection levels and white blood cell profiles over six weeks. Average weekly infection loads were consistently higher in 26°C-history frogs, a group that experienced a 5 °C temperature decrease, than in 16°C-history frogs, a group that experienced a 5 °C temperature increase, but this pattern only approached statistical significance. The 16°C-acclimated frogs had high neutrophil:lymphocyte (N:L) ratios (suggestive of a hematopoietic stress response) at baseline, which were conserved post-treatment. In contrast, the 26°C-acclimated frogs had low N:L ratios at baseline which reversed to high N:L ratios post-treatment (suggestive of immune system activation). Our results suggest that infections were less physiologically taxing for the 16°C-history frogs than the 26°C-history frogs because they had already adjusted immune parameters in response to challenging conditions (cold). Our findings provide a possible mechanistic explanation for observations that amphibians are more susceptible to Bd infection following temperature decreases compared to increases and underscore the consensus that increased temperature variability associated with climate change may increase the impact of infectious diseases.

Major histocompatibility complex variation and the evolution of resistance to amphibian chytridiomycosis.

Chytridiomycosis, caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd), has been implicated in population declines and species extinctions of amphibians around the world. Susceptibility to the disease varies both within and among species, most likely attributable to heritable immunogenetic variation. Analyses of transcriptional expression in hosts following their infection by Bd reveal complex responses. Species resistant to Bd generally show evidence of stronger innate and adaptive immune system responses. Major histocompatibility complex (MHC) class I and class II genes of some susceptible species are up-regulated following host infection by Bd, but resistant species show no comparable changes in transcriptional expression. Bd-resistant species share similar pocket conformations within the MHC-II antigen-binding groove. Among susceptible species, survivors of epizootics bear alleles encoding these conformations. Individuals with homozygous resistance alleles appear to benefit by enhanced resistance, especially in environmental conditions that promote pathogen virulence. Subjects that are repeatedly infected and subsequently cleared of Bd can develop an acquired immune response to the pathogen. Strong directional selection for MHC alleles that encode resistance to Bd may deplete genetic variation necessary to respond to other pathogens. Resistance to chytridiomycosis incurs life-history costs that require further study.

Drivers of salamander extirpation mediated by Batrachochytrium salamandrivorans.

The recent arrival of Batrachochytrium salamandrivorans in Europe was followed by rapid expansion of its geographical distribution and host range, confirming the unprecedented threat that this chytrid fungus poses to western Palaearctic amphibians. Mitigating this hazard requires a thorough understanding of the pathogen's disease ecology that is driving the extinction process. Here, we monitored infection, disease and host population dynamics in a Belgian fire salamander (Salamandra salamandra) population for two years immediately after the first signs of infection. We show that arrival of this chytrid is associated with rapid population collapse without any sign of recovery, largely due to lack of increased resistance in the surviving salamanders and a demographic shift that prevents compensation for mortality. The pathogen adopts a dual transmission strategy, with environmentally resistant non-motile spores in addition to the motile spores identified in its sister species B. dendrobatidis. The fungus retains its virulence not only in water and soil, but also in anurans and less susceptible urodelan species that function as infection reservoirs. The combined characteristics of the disease ecology suggest that further expansion of this fungus will behave as a 'perfect storm' that is able to rapidly extirpate highly susceptible salamander populations across Europe.

Disease dynamics of red-spotted newts and their anuran prey in a montane pond community.

Long-term monitoring of amphibians is needed to clarify population-level effects of ranaviruses (Rv) and the fungal pathogen Batrachochytrium dendrobatidis (Bd). We investigated disease dynamics of co-occurring amphibian species and potential demographic consequences of Rv and Bd infections at a montane site in the Southern Appalachians, Georgia, USA. Our 3-yr study was unique in combining disease surveillance with intensive population monitoring at a site where both pathogens are present. We detected sub-clinical Bd infections in larval and adult red-spotted newts Notophthalmus viridescens viridescens, but found no effect of Bd on body condition of adult newts. Bd infections also occurred in larvae of 5 anuran species that bred in our fishless study pond, and we detected co-infections with Bd and Rv in adult newts and larval green frogs Lithobates clamitans. However, all mortality and clinical signs in adult newts and larval anurans were most consistent with ranaviral disease, including a die-off of larval wood frogs Lithobates sylvaticus in small fish ponds located near our main study pond. During 2 yr of drift fence monitoring, we documented high juvenile production in newts, green frogs and American bullfrogs L. catesbeianus, but saw no evidence of juvenile recruitment in wood frogs. Larvae of this susceptible species may have suffered high mortality in the presence of both Rv and predators. Our findings were generally consistent with results of Rv-exposure experiments and support the purported role of red-spotted newts, green frogs, and American bullfrogs as common reservoirs for Bd and/or Rv in permanent and semi-permanent wetlands.

