Infectious pancreatic necrosis virus isolated from farmed rainbow trout and tilapia in Kenya is identical to European isolates.

Infectious pancreatic necrosis virus (IPNV) is an aquabirnavirus that causes serious diseases in a variety of fish species worldwide. It has been isolated from a large number of healthy fresh and marine water fish. Prior to this study, there was no record of the presence of IPNV infection in Kenya. Here, the presence of IPNV in farmed rainbow trout and tilapia was examined in Nyeri County of central Kenya. Head kidney samples taken from five rainbow trout and three tilapia farms and stored in RNALater® were processed by PCR followed by sequencing of a segment A fragment covering nucleotide positions 2,120-2,343 bp. IPNV was detected in all the farms sampled with infection ratios ranging from 0.3 to 0.78 although the infections were not associated with any specific clinical signs of disease. These findings were supported by immunohistochemistry staining of the virus in the kidney and exocrine pancreas of rainbow trout. Sequence alignment and phylogenetic analysis revealed that the Kenyan isolates were identical to European isolates, suggesting a common origin. These findings highlight the need for better biosecurity procedures with more stringent surveillance programmes and control for fish diseases, especially focusing on imported breeding materials to Kenya.

Detection of tilapia lake virus (TiLV) infection by PCR in farmed and wild Nile tilapia (Oreochromis niloticus) from Lake Victoria.

Tilapia lake virus disease (TiLVD) has emerged to be an important viral disease of farmed Nile tilapia (Oreochromis niloticus) having the potential to impede expansion of aquaculture production. There is a need for rapid diagnostic tools to identify infected fish to limit the spread in individual farms. We report the first detection of TiLV infection by PCR in farmed and wild Nile tilapia from Lake Victoria. There was no difference in prevalence between farmed and wild fish samples (p = .65), and of the 442 samples examined from 191 fish, 28 were positive for TiLV by PCR. In terms of tissue distribution, the head kidney (7.69%, N = 65) and spleen (10.99%, N = 191), samples had the highest prevalence (p < .0028) followed by heart samples (3.45%, N = 29). Conversely, the prevalence was low in the liver (0.71%, N = 140) and absent in brain samples (0.0%, N = 17), which have previously been shown to be target organs during acute infections. Phylogenetic analysis showed homology between our sequences and those from recent outbreaks in Israel and Thailand. Given that these findings were based on nucleic acid detection by PCR, future studies should seek to isolate the virus from fish in Lake Victoria and show its ability to cause disease and virulence in susceptible fish.

Contrasting expression of immune genes in scaled and scaleless skin of Atlantic salmon infected with young stages of Lepeophtheirus salmonis.

Atlantic salmon skin tissues with and without scales were taken from two preferred sites of salmon louse (Lepeophtheirus salmonis) attachment, behind the dorsal fin (scaled) and from the top of the head (scaleless), respectively. Tissues were profiled by qPCR of 32 genes to study responses to copepodids, 4 days post infection (dpi), and during the moult of copepodids to the chalimus stage, at 8 dpi. Basal/constitutive differences were found for many immune-related genes between the two skin sites; e.g., mannose binding protein C was over 100 fold higher expressed in the scaled skin from the back in comparison to the skin without scales from the head. With lice-infection, at 4 dpi most genes in both tissues showed lower values than in the non-infected control. By 8 dpi, the majority of responses increased towards the control levels, including cytokines of Th1, Th17 and Th2 pathways. Immunohistochemistry of three immune factors revealed an even distribution of MHC class II positive cells throughout epidermis, including the top layer of keratinocytes, marked compartmentalization of Mx+ and CD8α+ cells close to stratum basale, and an increase in numbers of CD8α+ cells in response to infection. In conclusion, suppression of immune genes during the copepodid stage likely sets off a beneficial situation for the parasite. At the moult to chalimus stage 8 dpi, only few genes surpassed the non-infected control levels, including CD8α. The gene expression pattern was reflected in the increased number of CD8α expressing cells, thus revealing a relatively minor activation of skin T-cell defenses in Atlantic salmon in response to L. salmonis infection.

