Challenge Models

Introduction

The susceptibility to IPNV infection is highly dependent on the genetic characteristics of the test fish. Atlantic salmon are susceptible to IPN by experimental infection as first feeding fry (fresh water) and as post-smolts (sea water). A Norwegian IPNV strain originating from an outbreak in post-smolts is used for all challenges. The virus is a serotype Sp/N1, characterized as highly virulent and carrying putative molecular virulence marker motifs.

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Cohabitation challenge of smolts (sea water)

Pre-smolts to be included in a bath or cohabitant challenge study are photoperiod-manipulated to smoltify. Challenges can be performed within 1-3 weeks after transfer to sea water. For cohabitant challenge studies, 20% i.p. injected shedders are introduced to the challenge tank. Fish are observed throughout a <35-day period.

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Immersion challenge of fry (fresh water)

Fish of different genetic characteristics can be kept in separate tanks or mixed in one tank during challenge. Possible tank effects can be reduced by mixing all families in one tank. The fish (0.1-0.2 grams in average weight) will be challenged by immersion 1-3 weeks after onset of start feeding. Mortality is recorded for 35-50 days. Subpopulations of fish from the challenged fish pool are typically identified by DNA fingerprinting.

Challenge test in Atlantic salmon populations with high (HR) and low (LR) resistance to IPN.

Challenge model

Piscine myocarditis virus (PMCV) is the causative agent of cardiomyopathy syndrome (CMS). VESO Aqualab has established an experimental challenge model for infection with PMCV. Smolts can be challenged through the intraperitoneal or intramuscular route. The diagnosis is typically based on qPCR and histopathology, and trials are run for 8 – 9 weeks post challenge. There is a strong correlation between viral load and histopathological changes in the heart.

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Inoculum

Cultivation of PMCV in cell line systems has so far not been successful. Experimental transmission of CMS has therefore been demonstrated using tissue homogenate from CMS-diagnosed fish. The challenge material produced at VESO Aqualab origins from a highly virulent field outbreak of CMS. The homogenate was screened for all known pathogens by qPCR. It was thereafter passed repeatedly through our pathogen free test fish to ensure a tissue homogenate containing PMCV only.

Histopathological changes in the heart after experimental infection with PMCV. High viral loads were detected by qPCR. Upper micrograph: 9 wpc by i.m. injection. Lower micrograph: 9 wpc by i.p. injection.

Detection of PMCV (red) in heart by in situ hybridization.

Introduction

Piscine orthoreovirus (PRV) is the causative agent of heart and skeletal muscle inflammation (HSMI). The virus is ubiquitous, but there is a significant increase in the viral load during an outbreak of HSMI. Experimental transmission of HSMI has been demonstrated using tissue homogenate, red blood cells and purified virus. The diagnosis is based on slowly emerging histopathological changes in the heart. qPCR and immunohistochemistry can be used to detect the virus at early stages of infection, before the onset of histopathological changes.

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Challenge model

A challenge model for experimental infection of Atlantic salmon has been developed at VESO Aqualab in close collaboration with researchers from the Norwegian School of Veterinary Science (NMBU). Parr and smolts can be challenged by i.p. injection of infectious material or by cohabitation, followed by serum and tissue sampling. Cohabitation trials are typically run for 8-10 weeks. There is a strong correlation between viral load and the following histopathological changes in the heart.

Detection of PRV after challenge by i.p. injection or cohabitation (n = 6, individual fish shown). Low Ct value = high viral load. From Wessel et al (2017), PLoS ONE 12(8).

Introduction

Infectious salmon anemia (ISA) is a systemic disease of farmed Atlantic salmon. Disease outbreaks have been reported in Norway, Canada, Scotland, the Faroe Islands, USA and Chile. Prophylactic measures are sought to prevent outbreaks of ISA in the salmon aquaculture industry. VESO Aqualab offers challenge models to evaluate the efficacy of vaccines, the effect of prophylactic feeding and the effect of selective breeding for resistance.

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Challenge models to evaluate the effect of vaccination

The fish will be vaccinated at the parr stage and either photoperiod-manipulated to smoltify or maintained as pre-smolts. After vaccination the fish are either challenged by i.p. injection of virus or by cohabitation with i.p. injected shedder fish. Evaluation of the protection provided by the vaccines is based on differences in mortality in vaccinated and unvaccinated fish after challenge.

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Challenge models to evaluate genetic resistance profiles

Fish of different genetic characteristics can be kept in separate tanks or mixed in one tank during challenge. Possible tank effects can be reduced by mixing all families in one tank. The fish will be acclimatized for minimum two weeks followed by challenge by i.p. injection of virus or cohabitation with i.p. injected shedder fish. Subpopulations of fish from the challenged fish pool are typically identified by DNA fingerprinting or PIT-tag readings.

