QASH workshop: abstracts & pdfs
Biomarkers session - Karin Pittman
M. Braceland1 M. Powell2
1 The Center For Aquaculture Technologies Canada, Souris, PE, COA 2BO, Canada
2 Havforskningsinstituttet, 5817 Bergen, Norway
Defining, and our abilities to do so, healthy populations is one of the greatest challenges faced to livestock industries generally. As such a multitude of scientific literature aims to establish biomarkers of infection, infestation, sub-clinical, clinical, and projected disease outcomes. However, few of these candidate biomarkers establish in a clinical setting. The pipeline of development includes several stages from discovery to implementation. However, due to the failures and issues with each of these few pass from discovery to implementation. Furthermore, the ability of a marker to pass through this pipeline is often blocked due to pre-conceived notions and the entrenchment of established practices which may not be appropriate and/ or better than that in development. This presentation aims to exemplify the process of a biomarker’s discovery, validation, translation, evaluation, and implementation with the pitfalls of these steps being explained.
Xavier A. Gutiérrez*, Tomás S. Mosquera and Felipe A. Briceño Norwegian Institute for Water Research in Chile (NIVA Chile)
Salmon farming productivity strongly relies on smolt adaptability success on seawater (SW) environment after transference from the freshwater (FW) phase, especially during the first weeks after the arrival. According to current understanding, sub-optimal water quality conditions at tank level are able to alter key smolt physiological traits (e.g osmoregulation) which can be critical for fish growth and survival. Even though important advances on smolt welfare from land-based farm exist, there is a lack of quantitative tools able to better link fish physiological traits with SW smolt performance (e.g smolt index). A risk assessment on key water quality parameters, as well as smolt physiological indicators has been proposed as a first step towards a physiological smolt welfare index in Chile.
The current study is based on data from an ongoing smolt physiological monitoring program undertaken by NIVA Chile from both RAS-based and flow-through (FT) fish farms since 2015. The risk assessment is based on three components: key water quality, blood parameters and metals accumulated in target organs (gills and liver). A total of 20 consecutive batches of smolts were examined under these components only days before the smolts are transferred to the sea farms. Batch sampling was based on 3 tanks in which water quality parameters from effluents were analyzed. For each tank, 6 individuals were sampled to measure blood parameters, from which 3 individuals were randomly chosen to collect gills and liver samples.
Using the database of previous projects conducted in fresh water salmon farms, in conjunction with revising scientific literature, limits and recommended levels were established for all components considered. This enabled the categorization of each parameter by providing ranges in which they represent low, medium, or high implications on fish welfare. The frequency (or probability of occurrence) at which each variable presented low, medium or high implications was also determined. The combination of implication/severity level and frequency level results in a qualitative matrix for risk assessment considering the most critical variables.
This matrix appears as a suitable tool for visualizing the main risks for smolt welfare depending on production system (RAS or FT). Outcomes from this matrix can serve as a guideline for decision making process to correct and minimize the risk of these variables. The producer, for example, is able to prioritize and improve aspects of water quality conditions which in turn, result in better fish welfare.
Barriers and stressors session – Mark Powell
Karin Pittman1, 2, Mearge Okubamichael2, Grigory Merkin2, Natalie Brennan1, Mark Powell3, Linda Andersen4 , Nini Sissener3, Arthur Lyngøy5 and Ole Jacob Myre2
1Department of Biosciences, University of Bergen, Thormøhlensgate 53, 5006 Bergen Norway firstname.lastname@example.org ; 2 Quantidoc AS, Thormøhlensgate 53, 5006 Bergen, Norway email@example.com ; 3Institute of Marine Research, Nordnesgaten 50, 5005 Bergen, Norway firstname.lastname@example.org ; 4Industrilaboratoriet, Thormøhlensgate 55, 5006 Bergen, Norway, email@example.com 5Fiskr AS Borgundveien 340, 6009 Ålesund firstname.lastname@example.org
Mucosal barriers of skin, gills and guts are the primary tissues protecting the fish from outside challenges like pathogens and parasites and the internal challenges of suboptimal feeds. The mean size and volumetric density of mucous cells producing this protective slime were first quantifiable in 2010 (Pittman et al. 2011, 2013) and the technique has since been applied in over 50 large and small scale trials in 6 countries, 6 species and 3 tissues with subdivisions. Now trademarked as Veribarr, the verification of the living barriers, results show that there is a reproducible “behaviour” from these tissues in response to a variety of inputs and as such may indicate “herd health” in addition to reflecting individual status. Mean cell size, volume of mucous in the skin epithelium and the combination of these factors shows how each tissue responds both in concert with the others and independently in response to eg. diet. The growing database allows the ascertainment of normal ranges for each tissue and species, while the objective measures allow comparison across species, time, treatment and tissue. Currently two projects are exploring the link between microarray data, RNA analyses and Veribarr results to look for reliable markers of healthy homeostasis in skin or guts. Results further show that a sufficiently sized non-lethal gill biopsy will give rise to reproducible results and may be used to possibly indicate general health. The method is complementary to all other existing methods investigating fish health.
