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 states 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 This research project was divided into three work packages, to study the 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 to improve fish health/welfare. Initially RNAseq analysis would be used to establish the host response to an important gill disease in Scotland, amoebic gill disease (AGD). Existing (archived) samples from an experimental challenge of salmon with AGD are available for use, and differentially-expressed genes will be determined using bioinformatics approaches. This data will be used to establish a suite of biomarkers 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 several seasons. qPCR analysis 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. Lastly, the potential to modulate the gill response will also be examined following administration of pathogen derived molecules or host pro-inflammatory molecules, as a possible means to moderate pathology or increase disease resistance locally. These proteins are also available in our lab ready for use and will be injected into fish and the responses examined by qPCR analysis of gill immune gene expression.

RNAseq of amoebic gill disease (AGD) samples

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 gave 1,027M filter reads and 76Gbp Q30 yield were obtained. The mean quality value across each base position was over 30 (Phred score). The RNA-seq reads were mapped to the latest Atlantic salmon genome using STAR. The number of reads mapping to annotated features in the reference genome (i.e. genes) was calculated using FeatureCounts. The count data from FeatureCounts was used for a gene-by-gene DE analysis with DESeq2 along with exploratory multivariate PCAs.

As an initial test, a DE analysis between control (n=7) and treatment (n=6) individuals at day 30 post-challenge was performed. This is where we had the most statistical power and expected the difference between treatment and controls to be most evident. This led to the identification of 65 DE genes ('ControlvsTreatmentDay30.txt) out of 48,597 features/genes with non-zero total read count, using an adjusted p-value < 0.05 (59 upregulated by treatment, 6 downregulated). This list of DE genes includes cytokines such as IL-4/13 and IL-8, along with several genes that seem appropriate in terms of the expected impact of AGD. The results were validated by RT-qPCR on selected genes.

Analyze gill samples with gill health issues

The above analysis was undertaken with a view to identify potential biomarkers to be tested in the gill samples with health issues supplied to us from fish farm sites. To date we have prepared cDNA from 172 gill samples sent to us from 6 fish farm sites. Of these 166 samples have passed our quality control criteria. These samples were stored at -20°C ready for testing with the new biomarkers and selected genes from past publications.

To date 70 genes have been analysed by RT-qPCR on a selection of 93 samples, to allow all samples (and controls) to be run in the same PCR plate. The major finding from this biomarker gene expression analysis was the divergent gene expression patterns that showed important site-effects probably due to multi-disease-causing factors. The genes upregulated in most disease samples across different sites included beta-defensin-3, a salmonid specific beta-defensin, MMP9 and MMP13. Samples from more sites may help to confirm these findings over the coming year. DNA samples have also been prepared from the same samples for detection of candidate pathogens, to verify the cause of the gill pathology.

In vivo stimulation with pathogen derived (PAMPs; flagellin, poly I:C) or host (IL-1beta, IFN-gamma) pro-inflammatory molecules.

Rainbow trout were injected with either flagellin, poly I:C, IL-1ß or IFN? at doses deemed optimal from past studies, with fish given the same volume of saline as control. Sampling occurred at 6 h and 24 h later. The gill samples were initially archived in TRI reagent. The samples from flagellin and poly IC injected fish have been processed and good quality cDNA has been obtained, ready for qPCR analysis. The TRI reagent lysate of samples from IL-1ß and IFN? injected fish have been archived at -80°C until processing. This experiment will confirm the best performing modulator of gill immune gene expression, for proposed further testing in salmon exposed naturally to a range of gill pathogens.
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

 
Description Coordinated meeting of the international 'Functional Annotation of All Salmonid Genomes' initiative 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact This meeting was attended by 30 salmonid biologists linked to the international FAASG initiative (https://www.faasg.org/). Key discussions were held on the future of the initiative, its links to UK infrastructure (EMBL-EBI) and future funding priorities, influencing funders in attendence (Norwegian Research Council and Genome Canada)
Year(s) Of Engagement Activity 2019
URL https://icisb.org/faasg-meeting/
 
Description Invited talk at International workshop Functional annotation of the Atlantic salmon genome, translation to improved health and performance in aquaculture. 'Advancing aquaculture by genome functional annotation: Memorial University, Canada. Aug 26-27th 2019. 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact Gave an invited talk at International workshop Functional annotation of the Atlantic salmon genome, translation to improved health and performance in aquaculture. 'Advancing aquaculture by genome functional annotation: Memorial University, Canada. Aug 26-27th 2019. The outcomes were an increased mutual understanding of research and collaborative activity with international collaborating scientists.
Year(s) Of Engagement Activity 2019
 
Description Lead coordinator of the fourth International Conference on the Integrative Biology of Salmonids (https://icisb.org/). 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact The fourth International Conference on Integrative Salmonid Biology (ICISB 2019), followed on from previous meetings in 2012 (Oslo, Norway), 2014 (Vancouver, Canada) and 2016 (Puerto Varas, Chile). The ICISB meetings have been core funded and organized by the International Cooperation to Sequence the Atlantic Salmon Genome (ICSASG), a trilateral effort between Canada, Chile and Norway. The theme of ICISB 2019 was'Beyond the genome: taking leaps forward in salmonid biology' to reflect the recent staggering progress in genomic resource development and exploitation since the Atlantic salmon reference genome was published in 2016.

There was an audience of ~200, which represented a mixture of researchers from Professors leading in the field, to undergraduate students. Many international collaborations and opportunities for further research, funding and meetings were explored with a range of stakeholders, including funders, media and industry.
Year(s) Of Engagement Activity 2019
URL https://icisb.org/