Ecdysis or skin sloughing in ectotherms can function as a constitutive innate immune defense mechanism by physically removing skin microbiota (73, 82). Sloughing usually occurs soon after dark, approximately every 3 or 4 days depending on species, and the frequency of sloughing increases with ambient temperature (73). Abnormal sloughing is a clinical sign of chytridiomycosis and corresponds with an increased frequency of sloughing and the production of smaller shed pieces (3, 83, 84). This may be the result of sporangia initiating premature keratinization and cell death in infected epidermal cells, in concert with hyperplasia and stimulated epidermal turnover as observed by electron microscopy (77). Using infra-red video recordings, Ohmer et al. (84) found that while sloughing rate increased with infection, chytridiomycosis did not affect sloughing behavior, duration or rhythmicity, although diseased frogs typically did not eat the sloughed skin as they normally would. Independent of temperature, the extent to which increased sloughing reduces Bd infection loads varies depending on species' intrinsic susceptibility, and sometimes results in clearance of infection (85). As such, Ohmer et al. (84) hypothesized that skin sloughing may be both beneficial and detrimental in the face of chytridiomycosis (for example, by removing pathogens or symbiotic bacteria, and disrupting skin homeostasis). Of interest, the most resistant species they studied, Limnodynastes tasmaniensis, demonstrated increased sloughing rates at lower infectious burdens, which Ohmer et al. (85) suggested may indicate an effective induced defense response. Increased rates of sloughing in warmer environments, induced as an immune defense, or in association with behavioral fever (86, 87) may partly explain the improved clearance of Bd infection at higher temperatures (88).

Naïve frogs (not previously exposed to Bd) are unlikely to express specific mucosal antibodies that bind to and inhibit zoospores (89). However, as occurs in other vertebrates, natural antibodies may be present and may limit initial pathogen burdens. Natural antibodies are polyreactive against highly conserved pathogen epitopes. They are typically encoded by germline genes, and in mammals, they are produced by innate-like B cells (90–92). Little is currently known about their efficacy against Bd in amphibians.

Lysozyme is a constitutively expressed antimicrobial enzyme found in body fluids and mucosal linings. Lysozyme from amphibian skin secretions has potent bactericidal activity (93, 94). Although typically considered an antibacterial enzyme, lysozyme has also been reported to possess antifungal properties (95, 96). Thus, amphibian lysozymes may have similar activity against pathogens such as Bd (11, 31, 97). To date there is little evidence for the role of lysozyme and other phagocyte- or keratinocyte-derived constitutive enzymes (e.g., antimicrobial lectins, secretory phospholipase A₂) in defense against Bd. However, Rosenblum et al. (98) reported an increase in expression of lysozyme genes in the skin of infected Xenopus tropicalis at 21 days post exposure in a transcriptomic study. Grogan et al. (18) similarly identified upregulation of lysozyme C genes in the skin of Litoria verreauxii alpina throughout infection, although the efficacy of this response for limiting Bd infection is currently unknown.

The antimicrobial peptides (AMPs) of vertebrate skin are typically small hydrophobic cationic peptides produced by serous glands that provide non-specific defense against pathogenic organisms (99, 100). Clarke (99) describes four main categories of defensive molecules including alkaloids, steroids, biogenic amines, and other peptides and proteins. The range and quantity of AMPs produced by amphibians is remarkable among vertebrates and has been the target of medical studies for decades, particularly for use in pharmaceutical applications [e.g., (101–103)]. The production of antimicrobial peptides can be induced and modulated by the presence of microbial flora (104) and chronic corticosteroid administration (105). Serous glands release antimicrobial peptides to the skin surface at a low continuous rate, however, mild activation of the sympathetic nervous system (such as alarm caused by a predator cue) is sufficient to stimulate the contraction of gland-associated muscle fibers and the release of larger quantities of AMPs to the skin surface (89, 106). It is unlikely that zoospore invasion alone would be sufficient stimulus to produce this response.

