With the availability of B. dendrobatidis’s full genome, genome based studies have led to an improved understanding of host-pathogen dynamics and the identification of several putative pathogenicity factors with high specificity for skin-related substrates, facilitating colonization or causing host damage. Nevertheless, processes taking place during the whole infection process at molecular and cellular level such as cell signaling, induction of cytoskeletal change and so on, are still less well or barely understood and definitely merit more attention. As research on the recently emerged B. salamandrivorans was launched only 2 years ago, our current knowledge is still at its infancy. For now, we are still groping in the dark about, for example, what factors make B. salamandrivorans specific to salamanders and newts, how infection is established and ultimately leads to mortality, and whether or not effective immune responses are elicited during infection. Identification of pathogenicity factors in B. salamandrivorans, involved in its pronounced clinical manifestation and its rapid disease progression, is still long coming. The genome of B. salamandrivorans is not yet fully sequenced but whenever available, comparative studies of both fungi’s expression profiles may considerably accelerate our insight in factors underlying their differential disease dynamics.

Colonization of amphibian skin

The different steps in the infection process by B. dendrobatidis comprise attraction to a suitable host, attachment of zoospores to the host skin, zoospore germination, germ tube development and penetration into the skin cells, followed by invasive growth in the host skin, finally resulting in the loss of the host cell cytoplasm. In this chapter, each of these crucial steps will be discussed in detail.

Interaction with the mucus barrier

Directed movement or chemotaxis of flagellated pathogens towards a suitable host and nutrient substrate are often crucial in the establishment of colonization (for review see [67]). There is proof of B. dendrobatidis responding positively to certain cues from host or vector origin. Chemotaxis to keratinous toe scales of geese [28] as well as to commercially available keratin and its main constituent amino acid, cysteine [68] has been reported. As numerous bacterial pathogens are found to be attracted to mucus, with some pathogens able to metabolize components of mucus [67], the question arises whether B. dendrobatidis could also migrate actively towards the mucous layer covering the amphibian epidermis or one of its components. In search of a suitable amphibian host, B. dendrobatidis zoospores indeed will first come into contact with skin mucus. The main component of mucus are mucins or mucin glycoproteins. In the African clawed-frog (X. laevis) the carbohydrate portion of mucins includes the sugars α-l-fucose, α-d-N-acetylgalactosamine, β-d-N-acetylglucosamine, N-acetylneuraminic acid or sialic acid, α-d-galactose and α-d-mannose. This spectrum of sugars, constituting the so-called integumental free sugars, was also found in its upper epidermis, and in the epidermises of the caecilian Ichtyophis kohtaoensis, the smooth newt (Lissotriton vulgaris) and the edible frog (Pelophylax kl. esculentus) [69]. Indeed, using a disc method, positive migration of B. dendrobatidis towards skin mucus isolated from X. laevis was observed (Table 1; also see Additional file 3). Using a capillary tube chemotaxis assay combined with real-time PCR quantification we found that the free sugars in mucus and amphibian skin are chemotactic (Fig. 6; also see Additional file 4). The odds of B. dendrobatidis zoospores being attracted to sugar were approximately 3–9 times higher compared to water (Table 2). Amphibian skin mucus not only offers protection against abrasive damage and dehydration, but is also thought to serve as a critical barrier against colonization by pathogens. Mucus contains several interdependent host factors including antimicrobial peptides (AMPs), lysozymes and mucosal antibodies as well as microbial-community factors, including symbiotic skin bacteria producing antifungal metabolites (for review see [70]), which will be discussed in a later part of this review (Sect. 4.3). This micro-ecosystem of the mucus is referred to as the mucosome. Before zoospores can establish a successful colonization of the host skin, they must first resist the defense factors of the mucosome. The mucosome in its entirety may reduce the infection load on the skin during the first 24 h that are critical for colonization and establishing skin infection [24]. Indeed, in vitro exposure of B. dendrobatidis zoospores to the skin mucus, covering the epidermis of X. laevis, with all residing defenses in physiological concentrations, causes up to a 3 to 20-fold reduction in the amount of viable zoospores within 2–24 h after exposure (Fig. 7; also see Additional file 5). All in all, these data suggest that skin mucus plays a dual role in pathogenesis: although mucus is attractive for zoospores, it constitutes a defensive barrier, limiting invasion to the underlying epithelium by trapping and reducing the amount of infective zoospores on the skin.

Table 1 Chemotaxis of B. dendrobatidis zoospores towards mucus and water Full size table

Fig. 6 Chemotaxis of Batrachochytrium dendrobatidis toward free integumental sugars. The sugars α-d-mannose (Man), α-d-galactose (Gal), α-l-fucose (Fuc), β-d-N-acetylglucosamine (GluNAc), α-d-N-acetylgalactosamine (GalNAc), N-acetylneuraminic acid (NeuNAc) or sialic acid were tested as attractans at a 0.1 M concentration, using a traditional capillary tube test. Water was used as vehicle and controle attractans. Genomic equivalents (GE) of B. dendrobatidis zoospores in the capillaries were quantified after a 90 min using quantitative real-time PCR. Mean ± standard error of three independent experiments are presented Full size image