Mountain Yellow-legged Frogs (Rana muscosa) did not Produce Detectable Antibodies in Immunization Experiments with Batrachochytrium dendrobatidis.

Chytridiomycosis is a devastating infectious disease of amphibians caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd). A growing number of studies have examined the role of amphibian adaptive immunity in response to this pathogen, with varying degrees of immune activation reported. Here we present immunologic data for the mountain yellow-legged frog, Rana muscosa, and the Sierra Nevada yellow-legged frog, Rana sierrae, which are two endangered and ecologically important species experiencing Bd-inflicted declines. Previous studies on these species that examined transcriptional response during Bd infection, and the effective of immunization, provided little evidence of immune activation to Bd. However, the studies did not directly assay immune effectors in the frog hosts. We performed experiments to examine antibody production, which is a hallmark of systemic adaptive immune activation. We used controlled laboratory experiments and enzyme-linked immunosorbent assays to examine the antibody response to Bd immunization and live Bd exposure. Rana muscosa and R. sierrae individuals did not produce detectable antibodies with the capacity to bind to denatured Bd antigens under our experimental conditions. While we cannot rule out antibody response to Bd in these species, our results suggest weak, poor, or inefficient production of antibodies to denatured Bd antigens. Our findings are consistent with susceptibility to chytridiomycosis in these species and suggest additional work is needed to characterize the potential for adaptive immunity.

Immunomodulatory metabolites released by the frog-killing fungus Batrachochytrium dendrobatidis.

Batrachochytrium dendrobatidis is a fungal pathogen in the phylum Chytridiomycota that causes the skin disease chytridiomycosis. Chytridiomycosis is considered an emerging infectious disease linked to worldwide amphibian declines and extinctions. Although amphibians have well-developed immune defenses, clearance of this pathogen from the skin is often impaired. Previously, we showed that the adaptive immune system is involved in the control of the pathogen, but B. dendrobatidis releases factors that inhibit in vitro and in vivo lymphocyte responses and induce lymphocyte apoptosis. Little is known about the nature of the inhibitory factors released by this fungus. Here, we describe the isolation and characterization of three fungal metabolites produced by B. dendrobatidis but not by the closely related nonpathogenic chytrid Homolaphlyctis polyrhiza. These metabolites are methylthioadenosine (MTA), tryptophan, and an oxidized product of tryptophan, kynurenine (Kyn). Independently, both MTA and Kyn inhibit the survival and proliferation of amphibian lymphocytes and the Jurkat human T cell leukemia cell line. However, working together, they become effective at much lower concentrations. We hypothesize that B. dendrobatidis can adapt its metabolism to release products that alter the local environment in the skin to inhibit immunity and enhance the survival of the pathogen.

Larval Environment Alters Amphibian Immune Defenses Differentially across Life Stages and Populations.

Recent global declines, extirpations and extinctions of wildlife caused by newly emergent diseases highlight the need to improve our knowledge of common environmental factors that affect the strength of immune defense traits. To achieve this goal, we examined the influence of acidification and shading of the larval environment on amphibian skin-associated innate immune defense traits, pre and post-metamorphosis, across two populations of American Bullfrogs (Rana catesbeiana), a species known for its wide-ranging environmental tolerance and introduced global distribution. We assessed treatment effects on 1) skin-associated microbial communities and 2) post-metamorphic antimicrobial peptide (AMP) production and 3) AMP bioactivity against the fungal pathogen Batrachochytrium dendrobatidis (Bd). While habitat acidification did not affect survival, time to metamorphosis or juvenile mass, we found that a change in average pH from 7 to 6 caused a significant shift in the larval skin microbial community, an effect which disappeared after metamorphosis. Additionally, we found shifts in skin-associated microbial communities across life stages suggesting they are affected by the physiological or ecological changes associated with amphibian metamorphosis. Moreover, we found that post-metamorphic AMP production and bioactivity were significantly affected by the interactions between pH and shade treatments and interactive effects differed across populations. In contrast, there were no significant interactions between treatments on post-metamorphic microbial community structure suggesting that variation in AMPs did not affect microbial community structure within our study. Our findings indicate that commonly encountered variation in the larval environment (i.e. pond pH and degree of shading) can have both immediate and long-term effects on the amphibian innate immune defense traits. Our work suggests that the susceptibility of amphibians to emerging diseases could be related to variability in the larval environment and calls for research into the relative influence of potentially less benign anthropogenic environmental changes on innate immune defense traits.

