Batrachochytrium dendrobatidis is a chytrid fungus that causes the lethal skin disease chytridiomycosis in amphibians. It is regarded as an emerging infectious disease affecting diverse amphibian populations in many parts of the world. Because there are few model amphibian species for immunological studies, little is known about immune defenses against B. dendrobatidis. We show here that the South African clawed frog, Xenopus laevis, is a suitable model for investigating immunity to this pathogen. After an experimental exposure, a mild infection developed over 20 to 30 days and declined by 45 days postexposure. Either purified antimicrobial peptides or mixtures of peptides in the skin mucus inhibited B. dendrobatidis growth in vitro. Skin peptide secretion was maximally induced by injection of norepinephrine, and this treatment resulted in sustained skin peptide depletion and increased susceptibility to infection. Sublethal X-irradiation of frogs decreased leukocyte numbers in the spleen and resulted in greater susceptibility to infection. Immunization against B. dendrobatidis induced elevated pathogen-specific IgM and IgY serum antibodies. Mucus secretions from X. laevis previously exposed to B. dendrobatidis contained significant amounts of IgM, IgY, and IgX antibodies that bind to B. dendrobatidis. These data strongly suggest that both innate and adaptive immune defenses are involved in the resistance of X. laevis to lethal B. dendrobatidis infections.Batrachochytrium dendrobatidis is a newly described chytrid fungus that causes the lethal skin disease chytridiomycosis in amphibians (29). Growing evidence links amphibian declines in Australia, Central America, the western United States, Europe, and Africa to this emerging infectious disease (4, 9, 12, 26, 29, 34-36, 45, 65). B. dendrobatidis colonizes skin cells of adults and the keratinized mouth parts of tadpoles (3, 4, 29, 34) but does not invade other tissues. It is spread by waterborne zoospores that attach to the skin and migrate to the basal layer of the epidermis (3). The pathogen replicates within the epidermal cells and moves to the surface as the cells mature. Emerging zoospores may infect the same host or another nearby host (3,4,29,34). Recent evidence supports the hypothesis that death results from impaired retention of essential ions by the skin resulting in eventual cardiac arrest (63, 64). Some species of amphibians are very resistant to lethal infections of B. dendrobatidis, whereas others are more susceptible (4,26,27,38,(66)(67)(68), and the factors that determine resistance or susceptibility are not well understood. Although much is known about amphibian immunity in general (9, 14, 41), there is limited information about specific immune responses against B. dendrobatidis.We hypothesized that resistant species have antimicrobial peptides or antibodies in the mucus that limit initial infections by B. dendrobatidis zoospores and prevent the further colonization of the same host by zoospores emerging from the skin. Previous work has shown th...

The chytrid fungus, Batrachochytrium dendrobatidis, causes chytridiomycosis and is a major contributor to global amphibian declines. Although amphibians have robust immune defenses, clearance of this pathogen is impaired. Because inhibition of host immunity is a common survival strategy of pathogenic fungi, we hypothesized that B. dendrobatidis evades clearance by inhibiting immune functions. We found that B. dendrobatidis cells and supernantants impaired lymphocyte proliferation and induced apoptosis; however, fungal recognition and phagocytosis by macrophages and neutrophils was not impaired. Fungal inhibitory factors were resistant to heat, acid, and protease. Their production was absent in zoospores and reduced by nikkomycin Z, suggesting that they may be components of the cell wall. Evasion of host immunity may explain why this pathogen has devastated amphibian populations worldwide.

Eco-immunology is the field of study that attempts to understand the functions of the immune system in the context of the host's environment. Amphibians are currently suffering devastating declines and extinctions in nearly all parts of the world due to the emerging infectious disease chytridiomycosis caused by the chytrid fungus, Batrachochytrium dendrobatidis. Because chytridiomycosis is a skin infection and remains confined to the skin, immune defenses of the skin are critical for survival. Skin defenses include secreted antimicrobial peptides and immunoglobulins as well as antifungal metabolites produced by symbiotic skin bacteria. Low temperatures, toxic chemicals, and stress inhibit the immune system and may impair natural defenses against B. dendrobatidis. Tadpoles' mouth parts can be infected by B. dendrobatidis. Damage to the mouth parts can impair growth, and the affected tadpoles maintain the pathogen in the environment even when adults have dispersed. Newly metamorphosing frogs appear to be especially vulnerable to infection and to the lethal effects of this pathogen because the immune system undergoes a dramatic reorganization at metamorphosis, and postmetamorphic defenses are not yet mature. Here we review our current understanding of amphibian immune defenses against B. dendrobatidis and the ability of the pathogen to resist those defenses. We also briefly review what is known about the impacts of temperature, environmental chemicals, and stress on the host-pathogen interactions and suggest future directions for research.