Atlantic salmon (Salmo salar L.) post-smolts challenged two or nine weeks after seawater-transfer show differences in their susceptibility to salmonid alphavirus subtype 3 (SAV3).

BACKGROUND: Pancreas disease (PD), caused by salmonid alphavirus (SAV), is an important disease affecting salmonid aquaculture. It has been speculated that Atlantic salmon post-smolts are more prone to infections in the first few weeks following seawater- transfer. After this period of seawater acclimatization, the post-smolts are more robust and better able to resist infection by pathogens. Here we describe how we established a bath immersion (BI) model for SAV subtype 3 (SAV3) in seawater. We also report how this challenge model was used to study the susceptibility of post-smolts to SAV3 infection in two groups of post-smolts two weeks or nine weeks after seawater - transfer. METHODS: Post-smolts, two weeks (Phase-A) or nine weeks (Phase-B) after seawater- transfer, were infected with SAV3 by BI or intramuscular injection (IM) to evaluate their susceptibility to infection. A RT-qPCR assay targeting the non-structural protein (nsP1) gene was performed to detect SAV3-RNA in blood, heart tissue and electropositive-filtered tank-water. Histopathological changes were examined by light microscope, and the presence of SAV3 antigen in pancreas tissue was confirmed using immuno-histochemistry. RESULTS: Virus shedding from the Phase-B fish injected with SAV3 (IM Phase-B) was markedly lower than that from IM Phase-A fish. A lower percentage of viraemia in Phase-B fish compared with Phase-A fish was also observed. Viral RNA in hearts from IM Phase-A fish was higher than in IM Phase-B fish at all sampling points (p < 0.05) and a similar trend was also seen in the BI groups. Necrosis of exocrine pancreatic cells was observed in all infected groups. Extensive histopathological changes were found in Phase-A fish whereas milder PD-related histopathological lesions were seen in Phase-B fish. The presence of SAV3 in pancreas tissue from all infected groups was also confirmed by immuno-histochemical staining. CONCLUSION: Our results suggest that post-smolts are more susceptible to SAV3 infection two weeks after seawater-transfer than nine weeks after transfer. In addition, the BI challenge model described here offers an alternative SAV3 infection model when better control of the time-of-infection is essential for studying basic immunological mechanisms and disease progression.

A thicker chorion gives ova of Atlantic salmon (Salmo salar L.) the upper hand against Saprolegnia infections.

Since the ban of malachite green in the fish farming industry, finding alternative ways of controlling Saprolegnia infections has become of utmost importance. Much effort has been made to elucidate the mechanisms by which Saprolegnia invades fish eggs. Little is known about the defence mechanisms of the hosts, making some eggs more prone to infection than others. One clue might lie in the composition of the eggs. As the immune system in the embryos is not developed yet, the difference in infection levels could be explained by factors influenced by the mother herself, by either transferring passive immunity, influencing the physical aspects of the eggs or both. One of the physical aspects that could be influenced by the female is the chorion, the extracellular coat surrounding the fish egg, which is in fact the first major barrier to be overcome by Saprolegnia spp. Our results suggest that a thicker chorion in eggs from Atlantic salmon gives a better protection against Saprolegnia spp. In addition to the identification of differences in sensitivity of eggs in a fish farm set-up, we were able to confirm these results in a laboratory-controlled challenge experiment.

Saprolegnia diclina IIIA and S. parasitica employ different infection strategies when colonizing eggs of Atlantic salmon, Salmo salar L.

Here, we address the morphological changes of eyed eggs of Atlantic salmon, Salmo salar L. infected with Saprolegnia from a commercial hatchery and after experimental infection. Eyed eggs infected with Saprolegnia spp. from 10 Atlantic salmon females were obtained. Egg pathology was investigated by light and scanning electron microscopy. Eggs from six of ten females were infected with S. parasitica, and two females had infections with S. diclina clade IIIA; two Saprolegnia isolates remained unidentified. Light microscopy showed S. diclina infection resulted in the chorion in some areas being completely destroyed, whereas eggs infected with S. parasitica had an apparently intact chorion with hyphae growing within or beneath the chorion. The same contrasting pathology was found in experimentally infected eggs. Scanning electron microscopy revealed that S. parasitica grew on the egg surface and hyphae were found penetrating the chorion of the egg, and re-emerging on the surface away from the infection site. The two Saprolegnia species employ different infection strategies when colonizing salmon eggs. Saprolegnia diclina infection results in chorion destruction, while S. parasitica penetrates intact chorion. We discuss the possibility these infection mechanisms representing a necrotrophic (S. diclina) vs. a facultative biotrophic strategy (S. parasitica).