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Challenge models to evaluate the effect of feeding

The fish will be acclimatized for minimum two weeks followed by a period of test feeding. After challenge by i.p. injection or cohabitation, mortality will be recorded throughout a five weeks observation period.

Mortality in groups of fish i.p. injected with ILAV (shedders) and in the naïve fish (cohabitants).

Introduction

The susceptibility to IPNV infection is highly dependent on the genetic characteristics of the test fish. Atlantic salmon are susceptible to IPN by experimental infection as first feeding fry (fresh water) and as post-smolts (sea water). A Norwegian IPNV strain originating from an outbreak in post-smolts is used for all challenges. The virus is a serotype Sp/N1, characterized as highly virulent and carrying putative molecular virulence marker motifs.

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Cohabitation challenge of smolts (sea water)

Pre-smolts to be included in a bath or cohabitant challenge study are photoperiod-manipulated to smoltify. Challenges can be performed within 1-3 weeks after transfer to sea water. For cohabitant challenge studies, 20% i.p. injected shedders are introduced to the challenge tank. Fish are observed throughout a <35-day period.

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Immersion challenge of fry (fresh water)

Fish of different genetic characteristics can be kept in separate tanks or mixed in one tank during challenge. Possible tank effects can be reduced by mixing all families in one tank. The fish (0.1-0.2 grams in average weight) will be challenged by immersion 1-3 weeks after onset of start feeding. Mortality is recorded for 35-50 days. Subpopulations of fish from the challenged fish pool are typically identified by DNA fingerprinting.

Challenge test in Atlantic salmon populations with high (HR) and low (LR) resistance to IPN.

Introduction

Yersiniosis or enteric red mouth disease (ERM) is caused by the gram-negative bacterium Yersinia ruckeri. The disease was historically observed in smaller fish in fresh water, but during the last years several outbreaks in larger fish in sea water have been reported. Fish may suffer from sub clinical infections and outbreaks can be seen after stressful situations such as transport or delousing.

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Challenge models

VESO Aqualab offers challenge models for fry, parr and smolts with Y. ruckeri serotype O1 isolates representative for fresh water and sea water outbreaks. Fish are challenged by i.p. injection or immersion.

Mortality in salmon parr bath challenged with Yersinia ruckeri.

Introduction

Bacteria belonging to the genus Vibrio are the causative agents of vibriosis. Vibriosis can cause significant mortality once an outbreak is in progress and vaccination is used to minimize the impact of the disease. VESO Aqualab offers challenge models in salmon with:

• Vibrio salmonicida (causative agent of cold-water vibriosis or Hitra-disease)

• Vibrio anguillarum, serotype O1 and O2a (causative agents of vibriosis)

• Vibrio ordalii (causative agent of vibriosis)

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Challenge models to evaluate the effect of vaccination

Salmon parr (pre-smolts) are acclimatized for a minimum of one week before vaccination. After the immunization period fish are challenged by i.p. injection or immersion in fresh water or sea water. After challenge, mortality is recorded during an observation period of two to four weeks. Evaluation of the potency of the vaccine is based on differences in mortality in vaccinated and unvaccinated fish.

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Vibrio ordalii:

Vibrio ordalii

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Vibrio Anguillarum:

Vibrio Anguillarum

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Mortality in groups of vaccinated and unvaccinated salmon i.p. injected with Vibrio anguillarum and Vibrio ordalii.

Introduction

Aeromonas salmonicida is the causative agent of furunculosis. This gram-negative bacterium infects salmonid species worldwide, with both typical and atypical strains causing disease. All salmonids are believed to be naturally susceptible to infection with Aeromonas salmonicida, and the bacterium has been isolated from a range of other species as well. VESO offers challenge models with Aeromonas salmonicida in Atlantic salmon and rainbow trout.

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Challenge models to evaluate the effect of vaccination

Salmonid parr (pre-smolts) are acclimatized for a minimum of one week before vaccination. After the immunization period fish are challenged by i.p. injection or cohabitation with i.p. injected shedder fish. After challenge, mortality is recorded during an observation period of three to four weeks. Evaluation of the potency of the vaccine is based on differences in mortality in vaccinated and unvaccinated fish.

Mortality in groups of vaccinated and unvaccinated salmon i.p. injected with Aeromonas salmonicida.

Information coming soon.

Introduction

Francisella is an intracellular bacterium that causes the systemic granulomatous inflammatory disease francisellosis in cod. VESO Aqualab offers challenge models with Francisella sp.