Through in vitro growth inhibition assays, many amphibian peptides and peptide mixtures (at concentrations likely to occur in vivo) have been found to inhibit the growth of various pathogens including Bd as well as other fungi [reviewed in (31, 107)]. Antimicrobial peptide defenses are considered reliable predictors of natural resistance of amphibians to chytridiomycosis. However, the efficacy of AMPs in defense against Bd appears to vary substantially by species and other factors, which may limit the value of AMP data for predicting and mitigating amphibian declines (108–113). These factors may include: (1) intrinsic peptide efficacy against Bd as demonstrated in vitro, (2) concentration, number and type of peptides produced, (3) rate and location of release to the skin surface in response to microbial pathogens, and (4) presence of host- and Bd-secreted proteases that may degrade AMPs (106, 114, 115). For example, depletion of AMPs led to increased infection probability in resistant amphibian species Xenopus laevis (89) and Rana pipiens (116), but not in Pelophylax esculentus and P. lessonae (117). Validation studies involving the functional modification of key AMP molecules are still needed to confirm these associations. Woodhams et al. (118) found that AMP expression differed between infected and uninfected wild-caught Litoria serrata, with infected frogs demonstrating reduced expression. However, they did not identify whether this was a cause or consequence of infection. Ribas et al. (119), Rosenblum et al. (120) identified AMP genes or precursors (including preprocareulein and cathelicidin) via microarray studies of the spleen and skin of frogs. More recently, comparing the anti-Bd activity of skin secretions collected from frogs before and after Bd emergence in Panama, Voyles et al. (15) found a significant increase in inhibitory efficacy post-emergence, consistent with an evolutionary shift in the host immune response. The mechanisms underlying this change in efficacy are unknown but may involve altered concentration and diversity of peptides or improved inhibitory function.

Commensal microbial communities are present in the mucus layer on amphibian skin and may provide another mechanism of constitutive innate immunity against Bd via several mechanisms (32, 61, 121). For example, Meyer et al. (82) reported the cultivation of approximately 0.5–1.7 × 10⁶ bacterial colonies and 1.6–2.6 × 10⁴ fungal colonies per square cm of dorsal skin of Rhinella marina. Interestingly, detected microbial loads were much lower on ventral skin, despite being in more frequent contact with moist substrates than the dorsum. Many bacteria and fungi secrete antimicrobial compounds with repellant and growth-inhibitive properties against pathogenic microbes (122–124). Furthermore, microbiota may also compete directly with Bd, and may functionally change host immune responses (121).

Numerous epibiotic bacterial species isolated from amphibian skin are growth inhibitive for Bd in vitro (125, 126). For example, the bacterial species Janthinobacterium lividum has shown particular promise for the anti-Bd properties of its secreted metabolite violacein, at concentrations greater than 15 μM (127). In clinical Bd exposure experiments, both frogs (Rana muscosa) and salamanders (Plethodon cinereus) inoculated with J. lividum did not become infected (128), whereas depletion of bacteria resulted in high Bd infection intensities (129). This effect also extended to soil augmentation and environmental transfer of bacterial species to some amphibian hosts, thereby inhibiting Bd infection, at least temporarily (130, 131). Despite these promising results, outcomes in other species are mixed. For example, inoculation of Panamanian Golden frogs (Atelopus zeteki; extinct in the wild) with probiotic bacteria was not associated with improved survival (132, 133).

There is some evidence for population-level correlation between the presence of (and proportion of individuals harboring) anti-Bd bacterial species and population declines (134, 135), although not all studies corroborate these findings (136). The presence of diverse bacterial communities in addition to anti-Bd bacterial species on amphibians, and synergy (or antagonism) between bacteria and AMPs, will affect the degree of Bd growth inhibition as demonstrated in vitro (137–139). Other field studies have shown that skin microbial communities differ consistently between Bd-susceptible and resistant/tolerant species, as well as between sites with differing infection histories (121, 140). It is unclear from these studies whether identified bacterial communities promote Bd tolerance or resistance, or are the result of it (121). Further applied work should target probiotic candidate selection and trial bioaugmentation approaches (32, 141, 142).

In the absence of targeted immune evasion, an invading microbe should prompt host recognition. Mechanisms responsible for pathogen recognition induce innate then adaptive immune responses in the host via antigens either secreted, expressed on the pathogen cell surface, or processed after phagocytosis. These antigens often contain widely recognized structural moieties known as pathogen-associated molecular patterns (PAMPs) that are common among different groups of microorganisms. These PAMPs bind to host germline-encoded pattern recognition receptors (PRRs) expressed within or on cells of the innate immune system (macrophages, dendritic Langerhans cells) and nonprofessional immune cells (keratinocytes, fibroblasts) [reviewed in (35)]. In mammals, four classes of PRRs include: (1) Toll-like receptors (TLRs) and (2) C-type lectin receptors (CLRs) within cell membranes, (3) NOD-like receptors (NLRs), and (4) Retinoic acid-inducible gene (RIG)-I-like receptors (RLRs) within the cytoplasm of host cells (148). Binding of PAMPs by PRRs leads to an innate amplifying inflammatory cascade that varies depending on the initial signaling pathways involved. Binding of PRRs may induce signaling pathways and the release of cytokines [pathways such as nuclear factor kappa-light-chain-enhancer of activated B cells [NFκB], and mitogen-activated protein [MAP] kinase]. Binding may also stimulate phagocytosis and destruction of extracellular microorganisms, or cell-mediated cytotoxicity and apoptosis of infected host cells [reviewed in (35)]. Patin et al. (149) presents an extensive review of existing knowledge about fungal recognition in mammals via PRRs, and lists common fungal ligands including β-glucans, zymosan, mannose, phospholipomannan, unmethylated DNA, and chitin.

