Rapid temperature-dependent wound closure following adipose fin clipping of Atlantic salmon Salmo salar L.

Three groups of Atlantic salmon were kept at a constant temperature of 4, 10 and 14 °C. The adipose fins were removed; six fish/group were sampled at 11 subsequent time points post-clipping. Samples were prepared for histopathological examination to study the course of re-epithelization. A score sheet was developed to assess the regeneration of epidermal and dermal cell types. Wounds were covered by a thin epidermal layer between 4 and 6 h post-clipping at 10 and 14 °C. In contrast, wound closure was completed between 6 and 12 h in fish held at a constant temperature of 4 °C. By 18 h post-clipping, superficial cells, cuboidal cells, prismatic basal cells and mucous cells were discernible in all temperature groups, rapidly progressing towards normal epidermal structure and thickness. Within the observation period, only minor regeneration was found in the dermal layers. A positive correlation between water temperature and healing rates was established for the epidermis. The rapid wound closure rate, epidermal normalization and the absence of inflammatory reaction signs suggest that adipose fin clipping under anaesthesia constitutes a minimally invasive method that may be used to mark large numbers of salmon presmolts without compromising fish welfare.

Association of spinal deformity and vaccine-induced abdominal lesions in harvest-sized Atlantic salmon, Salmo salar L.

Spinal deformities in Atlantic salmon, Salmo salar L., have been described as a disease of multifactorial origin for which vaccines and time of vaccination have been suggested as risk factors. A vaccine efficacy trial where spinal deformity became evident was continued by the observational study reported here. In the preharvest part of the study 17 months post-sea transfer, there was a prevalence of 11.3% spinal deformity, with deformities present only in one vaccine group indicating a strong vaccine involvement. At slaughter, the prevalence of spinal deformities was 11.7%, and deformed fish had only 62% of normal slaughter weight. Visual analogue scales (VAS) were used for continuous recordings of vaccine-induced abdominal lesions and deformity. A logistic regression model associating presence of spinal deformity with markers of abdominal lesions was developed. The odds ratio for spinal deformity was 5.7 (95% CI: 3.4-9.4) for each unit increase in adhesion score (0-6) and 4.9 (2.9-3.4) for each unit increase in melanin on abdominal organs (0-3). Lesions in the dorsal caudal part of the abdomen gave an odds ratio for spinal deformity of 2.2.

Strong genetic influence on IPN vaccination-and-challenge trials in Atlantic salmon, Salmo salar L.

Two series of experimental challenge trials were performed for evaluation of multivalent oil-adjuvanted vaccines with and without an infectious pancreatic necrosis virus (IPNV) antigen component. In both the trial series, Atlantic salmon were hatched, reared, vaccinated and subjected to temperature and light manipulation to induce smoltification. When ready for sea the fish were transported to the VESO Vikan experimental laboratory for bath or cohabitant challenge with IPNV. In the first series, four vaccination and bath challenge trials involving 2-year classes of experimental fish were conducted. In the second series, three groups of eyed eggs of Atlantic salmon allegedly differing in their innate resistance to IPNV were used (Storset, Strand, Wetten, Kjøglum & Ramstad 2007). Hatching, rearing and smoltification were synchronized for each group, and fish from each genetic group were randomly allocated IPN vaccine, reference vaccine or saline before being placed into parallel tanks for bath or cohabitant challenge. In the first series of trials, IPN-specific mortality commenced on day 10-12 after bath challenge. Replicates showed similar results. In trials 1 and 2 belonging to the same experimental fish year class, the average cumulative control mortality reached 60.6% and 79.5%, respectively, whereas in trials 3 and 4 belonging to the following year class the control mortality was consistently below 50%. In the second series of trials, the experimental fish originating from allegedly IPN susceptible parents consistently showed the highest cumulative mortality among the unvaccinated controls (>75%) whereas smolts derived from allegedly IPNV resistant parents showed only 26-35% control mortality. The IPN-vaccinated fish experienced significantly improved survival vs. the fish immunized with reference vaccine, with RPS values above 75% in the IPN susceptible strain. In the IPN resistant strain, the protection outcomes were variable and in part non-significant. The outcome of both the trial series suggests that control mortalities above 50% are necessary to reliably demonstrate specific protection with IPN vaccines.

Field validation of experimental challenge models for IPN vaccines.