A powerful mechanism for protection against disease in animals is synergy between metabolites present in the natural microbiota of the host and antimicrobial peptides (AMPs) produced by the host. We studied this method of protection in amphibians in regard to the lethal disease chytridiomycosis, which is caused by Batrachochytrium dendrobatidis (Bd). In this study, we show that the AMPs of Rana muscosa, as well as the metabolite 2,4-diacetylphloroglucinol (2,4-DAPG) from Pseudomonas fluorescens, a bacterial species normally found on the skin of R. muscosa, were inhibitory to the growth of Bd in vitro. When both AMPs and 2,4-DAPG were used in growth inhibition assays, they worked synergistically to inhibit the growth of Bd. This synergy resulted in reduced minimum concentrations necessary for inhibition by either 2,4-DAPG or AMPs. This inhibitory concentration of AMPs did not inhibit the growth of a P. fluorescens strain that produced 2,4-DAPG in vitro, although its growth was inhibited at higher peptide concentrations. These data suggest that the AMPs secreted onto frog skin and the metabolites secreted by the resident beneficial bacteria may work synergistically to enhance protection against Bd infection on amphibian skin. These results may aid conservation efforts to augment amphibian skins' resistance to chytridiomycosis by introducing anti-Bd bacterial species that work synergistically with amphibian AMPs.

dClostridium difficile infection (CDI) represents the most prevalent cause of antibiotic-associated gastrointestinal infections in health care facilities in the developed world. Disease symptoms are caused by the two homologous exotoxins, TcdA and TcdB. Standard therapy for CDI involves administration of antibiotics that are associated with a high rate of disease recurrence, highlighting the need for novel treatment paradigms that target the toxins rather than the organism itself. A combination of human monoclonal antibodies, actoxumab and bezlotoxumab, directed against TcdA and TcdB, respectively, has been shown to decrease the rate of recurrence in patients treated with standard-of-care antibiotics. However, the exact mechanism of antibodymediated protection is poorly understood. In this study, we show that the antitoxin antibodies are protective in multiple murine models of CDI, including systemic and local (gut) toxin challenge models, as well as primary and recurrent models of infection in mice. Systemically administered actoxumab-bezlotoxumab prevents both the damage to the gut wall and the inflammatory response, which are associated with C. difficile in these models, including in mice challenged with a strain of the hypervirulent ribotype 027. Furthermore, mutant antibodies (N297Q) that do not bind to Fc␥ receptors provide a level of protection similar to that of wild-type antibodies, demonstrating that the mechanism of protection is through direct neutralization of the toxins and does not involve host effector functions. These data provide a mechanistic basis for the prevention of recurrent disease observed in CDI patients in clinical trials. Clostridium difficile is an anaerobic, spore-forming, Gram-positive bacterium that causes infections in the lumen of the colon and is the most frequent cause of nosocomial diarrhea in the developed world (1, 2). C. difficile infections (CDI) contribute to thousands of deaths and are associated with over $1 billion in health care-related costs in the United States each year (3-5). The symptoms of CDI range from asymptomatic carriage or mild diarrhea to fatal pseudomembranous colitis, colonic rupture, and death (6, 7). The disease occurs mainly in patients undergoing (or who have recently undergone) a course of broad-spectrum antibiotics; in such patients, composition of the gut microbiota is altered, disrupting the body's natural defense against C. difficile infections. Clinical management of CDI consists of discontinuation of the offending antibiotic and treatment with either metronidazole, vancomycin, or the newly approved fidaxomicin (8). A major concern with CDI is that even when treatment of a primary infection is successful, 20 to 30% of patients experience a recurrence of the disease within days or weeks of symptom resolution. Disease recurrence results from continued disruption of the gut microbiota by standard-of-care antibiotics (9) combined with persistence of resistant C. difficile spores (relapse) or reacquisition of new spores from the environment (rein...