Table 2 Predictors of chemotaxis Full size table

Fig. 7 Zoosporicidal activity of Xenopus laevis skin mucus at physiological concentrations. Killing activity is expressed as log(10) viable spores added to the skin secretions—log(10) viable spores recovered after 2 and 24 h incubation. Results are presented as mean genomic equivalents of B. dendrobatidis ±standard error (SEM). Sample size (n) for time point (T) = 3 Full size image

Adherence to host surfaces

So far, the mechanisms and kinetics of adhesion of B. dendrobatidis to amphibian skin have only received limited attention. Adhesion has been documented in explanted amphibian skin and occurs within 2–4 h after exposure to zoospores [24]. Zoospores mature into thick-walled cysts on the host epidermis and often cluster in foci of infection. Cysts are anchored to the skin surface by fine fibrillar projections as shown in Fig. 8 (also see Additional file 6) that resemble the fibrillar adhesins documented for pathogenic fungi affecting human skin e.g. Trichophyton mentagrophytes (reviewed in [71]). However, the composition of these fibrils remains to be defined. Several genes encoding proteins involved in cell adhesion such as vinculin, fibronectin and fasciclin have been identified in the B. dendrobatidis genome, and are brought more to expression in sporangia than in zoospores [72]. When grown on pulverized host tissue, at least 11 potential adhesion genes which are almost all specific to B. dendrobatidis, show an increased expression [73] and require further characterization. The gene expression of B. dendrobatidis has not yet been mapped during its early interactions with amphibian skin (neither in vivo nor in vitro) and as such the exact factors mediating adhesion remain uncertain. Adherence mechanisms may include the action of mainly agglutinin-like and lectin-like proteins as described for numerous pathogens. Further research should focus on the identification of adhesins and their respective receptors on the host surface, especially since these insights could open new perspectives for prevention and treatment of chytrid infection.

Fig. 8 Adhesion of Batrachochytrium dendrobatidis to Xenopus laevis skin. Adhesion to the epidermal surface is established both by tubular projections, possibly adhesins (black arrow) and rhizoids (white arrow). Some encysted zoospores have collapsed (asterisk) due to cell hollowing; scale bar 5 µm Full size image

B. dendrobatidis is equipped with a chitin binding module (CBM18) that is hypothesized to facilitate survival on its amphibian host. Chitin, a polymer of N-acetylglucosamine is the main component of the fungal cell wall. CBM’s found in other pathogenic fungi function as competitor of, and limit access for foreign chitinases by binding to the chitin of their proper cell wall. In analogy, a key role of CBM18 in the pathogenesis and protection against host-derived chitinases is suggested. In addition, CBM18 would also allow attachment of the pathogen to non-host chitinous structures (e.g. insect or crustacean exoskeletons) allowing vectored disease spread [30, 74].

Invasion of the epidermis

The mechanism of host cell entry, intracellular development and spread within the skin has been documented in a skin explant model [24, 75] as well as for experimentally infected frogs [25]. In general, B. dendrobatidis develops endobiotically, i.e. with sporangia located inside the host cell and is achieved within 24 h after initial exposure. Colonization is established via a tubular extension or germ tube arising from the zoospore cyst that penetrates the host cell membrane and enables transfer of genetic material into the host cell. Then, the distal end of the germ tube swells and gives rise to a new intracellular chytrid thallus. The pathogen then uses the same tactics to spread to deeper skin layers: older “mother” thalli develop rhizoid-like structures spreading to deeper skin layers, form a swelling inside the host cell to finally give rise to a new “daughter” thallus. Figure 9 presents the putative lifecycle of B. dendrobatidis in the skin of susceptible amphibian species, resulting from compilation of all available data [23–25]. The presence of intracellular chytrid thalli clearly contributes to the disease progression in susceptible animals, but the question remains whether “internalization” of the chytrid fungus aids to evade the innate host defences. As such, more research is needed to define the biological advantage of spreading chytrid propagules to deeper skin layers. Conversely, in explanted skin of the infection tolerant X. laevis the pathogen develops merely epibiotically, i.e. with sporangia developing upon the skin (shown in Fig. 10). Here, the affected epidermal cells seem to be solely used as nutrient source for the growing sporangium upon the epidermis [24]. Due to the lack of conclusive histological evidence, it is not clear how infections manifest in this species under natural conditions. As this “saprobic” type of development has only been observed in vitro, more observations are necessary.

Fig. 9 Infection cycle of Batrachochytrium dendrobatidis in a susceptible host. The endobiotic lifecycle includes successively germ tube mediated invasion, establishment of intracellular thalli, spread to the deeper skin layers, upward migration by the differentiating epidermal cell to finally release zoospores at the skin surface Full size image

Fig. 10 Epibiotic lifecycle of Batrachochytrium dendrobatidis. The epibiotic lifecycle observed in skin explants of Xenopus laevis includes germ tube mediated invasion, outgrowth of a rhizoidal network, uptake of host cell cytoplasm as nutrient for the growing and maturing chytrid thallus upon the skin surface Full size image

B. dendrobatidis only colonizes keratinized, stratified epidermis. In anuran larvae, colonization is limited to the keratinized mouthparts, i.e. tooth rows and jaw sheats, and is absent in the epithelia fated to keratinize at metamorphosis, i.e. body, limbs, tail, mouth and gills [3, 66]. Studies in Mixophyes fasciolatus and Osteopilus septentrionalis larvae learn that during metamorphosis colonization of the skin by B. dendrobatidis progresses following the distribution of keratin. Shortly before metamorphosis (approx. Gosner stage 38) the epithelia on the feet begin to stratify and keratinize. Then, at metamorphosis (approx. Gosner stage 40), keratin degrades from the mouthparts before the epithelia on the rest of the froglet’s body begin to keratinize. At that time B. dendrobatidis infection transitions from the mouthparts to the hindlimbs [66, 76].

