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Article

The Effect of a Polypeptide Based Vaccine on Fish Welfare and Infestation of Salmon Lice, Lepeophtheirus salmonis, in Sea Cages with Atlantic Salmon (Salmo salar L.)

by
Ragnar Nortvedt
1,*,
Erik Dahl-Paulsen
1,
Laura Patricia Apablaza Bizama
2,
Amritha Johny
3 and
Erik Slinde
4
1
Matre Research Station, Institute of Marine Research, N-5984 Matredal, Norway
2
Department of Biological Sciences, University of Bergen, N-5007 Bergen, Norway
3
Department of Fish Health, Nofima AS, Osloveien 1, N-1430 Ås, Norway
4
Faculty of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, N-1433 Ås, Norway
*
Author to whom correspondence should be addressed.
Fishes 2025, 10(8), 405; https://doi.org/10.3390/fishes10080405
Submission received: 28 May 2025 / Revised: 17 July 2025 / Accepted: 6 August 2025 / Published: 13 August 2025
(This article belongs to the Special Issue Healthy Aquaculture and Disease Control)
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Abstract

A new polypeptide vaccine towards salmon lice (Lepeophtheirus salmonis) was given to experimental groups of 2 × 8000 Atlantic salmon parr (Salmo salar L.), following the vaccination of a total of 4 × 8000 parr with a common set of vaccines used in Norwegian aquaculture to prevent infestation in salmon growing at sea. The remaining 2 × 8000 salmon served as control. The trial was conducted at a sea farm research facility at Knappen-Solheim in Masfjorden, Norway. Natural infestation with sea lice were staged and counted once a week from January–December 2023. The infestation was never above two mature female lice per salmon, the maximum limit set specifically for the present trial by the Norwegian Food Safety Authorities, thus delousing with chemicals or other methods was avoided. Mortality, growth, feed consumption, sexual maturation, slaughter quality, and welfare quality parameters were not significantly different between vaccinated and control salmon. The effect size showed a moderate positive difference of 0.07 mature female salmon lice per salmon in favor of the vaccinated groups from a fish size above 600 g in May until November. All fish were slaughtered and marketed at a size of 5.8 kg (>83% superior quality).
Keywords:
salmon lice; peptide vaccine; sea trial; fish welfare; salmon growth
Key Contribution: This semi-commercial scale experiment demonstrated by a range of welfare-related parameters that no adverse effects could be attributed to the vaccine candidate or to the vaccination per se. On the other hand, the vaccine had only a moderate positive effect on mature female salmon lice attachment to the host, so further vaccine development should be encouraged.

1. Introduction

The government of Norway aims for growth of the salmon industry, but is faced with several challenges, including an annual large loss of money for the farmers due to disease and other production-related problems. Salmon lice infestation remains one of the great challenges for Atlantic salmon aquaculture during the seawater phase of production. Sea lice levels in salmon farms were under surveillance by The Norwegian Food Safety Authorities throughout the production period. It is a welfare issue for the fish and makes the fish vulnerable towards other diseases. Lepeophtheirus salmonis is the species of greatest concern [1,2,3] and the infestation status in each Norwegian fish farm is weekly reported on an open access website [4].
After hatching, L. salmonis free swimming nauplii molt into the infective copepodite stage in about 2 to 5 days. The nauplii and copepodites display different vertical migration patterns in response to light [5] before infestation. This is followed by the chalimus stage that undergoes two molting stages before becoming a pre-adult or mobile stage [3,6,7,8]. Then they can move around on the surface of the fish and may also swim in the water column. Finally, mature female lice develop egg strings that hatch and propagate the lice infestation [7]. Medicinal treatments to combat L. salmonis have increased, as well as resistance towards the chemicals [9].
The development of an effective vaccine still remains a key target, and initial approaches have been carried out to do so [10,11,12]. Contreras et al. [10] targeted the salmon louse midgut function and blood digestion for the identification of candidate target proteins (P30 and P33), where the observed vaccine efficacy was moderate for the chosen antigens. However, the same research partners suggested a dose–response effect from recombinant P33 vaccination [11], also pointing out the need for vaccination trials under field conditions typical of salmon farms.
Swain et al. [12] also found promising efficacy for a vaccine based on a peptide of 35 amino acids ribosomal P0 antigen from sea lice [13]. The authors pointed out that their challenge experimental infestation load of 35 copepodites per fish in land-based tanks was far higher than the natural conditions in the field. Therefore, new approaches must be introduced, and one possibility is a vaccine based on what proteins the lice inject into the blood stream of the salmon under natural field conditions.
The protein peroxiredoxin-2 was found to be a possible vaccine target, since it belongs to a family of ubiquitous multifunctional antioxidant proteins. Its main function is to eliminate peroxides generated during metabolism. Manufacturing of peptide vaccines is generally considered as safe and cost effective when compared to conventional vaccines [14,15]. From our previous results a peptide vaccine gives high protection towards both the Pacific and the Atlantic salmon lice [16]. The aim of the present study was to test a peptide vaccine from the protein peroxiredoxin-2 that has proven to be effective towards lice in land-based seawater tanks [16]. The protein is assumed to have a role in antioxidative mechanisms facilitating the feeding process of the parasite. The test was performed in semi-commercial scale at a sea farm research facility where the natural occurrence of salmon lice is a frequent problem.