Little is known about PRRs and their interactions with fungal pathogens in amphibians, although genes homologous to mammalian PRRs are present in model amphibian genomes (e.g., Xenopus spp.). The expression pattern and inducibility of TLRs has been studied to some extent in Xenopus spp. and Rana catesbeiana (51, 150). To date there is little evidence for early upregulation of PRR-coding genes in frogs infected with any pathogens (including Bd), although this may be the result of a lack of early immune cell infiltration rather than a lack of constitutive recognition by nonprofessional immune cells per se. However, studies with detailed kinetic analysis appropriate for examining early innate immune responses in amphibians are currently lacking. For example, Grogan et al. (18) did not find any evidence of differentially expressed Dectin genes (key members of the CLR family for detecting common fungal pathogens) at any point during infection, compared with uninfected control frogs. However, several other putative PRRs (TLRs and NLRs) and their downstream signaling pathways were found upregulated in the skin of frogs during late stage infections (18, 98, 151–153). Representatives of these pathways included mannose-associated genes, fc receptor 5 genes, NFκB subunit genes, caspase 6, 7, and 10 analog genes, and MyD88 pathway genes (18). These results from across the several species studied suggest that putative PRR-encoding genes are present and are likely activated. Although, it is possible that the pathogen actively interferes with these pathways, such as the inhibition of NFκB detected by Rosenblum et al. (120). We currently lack sufficient evidence to determine the efficacy of the early innate immune response to Bd. The detected late gene activation of these downstream signaling pathways may alternatively be associated with cellular stress and trauma or secondary bacterial infections (151), producing damage associated molecular patterns (DAMPs) that are similarly recognized by PRRs and protease-activated receptors (PARs) (154). These findings are consistent with the results of Brem and Parris (155) who showed that toads were less likely to be become infected if the epidermis was scraped (causing erosions) prior to Bd exposure. Priming of the innate immune system in response to trauma (Bd-associated epidermal erosions) may contribute to amplification of the innate response, leading to an exacerbated late-stage response. Similar immune priming may have applications for infection mitigation strategies. The precise characteristics of pathogen recognition and signaling may be further elucidated by detailed kinetic studies.

The complement system constitutes a set of receptors and soluble plasma proteins and enzymes with an important role in defense against pathogens. Complement activation stimulates a rapid cascade of molecular interactions triggered by bound antibodies and PAMPs that results in formation of the membrane attack complex (MAC) and plays a central role in innate defense against fungal infections [reviewed in (156)]. Bound complement components function to agglutinate extracellular pathogens and lyse their cell membranes, as well as attract phagocytes to the locality and enhance their phagocytosis of pathogens via opsonization. Although considered most capable at neutralizing extracellular pathogens, complement C3 binding prior to pathogen intracellularization can activate autonomous immunity within infected cells (via NFκB signaling), ultimately leading to cell destruction via apoptosis (157). The complement cascade may be activated via three mechanisms. The first mechanism is the classical pathway (triggered by antigen-antibody complexes, bacterial lipopolysaccharide, pentraxins such as C-reactive protein [CRP] and serum amyloid, etc.). A second mechanism is the alternative pathway (recognizes pathogen associated patterns or PAMPs). The third mechanism of activation is via the lectin pathway (recognizes carbohydrate structures via mannan-binding lectin [MBL], MBL-associated serine proteases [MASPs], and ficolins) [reviewed in (156)]. It is noteworthy that all elements of the complement system are well conserved across jawed vertebrates (158), such that with Xenopus spp. antiserum, it is possible to use purified mammalian complement to lyse amphibian red blood cells (159).

