Early exposure to <Emphasis Type="Italic">Batrachochytrium dendrobatidis</Emphasis> causes profound immunosuppression in amphibians

Fungal pathogens have evolved a broad suite of strategies aiming at evading the host immune response. Amphibians are globally infected by the panzootic chytrid fungus Batrachochytrium dendrobatidis (Bd) and, while robust innate immune defences have been characterised, there is little evidence for the existence of effective adaptive immunity. We determine the immune response of the common midwife toad following challenge by Bd as larvae. Immune function was described for both the cell-mediated and antibody-mediated immune responses following infectious challenge as larval amphibians. While there were no significant differences in the ratio of neutrophils/lymphocytes between infected and uninfected individuals, early exposure of tadpoles to Bd significantly dampened the levels of circulating immunoglobulins (IgM and IgY) in the serum of juveniles after metamorphosis. Our results show that Bd immunosuppresses amphibians when infection occurs as larvae with potentially broad effects on the remodelling of immunity during metamorphosis.     

Prior Infection Does Not Improve Survival against the Amphibian Disease Chytridiomycosis

Many amphibians have declined globally due to introduction of the pathogenic fungus Batrachochytrium dendrobatidis (Bd). Hundreds of species, many in well-protected habitats, remain as small populations at risk of extinction. Currently the only proven conservation strategy is to maintain species in captivity to be reintroduced at a later date. However, methods to abate the disease in the wild are urgently needed so that reintroduced and wild animals can survive in the presence of Bd. Vaccination has been widely suggested as a potential strategy to improve survival. We used captive-bred offspring of critically endangered booroolong frogs (Litoria booroolongensis) to test if vaccination in the form of prior infection improves survival following re exposure. We infected frogs with a local Bd isolate, cleared infection after 30 days (d) using itraconazole just prior to the onset of clinical signs, and then re-exposed animals to Bd at 110 d. We found prior exposure had no effect on survival or infection intensities, clearly showing that real infections do not stimulate a protective adaptive immune response in this species. This result supports recent studies suggesting Bd may evade or suppress host immune functions. Our results suggest vaccination is unlikely to be useful in mitigating chytridiomycosis. However, survival of some individuals from all experimental groups indicates existence of protective innate immunity. Understanding and promoting this innate resistance holds potential for enabling species recovery.     

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.     

Mapping the Global Emergence of Batrachochytrium dendrobatidis,the Amphibian Chytrid Fungus

The rapid worldwide emergence of the amphibian pathogen Batrachochytrium dendrobatidis (Bd) is having a profound negative impact on biodiversity. However, global research efforts are fragmented and an overarching synthesis of global infection data is lacking. Here, we provide results from a community tool for the compilation of worldwide Bd presence and report on the analyses of data collated over a four-year period. Using this online database, we analysed: 1) spatial and taxonomic patterns of infection, including amphibian families that appear over- and under-infected; 2) relationships between Bd occurrence and declining amphibian species, including associations among Bd occurrence, species richness, and enigmatic population declines; and 3) patterns of environmental correlates with Bd, including climate metrics for all species combined and three families (Hylidae, Bufonidae, Ranidae) separately, at both a global scale and regional (U.S.A.) scale. These associations provide new insights for downscaled hypothesis testing. The pathogen has been detected in 52 of 82 countries in which sampling was reported, and it has been detected in 516 of 1240 (42%) amphibian species. We show that detected Bd infections are related to amphibian biodiversity and locations experiencing rapid enigmatic declines, supporting the hypothesis that greater complexity of amphibian communities increases the likelihood of emergence of infection and transmission of Bd. Using a global model including all sampled species, the odds of Bd detection decreased with increasing temperature range at a site. Further consideration of temperature range, rather than maximum or minimum temperatures, may provide new insights into Bd-host ecology. Whereas caution is necessary when interpreting such a broad global dataset, the use of our pathogen database is helping to inform studies of the epidemiology of Bd, as well as enabling regional, national, and international prioritization of conservation efforts. We provide recommendations for adaptive management to enhance the database utility and relevance.     

