Refining, reducing and replacing non-model amphibian experiments on amphibian infectious diseases

With the urgent conservation issue posed by infectious diseases, licensed experimental research on non-model amphibian (NMA) species is expanding exponentially. In the UK alone this involves thousands of non-model amphibians (NMAs) p.a. and tens of thousands globally.

For example, the Garner Home Office returns for 2015 exceeded 1,800 individuals, and four US based laboratories report using, on average 8,000, and up to a maximum of 14,000 NMAs per lab per annum.

These experiments have provided key insights into the causes of NMA diseases and how to mitigate their impact, saving several amphibian species threatened with disease-driven extinction. As disease threats increase in number and distribution, we expect the use of NMAs in experiments to increase. However, use of the 3Rs as guidance for this global research initiative either lacks scientific justification (refinement) or application (reduction & replacement). What limited 3Rs progress has been made is relevant only for model amphibians which cannot be generalised due to significant differences in species biology. In recognizing this shortcoming, the NC3Rs is convening an international Amphibian Welfare Symposium October 2017, but there is immediate need to determine how the 3Rs can improve the welfare of amphibians in experimental disease research. Here we propose to address this for all 3Rs using a tripate approach.

The student will develop new protocols to significantly REPLACE and REDUCE animal use with non-animal cell and organ culture systems and develop validated welfare indicators that will REFINE existing animal experimental protocols that cannot be replaced with validity. To do this, the student will exploit nearly 15 years of field and experimental research on the two pathogen groups [amphibian-associated chytridiomycete fungus Batrachochytrium dendrobatidis (Bd) and ranaviruses (Rv)] that are global drivers of amphibian declines. This extensive history has shown that 3 factors currently dictate the severity of the disease outcome: environmental temperature, pathogen genotype and host species. Systems and pathogen isolates for examining the effects of all 3 of these factors are available to the student within the Garner lab, the Fisher lab or through established collaborations at London HEIs. The student will therefore examine how application of the 3Rs affects the research community’s ability to explore how interactions amongst these three determinants of virulence determine disease outcomes through three objectives:

1) to use cell and media culture of amphibian pathogens to determine the effect of pathogen genotype and environmental temperature on pathogen growth and gene expression and in so doing effect REPLACEMENT of animals in experimental studies of these;

2) to determine if organ culture can be used to study infection dynamics and gene expression of amphibian pathogens, and in so doing effect REDUCTION in overall animal use and the need for live animals in experiments, and;

3) to (i) establish urgently needed, valid welfare indicators for use in welfare monitoring that can discriminate between general ill health and ill health due to infectious disease and, through this, (ii) establish early predictors of outcomes that allow REFINEMENT of experimental protocols and implementation of improved humane endpoints to decrease the number of animals that reach current, relatively crude humane endpoints.

These three components scale up: from host-independent non-animal models to tissue-level analyses of pathogen dynamics that capture part of the host/pathogen interaction through to refined animal model experimental systems that can effectively discriminate between indicators that identify general ill health versus ill health caused by infectious disease. In so doing, we seek to provide increasingly complex replacement, reduction and refinement systems or methods that the researcher can use based on research need and the complexity of the question.

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PhD Studentship


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Principal investigator

Dr Trenton Garner


Zoological Soc London Inst of Zoology


Dr Siobahn Abeyesinghe
Professor Matthew Fisher

Grant reference number


Award date:

Jan 2018 - Dec 2020

Grant amount