Understanding the relationship between gill disease and immune status

Lead Research Organisation: University of Aberdeen
Department Name: Inst of Biological and Environmental Sci

Abstract

Fish are a major source of protein and omega-3 fatty acids. With the decline in fisheries worldwide, fish farming has increased to fill the gap and currently supplies around half of all fish consumed. Production must continue to increase in order to supply the increasing human population. The Atlantic salmon farming industry has been a major success story in Scotland, the third largest producer of Atlantic salmon globally, and is a major employer in rural areas. The sustainability of the aquaculture industry relies on good management of fish health and effective control of diseases. Within Scottish salmon farming, control of sea lice and improvement of gill health are currently the two most important health issues that the sector faces. Gill health impacts on the performance of fish at sea, and the need for expensive and often poorly efficacious therapeutic treatments.

Lack of information of host (salmon) responses in gills in different disease states has been highlighted at several recent industry-led workshops in Scotland. Therefore this application proposes to address this issue by undertaking an in-depth study of the genes expressed in gills following exposure to a major gill parasite that causes amoebic gill disease (AGD), using archived samples from a past study. We will determine whether the gill, as a multifunctional organ responsible for oxygen uptake, osmoregulation, as well as defence at a major point of entry for pathogens, has a limited scope to react to pathogens in fish. We will focus on elucidating the wound healing pathways activated and the growth factors potentially released that cause tissue remodelling, since many gill diseases cause similar pathologies, in terms of lamellar fusion and epithelial cell hyperplasia. The data from this initial study will also be used to identify candidate biomarkers relevant for gill health, to be used for further study

Next we will undertake qPCR analysis of the biomarkers in a variety of gill disease states. Gill samples from farmed fish will be collected during the late summer/autumn of 2017/18 by our collaborators, when gill disease is most prevalent. Initially the gill samples will be screened for pathogen presence to confirm the species causing the pathology and whether single or co-infections are involved. The biomarker analysis will reveal if common pathways are seen in different disease states, allowing the potential to alter these pathways to improve gill health against multiple diseases.

Lastly, we will try to modulate the gill responses using different immunomodulators to establish if this approach can improve gill health/ disease protection. We will focus on administration of a 4 molecules that we know are potent inducers of pro-inflammatory pathways or anti-viral defences. They will include flagellin, poly I:C and two cytokines (IL-1beta and IFN-gamma, from a related salmonid the rainbow trout) that are available in our lab as recombinant protein ready for use, or can be purchased (poly I:C). Following administration we will sample gill tissue over a time course and examine the impact on the biomarkers identified earlier in the programme, as well as on known antimicrobial and anti-viral pathways. The best modulator of gill responses will be trialled on a salmon site by our industrial collaborator to assess the impact on gill health.

Technical Summary

The sustainability of aquaculture relies on good health management. Gill disease is an important health issue in Scottish Atlantic salmon farming, and impacts on fish performance at sea. Gills are multifunctional, responsible for oxygen uptake and osmoregulation, as well as defence. Knowledge of gill responses is limited, yet crucial to understand the risk of life-threatening complications from asphyxia and ion imbalance in disease. Hence, this proposal will study host responses in complex gill disease states in salmon, to gain a better understanding of pathways that are elicited with a view to future interventions.

Initially we will use RNAseq to establish the host response to an important gill disease in Scotland, amoebic gill disease (AGD). Existing samples from experimental challenge of salmon with AGD will be used to generate Ilumina TruSeq Stranded libraries. Indexed libraries will be sequenced on two HiSeq4000 lanes, generating ~600 million paired-end 150bp reads (~15M reads per sample). Differentially-expressed genes will be identified using EdgeR, with a GLM accounting for group (i.e. treatment vs. control), time (day 0, 10, 30) and their interaction. This data will be used to establish a suite of biomarkers of value as indicators of gill health during disease states. This will be tested using samples from marine reared salmon with gill health issues, collected on farms over two seasons. qPCR will be used to determine the pathogens present and a qPCR-array to assess whether common or unique biomarkers of gill health can be identified.

The potential to modulate the gill response will also be examined following administration of pathogen derived (PAMPs; flagellin, poly I:C) or host (IL-1beta, IFN-gamma) pro-inflammatory molecules, as a possible means to moderate pathology or increase disease resistance locally. The proteins have been produced as recombinants, and shown to be bioactive. Optimal doses will be injected and the responses examined by qPCR.