IPS-1 Is Essential for the Control of West Nile Virus Infection and Immunity

The innate immune response is essential for controlling West Nile virus (WNV) infection but how this response is propagated and regulates adaptive immunity in vivo are not defined. Herein, we show that IPS-1, the central adaptor protein to RIG-I-like receptor (RLR) signaling, is essential for triggering of innate immunity and for effective development and regulation of adaptive immunity against pathogenic WNV. IPS-1^−/− mice exhibited increased susceptibility to WNV infection marked by enhanced viral replication and dissemination with early viral entry into the CNS. Infection of cultured bone-marrow (BM) derived dendritic cells (DCs), macrophages (Macs), and primary cortical neurons showed that the IPS-1-dependent RLR signaling was essential for triggering IFN defenses and controlling virus replication in these key target cells of infection. Intriguingly, infected IPS-1^−/− mice displayed uncontrolled inflammation that included elevated systemic type I IFN, proinflammatory cytokine and chemokine responses, increased numbers of inflammatory DCs, enhanced humoral responses marked by complete loss of virus neutralization activity, and increased numbers of virus-specific CD8+ T cells and non-specific immune cell proliferation in the periphery and in the CNS. This uncontrolled inflammatory response was associated with a lack of regulatory T cell expansion that normally occurs during acute WNV infection. Thus, the enhanced inflammatory response in the absence of IPS-1 was coupled with a failure to protect against WNV infection. Our data define an innate/adaptive immune interface mediated through IPS-1-dependent RLR signaling that regulates the quantity, quality, and balance of the immune response to WNV infection.     

c-di-GMP Enhances Protective Innate Immunity in a Murine Model of Pertussis

Innate immunity represents the first line of defense against invading pathogens in the respiratory tract. Innate immune cells such as monocytes, macrophages, dendritic cells, NK cells, and granulocytes contain specific pathogen-recognition molecules which induce the production of cytokines and subsequently activate the adaptive immune response. c-di-GMP is a ubiquitous second messenger that stimulates innate immunity and regulates biofilm formation, motility and virulence in a diverse range of bacterial species with potent immunomodulatory properties. In the present study, c-di-GMP was used to enhance the innate immune response against pertussis, a respiratory infection mainly caused by Bordetella pertussis. Intranasal treatment with c-di-GMP resulted in the induction of robust innate immune responses to infection with B. pertussis characterized by enhanced recruitment of neutrophils, macrophages, natural killer cells and dendritic cells. The immune responses were associated with an earlier and more vigorous expression of Th1-type cytokines, as well as an increase in the induction of nitric oxide in the lungs of treated animals, resulting in significant reduction of bacterial numbers in the lungs of infected mice. These results demonstrate that c-di-GMP is a potent innate immune stimulatory molecule that can be used to enhance protection against bacterial respiratory infections. In addition, our data suggest that priming of the innate immune system by c-di-GMP could further skew the immune response towards a Th1 type phenotype during subsequent infection. Thus, our data suggest that c-di-GMP might be useful as an adjuvant for the next generation of acellular pertussis vaccine to mount a more protective Th1 phenotype immune response, and also in other systems where a Th1 type immune response is required.     

The Influence of Temperature on Chytridiomycosis In Vivo

Chytridiomycosis, an amphibian disease caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd), is an ideal system for studying the influence of temperature on host–pathogen relationships because both host and pathogen are ectothermic. Studies of Bd in culture suggest that optimal growth occurs between 17 and 23°C, and death of the fungus occurs above 29 or below 0°C. Amphibian immune systems, however, are also temperature dependent and often more effective at higher temperatures. We therefore hypothesized that pathogen load, probability of infection and mortality in Bd-exposed frogs would peak at a lower temperature than that at which Bd grows best in vitro. To test this, we conducted a study where Bd- and sham-exposed Northern cricket frogs (Acris crepitans) were incubated at six temperatures between 11 and 26°C. While probability of infection did not differ across temperatures, pathogen load and mortality were inversely related to temperature. Survival of infected hosts was greatest between 20 and 26°C, temperatures where Bd grows well in culture. These results demonstrate that the conditions under which a pathogen grows best in culture do not necessarily reflect patterns of pathogenicity, an important consideration for predicting the threat of this and other wildlife pathogens.     

