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Prosjektnummer

901472

Prosjektinformasjon

Prosjektnummer: 901472
Status: Avsluttet
Startdato: 15.01.2018
Sluttdato: 30.04.2022

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

• Nedbrytning av peredikksyre (PAA) i sjøvann fulgte en eksponentiell reduksjonskurve med halveringstider fra minutter til timer. Reduksjonshastigheten økte med økt temperatur, salinitet og UV dose. Denne informasjonen er viktig både for dosering av virkestoff og håndtering av utslipp etter behandling.
• Frisk Atlantisk laksesmolt tolererte PAA doser på 0,6-10 ppm, men med økt risiko jo høyere dose. Fiskens respons var avhengig av eksponeringstid og -frekvens, stresstilstand og produkttype (PAA kilde). Disse faktorene utgjør et avgjørende rammeverk for optimal behandlingsprotokoll ved bruk av PAA.
• PAA hemmet aktiviteten av amøben som forårsaker AGD under cellekultur betingelser (in vitro). Behandling av AGD-infisert laks syntes å redusere parasittmengden, med ulikt utfall avhengig av behandlingsprotokoll. Resultatene var noe utvetydige da gjelleskår fortsatte å stige etter infeksjon og histologiske skader vedvarte, men en lengre rekonvalesensperiode er ofte nødvendig for å påvise effekt.
• Prosjektet har identifisert mekanismene for hvordan PAA påvirker fisken, samt fiskens responser for å motvirke det fysiologiske presset fra denne potente oksidanten. Selv om PAA anses som en stressor, responderte laksen effektivt mot virkestoffet under de konsentrasjonene som ble testet i dette studiet.

Key project achievements
• The degradation kinetics of peracetic acid (PAA) in seawater follows exponential first-order decay with half-lives on the order of minutes to hours. Temperature, salinity and UV significantly increased PAA decay. This information is important in dosing as well as in handling discharges after treatment.
• Healthy Atlantic salmon smolts could tolerate PAA doses from 0,6 to 10 ppm, though the risks are higher at higher doses. Moreover, the responses were dependent on exposure duration, stress status, frequency of exposure and PAA trade products. These factors are crucial in defining the frameworks of optimal treatment protocol using PAA.
• Under in vitro conditions, PAA exhibited amoebicidal activity. PAA treatment of AGD-affected fish appeared to reduce the parasitic load which was influenced by the treatment protocols. PAA treatment of AGD-affected fish did not provide an unequivocal treatment resolution. The gross gill scores continued to increase following infection. Histological lesions associated with infection likewise persisted, though in some cases, a longer recovery period may be necessary.
• The project identified the mechanisms of how PAA affects the fish and the countermeasures the fish mount to address the physiological pressures from this potent oxidant. Although PAA is considered a stressor, Atlantic salmon could effectively respond to the demands of PAA, at least within the concentrations tested in the study.
Sammendrag av resultater fra prosjektets faglige sluttrapport (English summary further below)
Prosjektet undersøkte potensialet til pereddiksyre (PAA) som behandling av amøbegjellesykdom (AGD) hos atlantisk laks. Flere studier ble gjennomført for å 1) undersøke nedbrytningstid til PAA og faktorene som påvirker denne iboende kjemiske egenskapen, 2) dokumentere konsekvenser på helse og velferd hos laks ved bruk av PAA, og 3) evaluere effekt av PAA på amøben og dens effekt på behandling av sykdommen. Temperatur, salinitet, lys og UV påvirket dynamikken og nedbrytning av PAA. Laksesmolt kunne tolerere PAA doser fra 0,6 til 10 ppm, men både atferdsmessige og fysiologiske responser ble signifikant påvirket av varighet og frekvens av PAA-eksponeringen. Hvilket kommersielt PAA-produkt som ble testet påvirket også resultatene. Stress og sykdomsstatus til fisken ble dokumentert å være avgjørende for fiskens respons mot PAA. Tilsetning av PAA ble identifisert å være som en mild stressor for fisken, som kunne indusere både temporære og systemiske oksidative systemer. Ulike fysiologiske adaptive responser ble avdekket hos fisken for å motvirke disse ytre påvirkningene. Bruk av PAA in vitro viste seg å hemme amøbens aktivitet. Når laks smittet med amøber ble eksponert for PAA (ulike behandlingsprotokoller), gav dette tvetydige sykdomsbilder. Behandlingen kunne føre til redusert parasittbelastning, samtidig som amøbeobservasjoner og mikroskopisk patologi vedvarte etter behandling. Ny innsikt om patofysiologien til AGD har blitt generert i prosjektet, spesielt med hensyn til oksidativt stress og sirkulære metabolitter. Det vil være behov for ytterligere optimalisering når det gjelder behandling av AGD med PAA. Men, prosjektet har ført til betydelige fremskritt angående kjemi og fysiologi i forbindelse med PAA-behandling på fisk, som vil være viktig i videre utvikling og bruk (erfaringsbasert) av dette desinfeksjonsmiddelet i akvakulturnæringen.