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Challenge models to evaluate the effect of vaccination

Cod are acclimatized for a minimum of one week before challenge by i.p. injection or immersion. After challenge, mortality is recorded during an observation period of two to three weeks. Evaluation of the potency of the vaccine is based on differences in mortality in vaccinated and unvaccinated fish.

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Diagram 1: Challenge by intraperitoneal injection

Diagram 2: Challenge by immersion

Mortality in groups of unvaccinated cod challenged with different doses of Francisella sp.

Introduction

Bacteria belonging to the family Pasteurellaceae are the causative agents of pasteurellosis. High mortality by pasteurellosis has been reported in Norway since 2012. Mortality related to the outbreak of Pasteurella sp. occurs often 2-4 weeks after episodes that involve a lot of stress for the fish, such as vaccination, transport and exposure in the cage. Mortality can accumulate to high levels if the disease is not treated. VESO Aqualab offers challenge models with Pasteurella sp.

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Challenge model to evaluate the efficacy of vaccination

Lumpfish are acclimatized for a minimum of two weeks before vaccination. After an immunization period of four weeks fish are challenged by i.p. injection or cohabitation. After challenge, mortality is recorded during an observation period of 2-4 weeks. Evaluation of the potency of the vaccine is based on differences in mortality in vaccinated and unvaccinated groups

Mortality in groups of unvaccinated lumpfish challenged with increasing doses of Pasteurella sp. by i.p. injection or cohabitation.

Introduction

Pasteurellosis is caused by the gram-negative and halophilic bacterium Photobacterium damselae. The disease affects a wide range of marine species, and fish can harbor the bacterium as subclinical infection for long periods of time. VESO Aqualab offers challenge models with Photobacterium damselae.

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Challenge model to evaluate the efficacy of vaccination

Seabass (average weight 4-10 grams) are acclimatized to 20°C for a minimum of two weeks before vaccination. After a four-week immunization period, fish are challenged by i.p. injection. After challenge, mortality is recorded during an observation period of 21 days. Evaluation of the potency of the vaccine is based on differences in mortality in vaccinated and unvaccinated groups.

Mortality in groups of vaccinated and unvaccinated seabass i.p. injected with Photobacterium damselae.

Introduction

Piscirickettsia salmonis is the etiological agent of Salmon rickettsial septicemia (SRS) or Piscirickettsiosis. The disease causes high mortality and significant economic losses to the Chilean salmon industry. Experimental trials are indispensable to evaluate the efficacy of prophylactic measures against Piscirickettsiosis, such as vaccination, selective breeding for resistance or functional feeds. VESO Aqualab offers experimental challenge models for pre-smolts (parr) and post-smolts. After challenge, the fish are observed daily, and mortality recorded. The diagnosis of Piscirickettsiosis is based upon presence of the characteristic external and internal signs of the disease and cultivation of the bacterium on agar plates. Serum and tissue can be sampled for subsequent analysis by ELISA and RT-qPCR.

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Challenge of parr

Parr will be acclimatized to 15°C before challenge. Fish are challenged by i.p. injection of bacteria, or cohabitation with i.p. injected shedders. After challenge the fish are kept under close observation and mortalities registered.

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Challenge of post-smolts

Pre-smolts to be included in an i.p. or cohabitant challenge study are photoperiod manipulated to smoltify before adaption to sea water. Fish will be acclimatised to 15°C before challenge. If the fish are included in a vaccination trial, vaccination will be performed during the smoltification period. The test fish are challenged after transfer to sea water, either by i.p. injection of bacteria or by cohabitation with i.p. injected shedder fish.

Mortality in groups of fish i.p. injected with Piscirickettsia salmonis (shedders), and in control fish (saline injected cohabitants).

Introduction

Bacteria belonging to the genus Vibrio are the causative agents of vibriosis, which can be a problem in cod farming. Vibriosis can cause significant mortality once an outbreak is in progress and vaccination is used to minimize the impact of the disease. VESO Aqualab offers challenge models with Vibrio anguillarum, serotypes O1, O2a and O2b.

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Challenge models to evaluate the effect of vaccination

Cod are acclimatized for a minimum of one week before challenge by i.p. injection or immersion. After challenge, mortality is recorded during an observation period of two to three weeks. Evaluation of the potency of the vaccine is based on differences in mortality in vaccinated and unvaccinated fish.

Mortality in groups of vaccinated and unvaccinated cod i.p. injected with Vibrio anguillarum.