2. Materials and Methods

2.1. Experimental Facility and Animal Ethics

The vaccine trial with Atlantic salmon was conducted at the Knappen-Solheim fish farm (Locality number 13567, Figure 1A), Matre Research Station, Institute of Marine Research, Norway, in accordance with relevant guidelines and approval granted by the Norwegian Food Safety Authority (FOTS ID: 29785, 13 July 2022). The Knappen-Solheim fish farm is located in the inmost part (16 km) of the Masfjord, a side branch from the Fensfjord along the coastline. The salmon lice infestation pressure was medium-high compared to the fish farms located in the main current along the coastline. The sea farm had 10 cages with experimental fish, where two experimental groups of salmon lice vaccinated postsmolt were reared in cages M4 and M9, and two control groups postsmolt were reared in cages M5 and M8 (Figure 1B) in the period of 21 November 2022–21 December 2023.
Given the known prevailing current direction and the forecasted higher infestation pressure from the innermost cages based on local experience, it might become a risk to randomize the positions of cages as it may potentially favor the vaccinated groups if they by random had been placed in the outermost part of the farm. On the other hand, by placing the parallel groups in a diagonal design (Figure 1B), it was ensured that the maximum spread of parallel vaccinated groups reduced the problem of too few cages available.
All groups were fed Skretting 9 mm feed (Premium 2500-50A), according to an automated feeding schedule during daylight. The surrounding cages kept other experimental groups of 2–5 kg salmon in cages M1–M3 and M6–M7 and 1–3 kg rainbow trout (Oncorhynchus mykiss) in cage M10, in the period of January until the middle of June 2023. Delousing fish with Salmosan™ was carried out in the surrounding fish groups M1–M3 and M10 on 13 January 2023 and on 13 February 2023, and fish groups M6–M7 on 22 February 2023, due to increasing levels of matured female salmon lice. These treatments were performed onboard a well boat and the treatment water was collected in onboard tanks and emptied in the open sea outside Masfjorden to avoid potential influence on fish farms and spawning areas for wild stocks.

2.2. Vaccination

A total of 32,000 salmon parr (hatched from AquaGen QTL-SHIELD roe) were fully vaccinated on 30 August 2022 with the ordinary vaccines (Alpha JECT micro 6 and Alpha JECT micro 1 PD) used in Norwegian aquaculture to prevent infestation in salmon growing at sea. The experimental subgroup of 2 × 8000 parr were then vaccinated on 20 September 2022 as described by [16] with a short peptide of 13 amino acids (NKEFKEVSLKDYT) at an average weight of 84 ± 5 g. The peptide was produced by ProImmune (Oxford, UK) with MontanideTM ISA 763 A VG as adjuvant and processed as a lyophilized powder with a purity of 98% with TFA (Tri-fluoric acid) as the counter ion. Each fish was vaccinated using a syringe needle, with 1 µg/g fish, 2 cm in front of the anus. The smoltified salmon were transferred to seawater on 21 November 2022 in land-based tanks and further transported to the sea cages in the period 7–9 December 2022 at an average weight of 140 g (136 ± 7 g in vaccinated and 143 ± 9 g in control groups).
During vaccination on the land-based farm, each group was given a secret code, only available to one land-based member of the staff. The leading scientist of the investigation then assigned a new code to the groups before transport to the sea cages, unknown to both the land-based and the sea-based staff. In this way, no one of the staff at the sea cages had information of which groups were vaccinated. Even the co-operating scientists and the financier did not know the group identities before slaughtering. After all sampling sessions and data recording, the lead scientist revealed the codes. In this way the study was blinded.

