Hydrogenperoksidresistens i lakselus (Lepeophtheirus salmonis)

In spite of a dramatic increase in the use of non-medicinal control, Norwegian salmon farming is still dependent on chemical delousing to keep the lice level below the maximum of 0.5 adult female lice per fish. Frequent use of a limited number of chemicals has led to resistance in the salmon louse. Hydrogen peroxide has for a long time been the "last chance" of chemicals for salmon lice control This project is a collaboration between a biotech. company (PatoGen AS), a university (NMBU, Faculty of Veterinary Medicine), a fish health service (Aqua Kompetanse AS) and two aquaculture companies (Marine Harvest AS and Sinkaberg-Hansen AS). Shortly before the start of the project, NMBU found that resistance to H2O2 was associated with increased expression of the enzyme catalase. An RNAseq survey of H2O2-sensitive and H2O2-resistant parasites showed that in addition to the catalase gene, the expression of other genes also correlated with resistance. These were genes coding for wound healing factors, factors that improve the ability of the parasites to cope with oxidative stress, and genes coding for transport proteins. The RNAseq data showed that the expression of the catalase gene was induced by H2O2 exposure. This makes monitoring in the field challenging. In 2018, it was verified that one of the other genes was not affected by exposure to H2O2. It was therefore interesting to see whether this was a suitable additional marker for H2O2 resistance. A number of samples from different salmon lice strains were examined for covariation of this gene's expression with H2O2 sensitivity, measured by bioassays. The covariance was excellent and these two markers provided an excellent prediction of louse's sensitivity to H2O2. The lab validation was done at NMBU, while PatoGen developed a Taq-Man assay that can be used commercially, and started validating the assay on field material. In 2018, PatoGen has worked to get the marker patented and will include it in its services. NMBU is in the process of compiling the results to a publication that is scheduled for submission to a peer-reviewed journal in the spring of 2019. We also studied behavioural and physical changes that the lice undergo when exposed to H2O2. The changes were documented by bioassays, video monitoring, histological- and chemical analyses. The results showed that with increasing H2O2 concentrations and/or exposure time, normal behaviour such as swimming ability and attachment to fish is first affected, leading to detachment. At higher H2O2 exposure, the lice become paralyzed and gas bubbles appear inside the body. These bubbles cause the lice to float and internal organs are damaged. Analyses showed that these bubbles were oxygen. The effects were the same in sensitive and resistant parasites, but were only seen at significantly higher concentrations in resistant parasites. NMBU is in the process of compiling the results to a publication that is scheduled for submission to a peer-reviewed journal in the spring of 2019. In 2018, the bioassay used in fields to estimate the parasites' sensitivity was reviewed and improved. Particular attention was made to temperature. It was demonstrated that water temperature was of great importance for the results. Even 1-2 degree deviation from the nominal temperature of 12 degrees resulted in significant differences, as more parasites died at higher temperatures and less at lower, although the H2O2 concentration was the same. Thus, the exposure solutions must be tempered when the parasites are exposed to H2O2 in bioassays. The protocols used are updated at this point. For other bioassays (azamethiphos, pyrethroids, emamectin), 1-2 degree temperature difference had no significant effect on the outcome. PatoGen has in 2018 worked with implementation of the molecular assays on salmon lice into a larger prediction tool for salmon lice infestation, cage variations, resistance dispersal and treatment effect. The goal is to be able to predict the effect of many different measures against salmon lice at individual sites and regions, both in the short and long term. The modelling work has been done by Norsk Regnesentral. It is expected that this will be an important tool in future Integrated Pest (salmon lice) Management (IPM). In 2018, there have been no hydrogen peroxide treatments suitable for special follow-up in participating aquaculture companies, Marine Harvest Norway and Sinkaberg-Hansen. This has created some problems for validating laboratory results in the field. Marine Harvest has, however, analysed a number of samples from its plants for the resistance markers. These data have been valuable in the validation and have, in addition to data from previous years, been used for a manuscript dealing with the decrease in sensitivity to H2O2 from 2013 to 2018. This script is ready for submission to a peer reviewed journal.

- De molekylære assays benyttes i stor grad av selskaper og fiskehelsetjenester i planlegging av kontroll med lakselus. Interessen fra andre land er stor, de benyttes allerede i Skottland, Færøyene og Canada, og skal lansert i Chile i 2019 - Bioassays for resistenstesting er forbedret. Testene brukes i Mattilsynets OK-program. Forbedringen gir sikrere resultater - Mattilsynets OK-program for lakselusresistens gir et unikt bilde av sensitivitetsstatus i Norge. Det benyttes både molekylære tester og forbedrede bioassaytester. Resultatene gir bedre planlegging av tiltak fra myndighetenes side - Impementeringen av testene i et større modelleringsverktøy for smittepress, spredning av resistensfaktorer og optimal kontroll forventes å bli meget nyttig for oppdrettere, fiskehelsetjenester og myndigheter når det lanseres i 2019 - PatoGen har etablert seg som den eneste leverandør av molekylære hurtigtester for resistens hos lakselus, både nasjonalt og internasjonalt

Norsk lakseoppdrett er foreløpig avhengige av kjemisk avlusing for å holde lusetallet nede på det myndighetsbestemte nivået. Hyppig bruk av få midler har ført til at lakselusa har utviklet resistens mot de fleste av de kjemikaliene som er tilgjengelig i dag. Dette gir store utfordringer for oppdrettsselskapene, i verste fall kan de bli tvunget til forsert utslakting. Hvis kontrollen på lusetallet tapes utsettes fisken for en helsepåkjenning og smittepresset til omkringliggende anlegg og villfisk øker. Hydrogenperoksid som lusemiddel har noen ulemper, det er kostbart å bruke, det angriper bare de bevegelige stadiene av lakselusa og sikkerhetsmarginen i forhold til toksisk dose er begrenset, og krymper jo varmere vannet er. Fram til nylig var det ikke registrert resistens mot hydrogenperoksid, dette midlet har derfor vært «the last man standing» i enkelte resistensutsatte områder. I 2012 begynte det å komme rapporter om enkelttilfeller med redusert behandlingseffekt. NMBU Veterinærhøgskolen utviklet i samarbeid med Aqua kompetanse AS en bioassaymetode som kunne skille mellom lakselusstammer med ulik sensitivitet mot hydrogenperoksid. Denne resistenstesten ble så brukt på en stamme som hadde vist redusert behandlingseffekt, og denne stammen hadde en sensitivitet som var 10 ganger lavere enn en fullt ut sensitiv stamme. Avkommet til den resistente stammen viste den samme høye resistensen som sine foreldre, egenskapen er derfor arvelig. Innledende studier har også blitt utført for å finne de molekylære mekanismene som ligger bak. De første resultater tyder på metabolske faktorer som årsak, og et første-generasjons assay er utviklet, selv om den egentlige genetiske "signatur" enda ikke er identifisert. Dette prosjektet tar sikte på å finne denne, utvikle et andre-generasjons molekylært assay og sette dette inn i en systematisk plan for overvåking av resistens og bekjempelse av lakselus.