Factors influencing Saprolegnia spp. spore numbers in Norwegian salmon hatcheries.

A quantitative survey of Saprolegnia spp. in the water systems of Norwegian salmon hatcheries was performed. Water samples from 14 salmon hatcheries distributed along the Norwegian coastline were collected during final incubation in the hatcheries. Samples of inlet and effluent water were analyzed to estimate Saprolegnia propagule numbers. Saprolegnia spores were found in all samples at variable abundance. Number of spores retrieved varied from 50 to 3200 L(-1) in inlet water and from 30 to >5000 L(-1) in effluent water. A significant elevation of spore levels in effluent water compared to inlet water was detected. The estimated spore levels were related to recorded managerial and environmental parameters, and the number of spores in inlet water and temperature was the factor having most influence on the spore concentration in the incubation units (effluent water). Further, the relative impact of spore concentration on hatching rates was investigated by correlation analysis. From this was found that even high spore counts did not impact significantly on hatching success.

Saprolegnia species in Norwegian salmon hatcheries: field survey identifies S. diclina sub-clade IIIB as the dominating taxon.

Saprolegnia isolates within the recognized clades encompassing the taxa S. parasitica and S. diclina act as opportunist and aggressive pathogens to both fish and their eggs. They are responsible for significant economic losses in aquaculture, particularly in salmonid hatcheries. However, the identity, distribution and pathogenic significance of involved species often remain unexplored. In this study, 89 Saprolegnia isolates were recovered from water, eggs and salmon tissue samples that originated from salmon (Salmo salar) hatcheries along the coast of Norway. The cultures were characterized morphologically and molecularly in order to provide an overview of the species composition of Saprolegnia spp. present in Norwegian salmon hatcheries. We demonstrate that S. diclina clearly dominated and contributed to 79% of the recovered isolates. Parsimony analyses of the nuclear ribosomal internal transcribed spacer (ITS) region split these isolates into 2 strongly supported sub-clades, S. diclina sub-clade IIIA and IIIB, where sub-clade IIIB accounted for 66% of all isolates. A minor portion of the isolates constituted other taxa that were either conspecific or showed strong affinity to S. parasitica, S. ferax, S. hypogyna and Scoliolegnia asterophora. The unique sub-clade IIIB of S. diclina was most prevalent in water and salmon eggs, while S. parasitica isolates were more frequently isolated from post hatching stages. The study demonstrated that morphological criteria in many cases were insufficient for species delimitation due to lack of sexual structures or incoherent morphological expression of such features within the tested replicates.

Distribution of EP4 receptor in different Atlantic salmon (Salmo salar L.) tissues.

Prostaglandin E2 (PGE2) is an important lipid mediator that plays diverse functions in mammals. Four receptor subtypes of PGE2, designated EP1-4, have been identified to mediate its signaling pathways. Extensive studies of PGE2 and its receptors have been carried out in mammals, but little is known in fish, including Atlantic salmon. In the current study, the distribution of Atlantic salmon EP4 receptor in different tissues was investigated using RT- and real-time PCR. A custom made antibody was used to investigate the distribution of this receptor in different tissues. Quantitative analysis by real-time PCR revealed that the expression was more abundant in the spleen followed by head kidney, skin and fin while it was least expressed in heart, muscles and brain. The staining intensity obtained by immunohistochemistry correlated with the RT-PCR results. EP4 expression was strongly associated with the immune cells in different tissues. To our knowledge, this is the first study to describe the distribution of EP4 receptor in Atlantic salmon tissues. Our findings suggest that EP4 may play a role in mediating immune responses as observed in mammals.