Atlantic salmon S1/2 pre-smolts from the VESO Vikan hatchery were assigned to study groups, i.p. immunized with commercially available, multivalent oil-adjuvanted vaccines with (Norvax Compact 6 - NC-6) or without (Norvax Compact 4 - NC-4) recombinant infectious pancreatic necrosis virus (IPNV) antigen. A control group received saline solution. When ready for sea, the fish were transported to the VESO Vikan experimental laboratory, where two identical tanks were stocked with 75 fish per group before being transferred to 10 degrees C sea water and exposed by bath to first passage IPNV grown in CHSE-214 cells. The third tank containing 40 fish from each group was challenged by the introduction of 116 fish that had received an i.p injection of IPNV-challenge material. The remaining vaccinated fish were transported to the VESO Vikan marine field trial site and placed in two identical pens, each containing approximately 53 000 fish from the NC-6 group and 9000 fish from the NC-4 group. In the experimental bath challenge trial, the cumulative mortality was 75% and 78% in the control groups, and the relative percentage survival (RPS) of the NC-6-immunized fish vs. the reference vaccine groups was 60% and 82%, respectively. In the cohabitation challenge, the control mortality reached 74% and the IPNV-specific vaccine RPS was 72%. In both models, the reference vaccine lacking IPNV antigen gave a moderate but statistically significant non-specific protection. In the field, a natural outbreak of infectious pancreatic necrosis (IPN) occurred after 7 weeks lasting for approximately 3.5 months before problems due to winter ulcers became dominating. During this outbreak, mortality in the NC-4 groups were 33.5% and 31.6%, respectively, whereas mortality in the NC-6 groups were 6.9% and 5.3%, respectively, amounting to 81% IPNV-specific protection. In conclusion, the IPN protection estimates obtained by experimental challenges were consistent between tanks, and were confirmed by the field results.

A user's inter-laboratory comparison of broodfish screening for infectious pancreatic necrosis virus using molecular and conventional diagnostic methods.

Individual testing and subsequent removal of eggs from infectious pancreatic necrosis virus (IPNV)-positive parents is required for export of salmonid eggs to some farming countries. Testing by cell culture requires more than three weeks before the eggs can be released from quarantine and incurs significant logistic problems and costs. The feasibility of the RT-PCR testing method as offered by several laboratories was therefore evaluated during the current inter-laboratory comparison study. Frozen kidney sub-samples from 100 motherfish of Atlantic salmon (Salmo salar L.) were shipped to three diagnostic laboratories (A, B and C) for testing by RT-PCR and cell culture. Of the 100 examined samples, all proved IPNV-negative by cell culture. Thirty samples were positive by RT-PCR analyses, but only four of these samples were RT-PCR positive in two laboratories and none in any of the three laboratories. From a disease management point of view, the RT-PCR test outcomes gave no reasonable guidance as to which fish were truly infected and which batches of fertilised eggs should be discarded. This is clearly an unacceptable situation and calls for new research to standardise sample conservation, RNA extraction procedures and amplification techniques, to estimate method sensitivity and specificity, and to validate the method's performance and robustness to support disease control measures in salmonid aquaculture.

Critical assessment of regulatory standards and tests for fish vaccines.

Within the European Economic Area (EU member states plus Norway, Iceland and Liechtenstein), veterinary vaccines must comply with EU regulations and relevant monographs of the European Pharmacopoeia. Since 1996, three European monographs specific for fish have been adopted; concerning oil-adjuvanted, injectable vaccine for salmonids against furunculosis, as well as bacterins against classical vibriosis (Listonella anguillarum) or cold-water vibriosis (Vibrio salmonicida) in salmonids. The regulatory requirements laid down in these monographs include the use of seronegative fish for in vivo safety testing; conduct of vaccination trials in which experimental challenge is administered by injection, and minimum relative protection to be achieved at a given level of control mortality. Several aspects of these requirements are being questioned. This concerns the relevance of injection challenge methods as opposed to waterborne challenge; the validity of relative protection assessed at 60 % control mortality (RPS6) as compared to protection calculated at the endpoint of mortality (RPSendpoint), and poor test power due to low numbers of fish per treatment group. There is a strong need for future efforts to refine the methods for documentation and testing of fish vaccines, and to assure their suitability for the intended purpose.

Light scattering in normal and cataractous lenses of farmed Atlantic salmon (Salmo salar): a slit lamp and Scheimpflug photographic study.