2.3. Sampling and Evaluation

The vaccination side-effects on the fish were quantified by X-ray imaging of the vertebra column [17,18] after slaughtering on 20 December 2023, and by observing melanin spots [19] in organs and body cavity sections and adherence in body cavity sections (Figure 2) at six sampling dates (n = 20 per cage) from 21 November 2022 until 20 December 2023, together with measurements of whole-body weight (n ≥ 100 per cage). The observations were undertaken blindly, without prior knowledge of the fish treatment group.
The scores from melanin spots and adherence were in the range from 0 to 3, where 3 is the most severe score (modified from [20]), which implied that the sum of scores from the three cavity sections could reach a maximum score of 9. Skin bleedings, external wounds, and other welfare-related scores like damaged fins, emaciated body, and external malformations were also quantified in the range of 0–3. The eye health was generally fine without signs of cataract and no tapeworms (Eubothrium sp.) were observed in the intestines during the experimental period. The overall mortality was 0.32% in land-based tanks and 4.8% in sea cages in the vaccinated groups, and 0.44% in land-based tanks and 7.9% in sea cages in the control groups, respectively. This shows that there was lower mortality in the vaccinated groups and there seemed to be no mortality side-effect from the salmon lice vaccination per se. Most of the mortality was recorded in December 2022–January 2023, due to predator attacks from cormorants (Phalacrocorax carbo) and a moderate incidence of mortality (0.47% in the vaccinated groups and 0.32% in the control groups) was observed on 20.07.2023, due to an episodic peak in aluminum from snow-melt water in the surrounding rivers.
Specific daily growth rate (SGR %) was calculated using Equation (1) according to [21]:
SGR (%) = {e[(ln W2−ln W1)/(T2−T1)] − 1} 100
where W1 and W2 denote fish wet weights at sampling times T1 and T2, respectively.
The effect of the salmon lice vaccine against sea lice infestation in each of the four experimental cages was evaluated weekly by counting the numbers of sessile, mobile, and mature female salmon lice (with or without egg strings) per fish in 20 salmon per cage (n = 100 per cage on 26 June and 26 October 2023). The fish were gently netted by a deeply submerged net, operated by a crane, and mildly sedated with 80 mg/L FinquelTM (100% Tricaine mesylate) for a period of 5–10 min in a bath before lice counting by experienced technicians with a head lamp. Lost lice in the sedation bath were also counted. The vaccination effect size in mature female salmon lice per salmon was simply calculated as the average difference in nos. of lice between the control and the vaccinated groups per month. The Norwegian Food Safety Authority gave a dispensation (30 December 2022) from the regulation to have up to 1 mature female salmon lice per salmon in the week nos. 16–21 and up to 2 mature female salmon lice per salmon for the rest of the trial period, in order to avoid delousing during the experimental period. The trial was terminated in December 2023.

2.4. Statistics

All statistical analyses [22] were carried out in Statistica Ver.13.4.D.14. Due to non-normally distributed data, outliers (xi) within groups for each sampling date were removed if they could be identified according to Equation (2), which is as follows:
xi < Q1 − 1.5 × IQR or xi > Q3 + 1.5 × IQR
where Q1 and Q3 are the 1st and 3rd quartiles and IQR is the interquartile range. When comparing the weekly count of mature female lice, outlier detection was performed by defining each cage as a class of data, running a partial least squares regression (PLS), and comparing the multivariate class distance for objects (lice counts at each week) between parallel cages. Outliers were omitted if they fell outside a 95% confidence interval. After removal of outliers, the data were analyzed with a Linear Mixed Model (LMM) to also account for within-cage correlations over time, where vaccination treatment, time, and their interaction are fixed effects and groups are a random effect. The intercept was not significant. Since we evidently had challenges with the varying periodic lice infestation pressure, we also applied a moving window ordinary least squares approach (OLS), taking into account the variation within each month. Standardization (dividing the means by the standard deviation) was applied to adjust for homoscedasticity, so that large abundance with a wide variance should not dominate over small abundances with lower variance. Autocorrelation between variables is a potential weakness of the present design and should be kept in mind when interpreting the results. We thus applied changes in values (yt’) instead of the values themselves in the time series, so that yt’= yt − yt−1, which taken together with the above-mentioned standardization partially overcame the challenge of autocorrelation. The p-values and confidence intervals are given along with the results.

3. Results

3.1. Welfare, Growth and Sexual Maturation

Neither of the vaccinated nor the control groups showed any appearance of vaccination injuries on the vertebral column, in contrast to earlier observations of vaccinated salmon [17,18]. Both the vaccinated and the control groups showed generally low and non-significant different internal melanin levels (50% between 1.0 and 2.0) in the belly (Figure 3A) in the autumn after vaccination and throughout the winter season. Thereafter the medians dropped from 1.5 in April to 1.0 in June and stayed low until slaughter in December. Melanin in organs (Figure 3B) were significantly higher in the vaccinated groups (median = 2.9) than in the control groups (median = 1.8) in December 2022, whereafter the level in the vaccinated groups gradually dropped and became equal to the control groups from June until December 2023 when slaughtered.
The adherence between the organs and the muscle segments in three internal body cavity regions were low and only appeared in Section 3 (mid belly region) during the autumn after vaccination (Figure 4). The levels of adherence increased significantly in all three evaluation sections in April 2023; however, no significant differences were observed between the vaccinated and the control groups, which showed stabilized adherence levels.
Both the vaccinated and the control groups showed generally low and non-significantly different levels of accumulated skin bleedings (Figure 5) and wounds (Figure 6) between the vaccinated and control groups. More than 95% of the fish had moderate occurrence (scores 0 and 1) of skin bleedings and almost 100% had moderate occurrence of wounds.
No significant differences were observed in welfare scores (fins, wounds, emaciated, scale loss, and external malformations) at slaughter in vaccinated vs. control groups (Figure 7). The highest accumulated median scores (1.9) were observed in fins, whereas median wounds and scale loss appeared between 0.5 and 0.8.
The smoltified salmon were on average 140 g at release into sea cages in December 2022, whereafter they showed a daily specific growth rate (SGR%) between 1.0 and 1.2% until October 2023 (Figure 8). From October to December 2023, the SGR dropped to 0.3% and the final average round slaughter weights were 5.7 kg in the vaccinated groups and 5.9 kg in the control groups, however, they were not significantly different. The FCR over the whole experimental period was 1.22 in the control groups and 1.23 in the vaccinated groups, without any significant differences between the groups. The maturation process had evidently started at the end of October in all groups when 20–30% of the males showed initial prolongation of the jaws and appearance of jaw crook (Score 1, Table 1), according to [23]. Between October and the beginning of December this incidence had increased to 30–37%, whereas 1–5% of all the salmon in both the vaccinated and the control groups showed conspicuous spawning colors or thicker posterior parts of their bodies at 4 December 2023 (Score 3, Table 1). The slaughterhouse reported 84.7 % superior quality in the control groups and 81.9% superior quality in the vaccinated groups on 20 December 2023, however they were not statistically significantly different.