Our knowledge about the interactions between B. salamandrivorans and its urodelan host is only a minute snippet. The limited number of publications concerning this novel pathogen evidence a rapid disease development in infected urodelans [13, 14]. Skin invasion correlates with host susceptibility. Inoculation of healthy susceptible salamanders is followed by invasion and intracellular colonization of the skin within 24 h and can cause mortality within 2 weeks [14]. As B. salamandrivorans develops germ tubes in culture, invasion and spread in the host epidermis is most likely also germ tube mediated, following the same pattern as B. dendrobatidis, but certainly merits further dissection. Due to its particular host specificity, the mechanisms of attraction to the host, host recognition and adherence require further study.

Impairment of the skin function

The skin of amphibians is of vital importance for their survival. Not only it functions as a sensory organ, but also plays an important role in osmoregulation, i.e. the regulation of the osmotic pressure of an amphibians’ body fluid, defense, thermoregulation, sex recognition and reproduction. Although most adult amphibians possess lungs, an important part of O 2 /CO 2 exchange takes place through the skin [77].

We have a quite clear picture of the physical and physiological changes resulting from infection by B. dendrobatidis and leading to (lethal) disease, while for its congener, B. salamandrivorans, the question remains hitherto unanswered. Severe chytridiomycosis interferes with the skin’s vital functions. Impairment starts with the physical disruption of the epidermis. In human pathogenic fungi causing skin infections, extracellular proteases e.g. serine-, aspartyl- and metallo-proteases play an important role in the invasion of the host skin [71]. These digestive enzymes not only cause damage to host tissue but also impair host defenses. B. dendrobatidis possesses a large number of protease encoding genes, that are lacking or far less prominent in its non-pathogenic congeners [21, 78]. In this fungus’ genome two gene families were found, encoding a serine-type protease and a fungalysin metallopeptidase, two candidates aiding in host cell invasion and dissolution of cellular cytoplasma. Indeed, in the laboratory, B. dendrobatidis secretes proteases capable of degrading casein, gelatin (a hydrolysed form of collagen) [26, 79] and elastin [79]. Additionally, Brutyn et al. [80] discovered that its zoospores secrete a complex mixture of virulence associated proteins including proteases, biofilm-associated proteins and lipases, compromising skin integrity by disturbing the hosts intracellular junctions. Furthermore, infection due to B. dendrobatidis triggers a decreased expression of host genes encoding for essential skin integrity components such as keratin, collagen, elastin and fibrinogen [73].

Physical disruption of the epidermis directly affects the osmoregulatory function of the skin: it impairs the electrolyte transport across the skin, accompagnied by a reduction in transepithelial resistance and leakage of ions, giving rise to ion imbalances, and a reduced ability of frogs to osmoregulate or rehydrate. In fact, in blood samples taken from amphibians with clinical chytridiomycosis significantly reduced plasma sodium, potassium and chloride ion concentrations as well as reduced overall blood plasma osmolality have been observed. Especially, low plasma potassium concentrations (or hypokalemia) that are linked to abnormal cardiac electrical activity and cardiac arrest, are thought to be the proximate cause of death in diseased amphibians (for review see [5]). Many fungal and bacterial pathogens are known to alter both structure and function of the host epidermis and induce changes in water and electrolyte transport by activation or inhibition of ion channels and transporters. Electrolyte transport across the amphibian epidermis is partially accomplished by epithelial sodium channels (ENaC) and sodium/potassium pumps. A study by Campbell et al. [5] shows that chytrid infection is likely to inhibit ENaC, leading to a severely reduced sodium absorption through the skin. Whether a toxin secreted by B. dendrobatidis itself or changes in enzyme function or protein expression induced by the fungus cause disruption of normal skin functioning, requires further research.

Host defenses against chytrid infection

Both innate and acquired immune components contribute importantly to the antimicrobial function of the mucus, as already pinpointed earlier, and will be elaborated in this chapter.

