Development and worldwide use of non-lethal, and minimal population-level impact, protocols for the isolation of amphibian chytrid fungi

Abstract

Parasitic chytrid fungi have emerged as a significant threat to amphibian species worldwide, necessitating the development of techniques to isolate these pathogens into culture for research purposes. However, early methods of isolating chytrids from their hosts relied on killing amphibians. We modified a pre-existing protocol for isolating chytrids from infected animals to use toe clips and biopsies from toe webbing rather than euthanizing hosts, and distributed the protocol to researchers as part of the BiodivERsA project RACE; here called the RML protocol. In tandem, we developed a lethal procedure for isolating chytrids from tadpole mouthparts. Reviewing a database of use a decade after their inception, we find that these methods have been applied across 5 continents, 23 countries and in 62 amphibian species. Isolation of chytrids by the non-lethal RML protocol occured in 18% of attempts with 207 fungal isolates and three species of chytrid being recovered. Isolation of chytrids from tadpoles occured in 43% of attempts with 334 fungal isolates of one species (Batrachochytrium dendrobatidis) being recovered. Together, these methods have resulted in a significant reduction and refinement of our use of threatened amphibian species and have improved our ability to work with this group of emerging pathogens.

Genetic analysis of post‐epizootic amphibian chytrid strains in Bolivia: Adding a piece to the puzzle

“…(1999) with slight modifications (Longcore, 2000). We used a hand lens or light microscope to screen larvae and adults for signs of chytridiomycosis using oral tissue dekeratinization (Fellers, Green, & Longcore, 2001; Fisher et al., 2018; Knapp & Morgan, 2006; Vieira et al., 2013) or thalli in excised skin pieces (toe webbing of adults). We dissected infected tissues for pathogen isolation on 1% tryptone agar with 0.2 mg/ml penicillin‐G and 0.4 mg/ml streptomycin sulphate (Longcore, 2000).…”

Arthropod–bacteria interactions influence assembly of aquatic host microbiome and pathogen defense

“…Using young, captivebred amphibians ensured that initial host microbiomes were homogeneous and that microbiomes were actively assembling. For the Bd inoculum, we used the isolate CLFT 196 (Global Panzootic Lineage-2), which originated from a tadpole collected in São Paulo state, Brazil [45]. To obtain Bd zoospores for the inoculum, we added a growing liquid Bd culture to Petri plates containing 1% tryptone agar.…”

Discriminating Lineages ofBatrachochytrium dendrobatidisusing quantitative PCR

“…Sites were surveyed along the Orange River at approximately 100 km apart from the source in the Drakensburg Mountains to near the estuary on the border with Namibia. At each site, we attempted to culture and isolate Bd using non-lethal tissue sampling from adult amphibians and lethal mouthpart sampling from tadpoles (Fisher et al, 2018). Any resulting chytrids were lineage-typed by WGS using the protocol described by O'Hanlon et al (O'Hanlon et al, 2018).…”

“…Any resulting chytrids were lineage-typed by WGS using the protocol described by O'Hanlon et al (O'Hanlon et al, 2018). All adult amphibians were also swabbed following 'Risk Assessment of Chytridiomycosis to European Amphibian biodiversity (RACE)' protocols (Biodiversa, 2013;Fisher et al, 2018). DNA extraction also followed the RACE protocol (supplementary information), following which swab samples were tested for Bd using the pan-lineage qPCR diagnostic.…”