3.2. Lice Infestations

The occurrence of salmon lice infestation showed four peaks of sessile stages throughout the experimental period within the control groups (Figure 9A). The occurrence of sessile stages was consecutively followed by time-delayed peaks or waves (red dotted lines in figures) of mobile and mature female stages, respectively (Figure 9 C,E), in line with lice development. A peak wave (0.6) of mature female stage per salmon was first observed in the second half of March 2023, followed by a peak in June and an increasing wave in October–November. The highest levels were observed by the end of November (single observations of >2.0 mature female salmon lice per fish), and the experiment was thus terminated in December.
The weekly trend lines for the vaccinated groups (Figure 9 B,D,F) followed the same general patterns as the control groups (Figure 9 A,C,E). More specifically, the monthly average nos. of mature female lice with egg strings were significantly lower in March in the control groups than in the vaccinated groups (p < 0.05). This trend in effect size changed in May, in favor of the vaccinated groups (Figure 10). The effect size showed a moderate positive difference of 0.07 mature female salmon lice per salmon in favor of the vaccinated groups from May until November (p < 0.09), whereas a more clear picture was seen from month to month (May; p = 0.01, June; p = 0.09, October; p = 0.02, November; p = 0.04). No differences between control groups and vaccinated groups were observed for females without egg strings.

4. Discussion

4.1. Lice Preventive Methods

Barrett et al. [24] have highlighted the benefits of the methods to prevent salmon lice infestation before they occur instead of treatment-focused methods, which all have negative effects on fish welfare [25]. A meta-analysis of studies trialing the efficacy of existing preventive methods concluded with a 76% reduction in infestation density in cages with plankton mesh ‘snorkels’ or ‘skirts’, while the development of an effective vaccine still remains a key target [24]. Lately, submerged cages with air domes [26] or access to air bubbles [27] show promising reductions in lice infestation; however, these methods might also create production and welfare challenges [28]. The alternative use of effective vaccines against salmon lice infestation would lower the costs for the farmers and the welfare issues for the fish.