In vitro passages impact on virulence of Saprolegnia parasitica to Atlantic salmon, Salmo salar L. parr.

The effect of serial in vitro subculturing on three pathogenic strains of Saprolegnia parasitica was investigated. The isolates were passed through Atlantic salmon, Salmo salar L. parr, and then re-isolated as single spore colonies. All strains caused infection. The isolate obtained from diseased fish served as a virulent reference culture and was designated 'AP' ('activated through passage'). Successive subculturing was made by obtaining an inoculum from AP to produce the 2nd subculture and then passaged to the 3rd subculture (from the 2nd), until the 15th passage was obtained. Spores used to produce storage cultures were collected at passages 5, 10 and 15. The different passages of each strain were used to artificially infect Atlantic salmon parr. Morphological characterization of growth patterns was performed to observe differences occurring due to serial in vitro subculturing. Two of the strains declined in virulence after 15 successive in vitro subcultures, whereas one did not. This study is the first to investigate attenuation of virulence in Saprolegnia and whether or not isolates of S. parasitica should be passed through the fish host prior to challenge experiments. It reveals that some strains degenerate more rapidly than others when subjected to successive in vitro subculturing on glucose-yeast extract.

Acute and sub-lethal effects in juvenile Atlantic salmon exposed to low µg/L concentrations of Ag nanoparticles.

Silver nanoparticles (Ag-NP) are components in numerous commercial products and are discharged into the environment in quantities that are largely unknown. In the present study, juvenile Atlantic salmon were exposed to 1, 20, and 100 µg/L (48 h, static renewal) of a commercially available Ag-NP colloidal suspension in natural (soft) lake water. A solution of AgNO(3) containing 20 µg/L Ag(I) ions was also included to discriminate the effect of NPs from that of ionic silver. Furthermore, the commercial Ag-NP suspension was compared to an in-house synthesised colloidal NP suspension prepared from AgNO(3) and NaBH(4) in citrate buffer. The size distribution of Ag in all exposure solutions was characterised by 0.22 µm filtration and 10 kDa hollow fibre cross-flow ultrafiltration in combination with ICP-MS. All exposures were characterised by a relatively high proportion of Ag-NP in the colloidal size fraction 3-220 nm. For assessment of biological effects, acute toxicity, gill histopathology, blood plasma parameters (Na, Cl, glucose, haemoglobin), and gene expression of a selection of gill biomarkers were measured. Results showed that the gills accumulated Ag in all exposure groups apart from the fish exposed to 1 µg/L Ag-NP. Accumulated Ag caused concentration-dependent response increases in general stress markers such as plasma glucose and gill gene expression of heat shock protein 70. Furthermore, induction of the metallothionein A gene indicated that Ag had been internalized in the gills, whereas a concentration-dependant inhibition of Na/K ATPase expression indicated impaired osmoregulation at as low as 20 µg/L concentrations of Ag-NP. The commercial Ag-NP suspension caused acute gill lamellae necrosis at high concentrations (100 µg/L), potentially giving rise to the substantial (73%) fish mortality at this concentration. The two different Ag-NP preparations gave comparable results for several endpoints measured, but differed in MT-A induction and mortality, thus emphasising the variation in effects that may arise from different Ag-NP preparations.

Slow release cortisol implants result in impaired innate immune responses and higher infection prevalence following experimental challenge with infectious pancreatic necrosis virus in Atlantic salmon (Salmo salar) parr.