As an early and rapid defense response, examining the extent of complement activation may be critical for assessing the efficacy of the innate immune response. Several studies thus far have indicated early downregulation of complement pathway genes (98, 119, 120) in the infected skin of several susceptible species. In contrast, Grogan et al. (18) found that gene analogs associated with the alternative complement pathway (venom factor 1 and complement factor B) were upregulated from the early infection stage in a phenotypically more resistant population of Litoria verreauxii alpina frogs, but only upregulated at later infection stages in more susceptible populations (18). This is consistent with studies in a variety of species where complement pathway genes were predominantly upregulated in late-stage infection (151–153). These findings may indicate that the alternative complement pathway plays an important role in defense against Bd in more resistant individuals. However, Bd may downregulate or fail to activate the complement cascade in susceptible individuals, at least until the late infection stage (98). Further research on activation of the alternative complement pathway may provide genetic markers for resistance and opportunities for selective breeding or genetic engineering.

Cytokines are endogenous inflammatory mediators and include lymphokines (such as macrophage activating factor [MAF]), interleukins (ILs), tumor necrosis factors (TNFs), interferons (IFNs), transforming growth factors (TGFs), chemokines, colony stimulating factors (CSFs), polypeptide growth factors (GFs), and stress proteins [including heat shock proteins (HSPs)]. Pro-inflammatory cytokines can act on adjacent cells or distant cells via the systemic circulation to amplify the inflammatory cascade, attract leukocytes to the site of infection, activate pathways involved in blood coagulation, and promote tissue repair [reviewed in (35)].

Experimental studies performed on chytridiomycosis thus far have lacked sufficiently early time-points (i.e., 6–24 h post-exposure) and infection-targeted tissue sampling strategies to evaluate expression of putative cytokines or their encoding genes. However, gene expression studies sampling tissues between 3 and 8 days post exposure have reported limited evidence for upregulation of putative cytokine-encoding genes. These included IL-17A/F-like gene, calcineurin IL-2 inducible gene, HSPs, TNF associated factor (TRAF) and guanylate binding protein IFN inducible gene in spleens of X. tropicalis (98, 119), IFN and IL-associated genes in Rana spp. (120), and IL-1B, IL-17C, and IL-17E, TNFα, IFN and IFN-induced genes, granulocyte colony-stimulating factor, and several chemokine-associated genes in Litoria verreauxii alpina (18). In contrast, studies of late-stage infections demonstrated changes across the spectrum of cytokines (numerous IFNs, ILs, TNFs, and chemokines), with the most dramatic responses observed in skin tissues from the most susceptible individuals (18, 151, 152). Of particular interest, several gene expression studies on multiple species identified significant upregulation of numerous IFN-induced very large GTPase gene analogs throughout infection (18, 151, 160). Interferon-induced GTPase signaling is important for eliminating intracellular pathogens in epithelial cells via their sequestration and destruction within inflammasomes [thought to be especially important for defense against skin fungal pathogens (161)], and thus could be a key mechanism of cell-autonomous immunity in chytridiomycosis (162). From these results, it appears that putative cytokine pathways are active in host response to Bd, although we currently have insufficient data to evaluate their relevance in the immediate post-exposure period. The reported upregulated late-stage cytokine responses may instead represent immunopathology from a dysregulated and non-protective immune response (18, 151).

Recruitment of leukocytes (immune effector cells) to the site of infection is a central component of the host immune response. Leukocytes of the innate immune system include circulating monocytes that differentiate into macrophages at the site of infection, polymorphonuclear phagocytes including neutrophils, eosinophils, and basophils, as well as natural killer cells and mast cells. These leukocytes contribute to recruit lymphocytes at the site of infection, amplify the inflammatory cascade, destroy extracellular pathogens via phagocytosis, and trigger apoptosis of damaged or infected host cells [reviewed in (35)].

The cellular immune response in chytridiomycosis appears inconsistent and is generally mild or decreased across species (48, 163). These studies largely compared skin and blood of Bd-infected and uninfected control frogs. For example, Woodhams et al. (111) found decreased circulating neutrophils and eosinophils, and increased numbers of basophils in infected adult Litoria chloris frogs. Davis et al. (164) and Peterson et al. (165) found increased circulating neutrophils and fewer eosinophils in infected Rana catesbeiana tadpoles and Litoria caerulea adults respectively [Peterson et al. (165) also found low circulating lymphocyte numbers [lymphopaenia]]. These results are consistent with a classic mammalian acute stress response [with the exception of the absence of lymphopaenia in the former study, (166)]. Young et al. (48) found lower circulating total white blood cell numbers in chronically infected adult L. caerulea, with overall impairment of responses on immune stimulation, and relatively higher neutrophil to lymphocyte ratios in infected frogs. These variable results may indicate that other unaccounted factors are playing a role (such as corticosteroid levels), or that species' responses differ.