Susceptibility to disease varies with ontogeny and immunocompetence in a threatened amphibian

Ontogenetic changes in disease susceptibility have been demonstrated in many vertebrate taxa, as immature immune systems and limited prior exposure to pathogens can place less developed juveniles at a greater disease risk. By causing the disease chytridiomycosis, Batrachochytrium dendrobatidis (Bd) infection has led to the decline of many amphibian species. Despite increasing knowledge on how Bd varies in its effects among species, little is known on the interaction between susceptibility and development within host species. We compared the ontogenetic susceptibility of post-metamorphic green and golden bell frogs Litoria aurea to chytridiomycosis by simultaneously measuring three host-pathogen responses as indicators of the development of the fungus—infection load, survival rate, and host immunocompetence—following Bd exposure in three life stages (recently metamorphosed juveniles, subadults, adults) over 95 days. Frogs exposed to Bd as recently metamorphosed juveniles acquired higher infection loads and experienced lower immune function and lower survivorship than subadults and adults, indicating an ontogenetic decline in chytridiomycosis susceptibility. By corresponding with an intrinsic developmental maturation in immunocompetence seen in uninfected frogs, we suggest these developmental changes in host susceptibility in L. aurea may be immune mediated. Consequently, the physiological relationship between ontogeny and immunity may affect host population structure and demography through variation in life stage survival, and understanding this can shape management targets for effective amphibian conservation.     

Comparative Genomics RNAi Screen Identifies Eftud2 as a Novel Regulator of Innate Immunity

The extent of the innate immune response is regulated by many positively and negatively acting signaling proteins. This allows for proper activation of innate immunity to fight infection while ensuring that the response is limited to prevent unwanted complications. Thus mutations in innate immune regulators can lead to immune dysfunction or to inflammatory diseases such as arthritis or atherosclerosis. To identify novel innate immune regulators that could affect infectious or inflammatory disease, we have taken a comparative genomics RNAi screening approach in which we inhibit orthologous genes in the nematode Caenorhabditis elegans and murine macrophages, expecting that genes with evolutionarily conserved function also will regulate innate immunity in humans. Here we report the results of an RNAi screen of approximately half of the C. elegans genome, which led to the identification of many candidate genes that regulate innate immunity in C. elegans and mouse macrophages. One of these novel conserved regulators of innate immunity is the mRNA splicing regulator Eftud2, which we show controls the alternate splicing of the MyD88 innate immunity signaling adaptor to modulate the extent of the innate immune response.     

Infection risk decreases with increasing mismatch in host and pathogen environmental tolerances

The fungal pathogen Batrachochytrium dendrobatidis (Bd) has caused the greatest known wildlife pandemic, infecting over 500 amphibian species. It remains unclear why some host species decline from disease‐related mortality whereas others persist. We introduce a conceptual model that predicts that infection risk in ectotherms will decrease as the difference between host and pathogen environmental tolerances (i.e. tolerance mismatch) increases. We test this prediction using both local‐scale data from Costa Rica and global analyses of over 11 000 Bd infection assays. We find that infection prevalence decreases with increasing thermal tolerance mismatch and with increasing host tolerance of habitat modification. The relationship between environmental tolerance mismatches and Bd infection prevalence is generalisable across multiple amphibian families and spatial scales, and the magnitude of the tolerance mismatch effect depends on environmental context. These findings may help explain patterns of amphibian declines driven by a global wildlife pandemic.     

The Essential,Nonredundant Roles of RIG-I and MDA5 in Detecting and Controlling West Nile Virus Infection

Virus recognition and response by the innate immune system are critical components of host defense against infection. Activation of cell-intrinsic immunity and optimal priming of adaptive immunity against West Nile virus (WNV), an emerging vector-borne virus, depend on recognition by RIG-I and MDA5, two cytosolic pattern recognition receptors (PRRs) of the RIG-I-like receptor (RLR) protein family that recognize viral RNA and activate defense programs that suppress infection. We evaluated the individual functions of RIG-I and MDA5 both in vitro and in vivo in pathogen recognition and control of WNV. Lack of RIG-I or MDA5 alone results in decreased innate immune signaling and virus control in primary cells in vitro and increased mortality in mice. We also generated RIG-I^−/− × MDA5^−/− double-knockout mice and found that a lack of both RLRs results in a complete absence of innate immune gene induction in target cells of WNV infection and a severe pathogenesis during infection in vivo, similar to findings for animals lacking MAVS, the central adaptor molecule for RLR signaling. We also found that RNA products from WNV-infected cells but not incoming virion RNA display at least two distinct pathogen-associated molecular patterns (PAMPs) containing 5′ triphosphate and double-stranded RNA that are temporally distributed and sensed by RIG-I and MDA5 during infection. Thus, RIG-I and MDA5 are essential PRRs that recognize distinct PAMPs that accumulate during WNV replication. Collectively, these experiments highlight the necessity and function of multiple related, cytoplasmic host sensors in orchestrating an effective immune response against an acute viral infection.     