A de novo Assembly of the Common Frog (Rana temporaria) Transcriptome and Comparison of Transcription Following Exposure to Ranavirus and Batrachochytrium dendrobatidis.

Amphibians are experiencing global declines and extinctions, with infectious diseases representing a major factor. In this study we examined the transcriptional response of metamorphic hosts (common frog, Rana temporaria) to the two most important amphibian pathogens: Batrachochytrium dendrobatidis (Bd) and Ranavirus. We found strong up-regulation of a gene involved in the adaptive immune response (AP4S1) at four days post-exposure to both pathogens. We detected a significant transcriptional response to Bd, covering the immune response (innate and adaptive immunity, complement activation, and general inflammatory responses), but relatively little transcriptional response to Ranavirus. This may reflect the higher mortality rates found in wild common frogs infected with Ranavirus as opposed to Bd. These data provide a valuable genomic resource for the amphibians, contribute insight into gene expression changes after pathogen exposure, and suggest potential candidate genes for future host-pathogen research.

Development of antimicrobial peptide defenses of southern leopard frogs, Rana sphenocephala, against the pathogenic chytrid fungus, Batrachochytrium dendrobatidis.

Amphibian species face the growing threat of extinction due to the emerging fungal pathogen Batrachochytrium dendrobatidis, which causes the disease chytridiomycosis. Antimicrobial peptides (AMPs) produced in granular glands of the skin are an important defense against this pathogen. Little is known about the ontogeny of AMP production or the impact of AMPs on potentially beneficial symbiotic skin bacteria. We show here that Rana (Lithobates) sphenocephala produces a mixture of four AMPs with activity against B. dendrobatidis, and we report the minimum inhibitory concentration (MIC) of synthesized replicates of these four AMPs tested against B. dendrobatidis. Using mass spectrometry and protein quantification assays, we observed that R. sphenocephala does not secrete a mature suite of AMPs until approximately 12 weeks post-metamorphosis, and geographically disparate populations produce a different suite of peptides. Use of norepinephrine to induce maximal secretion significantly reduced levels of culturable skin bacteria.

Defects in host immune function in tree frogs with chronic chytridiomycosis.

The amphibian chytrid fungus Batrachochytrium dendrobatidis (Bd) has caused mass mortality leading to population declines and extinctions in many frog species worldwide. The lack of host resistance may be due to fungal immunosuppressive effects that have been observed when Bd is incubated with cultured lymphocytes, but whether in vivo host immunosuppression occurs is unknown. We used a broad range of hematologic and protein electrophoresis biomarkers, along with various functional tests, to assess immune competence in common green (Litoria caerulea) and white-lipped (L. infrafrenata) tree frogs experimentally infected with Bd. Compared with uninfected frogs, Bd infection in L. caerulea caused a reduction in immunoglobulin and splenic lymphocyte responses to antigenic stimulation with sheep red blood cells, along with decreased white blood cell and serum protein concentrations, indicating possible impaired immune response capability of Bd-infected frogs. This is the first in vivo study suggesting that infection with Bd causes multiple defects in systemic host immune function, and this may contribute to disease development in susceptible host species. Although L. infrafrenata failed to maintain Bd infection after exposure, white blood cell and serum globulin concentrations were lower in recovered frogs compared with unexposed frogs, but antigen-specific serum and splenic antibody, and splenic cellular, responses were similar in both recovered and unexposed frogs. This may indicate potential systemic costs associated with infection clearance and/or redirection of host resources towards more effective mechanisms to overcome infection. No clear mechanism for resistance was identified in L. infrafrenata, suggesting that localized and/or innate immune defense mechanisms may be important factors involved in disease resistance in this species.