The cellular immune response within skin tissue appears inconsistent from studies thus far performed. For example, histopathology has revealed a variable mild inflammatory response in 10–40% of skin sites, involving foci with macrophages and few neutrophils. This mild response is also often present near areas of ulceration suggesting a possible association with secondary bacterial infections (77, 151, 167, 168). No evidence of specific leukocyte-associated gene upregulation has been reported during early infections in gene expression studies performed thus far. However, during late-stage infection, Rosenblum et al. (98) found a mild increase in neutrophil-associated genes in the skin and liver of infected Xenopus tropicalis frogs, while Ellison et al. (151) and Grogan et al. (18) found predominantly increased expression of several macrophage and neutrophil associated genes. Taken together, these results indicate that an early leukocyte response is weak or lacking with Bd infection, and furthermore, that the late-stage response is inconsistent and likely insufficient to limit Bd infection (and may alternatively be associated with epidermal damage or secondary bacterial infection). This overall observed poor inflammatory response with Bd infection could be the result of inadequacy of innate immune activation with minimal cytokine-mediated leukocyte recruitment toward Bd. This could possibly be associated with immunoevasion and/or active suppression of immune responses by Bd. However, a limited innate immune response, particularly in late stage infections, may also be symptomatic of an inadequate or impaired adaptive immune response to Bd (discussed below).

The primary components of the adaptive immune system include lymphocytes (T and B cells) and their respective mature effector forms responsible for enacting pathogen-specific cell-mediated immunity (cytotoxic or helper T cells) and humoral immunity (antibody-secreting plasma cells) together with secreted or membrane-bound antibodies (immunoglobulin). Unlike the germline-encoded components of the innate immune system, the adaptive immune system is characterized by unique antigen receptors. T and B cell receptors are generated when segments of immunoglobulin genes are rearranged by a unique process called somatic recombination. This can occur via V, D, and J (variable, diversity, joining) mechanisms, which require products from recombination-activating genes (RAGs). This gives rise to millions of naïve T and B cells during the development of an individual, with numerous distinct cell clones bearing unique surface receptors that together constitute the unique lymphocyte receptor repertoire of the host. Lymphocytes activated by antigen binding in combination with co-stimulatory molecules (Figure 1C) undergo clonal expansion and differentiation into their effector forms (Figure 1D). B cell receptors additionally undergo further changes after activation. This involves somatic hypermutation mediated by activation-induced cytidine deaminase (AICDA), which is accompanied by class switching during affinity maturation. During this clonal expansion process, large numbers of antigen-specific long-lived memory lymphocytes are produced (Figure 1E), and upon re-exposure to the pathogen or antigen, these memory lymphocytes respond more rapidly and effectively than the initial response (Figure 1F). Thus, not only is the adaptive immune system able to respond in a specific way to novel pathogens, but it adapts to those pathogens during the course of an infection. Assuming that the individual survives initial infection, a functioning adaptive immune response should increase in efficacy with subsequent exposures to a pathogen (or antigen) during an individual's lifetime (unlike the innate immune response), leading to the concept of immunization [or vaccination; reviewed in (35)].

Initial activation of the adaptive immune system involves the detection of pathogen-derived antigens by binding to PRRs expressed by host cells. Antigen binding stimulates endocytosis, degradation of the pathogen, and subsequent presentation of the antigen peptide on the cell surface via major histocompatibility complex (MHC) proteins. The two classes of MHC molecules, classes I and II, are expressed differently by cells of the body, and interact with different subsets of lymphocytes. MHC class I molecules are expressed by most nucleated somatic cells (for example, non-immune epithelial cells) and interact with CD8 cytotoxic T cells, leading to cytotoxicity and death of the host cell expressing antigen with the MHC class I molecule. MHC class I molecules are particularly important for recognition and elimination of intracellular pathogens via cell killing. MHC class II molecules are mainly expressed by professional immune cells such as dendritic cells and macrophages. These cells also recognize pathogens, but can present the antigen via the MHC class II molecules at the cell surface [reviewed in (35)]. Antigen binding then promotes differentiation of dendritic cells into antigen presenting cells (APC), and TNF-α stimulates APC migration to the spleen via the lymphatic or circulatory system where they contact lymphocytes with a variety of antigen-specific receptors (Figure 1C). MHC class II molecules bound to antigen on the surface of APCs interact with CD4 T helper cells in the presence of other co-stimulatory molecules, and their main function is to activate other immune effector cells (such as B cells; Figure 1C). Co-stimulatory molecules are expressed on APCs in response to mediators of the innate immune system (such as TLRs and NFκB), and they are essential for the activation of the naïve CD4 T helper cells.