4.2. The Semi-Commercial Approach

The nos. of cages in a commercial farm are low, whereas the nos. of fish in each cage are high. Therefore, the present field trial mimicking a similar semi-commercial scale had to be designed carefully. The four available cages for the current experiment were delimited by other concurrently ongoing experiments at the sea farm. Given that only four cages (two per group) were used, cage effects could confound the results. This is why simultaneously one cage per treatment was placed in the innermost zone with higher lice infestation pressure and one cage per treatment was placed in the outermost zone with lower lice infestation pressure. From the two holding tanks on land, four transport sessions over to the sea cages were performed in closed tanks onboard the transport vessel over two days. The group order within the control group and within the vaccinated group were randomly assigned their two respective sea cages, thus the assignment of fish to cages should not generate any unwanted effects. Moreover, no fish died under transport.
In the present semi-commercial scale trial with 8000 fish per 2016 m3 cage (maximum biomass of 23.4 kg/m3), there were no significant differences in welfare characteristics (melanin in belly or organs, adherence, skin bleedings or wounds, scale loss, fin or other external damages) between the vaccinated and control groups, in contrast to other vaccination studies [17,19]. Neither were there observed any side effects more common in one group than the other parallel group at any time point. Moreover, no differences in the occurrence of emaciated fish, growth, or incidence of maturation were observed between the groups. The X-ray images confirmed that neither of the vaccinated nor the control groups showed any appearance of vaccination injuries on the vertebral column, in contrast to what has been observed in other vaccination studies of Atlantic salmon [17,18]. We were thus not able to observe any negative effects from the vaccine or the vaccination procedure per se over the 12 months trial in the sea cages.
Other fish trials with salmon lice vaccines [10,11,12,16] have been carried out in lab-scale, pointing out the necessity of validating the efficacy in large-scale trials and applications under field conditions. As the first vaccine study mimicking industrial conditions, we have also faced the challenges that are naturally occurring in a fish farm. First of all, the salmon were infested with copepodites of wild salmon lice when they naturally appeared, just as the salmon in commercial fish farms do. This is in contradiction to the controlled infestation rates in a lab. The infestation rates were generally lower in the present fjord system (Figure 1A) with periodic peaks of fresh water supply from rivers than is recorded in salmon farms in high-salinity coast habitats [4]. It has previously been shown that the abundance and distribution of sea lice increase in numbers under increased temperatures [29,30] and salinity [31], and that the protective effects of snorkel barriers are strongly influenced by both salinity and temperature [32]. Secondly, infestation and delousing of the other co-occurring fish groups in the present fish farm have probably influenced the copepodite infestation pressure towards our four experimental groups. However, this pressure was partly removed after delousing and finally removed when the other groups were slaughtered in June.
Water currents probably had a more significant impact on the copepodite infestation pressure between the four experimental cages than natural variation in salinity and temperature. Nelson et al. [33] also observed major effects on the horizontal and vertical distribution of sea lice larvae in and around salmon farms. Salmon lice eggs and nauplii are spread from fish farms along the coast and from the outer part of the fjord (Figure 1A) into the present inner part where local current gyros appear and are partly mixing with the outward freshwater transport from the rivers and the hydroelectric power plants. Particularly the latter freshwater source has lately become quite unpredictable due to varying diurnal electricity production schemes. Locally, the dominant sea current direction from land side (Figure 1B) supplied pulses of salmon nauplii to the experimental cages. This resulted in peaks in copepodite infestation in the inner cages M4 and M5 before similar peaks were observed in M8 and M9, irrespective of vaccination status. Still, the experimental design with a diagonal spread of the experimental groups [34] ensured that some significant results were obtained.
A cost–benefit evaluation made it clear that an alternative tagging of 16,000 salmon to cohabitate tagged and untagged fish, and thus controlling for the cage effect, would be too costly in relation to the effects seen. From the present study it already seems safe to conclude that a change in nos. of mature female lice per salmon occurred in May, in favor of the vaccinated groups (Figure 9 and Figure 10). Moreover, sampling n = 20 out of 8000 salmon ensured the independency between sampled individuals in the same cage.
Another challenge with experimental groups in sea cages is the risk of fish mortality due to unplanned incidences, ranging from varying weather conditions and shifting water quality to predator attacks. Fortunately, the only significant mortality observed was attacks towards newly populated smolts from diving cormorants, which was restricted by mounting an extra preventive net outside the cages during the initial phase of the experiment, and mortality due to an episodic peak in aluminum from snow-melt water in the surrounding rivers. Weekly sampling of salmon lice stages under the prevailing conditions was based on predetermined variability and statistical power analysis [35]. A Standard Operating Procedure for the validation of automatic louse counts has been developed, and it was suggested to have a sampling size of 20 fish under high prevalence [36]. This sampling size was applied weekly in the present trial and an increased sample size of 100 fish at the end of June and in October confirmed the results in line with the sample size of 20 fish. Low fish mortality and an appropriate sample size should thus ensure that the harvested data from the semi-commercial approach were reliable.

4.3. The Vaccine Candidate

The novel approach of using a peptide from a protein as a vaccine instead of a cloned protein give new challenges. Vaccination with peptides always has a possibility of generating side effects, and an extra injection with a vaccine might also be harmful. However, as is shown through the figures compared with the control, growth rates are similar; melanin deposits are slightly and temporarily elevated due to the extra injection; and other welfare scores are also low and give indiscriminate differences in fish welfare scores.
From the proof-of-concept article [16] it is shown that the 70% purified vaccine gave better protection towards lice infestation than the 98% purified one. Upon high-purification, basic and acid side chains may react with each other to form an amide bond. This might change the structure of the peptide so much that the right immune stimulation was not achieved. The solubility of the peptide, i.e. the HLB (hydrophile–lipophile balance), in the adjuvant is also of importance. The peptide will have a distribution in the water-in-oil emulsion, making it prone to attack from proteases in the blood stream.
The length of the peptide might also be important for its immunogenic potential. The bioinformatic model showed that the selected peptide is from a surface sequence of peroxiredoxin-2 [16]. We believe this to be a linear sequence, but a 13 amino acid sequence may as well be coiled and does not necessarily mimic the surface sequence inducing the wanted ideal cellular immune response.
Preliminary experiments using the ELISA technique have so far given no indication of a humoral response, and we believe that a cellular MHC I response [37,38] is more likely. This is based on the fact that Atlantic salmon is an evolutionary old species [39], and MHC I is an old immune response system as well. According to this even shorter peptide chains, 6–12 amino acids, may stimulate the immune response. A shorter peptide sequence is also less expensive to produce, an advantage from a business point of view. Use of peptides as immune stimulants is a new technique, and the importance of the length of peptides as well as the sterically conformation are not known. From the above standing arguments, held together with the promising lab scale results [16], it seems clear that further development of the vaccine should be a goal.