Innate immunity to Paracoccidioides brasiliensis infection

Innate immunity is based in pre-existing elements of the immune system that directly interact with all types of microbes leading to their destruction or growth inhibition. Several elements of this early defense mechanism act in concert to control initial pathogen growth and have profound effect on the adaptative immune response that further develops. Although most studies in paracoccidioidomycosis have been dedicated to understand cellular and humoral immune responses, innate immunity remains poorly defined. Hence, the main purpose of this review is to present and discuss some mechanisms of innate immunity developed by resistant and susceptible mice to Paracoccidioides brasiliensis infection, trying to understand how this initial host-pathogen interface interferes with the protective or deleterious adaptative immune response that will dictate disease outcome. An analysis of some mechanisms and mediators of innate immunity such as the activation of complement proteins, the microbicidal activity of natural killer cells and phagocytes, the production of inflammatory eicosanoids, cytokines, and chemokines among others, is presented trying to show the important role played by innate immunity in the host response to P. brasiliensis infection.     

After the epizootic: Host–pathogen dynamics in montane tropical amphibian communities with high prevalence of chytridiomycosis

The amphibian fungal disease chytridiomycosis, caused by Batrachochytrium dendrobatidis (Bd), poses a great threat to global amphibian biodiversity. In Peruvian cloud forests of the Kosñipata Valley of Manu National Park where chytrid infection is highly prevalent, we have monitored species-rich amphibian communities since 1996. An epizootic of chytridiomycosis is thought to have caused the disappearance of 35% of species richness in the early 2000s. We investigated the post-epizootic Bd prevalence and infection intensity within the remnant amphibian community from 2008 to 2015, and modeled Bd dynamics as a function of species, season, reproductive mode, life stage, and elevation. Prevalence was higher in 2012–2015 than in 2008–2009, but overall prevalence has remained fairly constant (~50%) post-epizootic. We also found that while prevalence decreased with elevation during the wet season, it generally increased with elevation during the dry season, potentially due to seasonal changes in temperature and precipitation. In aquatic habitats, Bd is likely maintained through a single, stream-breeding, putative reservoir species (which survived epizootics, in contrast to other aquatic-breeding species). The now-dominant terrestrial-breeding species allow Bd to persist and spread in terrestrial habitats, possibly through individual dispersal into naïve areas. We conclude that Bd prevalence in the Kosñipata Valley has stabilized over time, suggesting that Bd is now enzootic. Long-term monitoring of host infection is important because temporal changes in prevalence and infection intensity can cause changes in host species richness and abundance, which in turn may alter the trajectory of host–pathogen dynamics.     

Heterogeneous Occupancy and Density Estimates of the Pathogenic Fungus Batrachochytrium dendrobatidis in Waters of North America

Biodiversity losses are occurring worldwide due to a combination of stressors. For example, by one estimate, 40% of amphibian species are vulnerable to extinction, and disease is one threat to amphibian populations. The emerging infectious disease chytridiomycosis, caused by the aquatic fungus Batrachochytrium dendrobatidis (Bd), is a contributor to amphibian declines worldwide. Bd research has focused on the dynamics of the pathogen in its amphibian hosts, with little emphasis on investigating the dynamics of free-living Bd. Therefore, we investigated patterns of Bd occupancy and density in amphibian habitats using occupancy models, powerful tools for estimating site occupancy and detection probability. Occupancy models have been used to investigate diseases where the focus was on pathogen occurrence in the host. We applied occupancy models to investigate free-living Bd in North American surface waters to determine Bd seasonality, relationships between Bd site occupancy and habitat attributes, and probability of detection from water samples as a function of the number of samples, sample volume, and water quality. We also report on the temporal patterns of Bd density from a 4-year case study of a Bd-positive wetland. We provide evidence that Bd occurs in the environment year-round. Bd exhibited temporal and spatial heterogeneity in density, but did not exhibit seasonality in occupancy. Bd was detected in all months, typically at less than 100 zoospores L⁻¹. The highest density observed was ∼3 million zoospores L⁻¹. We detected Bd in 47% of sites sampled, but estimated that Bd occupied 61% of sites, highlighting the importance of accounting for imperfect detection. When Bd was present, there was a 95% chance of detecting it with four samples of 600 ml of water or five samples of 60 mL. Our findings provide important baseline information to advance the study of Bd disease ecology, and advance our understanding of amphibian exposure to free-living Bd in aquatic habitats over time.     