To investigate normal light scattering and cataract formation, the anterior eye segments of farmed Atlantic salmon (Salmo salar) reared in fresh water and sea water were documented in vivo for the first time with a Topcon SL-45 Scheimpflug camera. A total of 40 fish from the fresh-water-rearing period, obtained from 2 groups of identical age but showing a different growth rate, and 24 fish from the sea-water-rearing period, sampled from 2 groups with identical age but being fed different food brands, were included in this study. The fish were anaesthetized before examination. Due to the naturally wide pupil, no mydriatic compound was applied. All fish were removed from the water for photography, which was performed for each eye in 0 degrees = vertical slit position. Images were recorded on Kodak Tmax 400 black-and-white film. Microdensitometric image analysis of all negatives was performed using a Joyce-Loebl online microdensitometer. In spite of the virtual absence of an anterior chamber gap between cornea and lens and very little light scattering in the normal fish lens, a small number of distinct layers could be reproducibly identified in the lens. While there was little abnormal light scattering which could point to cataract development in young fish from the fresh water period, the evaluation of the lenses from the 2 sea water groups showed the presence of specific forms of cataract especially in the cortical and supranuclear layers. There were significant differences between the groups fed different food brands at the sea water site. In conclusion, Scheimpflug photography proved to be applicable to eye research in fish in vivo. It is suggested that this method should be employed for reproducible documentation as an extension to slit lamp monitoring in experimental research to reveal causative factors for cataracts in farmed fish.

An epidemiological study of cataracts in seawater farmed Atlantic salmon Salmo salar.

Cataracts in farmed Atlantic salmon have been known for many years, but the aetiology and importance of the disease have not been clarified. A cross-sectional field study of 51 cages of Atlantic salmon at 49 randomly selected sea sites was performed during the summer of 1998. The target population was spring and autumn entry groups of the 1997 generation salmon. Approximately 15 fish from each cage, altogether 777 fish, were autopsied by the same person. Each eye of the fish was scored for cataracts on a scale from 0 to 4 using an otoscope lamp with magnification. The weight and length of each fish were measured. The prevalence of cataracts was 83 % and 79% in spring entry groups and autumn entry groups, respectively. The overall mean cataract index (mean score of both eyes) was 1.23, being significantly higher in the spring entry groups (1.36) than the autumn entry groups (0.85). The final results in the spring entry groups showed that the fish groups with higher weight at sea transfer also had a higher cataract index at inspection. The risk of development of cataracts varied significantly among the offspring from the 5 strains represented in the study. Fish from sites located in 2 counties in the southern part of Norway had a significantly higher cataract index than fish farmed in the northernmost county in the study. For the autumn entry groups none of the explanatory variables was significant. In the spring entry groups a significant negative relationship was observed between the cataract score and the weight of the fish at the time of inspection (Pearson's r = -0.17), while the corresponding correlation for the autumn released groups was r = -0.10. Among the spring entry groups the average weight of the fish with the highest cataract score was estimated to about a third of the weight of the fish with no visible cataracts.

Growth of Atlantic salmon Salmo salar after intraperitoneal administration of vaccines containing adjuvants.

Growth of Atlantic salmon after intraperitoneal (i.p.) administration of adjuvanted vaccines was studied using groups of individually tagged fish held together in one tank or pen under commercial farming conditions. Parallel experiments were initiated at 2 freshwater sites and 1 marine site. Trivalent (vibriosis, cold water vibriosis and furunculosis) vaccines containing oil or beta-1, 3 glucan as adjuvants were used for immunisation of pre-smolts, whereas identical formulations containing furunculosis antigens only were used in growers. Control fish remained unvaccinated. No outbreak of bacterial or viral disease was experienced at any of the sites. At all sites, the highest daily growth rate was recorded in unvaccinated fish. At one site, the average weight of post-smolts that had received oil-adjuvant vaccine was significantly reduced by 345 g (23%) after 15 mo. Impaired growth rate was associated with increasing severity of intra-abdominal lesions as determined during necropsy. At the second post-smolt site and in growers, weight development and growth rates were non-significant between groups throughout the study. The results indicate that intraperitoneal administration of oil-adjuvanted vaccines may retard growth of farmed Atlantic salmon, although the extent of this effect may vary between sites. Unidentified factors coinciding with vaccination are thought to have caused the highly variable results seen between parallel sites in this study.

Vaccinated fish welfare: protection versus side-effects.

Active immunisation of fish involves a number of potentially harmful procedures like handling, anaesthesia or injection of more or less toxic substances. Adjuvanted vaccines may cause inflammation, granuloma and pigmentation at the site of injection. Intraperitoneal administration of oil-adjuvanted vaccines to Atlantic salmon pre-smolts has occasionally resulted in impaired growth and reduced carcass quality. The consequences of such vaccination for fish welfare may therefore be questioned. With respect to furunculosis caused by Aeromonas salmonicida, scientific data suggest, however, that only oil-adjuvanted vaccines are protective throughout the production cycle of farmed salmon. Data are presented to show that salmonids are highly at risk to epizootics if left unprotected against this or other endemic diseases. A panel of parameters partly adopted from experimental animal medicine is proposed to assess the impact of vaccine side-effects in farmed fish. In intensive salmon aquaculture systems, reduced disease risks are thought to justify the observed level of side-effects following current vaccination practices. For future fish vaccines, reduction of side-effects without compromising long-term protective immunity constitutes a challenging goal.