4.4. Effects on Salmon Lice

The weekly catch-and-release sampling strategy of 20 out of 8000 salmon per sea cage by gently netting them with a deeply submerged net ensured a low probability for repeated sampling of the same individual from month to month, thus a minimum influence on lice levels from repeated sampling was expected. After the neighbor groups outside the present experiment were deloused and removed, a clearer effect was observed in May–June in favor of the vaccinated groups. In the following summer period (July–September) the natural lice infestation pressure was generally decreasing, probably due to the dominating melt water from the surrounding rivers, resulting in low infestation rates in all four groups and thus no effects detectable from the vaccine. However, during increasing natural infestation pressure in late September–November, the control groups showed higher levels of mature female lice.
Despite the before-mentioned challenges with a semi-commercial approach and the challenges related to the chosen peptide, the current trial has demonstrated the preventive effects of the vaccine. The effect size (Figure 10) showed a moderate positive difference of 0.07 mature female salmon lice per salmon in favor of the vaccinated groups from May until November, indicating that the vaccine did not show an effect until the salmon had passed 600 g in April, under the prevailing lice infestation pressure. This effect should be held together with the upper limit from the Norwegian Food Safety Authorities of 0.20 mature female salmon lice per salmon in the most critical period for smolt migration (April–May). Even if a moderate statistical difference was observed, the biological implications at farm level should be considered as a small brick in the collection of several preventive methods. By pointing at product development of the vaccine, it might be possible to increase its efficacy and biological significance, especially in farms located in the outer coastline with a heavier lice infestation pressure.
Contreras et al. [10] and Tartor et al. [11] have demonstrated immunization of Atlantic salmon against salmon lice infestation after vaccination with a salmon lice-gut recombinant protein P33. This protection seemed to be vaccine dose-dependent, where higher doses resulted in lower parasitic infestation rates. Both studies [10,11] showed a 35% reduction in adult salmon lice females after P33 vaccination. The present study did, however, not test different vaccine doses.
The effect of the vaccine (Figure 9 and Figure 10) was lower than expected. At present we do not know enough about the immune system of Atlantic salmon and how lice inhibit the immune system, although it has been shown that secreted salmon louse labial gland proteins have immune dampening functions in salmon [40]. The antigenicity of the peptide used must be increased, the purification optimized, a better adjuvant should be found, and different doses should be tested at lab-scale. The current effect of the vaccine might be combined with other preventive [24,25,26,27,28] methods, from which applications of acoustic lice treatment currently seems to be the most promising method [41], creating a combined effect to fight the challenges of salmon lice.

5. Conclusions

  • No adverse effects were observed on a range of salmon welfare-related parameters, mortality, growth, sexual maturation, or slaughter quality from the vaccination towards salmon lice per se.
  • The effect size showed a moderate positive difference of 0.07 mature female salmon lice per salmon in favor of the vaccinated groups from a fish size above 600 g in May until November, under natural lice infestation pressure in a fjord system.
  • Further product development of the vaccine should pay attention to the antigenicity of the peptide, optimization of the purification, finding a better adjuvant, and different doses should be tested at lab-scale.

Author Contributions

Conceptualization, R.N., E.D.-P., A.J. and E.S.; Data curation, R.N. and E.D.-P.; Formal analysis, R.N.; Funding acquisition, E.S.; Investigation, R.N., E.D.-P. and L.P.A.B.; Methodology, R.N., E.D.-P., A.J. and E.S.; Project administration, R.N. and E.S.; Resources, E.S.; Supervision, R.N. and E.S.; Validation, R.N. and E.D.-P.; Visualization, R.N. and E.D.-P.; Writing—original draft, R.N. and E.S.; Writing—review and editing, R.N., L.P.A.B. and E.S. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by Cape of good hope AS, Norway. Funding number: 001.

Institutional Review Board Statement

The animal study protocol was conducted in accordance with the guidelines provided by the Experimental Animal Administration Supervision and Application System (FOTS), under the Norwegian Food Safety Authorities. The Norwegian Computing Center calculated, based on the probability of salmon lice infestation of farmed salmon in a fjord system, the need for 8000 individual salmon in each sea cage to be able to find eventual differences between vaccinated and control groups. Power analysis was applied to calculate the need for weekly counting in each sea cage of at least 20 salmon for salmon infestation, 20 salmon for welfare aspects, and 80–100 salmon for growth monitoring, depending on current population variance.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in the study are included in the article, further inquiries can be directed to the corresponding authors.

Acknowledgments

The authors would like to acknowledge the staff and laboratory technicians at Matre Research Station, Institute of Marine Research, Norway, for their assistance throughout the challenge study, specifically Trond Asheim, Magnus Fjelldal, Simon Flavell, Jan Olav Fosse, Marius Lund Halland, Karen Anita Kvestad, Truls Marøy, Ivar Helge Matre, Linda Neset, Kris Oldham, Audun Østby Pedersen, Håkon Torvik, and Jan Even Østerbø.

Conflicts of Interest

The authors A.J. and E.S. are shareholders in the Cape of good hope AS, Norway. The other authors declare no conflicts of interest.