The diversity of MHC proteins expressed by cells is generated by polygeny (the presence of multiple interacting genes), allele codominance, and gene polymorphism [reviewed in (35)]. The evolution of MHC genes has been widely demonstrated to occur under selection by infectious diseases (171). Their inter-generational heritability (unlike T and B cell receptors) makes them important bridging elements between the innate and adaptive immune systems, and potential markers for selection of either resistance or tolerance to infection (14, 34, 172). Genes encoding MHC classes I and II molecules have been found to be upregulated throughout Bd infection, particularly within skin tissues (18, 34, 120, 151, 153). A variety of studies have demonstrated associations between characteristics of MHC alleles (allelic diversity, degree of heterozygosity, presence of certain alleles, presence of certain protein conformational elements) and degree of Bd susceptibility as it differs between species, populations and individuals (17, 20, 21, 173). Furthermore, several studies have demonstrated signals of positive selection at certain MHC loci in populations persisting with enzootic Bd, when compared with background levels of neutral genetic change (17, 20, 21). These findings suggest that certain MHC genes and alleles may play an important role in determining degree of Bd susceptibility, and that these are under directional selection for resistance or tolerance to Bd. However, recent recognition of the expansion of MHC class I-like genes in Xenopus spp. and presumably other ectothermic vertebrates [see Box 3; 64, 65] may require a revisit of some of the reported studies, especially transcriptomics. Indeed, MHC class I-like genes encode molecules with typical MHC primary structures but are polygenic and not or minimally polymorphic (66).

When lymphocytes are activated in the presence of peptidic antigen bound to MHC with appropriate co-stimulation, they proliferate by clonal expansion, differentiate into their effector type and migrate to the site of infection (Figure 1D). CD8 T lymphocytes stimulated by MHC class I-bound antigens differentiate into cytotoxic T cells that recognize and kill infected host cells. This form of cell-mediated immunity is likely to be especially important for intracellular pathogens such as Bd, as the most efficient means to eliminate the reproductive stage of the pathogen (zoosporangium) is to destroy infected host cells (31). However, B cells and their differentiated effector type (plasma cells) are likely to be similarly important for eliminating Bd. Unlike in mammals, amphibian B cells demonstrate phagocytic capabilities (58). B cells may also act as antigen-presenting cells for T helper cells, and their effector plasma cells produce antibodies (immunoglobulin, either membrane-bound or secreted) that may be capable of targeting and destroying extracellular pathogen stages such as zoospores and secreted toxins, as well as killing infected host cells. Antibodies target pathogens and kill infected host cells via a suite of mechanisms including (1) binding specifically with the epitope of the antigen and causing the antigens to agglutinate, inactivating them, (2) activating the classical complement cascade, leading to the membrane attack complex to lyse pathogens directly, and (3) antibody-dependent cell-mediated cytotoxicity (ADCC) involving tagging antigens for destruction by natural killer cells or phagocytes [reviewed in (35)]. Thus, both cell-mediated adaptive immunity (T cell elimination of the intracellular reproductive stage of Bd and B cell phagocytosis) and antibody-dependent humoral immunity [destroying zoospores and secreted products) are likely to be important for controlling Bd burdens (31)].

Although it has been suggested that herd immunity may protect populations if > 80% of frogs are immune (or resistant, through the effects of symbiotic bacteria) to Bd (134, 135, 174), there is currently little evidence to suggest that herd immunity operates in wild amphibian populations. Indeed, the existence of a herd immunity threshold relies on infection transmission being density-dependent, rather than frequency-dependent, as is expected for amphibian breeding aggregations (175). Similarly, herd immunity thresholds are unlikely to occur where the force of infection is unaffected by the presence of resistant individuals, as is the case for indirectly transmitted pathogens and those with multiple host species (or life-stages) with differing tolerance and susceptibility to Bd infection. Instead, the temporal patterns of enzootic Bd infection appear regulated by season and temperature rather than adaptive immunity in field populations (22). The few laboratory studies performed to date support these findings, suggesting limited activation of a protective adaptive immune response to chytridiomycosis. Young et al. (48) reported a decrease in total and IgY serum antibody responses [via anti-sheep red blood cells [SRBC] haemagglutination assay] in Bd-infected L. caerulea compared with uninfected frogs, while circulating numbers of lymphocytes were greatly reduced in infected frogs (48, 165). Results from histopathology of the skin showed only a mild response with foci of lymphocytes associated with regions of ulceration, or no evidence of lymphocytes (77, 163, 167, 168). In terms of gene expression results, Rosenblum et al. (98) found no change in lymphocyte markers or MHC genes in X. tropicalis. Results were similar in their second study on Rana spp. (120) despite mild upregulation of MHC class II genes in the skin during late-stage infection. Ribas et al. (119) found that adaptive immune genes were generally down-regulated in the spleen of X. tropicalis throughout infection. In contrast, other studies demonstrated upregulation of numerous adaptive immune genes associated with B and T lymphocytes, immunoglobulins and MHC genes, particularly in skin tissues at late stages of infection (18, 151, 160, 176). However, a countering signal of downregulated T cell associated genes was also detected in several studies (18, 151, 152). These conflicting results indicate a more complex set of interactions operating within the adaptive immune system, which may be associated with the different temperatures at which animals were exposed or housed as well as the timing of collection of samples. The latter finding of downregulated T cell responses is particularly important and will be discussed in detail in its own section below.

