Development of a method for non-lethal sampling from individual fish to investigate host responses to ectoparasites

Aims

This project aims to extend a method developed for the non-lethal collection and analysis of blood and mucus from fish to the study of the infection by the ectopic parasite, Paramoeba perurans.

Background

To develop control strategies for diseases of farmed fish, experimental infection in contained facilities is a common requirement. Traditional experimental fish infection studies are based on the sequential culling of infected animals at different time points. These use large numbers of fish because of the need for cohorts for each time point and the high inter-individual variability. A new method of collecting small blood samples from the same individual fish over the course of infection has previously been funded by the NC3Rs to reduce the number of animals used and is currently being developed to analyse fish responses to viral and bacterial infections. Extending this method to the study of fish infected by the ectopic parasite, Paramoeba perurans, which causes Amoebic Gill Disease, would reduce the numbers of fish required for such research by 80%.

Research details and methods

This project will investigate whether small amounts of mucus from gills can be used to detect localised responses to ectoparasites, and to correlate these with levels of chemokines and cytokines found in underlying tissues collected by lethal sampling for validation purposes. A range of assays will be used to identify key immune responses. The feasibility of monitoring disease progression through the quantification of parasitic load in water, and disease progression in fish through digital analyses of lesions will also be investigated. 

Publications

Chance RJ, Alcock Z, Secombes CJ, Collet B, Collins C. (2017). Effect of repeated exposure to AQUI-S® on the viability and growth of Neoparamoeba peruransJ Fish Dis. 00:1–8. doi: 10.1111/jfd.12712

Amoebic Gill Disease (AGD), a salmon disease caused by the amoeba Paramoeba perurans, has recently emerged in Europe and is a serious threat to the salmon aquaculture industry. To date too litle is known about the biology of the pathogen and its interaction with the host immune system to define any effective control strategies. We can predict that in the coming years numerous academic laboratories and/or commercial RTD departments will undertake experimental infestation to understand the host response or evaluate treatments. Mucosal responses in particular are of interest due to the disease manifesting in fish gills. Studies on fish mucosal immunity are increasing but there is still much to learn e.g. fish IgT immunoglobulin, thought homologous to IgA in mammals, was only recently found associated to mucosal tissue in fish. Traditional experimental infections are based on the sequential sacrifice of a number of infected animals, often resulting in high inter-individual variability. A new experimental model where small blood samples are collected from the same individual after viral or bacterial infection is currently being developed under the NC3Rs project G1100675. This PhD project aims to extend it to AGD by non-lethal collection of blood and mucous. The expected outcome will be i) the generation of more robust data with more accurate correlation between infection dynamics and host immune parameters and ii) a reduction in the number of animals used of over 80 %. Gill and kidney tissues will be collected during lethal sampling, conducted alongside for comparative purposes. The ability to utilise small amounts of mucous from gill (to reduce any impact on disease development/parasite load), to detect localised responses, and to correlate to levels of chemokines and cytokines found in underlying tissues will be investigated. Methods such as RT-QPCR, IHC, ISH, FACS, histology, enzymatic assays will be used to identify key immune responses and changes therein.

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PhD Studentship

Status:

Active

Principal investigator

Professor Chris Secombes

Institution

University of Aberdeen

Co-Investigator

Dr Catherine Collins
Dr Bertrand Collet

Grant reference number

NC/L001489/1

Award date:

Oct 2014 - Sep 2017

Grant amount

£90,000