Effectiveness of fusion peptide-based vaccine TT-P0 on the dynamics of salmon lice (Lepeophtheirus salmonis) infection in Atlantic salmon (Salmo salar L.)

Infection with parasitic copepod salmon louse Lepeophtheirus salmonis, represents one of the most important limitations to sustainable Atlantic salmon (Salmo salar L.) farming today in the North Atlantic region. The parasite exerts negative impact on health, growth and welfare of farmed fish as well as impact on wild salmonid populations. It is therefore central to ensure continuous low level of salmon lice with the least possible handling of the salmon and drug use. This necessitates development of an alternative preventive strategy that can document both effect on lice and that fish welfare is maintained in a satisfactory manner with high economic impact. To address this, vaccination is a cost-effective and environmentally free control approach avoiding the disadvantages of chemical and mechanical treatments. In this study, efficacy of a vaccine candidate (TT-P0), encompassing a peptide derived from ribosomal protein P0 and promiscuous T cell epitopes from tetanus toxin and measles virus, was validated post infestation with L. salmonis, at the lab-scale. The sampling results showed good potential of the TT-P0 vaccine in limiting the ectoparasite load, when administered intraperitoneal in the host, by affecting the total adult lice female counts and fecundity, with greater presumptive effect in F1 lice generation. This consequently speculate vaccine’s potential to reduce the amount and frequency of chemical drug, mechanical treatment and handling stress, currently used in salmon farming practices, thus improving the fish welfare, environment and economy. On the other hand, the vaccine showed minimal secondary effects and differential modulation of pro-inflammatory, Th1, Th2 and T regulatory mediators at the transcript level with respect to different lice stages in the vaccinated groups as compared to control. Overall, the results indicated potential effectiveness of TT-P0 antigen as a good and safe vaccine candidate against salmon lice. This is a very important preliminary documentation of the TT-P0 vaccine, as a preventive measure, for sustainable and profitable growth of the salmon industry. However, further validation is necessary under field conditions. Author summary Reducing the impact of salmon lice is a major concern for salmon producers around the globe. These parasitic copepods feed on host mucus, skin and blood, causing a negative impact due to reduction in host immune competence and making them more susceptible to other infections or by transmitting pathogens to the host. Farmed salmon populations are the main reservoirs and increasing numbers of salmon lice in the farms, negatively impacts’ wild salmon populations. The available control methods rely mainly on pesticides and other physical and biological treatment methods with their own limitations. In this context, development of an efficient vaccine would represent a significant advancement in sea lice control strategy, providing a practical, eco-friendly and sustainable solution with good fish welfare. However, identification of proper vaccine candidates and demonstration of their efficacy have been the main constraints for vaccine development. In the present research, we evaluated the effectiveness of a novel vaccine candidate in a laboratory trial and demonstrated that immunization with this formulation by intraperitoneal injection route, reduced total adult female counts and fecundity with minor secondary effects on the salmon. The results suggest the potential of this novel vaccine candidate against salmon lice by reducing the parasite load and minimizing the current treatment frequencies and handling stress and thus supports further investigations under field conditions as an important next step to demonstrate the effectiveness of the vaccine candidate to control lice infestations in salmon aquaculture.

Although there were no great differences between the total lice counts per fish on the immunized  Table 1). The results mentioned above clearly showed reduced number of eggs 181 produced by females in group 2 (42 % reduction, P<0.03) and thus supports significantly reduced 182 fecundity in terms of reduced egg string data and less gravid females in group 2 ( Table 1). Overall,  Table 1). The percentage reduction of 198 copepodids on day 10 was not high, as expected based on observation made on day 8 ( Fig 3A). This 199 was due to some unseen or technical problem occurring during the weekend, resulting in some 200 unexpected mortality of the copepodids before counting on day 10. The experiment was not 201 possible to repeat due to limited time and resources available.

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Overall, the results from lice counting and analysis of different parameters at different lice stages 203 post infestation, showed that the vaccine efficacy of group 2 was the best among the groups with 204 an efficacy of 86 %. However, group 3 efficacy was negative compared to control since some of the 205 parameters were lower than the control group (group 1). The terminology "vaccine efficacy" used 206 here should not be interpreted as protection obtained. This is the overall vaccine effects based on 207 different parameters studied, as described in the materials and methods section. showed an average score of 2.8, i.e below 3, which is in an acceptable range ( Fig 4B). On the other 217 hand, pigmentation score was significantly less in the immunized groups compared to the control 218 group, as shown in Fig 4B. Moreover, pigmentation was observed only on the epithelial lining and 219 not in muscle or tissue within the peritoneum. In most fish from group 2 and 3, the pigment spots 220 were extended to the anterior abdomen, which was related to the spread of vaccine pockets. 28 dpi (group 2) in spleen and at 17 dpi (group 2 and 3) as well as 28 dpi (group 2) in head kidney.

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Consequently, evaluating the two-way hierarchical clustering analysis for all the tissues, vaccinated 253 group 2 at 28 dpi showed the highest number of upregulated genes compared to the control group.

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However, vaccinated group 3 showed higher number of upregulated genes at 17 dpi in spleen and head kidney and at 28 dpi in skin. Heat map with two-way clustering of genes studied in the 256 individual tissue is given in S1 Fig

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The same results were obtained for metalloproteinase 9 (MMP-9) except at 28 dpi in spleen (S4A   To analyze the effect of vaccine on F1 generation copepodids production, the first reproductive egg 532 strings, obtained from gravid females at 50 dpi were incubated in well-aerated filtered seawater.

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This was to determine the effect, vaccine candidate had on hatching efficiency of the F1 generation 534 copepodids. Fifty egg strings (sampled from the first reproductive event at 50 dpi) from each 535 experimental group were incubated in 5 parallel aerated flow-through incubators (containing 500 536 mL filtered seawater/incubator at ~10 °C) for 8 days, to study the hatching success to F1 generation 537 copepodids. First visual observation was done on day eight post incubation and final counting was 538 performed at day ten. Copepodid density was estimated by taking 10 mL water samples from each 539 replicate and counting of copepodid was performed using dissecting microscope. This observation 540 was repeated four times for each replicate.

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The overall efficacy of the candidate vaccine (in percentage) was calculated using lice count data 543 collected from different dpi, including female lice fecundity parameters and F1 generation 544 copepodid count compared to control group, using a similar approach as used to assess vaccine   Table 2. The results were analyzed and expressed as mean ± standard deviation (SD) unless otherwise stated.

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Statistical analysis was performed and graphs were made using the Prism 6.01 software for 578 Windows (GraphPad software, San Diego, CA, USA). Experimental groups were conducted in 579 triplicates. Prior to data analysis, outliers were identified and removed from subsequent analyses. We would like to thank staffs at Aquaculture Research Station in Tromsø for assistance in fish 593 maintenance, copepodid production, performing lice challenge and lice counting. We also thank Dr.