Elvis M. Chikwati1,2, Paul J. Midtlyng1,3*, Åshild Krogdahl2
1Aquamedic AS, Gaustadallèen 21, 0349 Oslo, Norway. email@example.com
2Norwegian University of Life Sciences, Faculty of Veterinary Medicine, Department of Basic Sciences and Aquatic Medicine, Ullevålsveien 72, 0454 Oslo. firstname.lastname@example.org
3Norwegian University of Life Sciences, Faculty of Veterinary Medicine, Department of Food Safety and Infection Biology, Ullevålsveien 72, 0454 Oslo. email@example.com
Optimization of feed resource utilization, production costs, fish growth performance, and environmental impact of Atlantic salmon industrial production is highly dependent on a healthy, optimally functioning gut. Overt clinical signs of gut dysfunction are rare in field conditions, yet subclinical gut health disorders can significantly diminish fish performance and health. Regardless, gut health of farmed fish populations at sea is not routinely monitored for impacts of feed, noxious, infectious or parasitic agents. As part of the ongoing GutMatters project funded by the Norwegian Seafood Research Fund, a national survey to establish the prevalence of gut health disorders and their incidence during a production cycle in sea farmed Atlantic salmon was therefore initiated in the autumn of 2017. Six sea farming sites along the Norwegian coast were monitored starting at about 5 weeks after sea-transfer until about 12 months of the fish at sea. Standardized procedures were developed for comprehensive sampling of up to 20 fish per site, including external and abdominal gross pathology, fish weight, length and blood plasma, content and tissue from intestine, liver, head kidney, spleen, and heart for histology and/or gene expression, microbiota, metabolomics, and digestive enzyme activity analyses. At each of 3 sampling events per farm, site physico-chemical data, fish stock feeding, growth, and health history were collected. Histology was used as the initial screening tool for gut health status of the sampled fish from which subsequent analyses will be based on. This presentation reports results from a semi-quantitative histology scoring for selected inflammatory and degenerative morphological changes in the mucosa of the pyloric caeca, mid-, and distal-intestine and the liver. Main findings from the histological evaluation of the fish sampled after 5 weeks post sea-transfer were mild to moderate inflammatory changes in the distal intestine of most of the fish sampled from one of the participating farms, as well as mild to marked enterocyte steatosis in the pyloric caeca in most of the groups evaluated. The inflammation resembled the well-documented soybean meal induced distal intestinal enteritis observed in salmonids fed diets containing soybean, or other legumes, as a protein source. The steatosis is thought to represent a lipid transport or metabolism disorder in enterocytes that in severe cases manifest as lipid malabsorption, steatorrhea and ‘floating faeces’. Both disorders of inflammation and steatosis may markedly diminish gut function, fish growth and health and are candidate indicators of reduced feed utilization in Atlantic salmon. Details from the histological assessment of the gut and liver from fish sampled during the first and second round of monitoring the participating sea farms will be presented.
Vanessa Mella1*, Paulo Salinas1,2, Manuel Ulloa1, Barbara Etcharren1, Carlos Sandoval1
2 Instituto de biología, Pontifica Universidad Católica de Valparaíso, Valparaíso, 2340000, CHILE. firstname.lastname@example.org.
Pathology diagnosis has been performed by pathologists observing the stained specimen on the slide glass using a microscope. In recent years, attempts have been made to capture the entire slide with a scanner and save it as a digital image (Whole slide image, WSI). Researchers both in the image analysis and pathology fields have recognized the importance of quantitative analysis of pathology images. Since most current pathology diagnosis is based on the subjective (but educated) opinion of pathologists, there is clearly a need for quantitative image-based assessment of digital pathology slides. In VeHiCe we adopt these analyses to be applied in farmed salmon industry in Chile. Currently this method is applying to assess pathologies evolution, health status, organs responses to drugs and diets among others. Allowing farmers take objectives measures regarding health and productions issues.
This study reports the assessment of the effects of anti-inflammatory drug in the evolution of an inflammatory process in the heart fibers caused by PRV. Two groups were tested; control group and T1. In total, the heart of 50 fish with were histologically processed, transverse sections of the ventricle were made and then the whole histopathological slide was capture with scanner and save as a digital image (WSI). The 85%-95% of total surface of the ventricle was analyzed in each slide. The morphometric analyze of the images was realized using ImageJ v1.49 (National Institut of Health, EE.UU.). In total 550 images were analyzed. In control group, there was 6,18% of the total heart surface presenting an inflammatory process, and in T1 group only a 0,47% of the total heart surface presented inflammation.
Using this method, we were able to measure precisely the % of inflammation in the different groups analyzed and consequently the evolution of the inflammatory process and the response to the tested drug.