Immunization against Bd was suggested early on as a management strategy for chytridiomycosis (177) given the highly successful examples from humans and domestic animals (178). Studies reported by Rollins-Smith et al. (31) and Ramsey et al. (89) attempted to immunize Xenopus laevis frogs against chytridiomycosis via an intraperitoneal injection with heat-killed Bd. They found promising results with a high-titer pathogen-specific IgM and IgY serum antibody response in the immunized frogs at 14 days post final immunization. In another experiment, Bd-binding mucosal antibodies (IgM, IgY, and IgX) were demonstrated after repeated exposure to Bd (89). These results are supported by the finding of upregulated immunoglobulin genes in re-exposed Atelopus zeteki frogs, suggesting production of memory lymphocytes (151). Furthermore, X-irradiation of frogs to impair T cells increased Bd infection loads in X. laevis (89). In contrast, a repeat experiment with killed-Bd injections into the dorsal lymph sac (days 0 and 14) and peritoneum (day 28) of X. laevis followed by splenocyte culture with Bd showed generally weak lymphocyte proliferation in comparison with samples cultured with phytohaemagglutinin (PHA) alone (31). In another experiment, young boreal toads (Bufo boreas) were immunized following a similar protocol and then exposed to Bd, however there was no evidence for a difference in survival between the immunized and sham-injected exposed frogs, suggesting that the immunization had not been successful in stimulating protective adaptive immunity in the young toads (31). Stice and Briggs (179) immunized Rana muscosa with formalin-killed Bd in combination with adjuvants [saline, Freunds Complete [FCA], and Incomplete Adjuvant [FIA]] by injection into the dorsal lymph sac and found no differences in the proportion of frogs infected nor time to infection. A study by Cashins et al. (180) did not detect any evidence for a protective effect of prior infection on re-exposure in Litoria booroolongensis. However, a study in B. boreas by Murphy et al. (181) found that previously exposed frogs survived slightly longer if they had a dry habitat option upon re-exposure. A study by McMahon et al. (182) found that multiple prior exposures to Bd slowed the rate of progression of chytridiomycosis, although this finding may instead be associated with repeated innate immune priming through trauma (155). The variable results of these studies may be associated with differing routes of immunization or dose-rates of Bd exposure. Furthermore, these results suggest that although the adaptive immune system may be activated during Bd infections in some species, the capacity for a robust and protective adaptive response appears limited, which may be associated with Bd-induced suppression (discussed below).

There is no evidence to suggest that immunosuppression is necessary to predispose amphibians to chytridiomycosis epizootics, particularly with numerous observations of disease emergence in abundant species in undisturbed naïve localities (3, 163, 183). Furthermore, signs indicative of generalized immunosuppression, such as secondary bacterial infections, appear to be largely lacking (8, 48, 88). However, stress-induced immunosuppression may play a role particularly in the infection of more resistant individuals and species [reviewed in (30)]. The extent to which environmental stressors and corticosterone mediate chytridiomycosis and its effects on amphibians is currently unclear. Environmental stressors (poor nutritional status, high densities and exposure to predator cues) have been putatively linked with elevated corticosterone and reduced immune capacity in some tadpole studies (184, 185), although it is unknown whether corticosterone is a direct mediator of these effects. Elevations in corticosteroids have been demonstrated to have a range of detrimental effects on the immune system of frogs, including inhibiting the humoral response, and reducing both numbers and viability of circulating lymphocytes [reviewed in (30)]. Indeed, exogenous application of corticosterone was found to increase Bd infection abundance in adult amphibians (105, 186), but only had sublethal effects on tadpoles (187). Gabor et al. (188) inhibited corticosterone synthesis (using metyrapone) and found that this did not prevent Bd-associated reductions in mass, although it did increase Bd loads. They concluded that the adverse effects of Bd on growth were not mediated by corticosterone.