Innate immune defenses

Antimicrobial peptides

A first innate immune defense mechanism involves the production of AMPs in dermal granular glands. AMPs are small (12–50 amino acid residues), cationic and hydrophobic peptides that can reorganize into an amphipathic (with both a hydrophobic and hydrophilic portion within the same molecule) α-helix when bound to charged residues on target cell membranes. Resulting peptide complexes interact with and penetrate into the cell membrane. The mechanisms of AMP action are under debate and both membrane disruption and cell internalization followed by disruption of intracellular targets have been proposed [81]. Most of our current knowledge concerning amphibian AMPs stems from studies on Anura. Although a vast number of well-studied species lack conventional AMPs (for review see [82]), there is a plethora of data underscoring the effectiveness of AMPs in skin secretions as first line defense against B. dendrobatidis, reducing the infection load on amphibian skin to tolerable levels or even clearing them from infection (e.g. [83, 84]). It is not known to which degree anuran AMPs are effective against B. salamandrivorans. To date approximately forty anuran AMPs inhibiting B. dendrobatidis have been characterized [84]. Both purified and natural mixtures of these AMP’s effectively inhibit in vitro growth of both B. dendrobatidis zoospores and sporangia [83–85]. As the infective spores of B. dendrobatidis lack a cell wall, disruption of the cellular membrane integrity has been hypothesized [70]. However, it is not clear to which extent these peptides provide protection against chytridiomycosis in vivo. Species with peptides active in vitro such as the mountain yellow-legged frog (Rana muscosa) may turn out to be very susceptible for infection in nature [86]. Moreover, the efficacy of skin peptide defenses may vary at species and population level [83, 87]. Also little is known about the activity of AMPs once they are secreted upon the skin. Degradation dynamics of skin peptide defenses in species of the Pelophylax complex and in the Northern leopard frog (L. pipiens) suggest that once peptides are secreted upon the skin they stay active up to 1–2 h, but are then degraded by host proteases [88, 89]. Unlike for anurans, information about the AMP arsenal in skin secretions of urodelans is scant. In several salamanders species, the antimicrobial action of skin secretions has been attributed to antimicrobial compounds, most probably including AMPs [90, 91]. However, to date only a single antimicrobial peptide (the defensin CFBD) has been described from Cynops fudingensis (Fuding fire belly newt) [92], leaving a wealth of novel AMPs to be discovered. To our knowledge, the antimicrobial action of CFBD against both Batrachochytrium species has not (yet) been evaluated. Although published data are virtually lacking, AMPs may be involved in the anti-B. dendrobatidis activity of salamander skin secretions [90, 91] and potentially play a role in defense against B. salamandrivorans.

Antifungal metabolites

A second innate immune defense against B dendrobatidis infections is provided by secondary metabolites secreted by symbiotic bacteria present on amphibian skin. So far, only 3 inhibitory metabolites secreted by the skin bacterial species Janthinobacterium lividum, Lysobacter gummosus and Pseudomonas fluorescens have been identified, i.e. 2,4-diacetylphloroglucinol (2,4-DAPG), indol-3-carboxaldehyde (I3C) and violacein [93]. These antifungal metabolites aid to maintain infection loads below a lethal threshold and exhibit a dual action. First, these metabolites can inhibit growth of B. dendrobatidis both in vitro and in vivo [93–95]. However, it is unknown to what extent these metabolites can inhibit B. salamandrivorans. Besides, co-culture of skin bacterial isolates can ultimately lead to secretion of new, more potent metabolites then when grown in monoculture. As such, the inhibitory metabolite tryptophol was found to emerge from co-culturing an unknown Bacillus skin bacterium and Chitinophaga arvensicola [96]. Similarly, Myers et al. [94] discovered that these metabolites work synergistically with AMPs to inhibit growth of B. dendrobatidis, at lowered minimal inhibitory concentrations (MIC) necessary for inhibition by either metabolites or AMP’s. In addition, the metabolites 2,4-DAPG and I3C seem to exert a repellent action on B. dendrobatidis zoospores [97]. As for AMPs, variation in pathogen susceptibility among populations is thought to result in part from differences in bacterial skin communities. By comparing bacterial communities on the skin of a declining Rana muscosa population and a population coexisting with B. dendrobatidis, researchers found a significantly higher number of individuals with culturable bacterial species displaying antifungal properties in coexisting populations than in those at decline. Alteration of this microbial community composition, for example by environmental factors, can considerably increase susceptibility to disease [95]. Conversely, the team of Harris [98] found that addition of the beneficial skin bacterium J. lividum to the skin of susceptible R. muscosa frogs before experimental exposure to B. dendrobatidis, considerably alleviated symptoms of chytridiomycosis and prevented morbidity and mortality. However, mitigation of chytridiomycosis using probiotics will prove challenging as not all symbiotic skin bacteria exhibit broad-spectrum inhibition across isolates of a hypervirulent, globally spread B. dendrobatidis lineage (BdGPL) [19, 99].

Lysozyme

Another compound with fungicidal potential in amphibian skin mucus is lysozyme [70], but has hitherto not been studied in detail. The natural substrate of lysozyme or murimidase is peptidoglycan, a major component of the bacterial cell wall and polymer of N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid units. By splitting the β-1,4 bonds lysozyme causes cell lysis. As the fungal cell wall consists mainly of chitin, a polymer of β-1,4 linked GlcNAc units, it is also a potential target for lysozyme. Although we detected lysozyme or lysozyme-like proteins in mucus from X. laevis, preliminary MIC assays using commercial lysozyme from chicken egg white (1–128 U/mL), failed to demonstrate any fungicidal effect against B. dendrobatidis (Additional file 7). More studies in this domain are necessary to draw further conclusions.

