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Prosjektnummer: 901472
Status: Pågår
Startdato: 15.01.2018
Sluttdato: 30.05.2021

Peredikksyre som behandling av amøbegjellesykdom (AGD) i laks (PERAGILL) / Peracetic acid as a potential treatment for amoebic gill disease (AGD) in Atlantic salmon

  • Report: Peracteic acid as a potential treatment for amoebic gill disease (AGD) in Atlantic salmon - Stage I
    Nofima. Rapport 21/2019. 14. juni 2019. Carlo C. Lazado (Nofima), Gerrit Timmerhaus (Nofima), Lars-Flemming Pedersen (Technical University of Denmark), Karin Pittman (Quantidoc AS, University of Bergen), Malene Soleng, Sindre Haddeland (Quantidoc AS, University of Bergen), Lill-Heidi Johansen (Nofima), Mette W. Breiland (Nofima), Lisbeth Rørmark (Lilleborg AS), Saima Nasrin Mohammed (The Norwegian Veterinary Institute) and Sigurd Hytterød (The Norwegian Veterinary Institute)
AGD is a significant health and disease problem in Atlantic salmon aquaculture. Since the first documented case of AGD in Norway, the threats have been quite persistent and the number of reported cases is showing an upward trajectory trend. The industry must be kept abreast with sustainable treatment options to combat this emerging threat. The use of freshwater and oxidative disinfectant bathing are the two strategies being applied in commercial production. Peracetic acid (PAA), an organic peroxide, is being considered as a more sustainable disinfectant in aquaculture compared with other conventional disinfectants. PAA offers some advantages that may address the challenges in the currently available treatments for AGD. Given the escalating threat that AGD is posing to the Norwegian salmon aquaculture industry, there is indeed a big challenge to develop alternative methods that have the potential in addressing issues on efficacy, practicality and most importantly, sustainability. The documented features of PAA make it a good candidate to be explored as a treatment for AGD. The exploration of this potential must be provided with a complementary toolbox that will assess treatment impacts and success through insights from different biological perspectives.
Main objective
To explore the potential of peracetic acid (PAA) as an alternative and sustainable treatment to amoebic gill disease (AGD) in salmon aquaculture.
Sub-objectives STAGE I (2017–2019)
• To investigate the amoebicidal activity of PAA and identify influencing factors (work package 1).
• To assess the impact of PAA treatment on fish health and welfare (WP1).
• To determine the potential environmental risk of PAA treatment (WP2).
• To develop integrative assessment toolbox to evaluate treatment impacts and efficacy (WP3).
Sub-objectives STAGE II (20192021)
• To identify additional factors that may affect PAA degradation and its associated residuals (WP2).
• To develop further the assessment toolbox related to salmon response to PAA and AGD (WP3).
• To evaluate the efficacy of PAA treatment against AGD-infected fish under different scenarios in the laboratory, as well as in the field (WP4).
Expected project outcome
The project will attempt to offer the salmon aquaculture industry an effective and eco-friendly therapeutic measure for AGD. The expected results will provide the industry significant long-term benefits because a new alternative solution to an existing problem is to be developed, especially that it may offer practical advantages in cases where the current treatments for AGD are hampered with logistical concerns. Lastly, fundamental knowledge will be generated, such as the identification of markers for treatment assessment that may be utilised in future endeavors, especially in the development of diagnostic tools and therapeutics for AGD.
Foreløpige resultater per juni 2019​​​​
Sammendrag av resultater fra prosjektets delrapport I (Stage I)
Det overordnede målet for PERAGILL var å etablere en alternativ behandling mot AGD. Eksisterende behandlinger har flere praktiske og miljømessige begrensninger. Pereddikksyre (PAA) er et potent oksidasjonsmiddel med bredspektret antimikrobiell aktivitet som brytes ned til relativt trygge komponenter og har derfor blitt anerkjent som et bærekraftig desinfeksjonsalternativ i akvakultur. Det har allikevel ikke blitt testet ut som potensiell behandling mot AGD. Dette prosjektet hadde derfor til hensikt å teste ut hvorvidt PAA kunne være et alternativt behandlingsmiddel. Fase 1 i prosjektet har undersøkt effekt av PAA eksponering på laksens helse og velferd, nedbrytningskinetikk og antiparasittisk effekt mot Paramoeba peruans, agenset som forårsaker AGD. Det ble gjennomført 3 in vivo-eksperimenter der laks ble eksponert for ulike nivå av PAA. Eksperiment 1 var designet for å evaluere hvorvidt tidligere eksponeringshistorikk ville dempe sensitivitetsresponsen ved gjentatt eksponering. Eksperiment 2 undersøkte hvorvidt stresshåndtering i forkant at PAA-behandlingen ville påvirke effekten av PAA. I eksperiment 3 ble effekt av PAA etter gjentatt eksponering undersøkt. Veksthastighet var ikke påvirket av noen av eksponeringsalternativene. Adferdsendring ble kun observert i eksperiment 3. Det ble ikke registrert økt dødelighet i noen av forsøkene og den eksponerte fisken kom seg raskt etter behandlingen. Fôringsaktiviteten ble raskt gjenopptatt. Eksterne velferdsforandringer var av mild karakter. Histologisk analyse av gjeller og skinn viste at selv om det var observert noe patologi hos den eksponerte fisken var de mukosale barrierene oppretthold. Gjentatte eksponeringer så derimot ut til å påvirke det mukosale laget i gjellene (eksperiment 3). Fiskens adaptive responser var robust og i de fleste tilfellene var fiskens basale konsentrasjon gjenopprettet kort tid etter eksponeringen. PAA eksponeringen i eksperiment 1 og 2 påvirket ikke plasma metabolomet i motsetning til gjentatt eksponering (eksperiment 3). Transkriptomanalyse av mukosale vev (skinn og gjeller) viste at PAA trigger en sterk immunologisk respons. Den etablerte gjellekulturmodellen egner seg til å teste mukosal respons mot oksidasjonsmidler (som PAA versus H2O2). PAA hadde amøbicid effekt mot P. perurans. Effekten av PAA var påvirket av flere faktorer (som tetthet, te​mperatur, lys). PAA ødelegger amøbens cellemembran. Nedbrytingen av PAA var påvirket av en rekke faktorer inkludert lys, fisketetthet og salinitet. Forsøkene viste at PAA nedbrytes signifikant raskere enn H2O2. Resultatene fra forsøkene indikerer at PAA er trygt for laksen og er et potensielt lovende medikament mot AGD med lav miljømessig risiko.

