Using terminal transferase-mediated dUTP Nick End-labelling (TUNEL) and Caspase 3/7 assays to measure epidermal cell death in frogs with Chytridiomycosis

Brannelly, Laura A., Roberts, Alexandra A., Skerratt, Lee F., and Berger, Lee (2018) Using terminal transferase-mediated dUTP Nick End-labelling (TUNEL) and Caspase 3/7 assays to measure epidermal cell death in frogs with Chytridiomycosis. Journal of Visualized Experiments, 135. e57345.

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Abstract

Amphibians are experiencing a great loss in biodiversity globally and one of the major causes is the infectious disease chytridiomycosis. This disease is caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd), which infects and disrupts frog epidermis; however, pathological changes have not been explicitly characterized. Apoptosis (programmed cell death) can be used by pathogens to damage host tissue, but can also be a host mechanism of disease resistance for pathogen removal. In this study, we quantify epidermal cell death of infected and uninfected animals using two different assays: terminal transferase-mediated dUTP nick end-labelling (TUNEL), and caspase 3/7. Using ventral, dorsal, and thigh skin tissue in the TUNEL assay, we observe cell death in the epidermal cells in situ of clinically infected animals and compare cell death with uninfected animals using fluorescent microscopy. In order to determine how apoptosis levels in the epidermis change over the course of infection we remove toe-tip samples fortnightly over an 8-week period, and use a caspase 3/7 assay with extracted proteins to quantify activity within the samples. We then correlate caspase 3/7 activity with infection load. The TUNEL assay is useful for localization of cell death in situ, but is expensive and time intensive per sample. The caspase 3/7 assay is efficient for large sample sizes and time course experiments. However, because frog toe tip biopsies are small there is limited extract available for sample standardization via protein quantification methods, such as the Bradford assay. Therefore, we suggest estimating skin surface area through photographic analysis of toe biopsies to avoid consuming extracts during sample standardization.

Item ID: 53833
Item Type: Article (Non-Refereed Research)
ISSN: 1940-087X
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https://www.jove.com/video/57345/

Date Deposited: 07 Aug 2018 02:05
FoR Codes: 07 AGRICULTURAL AND VETERINARY SCIENCES > 0707 Veterinary Sciences > 070705 Veterinary Immunology @ 50%
07 AGRICULTURAL AND VETERINARY SCIENCES > 0707 Veterinary Sciences > 070707 Veterinary Microbiology (excl Virology) @ 50%
SEO Codes: 96 ENVIRONMENT > 9608 Flora, Fauna and Biodiversity > 960807 Fresh, Ground and Surface Water Flora, Fauna and Biodiversity @ 50%
96 ENVIRONMENT > 9604 Control of Pests, Diseases and Exotic Species > 960405 Control of Pests, Diseases and Exotic Species at Regional or Larger Scales @ 50%
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