Acquired immune defenses

Unlike the innate immune system, the acquired or adaptive immune system provides highly specific protection against pathogens. This immunity strategy involves both cell-mediated and antibody responses. However, what has puzzled many researchers is the apparent absence of a robust immune response in susceptible amphibian species. Mapping of transcriptomic changes in immunological important tissues (skin, liver, spleen, small intestine) from frogs diseased with B. dendrobatidis, evidence a decreased expression of immunity-related genes (associated with e.g. lymphocytes, Toll-like receptors, complement pathways) [100, 101] or even a lack of protective response by the adaptive immune system [102]. Besides, there is conflicting information about whether or not a protective immune response can be elicited in amphibians. So far, attempts to immunize frogs by subcutaneous or intraperitoneal injection of formalin (Rana muscosa, see [103]) or heat-killed B. dendrobatidis (Bufo boreas, see [70]) failed to elicit an acquired immune response against B. dendrobatidis. Only in X. laevis, B. dendrobatidis-specific IgM, IgX (mammalian IgA-like) and IgY (mammalian IgG-like) antibodies were found in skin mucus after injection with heat-killed zoospores [85]. Usually, antibodies play an important role in neutralizing pathogens or presenting them to other components of the immune system for destruction [70]. In vitro, the mucosal antibodies elicited in X. laevis frogs indeed bind with B. dendrobatidis zoospores and are suggested to limit colonization of the skin to mild and non-lethal infections [85], but their contribution to real-time protection remains to be determined. As B. dendrobatidis infections naturally occur in the skin, it seems likely that introduction of B. dendrobatidis antigens directly into the skin and targeting immune effectors in the skin may be more effective [70]. However, evidence for effectiveness of vaccination in form of prior infection by topical exposure of the skin to live or heat-killed B. dendrobatidis is mixed. Several studies have reported a higher survival, reduction in the infection load or complete clearance in frogs that have been repeatedly exposed to B. dendrobatidis than in immunologically naïve frogs [85, 104], while in others pre-exposure had no such effect [105]. To our knowledge, immunization of salamanders against B. salamandrivorans has not yet been attempted.

Immune evasion by chytrid fungi

Evasion of host immune recognition and inhibition of antifungal defenses are commonly seen in fungal pathogens. Also for B. dendrobatidis there is ample proof of active suppression of host responses coming from genetic, peptidomic, in vitro and in vivo-immune studies [100–102, 106–109].

Ellison et al. [101] found that in highly susceptible harlequin frogs (Atelopus zeteki) B. dendrobatidis-specific immune responses are indeed elicited, but are not effective. Ineffective immune pathway activation and antibody production have been suggested as underlying mechanisms [101]. A breakthrough in research was the discovery that B. dendrobatidis cripples the lymphocyte mediated response [108, 109]. Apparently, soluble factors in B. dendrobatidis culture supernatant inhibit lymphocyte proliferation and induce apoptosis, most probably by activating apoptosis signaling pathways. These inhibitory factors have not yet been fully characterized, but seem of non-protein nature, and broadly cytotoxic. Soon after that, the same research team found that this immunosuppression is not absolute. There is proof that increased lymphocyte proliferation and abundance in the spleen can be achieved by repeated pathogen exposure and temperature-induced clearance of infection (i.e. exposure of infected amphibians for longer than 24 h to 30 °C which is the critical thermal maximum for B. dendrobatidis). As such, at least Oak toads (Bufo quercicus), Cuban treefrog (Osteopilus septentrionalis) and booroolong frogs (Litoria booroolongensis) do acquire immunity to the chytrid fungus, that overcomes B. dendrobatidis-induced immunosuppression [104].

Also inhibition of AMP release from dermal granular glands and selective degradation of AMP’s by fungal proteases have been suggested to contribute to reduced skin defenses against B. dendrobatidis (e.g. [106]). Thekkiniath et al. [110] discovered a particular subtilisin-like serine protease secreted by B. dendrobatidis, able to cleave certain amphibian antimicrobial peptides (AMP). Inactivation of the protective function of AMPs by pathogen-derived proteases, is a common strategy in pathogenic bacteria and fungi and contributes significantly to pathogenesis.

Concepts of susceptibility, tolerance and resistance in chytridiomycosis

Not all amphibians respond equally to a chytrid infection and host responses can be roughly categorized as susceptible (infection resulting in disease, either followed by clinical recovery or by mortality), tolerant (persistent infection in absence of disease) or resistant (inhibition or fast clearance of infection). In chytrid-literature the term resistant (pathogen-inhibiting or pathogen-limiting) is often used for describing species that are actually tolerant (damage-limiting) and definitions may vary according to the author. More importantly, this classification is rather controversial as host susceptibility is more likely to fall along a continuum where the response of a species, population or individual host to B. dendrobatidis (and probably also B. salamandrivorans) is dictated by myriad other factors inherent to host, pathogen and environment than infection dose only. In the next paragraphs we will give some examples for each host response category. However, bearing in mind that susceptibility may vary within a species, this representation may oversimplify reality and the distinction drawn between resistant and tolerant may be disputable.

