Pathogenesis
Whereas the pathogenesis of Bd infections is relatively well known, Bsal has only recently been discovered, and its pathogenesis is incompletely understood. This chapter will focus on the pathogenesis of Bd, while highlighting any known differences to Bsal.
14.4.1 Skin Colonization
The different steps in the infection process comprise attraction of the free swimming zoospore to a suitable host with subsequent attachment to and invasion into the host skin, leading to impairment of the skin function. Bd is attracted to keratin and to carbohydrate components of the mucus and epidermis (Meyer et al. 2007; Moss et al. 2008; Van Rooij et al. 2015). If the zoospores resist the defence mechanisms of the mucus (see below), adhesion to the skin surface occurs within 2-4 h after exposure (Van Rooij et al. 2012), after which the zoospores mature into thick-walled cysts. Cysts are anchored to the skin surface by fine fibrillar projections. Several adhesion proteins such as vinculin, fibronectin and fasciclin are expected to contribute to this process (Rosenblum et al. 2008, 2012). Besides, Bd is equipped with a chitin binding module (CBM18) that is hypothesized to facilitate survival on its amphibian host by limiting access for foreign chitinases by binding to the chitin of their proper cell wall. 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 (Abramyan and Stajich 2012; McMahon et al. 2013; Liu and Stajich 2015; Van Rooij et al. 2015).
Bd further develops endobiotically (with sporangia located inside the host cell). 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. 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 dig its way to deeper skin layers: older “mother” thalli develop rhizoid-like structures spreading to deeper skin layers, forming a swelling inside the host cell to finally give rise to a new “daughter” thallus (Berger et al. 2005; Van Rooij et al. 2012; Greenspan et al. 2012; Van Rooij et al. 2015). This intracellular proliferation occurs within the cells of the stratum corneum and the stratum granulosum. Immature sporangia are carried from the deeper skin layers to the skin surface by differentiating epidermal cells. At the time sporangia have developed discharge tubes and contain mature zoospores, they occur in the stratum corneum, where the zoospores are released in the environment (Longcore et al. 1999; Berger et al. 2005; Van Rooij et al. 2015).Conversely, in explanted skin of the infection tolerant X. laevis, the pathogen develops merely epibiotically (with sporangia developing upon the skin) (Van Rooij et al. 2015). Here, the affected epidermal cells seem to be solely used as nutrient source for the growing sporangium upon the epidermis (Van Rooij et al. 2012). Due to the lack of conclusive histological evidence, it is not clear how infections manifest in this species under natural conditions (Van Rooij et al. 2015). As this “saprobic” type of development has only been observed in vitro, further in vivo evidence is needed. Skin invasion by Bsal was also shown to occur within 24 h (Martel et al.
2014), although the mechanism of Bsal invasion is not known.
Bd selectively colonizes keratinized, stratified epidermis. In anuran larvae, colonization is limited to the keratinized mouthparts, i.e. tooth rows and jaw sheaths, but absent from the body, limbs, tail, mouth and gills. Studies in Mixophyes fasciolatus and Osteopilus septentrionalis tadpoles show that during metamorphosis, colonization of the skin by Bd progresses following the distribution of keratin (Marantelli et al. 2004; McMahon and Rohr 2015).
In contrast with anuran larvae, no colonization of salamander larvae by Bd has been demonstrated so far (Van Rooij et al.2015).
Susceptibility to Bsal infection was equally shown to be life stage dependent. Whereas Bsal infects all fire salamander (Salamandra salamandra) life stages post metamorphosis, larvae seem to be refractory to infection (Van Rooij et al. 2015).
14.4.2 Pathophysiology
Severe chytridiomycosis causes functional disruption of the epidermal barrier. Bd possesses a large number of proteolytic enzymes, including serine-type proteases and fungalysin metallopeptidases, which can cause damage to skin integrity by disturbing the host's intracellular junctions (Joneson et al. 2011; Brutyn et al. 2012 Rosenblum et al. 2013). Furthermore, infection due to Bd triggers a decreased expression of host genes encoding essential skin integrity components such as keratin, collagen, elastin and fibrinogen (Rosenblum et al. 2012).
Physical disruption of the epidermis directly affects the osmoregulatory function of the skin: it impairs the electrolyte transport across the skin, accompanied by a reduction in transepithelial resistance and leakage of ions, giving rise to ion imbalances, and a reduced ability of amphibians to osmoregulate or rehydrate. Blood samples from amphibians with clinical chytridiomycosis show significantly reduced plasma sodium, potassium and chloride ion concentrations and reduced overall blood plasma osmolality. The low plasma potassium concentrations (or hypokalaemia) are linked to abnormal cardiac electrical activity and cardiac arrest and are thought to be the proximate cause of death in diseased amphibians (Campbell et al. 2012, Van Rooij et al. 2015).
Besides the detrimental effect on the skin, Bd also actively suppresses host responses (Rosenblum et al. 2009; Ellison et al. 2014; Ribas et al. 2009; Woodhams et al. 2012a, b; Young et al. 2014). Bd culture supernatant inhibits lymphocyte proliferation and induces apoptosis (Fites et al. 2013, 2014). Fungal proteases, such as a subtilisin-like serine protease, inhibit antimicrobial peptide (AMP) release from dermal granular glands and/or selectively degrade AMPs (Woodhams et al. 2012a, b; Thekkiniath et al. 2013).
14.5