Introduction

Bacteria belonging to the genus Vibrio are the causative agents of vibriosis. Vibriosis can cause significant mortality once an outbreak is in progress and vaccination is used to minimize the impact of the disease. VESO Aqualab offers challenge models in salmon with:

• Vibrio salmonicida (causative agent of cold-water vibriosis or Hitra-disease)

• Vibrio anguillarum, serotype O1 and O2a (causative agents of vibriosis)

• Vibrio ordalii (causative agent of vibriosis)

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Challenge models to evaluate the effect of vaccination

Salmon parr (pre-smolts) are acclimatized for a minimum of one week before vaccination. After the immunization period fish are challenged by i.p. injection or immersion in fresh water or sea water. After challenge, mortality is recorded during an observation period of two to four weeks. Evaluation of the potency of the vaccine is based on differences in mortality in vaccinated and unvaccinated fish.

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Diagram 1:

Diagram 2:

Mortality in groups of vaccinated and unvaccinated salmon i.p. injected with Vibrio anguillarum (diagram 1) and Vibrio ordalii (diagram 2).

Introduction

Bacteria belonging to the genus Vibrio are the causative agents of vibriosis, which can be a problem in cod farming. Vibriosis can cause significant mortality once an outbreak is in progress and vaccination is used to minimize the impact of the disease. VESO Aqualab offers challenge models with Vibrio anguillarum, serotypes O1, O2a and O2b.

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Challenge models to evaluate the effect of vaccination

Cod are acclimatized for a minimum of one week before challenge by i.p. injection or immersion. After challenge, mortality is recorded during an observation period of two to three weeks. Evaluation of the potency of the vaccine is based on differences in mortality in vaccinated and unvaccinated fish.

Mortality in groups of vaccinated and unvaccinated cod i.p. injected with Vibrio anguillarum.

Introduction

Bacteria belonging to the genus Vibrio are the causative agents of vibriosis. Vibriosis can cause significant mortality once an outbreak is in progress and vaccination is used to minimize the impact of the disease. VESO Aqualab offers challenge models with Vibrio anguillarum.

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Challenge model to evaluate the efficacy of vaccination

Lumpfish are acclimatized for a minimum of two week before vaccination. After an immunization period of five to six weeks, fish are challenged by i.p. injection. After challenge, mortality is recorded during an observation period of maximum 21 days. Evaluation of the potency of the vaccine is based on differences in mortality in vaccinated and unvaccinated groups.

Mortality in groups of unvaccinated lumpfish i.p. injected with increasing doses of Vibrio anguillarum.

Introduction

Vibrio salmonicida is the causative agent of cold water vibriosis or so-called Hitra disease. During 2012 VESO Aqualab received several requests regarding outbreaks of this disease in northern Norway. We brushed the dust off our old challenge model, and further developed it into a clinically relevant bath challenge model. Mortality in control smolts typically reaches 70% and a new field isolate from northern Norway is available for challenge.

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Challenge models to evaluate the effect of vaccination

Salmon parr (pre-smolts) are acclimatized for a minimum of one week before vaccination. After the immunization and smoltification period fish are challenged by i.p. injection or immersion in fresh water or sea water. After challenge, mortality is recorded during an observation period of two to four weeks. Evaluation of the potency of the vaccine is based on differences in mortality in vaccinated and unvaccinated fish.

Mortality in groups of unvaccinated salmon smolts bath challenged with Vibrio salmonicida.

Introduction

Bacteria belonging to the genus Vibrio are the causative agents of vibriosis. Vibriosis can cause significant mortality once an outbreak is in progress and vaccination is used to minimize the impact of the disease. VESO Aqualab offers challenge models in salmon with:

• Vibrio salmonicida (causative agent of cold-water vibriosis or Hitra-disease)

• Vibrio anguillarum, serotype O1 and O2a (causative agents of vibriosis)

• Vibrio ordalii (causative agent of vibriosis)

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Challenge models to evaluate the effect of vaccination

Salmon parr (pre-smolts) are acclimatized for a minimum of one week before vaccination. After the immunization period fish are challenged by i.p. injection or immersion in fresh water or sea water. After challenge, mortality is recorded during an observation period of two to four weeks. Evaluation of the potency of the vaccine is based on differences in mortality in vaccinated and unvaccinated fish.

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Diagram 1:

Diagram 2:

Mortality in groups of vaccinated and unvaccinated salmon i.p. injected with Vibrio anguillarum (diagram 1) and Vibrio ordalii (diagram 2).

Introduction

Moritella viscosa is a gram-negative bacterium that is the causative agent of winter ulcer disease. Winter ulcers often appear during periods of cold water and high salinity and the disease is characterized by the formation of dermal and subdermal ulcers. When the temperature increases, or salinity decreases, fish may recover from an outbreak of winter ulcers.