Preliminary results as of June 2019
Summary of results from the project’s report I (Stage I):
PERAGILL is an initiative that ultimately aims to develop an alternative treatment for the currently available therapies for amoebic gill disease (AGD) that have several practical and environmental issues. Peracetic acid (PAA) is a potent oxidant with a broad spectrum of antimicrobial activity and decays into relatively safe residuals, thus, has been widely recognised as a sustainable disinfectant in aquaculture. Earlier reports on PAA underscore its potential to address the challenges of the current AGD treatments. Hence, this project aimed to establish its credentials as a chemotherapeutant for AGD. Stage 1 documented the impacts of PAA exposure on the health and welfare of salmon, its degradation kinetics and its antiparasitic activity against the Paramoeba perurans, the causative agent of AGD. There were 3 in vivo exposure experiments performed where salmon were exposed to varying levels of PAA. Experiment 1 was designed to evaluate whether previous exposure history might desensitise the responses upon re-exposure. Salmon were exposed to different nominal concentrations (0, 0.6, and 2.4 ppm) of PAA for 5 minutes, followed by a re-exposure to the same concentrations for 30 minutes 2 weeks later. Experiment 2 explored how a stressful episode before exposure might interfere with the adaptive responses to PAA. Fish were subjected to crowding stress prior to PAA exposure at 4.8 ppm for 30 minutes. And lastly, Experiment 3 investigated the impacts of repeated exposures to PAA. Salmon were exposed to 10 ppm PAA either for 15 minutes to 30 minutes every 3 weeks, with 3 exposures in total. Growth performance was not affected in all exposure trials. Behavioural changes such as agitation, erratic swimming, increased ventilation and loss of balance during exposure were only observed in experiment 3. No significant mor​tality was recorded in all experiments, and exposed fish recovered quickly after exposure as evidenced by unaffected feeding patterns. Though there were external welfare changes (e.g. skin damage, fin damage) following exposure, the degree of alterations was not dramatically high. Histological analyses of gills and skin revealed that despite the presence of some pathologies in PAA-exposed fish, mucosal barriers can still be categorised as healthy. Repeated exposure, however, may compromise the barrier status of the gills as observed in experiment 3. PAA could trigger oxidative stress. In addition, classical players of systemic stress responses were activated by PAA exposure. The adaptive responses were robust and, in most cases, the level returned to basal concentrations hours after exposure. Crowding stress prior to exposure could interfere with the normal systemic stress and antioxidant responses to PAA. Metabolic profiling revealed that PAA concentrations in experiments 1 and 2 did not substantially alter the plasma metabolomes. Recurrent exposures, however, have a significant impact. Metabolites that were differentially affected by PAA exposure were known to be involved in protecting the cells from oxidative stress damage, suggesting that salmon were able to mount a strong protective response against PAA-induced oxidative stress. Transcriptomic profiling of the mucosal tissues (i.e., skin and gills) demonstrated that PAA could trigger a strong immunological response as several differentially expressed genes following PAA exposure have known roles in immunity. Skin transcriptome was more responsive than the gills at lower PAA dose. However, the opposite trend was identified at a higher dose. The developed gill explant culture could be used as a model to compare mucosal responses to oxidants (i.e., PAA vs. H2O2). PAA exhibited amoebicidal activity against P. perurans. Viability of the amoeba can be reduced by 50 % following exposure to 4.8 ppm PAA and higher. Toxicity of PAA towards the amoeba was influenced by different factors (i.e., density, temperature, light, culture age) at varying degrees. Toxic effect of PAA against the amoeba is rendered by disruption of the cell membrane. The decay of PAA was affected by several factors including light, fish density and salinity. It was demonstrated that PAA degrades significantly faster compared with H2O2 in seawater. Taken together, the results indicate that PAA is safe for use in salmon, with promising potential as a chemotherapeutant for AGD with low environmental risk.