Results achieved
Summary of results from the project's final report
The project investigated the potential of peracetic acid (PAA) as a treatment for amoebic gill disease (AGD) in Atlantic salmon. A series of studies were performed to 1) investigate the decay of PAA and the factors that influence this inherent chemical behaviour, 2) document the health and welfare consequences of using PAA in salmon smolts, and 3) evaluate the amoebicidal activity of PAA and its efficacy to treat AGD. Temperature, salinity, light and UV affected the decay dynamics of PAA. Salmon smolts could tolerate PAA doses of 0.6 to 10 ppm, however, behavioural and physiological responses were significantly influenced by duration and frequency of exposure, as well as with PAA commercial products. Stress and disease status of fish were documented to influence the responses of salmon to PAA. The application of PAA was identified to be a mild stressor and could induce both transient and systemic oxidative systems. Different physiological adaptive responses were mounted by the fish to counteract these impacts. PAA exhibited amoebicidal activity in vitro. Exposure of AGD-affected salmon to PAA using different treatment protocols resulted in an equivocal disease resolution. Though treatment could reduce parasite load, gross and microscopic pathologies persisted after the treatments. New insights about the pathophysiology of AGD had been generated particularly on the role of oxidative stress and circulating metabolites. Additional treatment optimisation is required for PAA as a treatment for AGD. Nonetheless, the project provided significant advancements in the chemistry and physiology of PAA in fish that will be crucial in developing the evidence-driven application of this disinfectant in aquaculture.

Vitenskapelige publikasjoner/ Scientific publications (noen av disse ligger også i fulltekst i faglig sluttrapport/ some of the following reports are also included in the final report)
– C. C. Lazado, M. W. Breiland, F. Furtado, E. Burgerhout, and D. Strand, ‘The circulating metabolome of Neoparamoeba perurans-infected Atlantic salmon (Salmo salar)’, Microbial Pathogenesis, 166 (2022), 105553.
– J. Osório, K. T. Stiller, B. K. Reiten, J. Kolarevic, L. H. Johansen, F. Afonso, and C. C. Lazado, ‘Intermittent administration of peracetic acid is a mild environmental stressor that elicits mucosal and systemic adaptive responses from Atlantic salmon post-smolts’, BMC Zoology, 7 (2022), 1.
– C. C. Lazado, G. Timmerhaus, M. W. Breiland, K. Pittman, and S. Hytterød, ‘Multiomics provide insights into the key molecules and pathways involved in the physiological adaptation of Atlantic salmon (Salmo salar) to chemotherapeutic-induced oxidative stress’, Antioxidants, 10 (2021), 1931.
– S. Haddeland, C. C. Lazado, G. Merkin, O. J. Myre, M. Okubamichael, L. F. Pedersen, and K. Pittman, ‘Dynamic morphometrics of mucous cells reveal the minimal impact of therapeutic doses of peracetic acid on Atlantic salmon gill health’, Aquaculture, 534 (2021), 736315.
– C. C. Lazado, L. Sveen, M. Soleng, L. F. Pedersen, and G. Timmerhaus, ‘Crowding reshapes the mucosal but not the systemic response repertoires of Atlantic salmon post-smolts to peracetic acid’, Aquaculture, 531 (2021), 735830.
– C. C. Lazado, V. Voldvik, M. W. Breiland, J. Osório, M. S. Hansen, and A. Krasnov, ‘Chemical oxidative stressors alter the physiological state of the nasal olfactory mucosa of Atlantic salmon’, Antioxidants, 9/11 (2020), 1144.
– C. C. Lazado, G. Timmerhaus, M. Soleng, K. H. Kirste, M. B. Breiland, and L. F. Pedersen, ‘Oxidant-induced modifications in the mucosal transcriptome and circulating metabolome of Atlantic salmon’, Aquatic Toxicology, 227 (2020), 105625.
– C. C. Lazado, S. Haddeland, G. Timmerhaus, R. S. Berg, G. Merkin, K. Pittman, and L. F. Pedersen, ‘Morphomolecular alterations in the skin mucosa of Atlantic salmon (Salmo salar) after exposure to peracetic acid-based disinfectant’, Aquaculture Reports, 17 (2020), 100368.
– L. F. Pedersen and C. C. Lazado, ‘Decay of peracetic acid in seawater and implications for its chemotherapeutic potential in aquaculture’, Aquaculture Environment Interactions, 12 (2020), 153–165.
– C. C. Lazado and V. Voldvik, ‘Temporal control of responses to chemically induced oxidative stress in the gill mucosa of Atlantic salmon (Salmo salar)’, Journal of Photochemistry and Photobiology B: Biology, 205 (2020), 111851.
– C. C. Lazado, ‘The 1st International Symposium on Mucosal Health in Aquaculture – MHA2019’, Tissue Barriers, (2020) 1712177.
– M. Soleng, L. H. Johansen, H. Johnsen, G. S. Johansson, M. W. Breiland, L. Rørmark, K. Pittman, L. F. Pedersen, and C. C. Lazado, ‘Atlantic salmon (Salmo salar) mounts systemic and mucosal stress responses to peracetic acid’, Fish & Shellfish Immunology, 93 (2019), 895–903.
​Prosjektet har gjort en utførlig dokumentasjon og evaluering av peredikksyre som en mulig ny kjemisk behandlingsmetode mot AGD-parasitten og potensielt også andre uønskede mikroorganismer, som et nødvendig kunnskapsgrunnlag for at næringen kan ta dette direkte i bruk. Næringen trenger et bredt repertoar av behandlingsmetoder for å håndtere parasitter som utvikler rask resistens. Med nye lukkede systemer for utslippsfri behandling med kjemiske preparater i sjø, vil PAA være ett av disse som kan gi bedre kontroll med AGD og andre uønskede gjelleagens.
Background
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.
Objectives
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 (2019–2022)
• 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.
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 organization
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.
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