Stress can affect the immune system and increase susceptibility to various diseases but knowledge of the underlying mechanisms is scarce. There is a complex interaction between the immune system and the endocrine system of vertebrates. In fish, cortisol is a key hormone regulating stress response and recent studies have also suggested that this hormone can affect the immune system, where cortisol is mainly regarded as an immunosuppressive factor. The aim of the present study was to examine the impact of chronically elevated levels of cortisol on the immune response and susceptibility to experimental infection with infectious pancreatic necrosis virus (IPNV). Further, the effect of IPNV challenge on circulating levels of cortisol was investigated. Atlantic salmon parr were implanted intraperitoneally with sustained-release implants of bovine of cortisol (50 µg cortisol g(-1) body weight in an implant based on vegetable lipids). Vehicle implants were used as control (sham-injected). At 45 days after implantation (DAI), fish were challenged with a low virulent isolate of IPNV (by immersion). Samples of plasma, liver and head kidney was taken from fish before and 24 h, 48 h, 7 days week and 21 days post infection (DPI). Cortisol level in plasma was measured using radioimmunoassay and gene expression in liver and head kidney was analyzed with real-time PCR (RT-PCR). Infection prevalence in infected fish was assessed by virus culture and RT-PCR of head kidney samples. Cortisol implantation compared with sham-implanted fish had increased levels of plasma cortisol at 45 DAI. The relative expression of Interferon alpha-1 (IFNα-1), Myxo virus-1 Mx, Heat-shock protein 70 (HSP70), Serum amyloid A (SAA), Glucocorticoid receptor (GR) and Heat-shock protein 90 (HSP90) tends to be down-regulated by cortisol implantation. There was a higher prevalence of fish with detectable levels of IPNV, as measured by cell culture and RT-PCR, in the cortisol-implanted group challenged with IPNV (0 = 0.0305) relative to the group that received a sham implantation. Further, cortisol seems to delay the induction of the antiviral IFNα-1 pathway and Mx mRNA expression. This study shows that elevated plasma cortisol level leads to an impaired innate immune response, and higher virus (IPNV) prevalence in Atlantic salmon parr.

Pathogenicity of Saprolegnia spp. to Atlantic salmon, Salmo salar L., eggs.

Live and dead Atlantic salmon eyed eggs were challenged with eight different Saprolegnia isolates, selected because of their varied origins, known morphological characteristics and growth/germination pattern. Some isolates were also tested for pathogenicity to Atlantic salmon parr. Challenge of eggs was performed by exposure to spores in suspension or by co-incubation of live eggs with infected dead eggs. The phenotypic characteristics of the isolates were evaluated in relation to their observed pathogenicity from the challenge experiment, to identify possible virulence factors leading to egg-infection by Saprolegnia. The results from the experiments confirm that live eggs are refractory to infection with Saprolegnia spores in suspension and that an infection of live eggs can only occur from an infection nucleus represented by dead eggs or debris. It was observed that strains pathogenic to salmon parr were not particularly infective towards eggs, and the isolates that gave the highest infection rates to eggs were species considered to be saprotrophs.

Quantitative genetics of disease resistance in vaccinated and unvaccinated Atlantic salmon (Salmo salar L.).

Furunculosis (Aeromonoas salmonicida) is an important disease in Atlantic salmon (Salmo salar) farming. Vaccination and selective breeding for increased resistance to the disease on the basis of challenge tests of unvaccinated fish are used as complementary prophylactic methods. An important issue is whether genetic predisposition to infection is consistent across vaccinated and unvaccinated fish. Hence, the main objective of this study was to determine the magnitude of the genetic associations (correlations) between resistance to furunculosis in vaccinated and unvaccinated fish, and to estimate the magnitude of the correlation of resistance to furunculosis with resistance to the viral diseases infectious pancreatic necrosis (IPN) and infectious salmon anaemia (ISA). Sub-samples of unvaccinated and vaccinated salmon from 150 full-sib families were subjected to separate cohabitation challenge tests. Substantial genetic variation was found in resistance to furunculosis in both the unvaccinated (heritabilities of 0.51 ± 0.05) and vaccinated (0.39 ± 0.06) fish. However, the genetic correlation between resistance to furunculosis in the two groups was low (0.32 ± 0.13), indicating a weak genetic association between resistance in the two groups. Hence, the current selection strategy on the basis of challenge tests of unvaccinated fish is likely to produce low genetic improvement in resistance to furunculosis under field conditions, where fish are vaccinated with an effective vaccine. Evidence was found of significantly favourable genetic associations of resistance to furunculosis in unvaccinated (but less so for vaccinated) fish with resistance to both IPN and ISA (unvaccinated fish), indicating that vaccination 'mask' genetic associations between resistance to different diseases.