Abbreviations

The following abbreviations are used in this manuscript:
FOTSExperimental Animal Administration Supervision and Application System in Norway
FCRFeed Conversion Efficiency
HLBHydrophile-Lipophile Balance
IQRInterquartile Range
LMMLinear Mixed Model
M1-M10Fish Cage Number 1–10
MHCMajor Histocompatibility Complex
NKEFKEVSLKDYTSpecific 13 Amino Acid Peptide Vaccine Candidate
OLSOrdinary Least Squares Regression
PLSPartial Least Squares Regression
SGRSpecific Daily Growth Rate (%)
TFATri-fluoric acid

References

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Figure 1. (A) Schematic map of Knappen Solheim fish farm, located in Masfjorden, north of Bergen, south of the Sognefjord in Norway (Position: N 60°53.120/E 5°27.696). (B) Experimental set-up at Knappen-Solheim fish farm, with 10 cages (12 × 12 × 17 m). The two salmon lice vaccinated fish groups were reared in cages M4 and M9, whereas the two control groups were reared in cages M5 and M8. The dominant sea current direction from the land side is indicated by the blue arrow.
Figure 1. (A) Schematic map of Knappen Solheim fish farm, located in Masfjorden, north of Bergen, south of the Sognefjord in Norway (Position: N 60°53.120/E 5°27.696). (B) Experimental set-up at Knappen-Solheim fish farm, with 10 cages (12 × 12 × 17 m). The two salmon lice vaccinated fish groups were reared in cages M4 and M9, whereas the two control groups were reared in cages M5 and M8. The dominant sea current direction from the land side is indicated by the blue arrow.
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Figure 2. Upper photo (A): Evaluation sections (1–3) of internal vaccination effects. Lower photo (B) by Audun Østby Pedersen: X-ray image of anterior and posterior whole-body parts of vaccinated Atlantic salmon.
Figure 2. Upper photo (A): Evaluation sections (1–3) of internal vaccination effects. Lower photo (B) by Audun Østby Pedersen: X-ray image of anterior and posterior whole-body parts of vaccinated Atlantic salmon.
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Figure 3. (A) Melanin in belly section and (B) melanin in organs in vaccinated (orange) and control (blue) groups (n = 40 fish in each pooled group) from the period 21 November 2022–20 December 2023. The data are presented by the median values (X), 50% interquartile range (IR in box), upper and lower quartile ± 1.5 interquartile range, respectively (whiskers), and eventual outliers.
Figure 3. (A) Melanin in belly section and (B) melanin in organs in vaccinated (orange) and control (blue) groups (n = 40 fish in each pooled group) from the period 21 November 2022–20 December 2023. The data are presented by the median values (X), 50% interquartile range (IR in box), upper and lower quartile ± 1.5 interquartile range, respectively (whiskers), and eventual outliers.
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Figure 4. Internal distribution of adherence (blue = section 1, orange = section 2, and green = section 3) in vaccinated and control fish (n = 40 fish in each pooled group) from the period 21 November 2022–20 December 2023.
Figure 4. Internal distribution of adherence (blue = section 1, orange = section 2, and green = section 3) in vaccinated and control fish (n = 40 fish in each pooled group) from the period 21 November 2022–20 December 2023.
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Figure 5. Accumulated skin bleedings (scores 0–3) in vaccinated vs. control groups (n = 40 fish in each pooled group) throughout the experimental period. Red = % score of 0 appearance, green = % score of 1, yellow = % score of 2 and, gray = % score of 3.
Figure 5. Accumulated skin bleedings (scores 0–3) in vaccinated vs. control groups (n = 40 fish in each pooled group) throughout the experimental period. Red = % score of 0 appearance, green = % score of 1, yellow = % score of 2 and, gray = % score of 3.
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Figure 6. Accumulated wounds (scores 0–3) in vaccinated vs. control groups (n = 40 fish in each pooled group) throughout the experimental period. Red = % score of 0 appearance, green = % score of 1, yellow = % score of 2, and gray = % score of 3.
Figure 6. Accumulated wounds (scores 0–3) in vaccinated vs. control groups (n = 40 fish in each pooled group) throughout the experimental period. Red = % score of 0 appearance, green = % score of 1, yellow = % score of 2, and gray = % score of 3.
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Figure 7. Welfare scores (blue = fins, orange = wounds, gray = emaciated, yellow = scale loss, green = external malformations) at slaughter (20 December 2023) in vaccinated vs. control groups (n = 40 in each pooled group).
Figure 7. Welfare scores (blue = fins, orange = wounds, gray = emaciated, yellow = scale loss, green = external malformations) at slaughter (20 December 2023) in vaccinated vs. control groups (n = 40 in each pooled group).
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Figure 8. Specific daily growth rate (SGR%) for vaccinated (orange) vs. control (blue) groups (n > 200 in each pooled group) in the period 9 December 2022–20 December 2023.
Figure 8. Specific daily growth rate (SGR%) for vaccinated (orange) vs. control (blue) groups (n > 200 in each pooled group) in the period 9 December 2022–20 December 2023.
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Figure 9. Dynamic, wild salmon lice infestation of sessile ((A), red lines), mobile (C), and mature female lice (E) stages of the two control groups in the period 9 December 2022–20 November 2023. Each legend represents the average lice level from 20 fish. Circles and squares represent parallel groups and the solid line with triangles represents the average levels. Dynamic, wild salmon lice infestation of sessile ((B), blue lines), mobile (D), and mature female lice (F) stages of the two vaccinated groups in the period 9 December 2022–20 November 2023. Each legend represents the average lice level 20 fish. Circles and squares represent parallel groups and the solid line with triangles represents the average levels.
Figure 9. Dynamic, wild salmon lice infestation of sessile ((A), red lines), mobile (C), and mature female lice (E) stages of the two control groups in the period 9 December 2022–20 November 2023. Each legend represents the average lice level from 20 fish. Circles and squares represent parallel groups and the solid line with triangles represents the average levels. Dynamic, wild salmon lice infestation of sessile ((B), blue lines), mobile (D), and mature female lice (F) stages of the two vaccinated groups in the period 9 December 2022–20 November 2023. Each legend represents the average lice level 20 fish. Circles and squares represent parallel groups and the solid line with triangles represents the average levels.
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Figure 10. Effect size (control minus vaccinated, with 95% confidence intervals) in mature female salmon lice per salmon in the period from January–November 2023. A logarithmic trend line y =−0.05 + 0.05 ln(x) is displayed. The LMM analysis reveals a significant increase in lice level over time (p < 0.001), whereas the general overall trend line of the effect size only shows a weak significance (p < 0.09). The OLS analyses for the varying levels within each month reveal lower mature female lice levels in the control groups in March (p = 0.01) and higher levels in the control groups in May (p = 0.01), June (p = 0.09), October (p = 0.02), and November (p = 0.04), compared to the vaccinated groups.
Figure 10. Effect size (control minus vaccinated, with 95% confidence intervals) in mature female salmon lice per salmon in the period from January–November 2023. A logarithmic trend line y =−0.05 + 0.05 ln(x) is displayed. The LMM analysis reveals a significant increase in lice level over time (p < 0.001), whereas the general overall trend line of the effect size only shows a weak significance (p < 0.09). The OLS analyses for the varying levels within each month reveal lower mature female lice levels in the control groups in March (p = 0.01) and higher levels in the control groups in May (p = 0.01), June (p = 0.09), October (p = 0.02), and November (p = 0.04), compared to the vaccinated groups.
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Table 1. Average% incidence (from two cages) of sexual maturation (predominantly in males) in vaccinated (V) and control (C) groups, by visual external categorization in four categories, where score 0 = no signs of maturation, 1 = initial signs of maturation like prolongation of jaws and appearance of jaw crook, 2 = the skin is darker, mainly silvery, conspicuous prolongation of jaws in males, and 3 = conspicuous spawning colors, brownish, the posterior part of the body is thicker than in immature salmon, according to [16]. n = 20 fish from each cage, except for October when n = 100.
Table 1. Average% incidence (from two cages) of sexual maturation (predominantly in males) in vaccinated (V) and control (C) groups, by visual external categorization in four categories, where score 0 = no signs of maturation, 1 = initial signs of maturation like prolongation of jaws and appearance of jaw crook, 2 = the skin is darker, mainly silvery, conspicuous prolongation of jaws in males, and 3 = conspicuous spawning colors, brownish, the posterior part of the body is thicker than in immature salmon, according to [16]. n = 20 fish from each cage, except for October when n = 100.
DateGroupScore 0Score 1Score 2Score 3
7 February 2023V100000
7 February 2023C100000
24 April 2023V100000
24 April 2023C100000
26 June 2023V100000
26 June 2023C100000
26 August 2023V97300
26 August 2023C100000
25 October 2023V752005
25 October 2023C673021
4 December 2023V603703
4 December 2023C653032
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Nortvedt, R.; Dahl-Paulsen, E.; Bizama, L.P.A.; Johny, A.; Slinde, E. The Effect of a Polypeptide Based Vaccine on Fish Welfare and Infestation of Salmon Lice, Lepeophtheirus salmonis, in Sea Cages with Atlantic Salmon (Salmo salar L.). Fishes 2025, 10, 405. https://doi.org/10.3390/fishes10080405

