DAO 103:77-85 (2013)  -  doi:10.3354/dao02560

Screening bacterial metabolites for inhibitory effects against Batrachochytrium dendrobatidis using a spectrophotometric assay

Sara C. Bell1,*, Ross A. Alford1, Stephen Garland2, Gabriel Padilla3, Annette D. Thomas4

1School of Marine and Tropical Biology, James Cook University, Townsville, Queensland 4811, Australia
2School of Public Health, Tropical Medicine and Rehabilitation Sciences, James Cook University, Townsville, Queensland 4811, Australia
3Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil
4Tropical and Aquatic Animal Health Laboratory, Department of Employment, Economic Development and Innovation, Oonoonba, Queensland 4811, Australia

ABSTRACT: Certain bacteria present on frog skin can prevent infection by the pathogenic fungus Batrachochytrium dendrobatidis (Bd), conferring disease resistance. Previous studies have used agar-based in vitro challenge assays to screen bacteria for Bd-inhibitory activity and to identify candidates for bacterial supplementation trials. However, agar-based assays can be difficult to set up and to replicate reliably. To overcome these difficulties, we developed a semi-quantitative spectrophotometric challenge assay technique. Cell-free supernatants were prepared from filtered bacterial cultures and added to 96-well plates in replicated wells containing Bd zoospores suspended in tryptone-gelatin hydrolysate-lactose (TGhL) broth medium. Plates were then read daily on a spectrophotometer until positive controls reached maximum growth in order to determine growth curves for Bd. We tested the technique by screening skin bacteria from the Australian green-eyed tree frog Litoria serrata. Of bacteria tested, 31% showed some degree of Bd inhibition, while some may have promoted Bd growth, a previously unknown effect. Our cell-free supernatant challenge assay technique is an effective in vitro method for screening bacterial isolates for strong Bd-inhibitory activity. It contributes to the expanding field of bioaugmentation research, which could play a significant role in mitigating the effects of chytridiomycosis on amphibians around the world.


KEY WORDS: Chytridiomycosis · Challenge assay · Bacterial metabolite · Amphibian disease · Bioaugmentation


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Cite this article as: Bell SC, Alford RA, Garland S, Padilla G, Thomas AD (2013) Screening bacterial metabolites for inhibitory effects against Batrachochytrium dendrobatidis using a spectrophotometric assay. Dis Aquat Org 103:77-85

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