Molecular and immunohistochemical studies on epidermal responses in Atlantic salmon Salmo salar L. induced by Gyrodactylus salaris Malmberg, 1957.

Various strains of Atlantic salmon exhibit different levels of susceptibility to infections with the ectoparasitic monogenean Gyrodactylus salaris. The basic mechanisms involved in this differential ability to respond to this monogenean were elucidated using controlled and duplicated challenge experiments. Highly susceptible East Atlantic salmon allowed parasite populations to reach up to 3000 parasites per host within 6 weeks, whereas less susceptible Baltic salmon never reached larger parasite burdens than 122 parasites per host during the same period. The present study, comprising immunohistochemistry and gene expression analyses, showed that highly susceptible salmon erected a response mainly associated with an increased expression of interleukin-1beta (IL-1beta), interferon-gamma (IFN-gamma), IL-10 and infiltration of CD3-positive cells in the epidermis of infected fins. Less susceptible salmon showed no initial response in fins but 3-6 weeks post-infection a number of other genes (encoding the immune-regulating cytokine IL-10, cell marker MHC II and the pathogen-binding protein serum amyloid A) were found to be up-regulated. No proliferation of epithelial cells was seen in the skin of less susceptible salmon, and IL-10 may play a role in this regard. It can be hypothesized that resistant salmon regulate the parasite population by restricting nutrients (sloughed epithelial cells and associated material) and thereby starve the parasites. In association with this 'scorched-earth strategy', the production of pathogen-binding effector molecules such as serum amyloid A (SAA) (or others still not detected) may contribute to the resistance status of the fish during the later infection phases.

Combined immersion and oral vaccination of Vietnamese catfish (Pangasianodon hypophthalmus) confers protection against mortality caused by Edwardsiella ictaluri.

Edwardsiella ictaluri septicemia occurs worldwide and causes high mortality and considerable economic damage to the catfish industry especially in Vietnam and the USA. To control Edwardsiella septicemia farmers extensively use antibiotics and various vaccination methods. Vaccination with inactivated vaccines has come with variable efficacy. In this trial the results of an approach of controlling Edwardsiella septicemia of Tra catfish (Pangasianodon hypophthalmus) in Vietnam through vaccination via mucosal surfaces are presented. The results show that a combination of primary vaccination by immersion with inactivated E. ictaluri followed by an oral boost with a formulated antigen preparation induces a statistically significant level of protection against mortality caused by experimental infection 4 weeks post-boost. Fish immunized by immersion only show significantly lower level of protection but significantly higher than the controls. Repeated boosts result in improved duration of immunity with a relative percent survival (RPS) of 47% at 90% control mortality. The immunization procedure provides an alternative for disease control through vaccination.

The effect of hyperoxygenation and reduced flow in fresh water and subsequent infectious pancreatic necrosis virus challenge in sea water, on the intestinal barrier integrity in Atlantic salmon, Salmo salar L.

In high intensive fish production systems, hyperoxygenation and reduced flow are often used to save water and increase the holding capacity. This commonly used husbandry practice has been shown to be stressful to fish and increase mortality after infectious pancreatic necrosis virus (IPNV) challenge, but the cause and effect relationship is not known. Salmonids are particularly sensitive to stress during smoltification and the first weeks after seawater (SW) transfer. This work aimed at investigating the impact of hyperoxygenation combined with reduced flow in fresh water (FW), on the intestinal barrier in FW as well as during later life stages in SW. It further aims at investigating the role of the intestinal barrier during IPNV challenge and possible secondary infections. Hyperoxygenation in FW acted as a stressor as shown by significantly elevated plasma cortisol levels. This stressful husbandry condition tended to increase paracellular permeability (P(app)) as well as translocation of Aeromonas salmonicida in the posterior intestine of Atlantic salmon. After transfer to SW and subsequent IPNV challenge, intestinal permeability, as shown by P(app), and translocation rate of A. salmonicida increased in the anterior intestine, concomitant with further elevation in plasma cortisol levels. In the anterior intestine, four of five fish displayed alterations in intestinal appearance. In two of five fish, IPNV caused massive necrosis with significant loss of cell material and in a further two fish, IPNV caused increased infiltration of lymphocytes into the epithelium and granulocytes in the lamina propria. Hyperoxygenation and reduced flow in the FW stage may serve as stressors with impact mainly during later stages of development. Fish with an early history of hyperoxygenation showed a higher stress response concomitant with a disturbed intestinal barrier function, which may be a cause for the increased susceptibility to IPNV infection and increased susceptibility to secondary infections.