Clinical chytridiomycosis due to B. dendrobatidis appears to mostly occur in anurans. In truly susceptible anuran species, exposure to B. dendrobatidis under laboratory conditions to initial low doses of 100 zoospores can lead to 100% mortality of the experimental animals [111] and in the wild, exposure to B. dendrobatidis can lead to sharp declines and even extinction of a given species. In particular species that live and/or breed in permanent water or streams at higher elevations seem most susceptible. Striking examples include the neotropical toad genus Atelopus (harlequin frogs) which is by far the most threatened clade of amphibians with at least 30 of the 97 species presumably extinct [1] and the family of the Myobatrachidae with several taxa that are critically endangered (Tautodactylus, Pseudophryne) or suspected to be driven extinct by B. dendrobatidis (gastric brooding frogs in the genus Rheobatrachus) Other taxa susceptible for chytridiomycosis and sufferning from population declines/crashes can be found within, but are not limited to, the families Alytidae (e.g. Alytes and Discoglossus), Bombinatoridae (e.g. Bombina), Bufonidae (e.g. Incilius periglenes, Epidalea calamita, Anaxyrus boreas), Craugastoridae, Dendrobatidae, Hylidae (e.g. Litoria caerulea, Litoria chloris, Litoria genimaculata), Leiopelmatidae (e.g. Leiopelma archeyi), Leptodactylidae (e.g. Leptodactylus fallax), and Ranidae (e.g. Lithobates chiricahuensis, Rana muscosa, Lithobates yavapaiensis, Lithobates tarahumarae) [1, 32]. Far less research has been conducted on salamanders. Lethal chytridiomycosis has been reproduced experimentally only in a very small number of urodelan species, e.g. Batrachoseps attenuatus [112], Bolitoglossa rufescens [34], Plethodon metcalfi [113] and Tylototriton asperrimus [114]. Although chytridiomycosis in wild salamander populations has been described, for example in fire salamanders (S. salamandra) in Central Spain [36] and the endangered Sardanian newt Euproctus platycephalus [1, 115], the impact on population level seems by far less obvious and long-lasting than in susceptible anuran populations. One potential exception are Central American populations of plethodontid salamanders (Plethodontidae), which may also be strongly declining due to chytridiomycosis [4, 34, 64, 65]. Regarding B. salamandrivorans, especially non-Asian Salamandridae seem highly susceptible. So far, one known exception to this rule is the palmate newt (Lissotriton helveticus), which is resistant to B. salamandrivorans while infections are lethal to its close relative, the italian newt (Lissotriton italicus), with mortality occurring approximately 10 days after exposure [14]. In B. salamandrivorans susceptible urodelans, disease can evolve in two opposite directions: clinical recovery or mortality. Although some Asian representatives of the Salamandridae family (i.e. Cynops pyrrhogaster, Cynops cyanurus, Paramesotriton deloustali) are classified as “susceptible”, they are capable of limiting clinical disease. In experimentally infected individuals, infection either persists for up to at least 5 months, with infection loads up to 103 zoospores and with recurring episodes of clinical disease, or is totally cleared [14].

Tolerant species are able to limit the fitness consequences of infection. Species belonging to this host response category do not succumb to B. dendrobatidis infection either in the wild, or under laboratory conditions, although they may be persistently infected. Therefore, they may act as carriers. While prevalence data for B. dendendrobatidis are abundant, information on the magnitude of the infection loads these carriers bear is scarce. From available data, the infection loads in naturally infected tolerant species such as Xenopus laevis (African clawed frog) [48, 116] and Lithobates pipiens (the Northern leopard frog) [117] are low (up to 200 zoospores). However, other tolerant species such as the widespread invasive Lithobates catesbeianus (bullfrog) and Pseudacris regilla (the pacific chorus frog) seem to be “supershedders”. In naturally infected bullfrogs, detected infection loads can run up to 105 zoospores [51], while in experimentally infected P. regilla infection loads amount up to 104 zoospores and are maintained over a 4-month period [54]. Both species may carry extremely high pathogen burdens without morbidity or mortality, which are lethal to most other species.

Truly resistant species for B. dendrobatidis infection are quite scarce. The European cave salamanders (Speleomantes spp.) seem to be a striking example of resistant species as B. dendrobatidis is not able to get grip on the skin, probably due to its highly efficient skin defences. In this species, experimental infections are cleared within 7–14 days and despite large sampling efforts there is zero prevalence of B. dendrobatidis infections in the wild despite the presence of an aggressive B. dendrobatidis lineage (BdGPL) within its geographical range [91, 115]. Truly resistant species for B. salamandrivorans include all so far surveyed anurans and caecilians, and several urodelan species belonging to Asian hynobiid, ambystomatid and North-American plethodontid families [14, 37–39].

Mediators of chytrid infection dynamics and disease outcome

As discussed in Sect. 4.3 the amphibian immune system plays a crucial role in confering resistance or tolerance to chytrid infection. However, there is still considerable variation in the response of a species, a population or an individual host to chytrid infection, that cannot be explained by variation in host defenses only. In this chapter we will highlight how slight changes in host, pathogen and environment, whether or not with direct repercussions on the immune system, may affect an individual’s or a population’s vulnerability to infection.