Lene Rydal Sveen a,b,*, Gerrit Timmerhausb, Aleksei Krasnov, Harald Takled, Sigurd Handelandc, Sigurd Stefanssona, Elisabeth Ytteborgb.
a University of Bergen, Postboks 7800, 5020 Bergen, Norway
b Nofima, Osloveien 1, 1430 Ås, Norway
c Uni Research, Thormøhlens Gate 55, 5008 Bergen, Norway
d Cermaq Group AS, Dronning Eufemias gate 16,0102 Oslo, Norway
The skin of Atlantic salmon (Salmo salar), is a coherent and dense barrier that protects the interior of the fish against the outer environment. The skin covers the entire outer surface, including the head, fins and eyes. Lesions in the skin are a major welfare issue for the fish.
Materials and methods
Atlantic salmon post-smolts (mean weight 120g) were divided into two identical tanks (500L), and two treatments were established. High production intensity, HPI (mean fish density 126 kg/m3) and normal production intensity, NPI (mean fish density 16 kg/m3) . Three cylinders of tissue were excised with a 5 mm biopsy punch. Samples (n=12 per treatment) for gene expression analyses (microarray), histology, immunohistochemistry and scanning electron microscopy were collected 1, 3, 7, 14, 36, 42 and 57 days post wounding (dpw).
In general, the wounds from both HPI and NPI followed the normal progression of wound healing, with hemostasis, re-epithelialization, inflammation, tissue formation and tissue remodeling. The first 14 days of the healing process was dominated by acute inflammation and epidermal repair as shown through imaging, histological evaluation and transcriptomics. In the early inflammatory phase a more adherent mucus layer was observed, which further correlated with altered transcription of glycosyl transferases and mucin genes. This may indicate different properties and functions of the mucus during the acute inflammatory phase. Formation of scales and granulation tissue started approximately at 14 days post wounding. This was followed by wound contraction and formation of dermal structures.
At the transcriptomic level the greatest differences between NPI and HPI were found at 2-14 dpw, with more than > 500 DEG at each sampling point. In general, inflammation was enhanced in the HPI wounds, while cell proliferation and tissue regeneration was repressed. Histological examinations showed transient delays in the formation of epidermis, mucus response, scale mineralization, wound pigmentation and formation of dense connective tissue in HPI wounds. The overall wound morphology was also altered in fish reared at HPI. Wounds from NPI fish contracted in an elongated manner, while the wounds from HPI fish were more circular. The fish reared at HPI had significant higher cortisol levels compared to NPI fish, thus we suggest that cortisol are one of the main factors contributing to the delayed wound healing responses in fish reared at HPI .
The presented description of the wound healing processes in Atlantic salmon and the effect of HPI, gives insight into comparative ulcerative biology in fish and provides both novel and updated knowledge that can be applied for improved best operational practices for fish welfare in aquaculture.
Biomarkers And Barriers – Criteria, Cutoff Levels, Long-Term Effects, And Remedial Actions? – Mark Powell and Karin Pittman
Available tool boxes session
Stian Nylund1* and Liv Sandlund2
1PHARMAQ Analytiq, Thormoehlensgate 53D, 5008, Bergen, Norway email@example.com
2PHARMAQ Analytiq, Thormoehlensgate 53D, 5008, Bergen, Norway firstname.lastname@example.org
Molecular methods for use in fish diagnostics have been around for quite some time, both as tools for detection of pathogens and for use in quantitative analyses of gene expression. It has to a large degree replaced tried and true diagnostic methods in veterinary medicine, both in universities and veterinary colleges, and is supplied as commercially available products by several private laboratories linked to the aquaculture industry.
In Norway, realtime RT-PCR has become the staple method for monitoring and detecting pathogens during production of Atlantic salmon, due to its advantages in high sensitivity of detection, high specificity and the possibility to perform high- throughput analyses on a population scale. As a result, many different laboratories strive to design the highest performing assay for detection, identifying the best method for fish tissue processing, and how to interpret the outcoming results for use in setting a diagnosis. This increase in method development and optimization of performance, while positive, also have some negative impacts. Since different laboratories can employ different methodology, results are often not directly comparable between laboratories, and the interpretation of the results and their relevance in setting the correct disease diagnosis, will not always be straightforward.
The topic of this talk is to highlight some of the key parameters that will affect results of a realtime RT-PCR run. This includes choice of tissue for sampling, method for nucleic acid extraction and relevant factors that have to be taken into account when designing an assay for use in fish diagnostics. How to interpret the results, and how they are used in decision making, alongside complementing methodology will also be discussed.
WORKSHOP 2 - Tool boxes available – ease of use, global applicability, time usage? Integrated pest management and threshold values to elicit a response? – Robin Shields
Overview of Results, Comments, Volunteers for Core International QASH Proposal Work
The Knowledge Gaps And Missing Tools In The Toolbox; Core QASH Objectives For 2019