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.

Development of intimal thickening of coronary arteries over the lifetime of Atlantic salmon, Salmo salar L., fed different lipid sources.

The objective of the present study was to investigate the development of intimal changes of coronary arteries over the lifetime of farmed Atlantic salmon, Salmo salar L., fed either a 100% fish oil or a 100% vegetable oil blend. The study was performed as a randomized observer blinded controlled trial with parallel group design. At the start of the project, the fish were divided in two groups and sampled at five different time points throughout their life span. The total study sample consisted of 259 healthy fish. Serial sections were taken from the coronary artery lying on the bulbus arteriosus for histopathological evaluation and for area measurements using semi-quantitative and quantitative methods. The earliest onset of vascular changes was detected in fish from both groups in the freshwater stage prior to smoltification. The mean range lesion (MRL), used to describe the severity of the lesions observed, increased significantly for both groups from sea transfer throughout the study period. Comparison of the two groups based on the overall material corrected for time of sampling did not show any difference (P = 0.20) between the two groups with regard to MRL. The percentage lumen loss (PLL) measured by a quantitative method and used as a measure to indicate lesion severity showed an incremental, non-significant increase from week 72 to week 92 and further to week 115 in both diet groups during the seawater phase. Comparison of the groups corrected for time of sampling indicated a difference of PLL in favour of VO (P = 0.02). Heart weight, body weight and body length were all positively and significantly correlated to Log MRL. The partial correlation analysis indicated that heart weight was the most dominant variable in the set. Early vascular changes were found in the major bifurcation of the coronary artery at the apex and beyond the flow divider into the daughter branches. The latter represented the dominant changes and were found throughout the entire lifecycle of the fish. Increasing in size over time they formed pads or cushions that were regularly located close to the outer walls of the bifurcation. The origin of the cells forming the intimal thickening has not been conclusively determined, but immunohistochemical findings indicate a smooth muscle cell origin, possibly of a myointimal type. Our findings suggest there is no correlation between diet and intimal changes. The severity of the changes, MRL and PLL, of the coronary vessels correlate with heart weight and fish weight growth and growth rate and mechanical factors are implicated in intimal development, but rather than being induced by external injury due to the location of the coronaries, haemodynamic factors and low shear stress are proposed as the main mechanism behind these changes.

Aquatic birnavirus infection activates the transcription factor NF-kappaB via tyrosine kinase signalling leading to cell death.

Our previous studies found that infectious pancreatic necrosis virus (IPNV) induces host apoptotic cell death, possibly through a newly synthesized protein trigger. Here, we examine whether IPNV infection can induce NF-kappaB activation through tyrosine kinase signalling of CHSE-214 cell death (host cell death). Using the electrophoretic mobility shift assay (EMSA) to detect transcription factor activation, we found that NF-kappaB is apparently activated 6-8 h post-IPNV infection. Using genistein (100 microg mL(-1); a tyrosine kinase inhibitor) to determine whether NF-kappaB activation requires tyrosine kinase activation, we found genistein blocks NF-kappaB activation at 8 h post-infection (p.i), and either enhances cell viability up to 50% at 12 h p.i. or blocks DNA fragmentation at 24 h p.i. Furthermore, the proteasome inhibitors PSI-I and PSI-II (both at 40 microm) also effectively blocked the NF-kappaB activation as well as stimulating a 30% increase in cell viability (30% decrease in apoptosis) at 8 and 12 h p.i. Taken together our data suggest that IPNV may induce NF-kappaB activation through tyrosine kinase signalling, which may be associated with induction of apoptosis.