In the field, non-invasive measures of corticosterone in free-living populations of tadpoles revealed that corticosterone levels correlated both with Bd infection and altitude, and that infections with a more virulent strain of Bd (BdGPL) led to higher corticosterone release (189, 190). Measuring urinary corticosterone, Graham et al. (191) and Kindermann et al. (192) similarly found higher levels in infected frogs as well as frog populations at higher altitudes. Furthermore, Peterson et al. (165) measured plasma corticosterone and found that diseased frogs (showing clinical signs of chytridiomycosis) demonstrated higher corticosterone levels than subclinically infected frogs. Thus, from current evidence it appears that elevated corticosterone correlates with infection in situ, and both predisposes to chytridiomycosis, and is a result of infection. However, the link between putative environmental stressors and elevated corticosterone is less robust, and elevated corticosterone does not appear to mediate the sublethal effects of chytridiomycosis (growth and mass).

Throughout this review, we have synthesized the results of numerous studies and highlighted the lack of a generally robust and protective immune response to Bd infection. We considered the epidemiology, general degree of inflammation, as well as markers of the innate immune response during early infection stages, and the adaptive immune response during late infection stages. This observed apparent lack of immune response may be the result of either (1) the failure of the host to recognize Bd as a pathogen, through low inherent antigenicity (possibly due to intracellular localization), immunoevasion, or masking antigens, or (2) Bd-induced immunosuppression or downregulation of key immune responses necessary for a protective immune response (145). Both of these mechanisms may occur in parallel in chytridiomycosis. For example, PRRs are generally not upregulated in early infection, suggesting a possible lack of pathogen recognition, whereas T cell responses appear actively suppressed or inhibited, as are complement-associated pathways.

Current evidence supports a specific role for Bd-induced immunosuppression, detected first via skin histopathology (77) and general immune function measures (31, 48), and corroborated via gene expression data (18, 98, 119, 120, 151, 153). Further experimental work has characterized at least one mechanism by which this might occur, via soluble Bd-secreted factors. Fites et al. (193) demonstrated that soluble factors released by Bd zoosporangia inhibited proliferation and/or caused apoptosis of T cells in vitro. For this work, they used in vitro immune experiments involving the proliferation of splenic lymphocytes (from X. laevis and R. pipiens) in culture. Interestingly, they found that macrophage phagocytosis was not similarly affected. Another study investigated apoptosis (via TUNEL and caspase assays) and found that programmed cell death was positively associated with infection load and morbidity (194). They speculated that apoptosis may thus be a pathogen virulence mechanism. In vivo studies also revealed immune inhibition activity associated with Bd supernatants by measuring delayed-type-hypersensitivity responses (33). Rollins-Smith et al. (195) then went on to characterize two metabolites (methylthioadenosine and kynurenine) produced by Bd that are capable of inhibiting lymphocyte proliferation and survival in vitro.

Despite relative Bd-associated immunosuppression, several gene expression studies on a variety of amphibian species have demonstrated that susceptible individuals express both greater number and variety of dysregulated immune genes during late stage infections than more resistant individuals (18, 151, 152). This negative correlation between extent of immune response and degree of phenotypic susceptibility suggests that susceptible individuals may mount massively dysregulated and non-protective immune responses. This immunopathology is likely associated with DAMPs induced late in infection as the pathogen damages skin cells in order to release subsequent generations of zoospores. Such a dysregulated response may rapidly disrupt cellular homeostatic mechanisms. Indeed, recent metabolomics findings support this hypothesis by demonstrating the significant depletion of the “immune nutrient factor,” alpha-ketoglutarate and its associated metabolite glutamate in severely infected animals (196, 197). This metabolic dysregulation has carry-on effects on numerous other aspects of cell homeostasis, particularly cellular energy metabolism (alpha-ketoglutarate is a key intermediate of the Krebs cycle). Furthermore, in vitro and gene expression studies suggest massive disruption of homeostatic mechanisms involved in epithelial stability, water and ion transport and musculoskeletal functions in susceptible individuals (18, 41, 98, 120, 151–153, 176). Therefore, immunopathology within susceptible amphibian species may not only cause their immune responses to be ineffective at eliminating the pathogen, but it may contribute to host morbidity and mortality due to the extensive disruption of cellular homeostasis and consumption of energy resources.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.