Host factors

The genetic make-up of the host may largely determine the outcome of B. dendrobatidis infection. In vertebrates, major histocompatibility complex (MHC) loci encode cell-surface receptors regulating the acquired immune response. In amphibians, individuals with specific MHC genotypes, seem to benefit from a higher survival rate when infected by B. dendrobatidis [118]. Indeed, specific conformations of the MHC molecules may promote binding to B. dendrobatidis antigens. Recently, Bataille et al. [119], found that at least one specific MHCII conformation (pocket 9) functions as adaptive marker for resistance to B. dendrobatidis. In contrast, a low genetic diversity within a species or population and consequent reduced biological fitness, may complicate the ability of a species or population to fight B. dendrobatidis infection [120].

Furthermore, there seems to be a direct correlation between the body temperature of frogs and their vulnerability to B. dendrobatidis infection. A temporary rise in body temperature above 25 °C may negatively affect B. dendrobatidis, since this thermal regime is suboptimal for the pathogen or, as suggested, since elevated body temperatures favour the immune response and thus promote survival. However, it is not clear whether these acute changes in body temperature are related to digestion, growth, reproduction or short exposure to a warm microhabitat [121] and coincidentally affect vulnerability for infection. Alternatively, this physiological response may be driven by pathogen recognition (so-called behavioural fever) which has been reported in a variety of invertebrates and ectothermic vertebrates [122]. There is also proof of amphibians acquiring behavioural resistance. In the study of McMahon et al. [104] frogs were more reluctant to avoid substrates infected with B. dendrobatidis after having been exposed to the fungus only once, followed by temperature induced clearance than naieve frogs.

Individual stress levels may also influence the outcome of infection. The glucorticoid stress hormone corticosterone increases due to physiological stress and is in charge for altering the host’s physiology and its susceptibility to a pathogen. Several studies have shown a positive correlation between increased stress levels and infection intensity [123]. However, the effect of stress seems to vary with the life history stage and species. In the study of Searle et al. [124] exogenous exposure of Anaxyrus boreas and Lithobates catesbeianus larvae and both larvae and post-metamorphs of Rana cascadae to corticosterone did not alter their susceptibility to infection. The interactions between environmental change, stress hormones and infectious diseases are complex, and it is not quite clear whether higher corticosterone levels due to e.g. changes in the environment, metamorphosis, breeding make individuals more susceptible to infection or if infection triggers higher corticosterone levels.

Differential susceptibility for infection is observed between larval, post-metamorphic, sub-adult and adult stages. For example, tadpoles of Rana muscosa can be infected by B. dendrobatidis without developing clinical symptoms, while in post-metamorphic animals infection induces morbidity and mortality [59]. Alternatively, B. dendrobatidis can negatively affect some species of amphibians at the larval stage and not others [125]. Also larvae and adult salamanders might display a differential susceptibility for infection with B. salamandrivorans. There are several plausible explanations for these phenomena. Just at metamorphosis the larval epidermis begins to stratify and keratinize, a process that is controlled by the thyroid hormone triidodothyronine (T3). Increased hormone levels during metamorphosis (e.g. T3, coricosteroid hormones), may trigger immune suppression and an increased susceptibility to chytrid infection [110, 126]. But also, at metamorphosis the immune system undergoes a dramatic reorganization, and in newly metamorphosed frogs immune defenses are not yet mature [126].

Finally, it is important to distinguish between individual and population level effects of chytridiomycosis. Infection may cause morbidity and mortality at individual level, ultimately leading to population decline but may just as well go unnoticed. In populations where B. dendrobatidis has a high impact on adult survival, increased recruitment (i.e. entry of new individuals into a population by reproduction) may be an important compensatory strategy allowing a population to recover from disease driven decline, even despite the endemic presence of B. dendrobatidis [127, 128]. Compensatory recruitment is only successful when B. dendrobatidis has a minimal impact on larvae and juveniles, combined with succesful mating by first time breeders before large increases in disease prevalence and intensity occur [128]. Moreover, populations left at low densities after disease-driven decline may recover due to altered disease dynamics; in the case of chytridiomycosis high population densities are likely to promote a rapid build-up of infection intensity and continuous reinfection of (infected) individuals [129]. More importantly, the complexity of the amphibian community may affect disease risk. This is known as the “dilution effect”: increased species richness, of both hosts and non-hosts, will reduce the impact of B. dendrobatidis (infection prevalence and intensity), by affecting host-pathogen contact rates and transmission [130].

Pathogen virulence

The virulence of B. dendrobatidis or its relative capacity to cause damage to its amphibian host, is isolate and genotype dependent [19, 131, 132]. So far, at least 6 major B. dendrobatidis lineages are being recognized, including a hypervirulent global panzootic lineage (BdGPL) [19, 20, 44]. Unlike non-GPL strains, this invasive lineage is associated with massive declines and extinctions that spread in a wave-like manner once introduced into a new area and was involved in the major epizootics in the Americas, Australia, and Europe (Spain, French Pyrenees). As there are currently no isolates available from endemic northern-european amphibian populations coexisting with B. dendrobatidis, the genotype(s) circulating in this area is/are unknown. Yet, further collection of isolates, of both B. dendrobatidis and B. salamandrivorans, is of vital importance for gaining insights in the evolution of virulence.