Planned Impact

Farmed fish are consumed worldwide and are an important source of protein in addition to meat and poultry. The UK has a sizeable salmon farming industry, and is the third largest producer globally after Norway and Chile. In Scotland, salmon is the largest food export, with a retail value >£1 billion annually, and in addition the industry supports employment of a large work force in rural areas. The Scottish Government aims to increase production of marine fish to 210,000 tons by 2020. The sustainability of the salmon industry is therefore vital for both the local economy and global food security. Disease issues threaten to undermine the sustainability of aquaculture, but if controlled may lead to improved fish health/welfare and increased production. The proposed research is focused on salmon gill health, as a major current problem affecting fish at sea. Therefore the work has the potential to impact on: 1) the aquaculture industry, to find solutions to gill health problems, 2) academia, in terms of new knowledge about the gill responses in health and disease, 3) government, to enable aquaculture targets to be met, and 4) the wider public, by contributing to a sustainable supply of fish for the table.

Gills are vulnerable to environmental change and pathogen infection, and gill health impacts on the welfare and productivity of farmed fish. Gill associated diseases have become a major issue for the industry in recent years. There are a number of known pathogens identified, often causing diseases of multifactorial aetiology. There is a huge knowledge gap in understanding the underlying mechanisms of pathology of gill disease and the immune responses that can be elicited in gills to the different pathogens encountered. This project will study these responses to a new level using deep sequencing approaches, initially in the context of a single gill disease (amoebic gill disease), but then extrapolating the results to look at gill responses during more complex disease tates on fish farms, with collaborators supplying samples from Ireland, Scotland and Norway. We anticipate the research will generate a wealth of knowledge on gill responses during disease, especially of host markers typically modulated during disease pathogenesis and associated with the wound healing process elicited to limit the damage and impact on the host.

The project will also assess the potential to modulate gill responses, to help control and manage salmon gill diseases. Both host-derived and pathogen-derived factors will be tested, and the best regime will be examined further on a farm site.

This project involves UK partners from academia (University of Aberdeen), the fish farming industry (Marine Harvest) and government (Marine Scotland Science), with additional partners in Ireland (Marine Institute, Oranmore, Co. Galway) and Norway (Norwegian Veterinary Institute, Oslo). The partnership will ensure that the research findings can be disseminated effectively to benefit the research community, advice policy and explore potential commercialisation.
 
Description Since this programme started we have undertaken RNAseq (deep sequencing) of archived samples of gills from salmon experimental challenged with P. perurans, the causative agent of amoebic gill disease. RNA quality/quantity was initially checked following the criteria proposed by the Centre for Genome Enabled Biology and Medicine (CGEBM - University of Aberdeen), including a RNA integrity number RIN>8 (Agilent 2200 TapeStation). Samples that passed the quality control then underwent rRNA depletion, prior to sequencing. The sequencing has recently been completed, giving 1,027M filter reads and 76Gbp Q30 yield have been obtained. The mean quality value across each base position is over 30 (Phred score). RNA-Seq raw reads have been processed using bioinformatics tools at the High Performance Computing center, University of Aberdeen. To identify differentially expressed genes (DEGs), data sets from infected samples will be compared to the corresponding control group. The Bioconductor package (including edgeR, DESeq2, and limma approaches) will be applied for the analysis and comprehension of RNAseq data. This analysis will be completed in the next six months, to identify potential biomarkers to be tested in the gill samples with health issues supplied to us from fishfarm sites.

To date we have prepared cDNA from 172 gill samples sent to us from 6 fishfarm sites during the summer of 2018. Of these 166 samples have passed our quality control criteria. These samples are stored at -20 °C ready for testing the new biomarkers.
Exploitation Route Gills are vulnerable to environmental change and pathogen infection, and gill health impacts on the welfare and productivity of farmed fish. Gill associated diseases have become a major issue for the industry in recent years. We anticipate the research undertaken in this project will generate a wealth of knowledge on gill responses during disease, especially of host markers typically modulated during disease pathogenesis and associated with the wound healing process elicited to limit the damage and impact on the host. Such markers will allow improved management of gill diseases in the future. The project will also assess the potential to modulate gill responses, to help control and manage salmon gill diseases.
Sectors Agriculture, Food and Drink