A Non-Invasive Stress Assay Shows That Tadpole Populations Infected with Batrachochytrium dendrobatidis Have Elevated Corticosterone Levels

Batrachochytrium dendrobatidis (Bd) is a fungus that causes the disease chytridiomycosis and is associated with widespread amphibian declines. Populations vary in their susceptibility to Bd infections, and the virulence of the infecting lineage can also vary. Both of these factors may manifest as a differential physiological stress response. In addition, variation in disease susceptibility across amphibian populations may be influenced by immunosuppression caused by chronic stress imposed by environmental factors. Here, we use a non-invasive water-borne hormone technique to assess stress levels (corticosterone) of free-living tadpole populations that are infected by Bd. We found that corticosterone release rates were higher in infected populations of two species of tadpoles (Alytes obstetricans and A. muletensis) than in an uninfected population for both species. The relationship between corticosterone and the intensity of infection differed between species, with only the infected A. obstetricans population showing a significant positive correlation. The higher corticosterone release rates found in A. obstetricans may be an outcome of infection by a highly virulent lineage of Bd (BdGPL), whereas A. muletensis is infected with a less virulent lineage (BdCAPE). These results suggest that different lineages of Bd impose different levels of stress on the infected animals, and that this may influence survival. The next step is to determine whether higher corticosterone levels make individuals more susceptible to Bd or if Bd infections drive the higher corticosterone levels.     

Variation in phenotype and virulence among enzootic and panzootic amphibian chytrid lineages

The Global Panzootic Lineage of the fungus Batrachochytrium dendrobatidis (Bd-GPL) is threatening amphibians worldwide. In contrast, four lineages (Bd-Brazil, Bd-CH, Bd-Cape, and Bd-Korea) that diverged early in the history of Bd have not yet been directly linked to amphibian declines. Bd likely evolves in response to strong selective pressure imposed by hosts and the environment, leading to differences among pathogen phenotypes and genotypes that may directly affect virulence. Here, we report on variation in phenotype, genotype, and virulence of Bd-Brazil and Bd-GPL. Specifically, we (i) used a controlled infection experiment to compare virulence between one Bd-Brazil and three Bd-GPL isolates on a North American amphibian host (Lithobates sylvaticus), (ii) tested for relative phenotypic and genotypic differentiation among Bd isolates from Brazil, and (iii) tested for possible correlations between environmental variables and Bd phenotypes. We found substantial variation in virulence among Bd-GPL isolates and found that our Bd-Brazil isolate showed virulence comparable to an average North American Bd-GPL. North American hosts infected with a Bd-GPL isolate from Panama did not show significant mortality. Bd phenotypes varied significantly across sampling locations; these phenotypes were neither spatially clustered nor correlated with any environmental variables. Additionally, we found a surprising lack of correlation between genotypic divergence and zoospore and zoosporangium sizes in our sample. Although Bd-Brazil was less virulent infecting L. sylvaticus than one Bd-GPL isolate, this endemic lineage still caused ∼50% mortality in our experimental North American hosts. This indicates that Bd-Brazil has the potential to kill amphibians if introduced to naïve wild populations. Our findings underscore that characterizing virulence of multiple Bd isolates and lineages is important for understanding the evolutionary history and diversity of Bd.     

Retrospective Survey of Museum Specimens Reveals Historically Widespread Presence of Batrachochytrium dendrobatidis in China

Chytridiomycosis, caused by the chytrid fungus Batrachochytrium dendrobatidis (Bd), has been implicated in amphibian population declines worldwide. However, no amphibian declines or extinctions associated with Bd have been reported in Asia. To investigate the history of this pathogen in China, we examined 1,007 museum-preserved amphibian specimens of 80 species collected between 1933 and 2009. Bd was detected in 60 individuals (6.0%), with the earliest case of Bd infection occurring in one specimen of Bufo gargarizans and two Fejervarya limnocharis, all collected in 1933 from Chongqing, southwest China. Although mainly detected in non-threatened native amphibians, Bd was also found in four endangered species. We report the first evidence of Bd for Taiwan and the first detection of Bd in the critically endangered Chinese giant salamander (Andrias davidianus). Bd appears to have been present at a low rate of infection since at least the 1930s in China, and no significant differences in prevalence were detected between decades or provinces, suggesting that a historical steady endemic relationship between Bd and Chinese amphibians has occurred. Our results add new insights on the global emergence of Bd and suggest that this pathogen has been more widely distributed in the last century than previously believed.