Virulence of B. dendrobatidis increases experimentally with ambient temperatures below 25 °C. As discussed earlier, optimal growth of B. dendrobatidis occurs within a temperature range of 17–25 °C. Within this range, zoospores encyst and develop into zoosporangia faster than at low temperatures. However, at low temperatures, a larger number of zoospores is produced per zoosporangium, with zoospores remaining active and thus infective for a longer period [133]. As a consequence, mortality in naturally infected amphibians will be considerably higher, in the cooler months of the year in tropical and subtropical areas, while warmer temperatures at other times of the year will promote survival [134]. B. salamandrivorans infection and disease dynamics are likewise dictated to great extent by environmental temperature. Infection intensities of 10 000 zoospore equivalents at which mortality occurs [135] are reached twice as fast at 15 °C than at 20 °C, while at 25 °C B. salamandrivorans is unable to colonize skin [29].

B. dendrobatidis seems liable to attenuation. Strains that have been successively passaged on culture media quite rapidly display a weakened infectivity and pathogenicity when exposed to amphibians, which can however be partially restored by passage through an amphibian host [136].

Impact of environmental factors

Differential susceptibility to B. dendrobatidis observed in natural populations may be due to several abiotic, environmental factors such as season, temperature, elevation [133, 134, 137] and intensity of ultraviolet B (UV-B) radiation [138]. Especially high-elevation areas or regions with cool temperatures entail an increased risk for B. dendrobatidis-related declines and extinctions (e.g. [139]). Plausibly, these environmental factors may increase the vulnerability of an individual considerably, by changing the virulence of B. dendrobatidis and/or by altering the immune function of the host of the amphibian host.

The work of Schmeller et al. [57] illustrates well how both abiotic and biotic factors influence the probability of infection by B. dendrobatidis at population level. They observed a variation in prevalence of B. dendrobatidis among populations of common midwife toads (Alytes obstetricans) at different amphibian breeding sites in the French Pyrenees. At the majority of the breeding sites, prevalence of B. dendrobatidis infection was less than 5%, while at only few sites prevalence ran up to more than 90%. Both altitude and temperature correlated positively with prevalence and mortality but were not conclusive, as at sites with equivalent temperature regimes still substantial variation in prevalence and mortality was observed. At these particular sites, Schmeller et al. [57] found that prevalence of B. dendrobatidis correlates with the abundance and diversity of the aquatic microfauna in the mountain lakes. In this particular case, ciliates and rotifers were found to predate on the aquatic infectious zoospores, and lowered the environmental abundance of B. dendrobatidis. Also microcrustacean zooplankton e.g. water fleas (Daphnia) [140] graze on the spores of this chytrid fungus and are known to reduce the risk on infection in aquatic environments. Variation in the occurrence of B. dendrobatidis might as well coincide with variation in other biotic factors including the macroinvertebrate community structure (e.g., midge larvae, dragonflies, waterbugs and snails) [141] and the presence of green algae that interfere with B. dendrobatidis, either physically or by allelopathy (the release of secondary metabolites that are detrimental for B. dendrobatidis) [140]. Additional research in this field is necessary to fully comprehend the impact of these biotic factors.

Co-infection with multiple pathogens

So far, this review has focused on single pathogen interactions. In reality, amphibian hosts may be exposed to various pathogens including viruses, bacteria, non-chytrid fungi or helminths that may also cause severe pathology and mortality. In captive amphibians, chytridiomycosis due to B. dendrobatidis has been found concomitantly with e.g. Ranavirus [142], Chlamydia pneumoniae [143], Aeromonas hydrophila [144] and Mycobaterium spp. [144] infection. Also in the wild, co-infection with B. dendrobatidis and Ranavirus has been observed [145]. In these cases, it is difficult to determine which pathogen contributes most to morbidity and mortality or to distinguish between primary and secondary pathogen. Indeed, information on how interactions between co-occurring pathogens affect disease severity are quite scarce. A positive correlation has been found between infection by Ranavirus and B. dendrobatidis in some neotropical Hylidae, Craugastoridae and Dendrobatidae. Particularly in Craugaster fitzingeri, the odds of finding Ranavirus were significantly higher in individuals infected with B. dendrobatidis [145]. Also lower survival is observed in Pseudacris regilla larvae experimentally exposed to both the nematode Ribeiroia and B. dendrobatidis than when exposed to one of either pathogens [146]. But as discussed earlier, also abiotic environmental stressors may strongly influence disease susceptibility and might control whether interactions between pathogens occur.

In the same light, the question what would happen if both potentially lethal fungi are present in the same amphibian population is quite worry some. In Brazil, the co-occurrence of the hypervirulent BdGPL and an endemic lineage (BdBz) resulted in a moderate but steady prevalence, suggestive for tempering of the most lethal lineage [46]. Besides, co-occurrence gave also rise to hybridization between both B. dendrobatidis lineages [20, 21]. In the Netherlands and Belgium, both B. salamandrivorans and B. dendrobatidis are present in native amphibian populations. While Dutch and Belgian amphibian communities are in coexistence with B. dendrobatidis [8], the newly emerged B. salamandrivorans has caused rapid mortality in Dutch fire salamander populations [12, 13]. The crucial question is whether co-infection by both fungi will reinforce or on the contrary temper the high lethality of B. salamandrivorans. In case of reinforcement, the native species richness is at stake and urges for appropriate measures to prevent and control local chytridiomycosis outbreaks.