Project design and implementation
The project is organised into four work packages (WPs). WPs 1–3 constitute activities for STAGE I (funded from 2017-2019) that are continued or expanded in STAGE II (funded from 20192022). WP4 is conducted in STAGE II. 

WP1 is the backbone of the project and together with WP2, will lay the foundation for WP4. WP1 will explore the effects of PAA on the causative agent and on the host. WP2 will evaluate the environmental risk associated with PAA treatment. WP4 will investigate different PAA application strategies to treat AGD infection. WP3 constitutes the toolbox that will be used to assess the effects and efficacy of the treatment and will bridge STAGES I and II.

WP1 – Amoebicidal activity and health-related impacts
This WP will explore the amoebicidal activity of different peracetic acid based disinfectants against several Norwegian isolates of N. perurans. Several factors will be tested to understand the susceptibility of the amoeba and the mechanisms of actions of PAA. The health-related impacts of PAA exposure will likewise be investigated.
WP2 – Environmental impacts 
Stage I: Based on literature review and PAA residual measurements in seawater exposure trials, degradation time (DT50) and half-lives of PAA will be identified. Degradation products will be quantified and the levels will be compared with existing data concerning safe limits in Norwegian setting. The work package will also include recommendations for solutions to minimise discharge of PAA residuals under different treatment scenarios.

Stage II: The potential practical solutions to increase the degradation of all three compounds (i.e., peracetic acid, hydrogen peroxide, acetate) will be tested in a commercial PAA product. Different options will be tested, for example chemical agents or organic matter. Specific batch test will be made where PAA is spiked to seawater containing different levels of commercial bacterial remediation mixture. These tests will give information on the microbial degradation of hydrogen peroxide and acetate as well as the impact of PAA degradation, and can potentially be of relevance as a tool to control residual discharge.

WP 3 – Integrative assessment toolbox
Stage I: WP3 will provide the tools and techniques to assess the effects and the efficacy of PAA treatments. The integration of traditional and modern approaches and tools would facilitate the development of a toolbox that will be valuable in future research concerning AGD treatments, especially with the use of oxidative disinfectants. At the end of the project, the predictive nature of the tools will be assessed. 

Stage II: The techniques that were employed in Stage I will still be applied in the samples in Stage II. These techniques will be combined to produce an array of biomarkers for assessing the health and disease status of salmon during AGD infection and after PAA treatment. Moreover, tools will be developed and verified on quantifying the amoeba and the efficacy of PAA treatment.

WP4 – Treatment of AGD-infected salmon (STAGE II)
This WP will explore different factors and scenarios that may influence the efficacy of PAA as a chemotherapeutant for salmon. Laboratory trials will investigate the effects of temperature, severity of infection, crowding stress and time of treatment on the efficacy of PAA against AGD. Possible development of resistance to the treatment will also be explored. It will also be attempted to apply PAA to an AGD outbreak in the field.

Project organisation
Nofima will lead the project and will carry out most of the experimental works involved. DTU Aqua will carry out part of WP1 and will lead WP2. Quantidoc AS will perform the histological examination of all samples collected. Lilleborg AS will provide all the disinfectants that will be used in the experiment.
Dissemination of project results
The results of the study will be made available through print and online communication channels. All peer-reviewed publications will be distributed under Open Access scheme. Popular science articles, both in Norwegian and English, will be published in communication media that are highly relevant to the industry. The dissemination and communication channels of Nofima and of the partner institutions will be maximized.

The results are expected to be presented at national and international conferences/symposia as poster and/or oral presentations. Conferences organised by FHF and the Research Council of Norway (e.g., Havbruk and Frisk Fisk) are excellent opportunities to establish the credential of the project at a national level. At the international scene, conferences with close connection with the industry (i.e., Aquaculture Europe, World Aquaculture) are priorities for result dissemination.