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Challenge by immersion

VESO Aqualab has long experience in conducting challenge trials with Moritella viscosa, and we recommend models based on bath challenge of smolts in sea water which mimic a natural disease situation. Pre-smolts are photoperiod-manipulated to smoltify, transferred to sea water and acclimatized at 8-10°C before challenge. Development of ulcers and mortality is observed throughout a 15-30-day period. The mortality rate may be controlled by adjusting the water temperature.

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Confirmatory diagnosis of Moritella viscosa is based on colony morphology and viscosity following growth on blood agar plates from smears from the pronephros of dead fish. Principal outcome parameters are prevalence and severity of ulceration and mortality.

Introduction

Renibacterium salmoninarum is the causative agent of Bacterial Kidney Disease - BKD. This disease is broadly distributed within salmon farming countries, including Europe, North and South America. BKD may occur as a chronic or acute clinical manifestation, producing important mortality levels causing economic losses in farming companies. In this scenario, it becomes critical to be able to test a variety of prophylactic measures such as vaccines, antimicrobials and genetic selection programs under controlled trial conditions. VESO offers an experimental challenge model fish at the stage of pre-smolts (parr). After challenge, the fish are observed daily and mortality is recorded. The diagnosis of the disease is based upon presence of the characteristic internal signs of the disease and cultivation of the bacterium in agar plates.

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Challenge model

In preparation to the challenge, fish are acclimated to 15°C for a minimum of 7 to 14 days. Then, fish are challenged via i.p. injection with the challenge material. After challenge, the fish are kept under a minimum disturbance policy and under close observation for recording mortalities. During this period, we can perform all type of samplings required by the clients.

Mortality in groups of fish i.p. injected with Renibacterium salmoninarum at different concentrations.

Introduction

Amoebic Gill Disease (AGD) has been a problem in Tasmanian aquaculture for years. The disease is caused by the amoeba Paramoeba perurans, which thrives very well in high salinity sea water at increasing temperatures. During the last years outbreaks have been reported in Ireland and Scotland, and also along the Norwegian coastline.

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Challenge model

To face this problem, VESO Aqualab established a challenge model for AGD. Through close collaboration with the Norwegian Veterinary Institute, we are able to cultivate the amoeba and have successfully infected salmon smolts in experimental trials. We have studied the dynamics of the challenge model at different temperatures and salinities, as well as during fresh water treatment. Large scale AGD trials with a maximum of ~4000 smolts in one tank can be conducted.

Introduction

Amoebic Gill Disease (AGD) has been a problem in Tasmanian aquaculture for years. The disease is caused by the amoeba Paramoeba perurans, which thrives very well in high salinity sea water at increasing temperatures. During the last years outbreaks have been reported in Ireland and Scotland, and also along the Norwegian coastline.

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Challenge model

To face this problem, VESO Aqualab established a challenge model for AGD. Through close collaboration with the Norwegian Veterinary Institute, we are able to cultivate the amoeba and have successfully infected salmon smolts in experimental trials. We have studied the dynamics of the challenge model at different temperatures and salinities, as well as during fresh water treatment. Large scale AGD trials with a maximum of ~4000 smolts in one tank can be conducted.

Paramoeba perurans (Terje Steinum, NVI).

Atlantic salmon gills‍

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Paramoeba perurans (Terje Steinum, NVI) and Atlantic salmon gills three weeks after bath challenge with Paramoeba perurans.

Introduction

VESO has broad experience in design and conduct of experimental sea lice trials. Sea lice larvae and motile development stages are cultivated at VESO Vikan. They are typically produced from egg strings that have been obtained from fish farms. Larvae are hatched in specially designed units.

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Challenge models

Sea lice challenge trials can be conducted to evaluate the efficacy of new generation anti parasitical drugs, vaccination, feeding or selective breeding for resistance against sea lice attachment. Challenges are typically performed using copepods.

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Effect of in vivo treatment

VESO has established several challenge models for testing and screening of chemotherapeutics. Trials are performed with sea lice of all developmental stages (copepodite – adult) attached to seawater adapted post-smolts. We can also evaluate the hatching ability of egg strings after exposure to test substances.

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Bioassay – in vitro

Bioassays are designed to measure the sensitivity of a sea lice population to a specific chemotherapeutic. Bioassays may be performed on planktonic larval stages or motile (pre-adult and adult) developmental stages. The sensitivity to test compounds will show a degree of variation depending on e.g. developmental stage and gender. Bioassays must therefore be individually designed according to each test substance.

Tendency of developmental drift in a laboratory sea lice population.