Towards the generation of gnotobiotic larvae as a tool to investigate the influence of the microbiome on the development of the amphibian immune system.

The immune equilibrium model suggests that exposure to microbes during early life primes immune responses for pathogen exposure later in life. While recent studies using a range of gnotobiotic (germ-free) model organisms offer support for this theory, we currently lack a tractable model system for investigating the influence of the microbiome on immune system development. Here, we used an amphibian species (Xenopus laevis) to investigate the importance of the microbiome in larval development and susceptibility to infectious disease later in life. We found that experimental reductions of the microbiome during embryonic and larval stages effectively reduced microbial richness, diversity and altered community composition in tadpoles prior to metamorphosis. In addition, our antimicrobial treatments resulted in few negative effects on larval development, body condition, or survival to metamorphosis. However, contrary to our predictions, our antimicrobial treatments did not alter susceptibility to the lethal fungal pathogen Batrachochytrium dendrobatidis (Bd) in the adult life stage. While our treatments to reduce the microbiome during early development did not play a critical role in determining susceptibility to disease caused by Bd in X. laevis, they nevertheless indicate that developing a gnotobiotic amphibian model system may be highly useful for future immunological investigations. This article is part of the theme issue 'Amphibian immunity: stress, disease and ecoimmunology'.

Cooperation between physiological defenses and immune resistance produces asymptomatic carriage of a lethal bacterial pathogen.

Animals evolved two defense strategies to survive infections. Antagonistic strategies include immune resistance mechanisms that operate to kill invading pathogens. Cooperative or physiological defenses mediate host adaptation to the infected state, limiting physiological damage and disease, without killing the pathogen, and have been shown to cause asymptomatic carriage and transmission of lethal pathogens. Here, we demonstrate that physiological defenses cooperate with the adaptive immune response to generate long-term asymptomatic carriage of the lethal enteric murine pathogen, Citrobacter rodentium. Asymptomatic carriage of genetically virulent C. rodentium provided immune resistance against subsequent infections. Immune protection was dependent on systemic antibody responses and pathogen virulence behavior rather than the recognition of specific virulent antigens. Last, we demonstrate that an avirulent strain of C. rodentium in the field has background mutations in genes that are important for LPS structure. Our work reveals insight into how asymptomatic infections can arise mechanistically with immune resistance, mediating exclusion of phenotypically virulent enteric pathogen to promote asymptomatic carriage.

Cooperation between physiological defenses and immune resistance produces asymptomatic carriage of a lethal bacterial pathogen.

Animals have evolved two defense strategies to survive infections. Antagonistic strategies include mechanisms of immune resistance that operate to sense and kill invading pathogens. Cooperative or physiological defenses mediate host adaptation to the infected state, limiting physiological damage and disease, without killing the pathogen, and have been shown to cause asymptomatic carriage and transmission of lethal pathogens. Here we demonstrate that physiological defenses cooperate with the adaptive immune response to generate long-term asymptomatic carriage of the lethal enteric murine pathogen, Citrobacter rodentium. Asymptomatic carriage of genetically virulent C. rodentium provided immune resistance against subsequent infections. Host immune protection was dependent on systemic antibody responses and pathogen virulence behavior, rather than the recognition of specific virulent factor antigens. Finally, we demonstrate that an avirulent strain of C. rodentium in the field has background mutations in two genes that are important for LPS structure. Our work reveals novel insight into how asymptomatic infections can arise mechanistically with immune resistance, mediating exclusion of phenotypically virulent enteric pathogen to promote asymptomatic carriage.

Investigating the potential use of an ionic liquid (1-Butyl-1-methylpyrrolidinium bis(trifluoromethylsulfonyl)imide) as an anti-fungal treatment against the amphibian chytrid fungus, Batrachochytrium dendrobatidis.

The disease chytridiomycosis, caused by the pathogenic chytrid fungus, Batrachochytrium dendrobatidis (Bd), has contributed to global amphibian declines. Bd infects the keratinized epidermal tissue in amphibians and causes hyperkeratosis and excessive skin shedding. In individuals of susceptible species, the regulatory function of the amphibian's skin is disrupted resulting in an electrolyte depletion, osmotic imbalance, and eventually death. Safe and effective treatments for chytridiomycosis are urgently needed to control chytrid fungal infections and stabilize populations of endangered amphibian species in captivity and in the wild. Currently, the most widely used anti-Bd treatment is itraconazole. Preparations of itraconazole formulated for amphibian use has proved effective, but treatment involves short baths over seven to ten days, a process which is logistically challenging, stressful, and causes long-term health effects. Here, we explore a novel anti-fungal therapeutic using a single application of the ionic liquid, 1-Butyl-1-methylpyrrolidinium bis(trifluoromethylsulfonyl)imide (BMP-NTf2), for the treatment of chytridiomycosis. BMP-NTf2 was found be effective at killing Bd in vitro at low concentrations (1:1000 dilution). We tested BMP-NTf2 in vivo on two amphibian species, one that is relatively tolerant of chytridiomycosis (Pseudacris regilla) and one that is highly susceptible (Dendrobates tinctorius). A toxicity trial revealed a surprising interaction between Bd infection status and the impact of BMP-NTf2 on D. tinctorius survival. Uninfected D. tinctorius tolerated BMP-NTf2 (mean ± SE; 96.01 ± 9.00 µl/g), such that only 1 out of 30 frogs died following treatment (at a dose of 156.95 µL/g), whereas, a lower dose (mean ± SE; 97.45 ± 3.52 µL/g) was not tolerated by Bd-infected D. tinctorius, where 15 of 23 frogs died shortly upon BMP-NTf2 application. Those that tolerated the BMP-NTf2 application did not exhibit Bd clearance. Thus, BMP-NTf2 application, under the conditions tested here, is not a suitable option for clearing Bd infection in D. tinctorius. However, different results were obtained for P. regilla. Two topical applications of BMP-NTf2 on Bd-infected P. regilla (using a lower BMP-NTf2 dose than on D. tinctorius, mean ± SE; 9.42 ± 1.43 µL/g) reduced Bd growth, although the effect was lower than that obtained by daily doses of itracanozole (50% frogs exhibited complete clearance on day 16 vs. 100% for itracanozole). Our findings suggest that BMP-NTf2 has the potential to treat Bd infection, however the effect depends on several parameters. Further optimization of dose and schedule are needed before BMP-NTf2 can be considered as a safe and effective alternative to more conventional antifungal agents, such as itraconazole.