AMA Style

Nortvedt R, Dahl-Paulsen E, Bizama LPA, Johny A, Slinde E. The Effect of a Polypeptide Based Vaccine on Fish Welfare and Infestation of Salmon Lice, Lepeophtheirus salmonis, in Sea Cages with Atlantic Salmon (Salmo salar L.). Fishes. 2025; 10(8):405. https://doi.org/10.3390/fishes10080405

Chicago/Turabian Style

Nortvedt, Ragnar, Erik Dahl-Paulsen, Laura Patricia Apablaza Bizama, Amritha Johny, and Erik Slinde. 2025. "The Effect of a Polypeptide Based Vaccine on Fish Welfare and Infestation of Salmon Lice, Lepeophtheirus salmonis, in Sea Cages with Atlantic Salmon (Salmo salar L.)" Fishes 10, no. 8: 405. https://doi.org/10.3390/fishes10080405

APA Style

Nortvedt, R., Dahl-Paulsen, E., Bizama, L. P. A., Johny, A., & Slinde, E. (2025). The Effect of a Polypeptide Based Vaccine on Fish Welfare and Infestation of Salmon Lice, Lepeophtheirus salmonis, in Sea Cages with Atlantic Salmon (Salmo salar L.). Fishes, 10(8), 405. https://doi.org/10.3390/fishes10080405

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