[Show abstract][Hide abstract] ABSTRACT: Previously, our group has shown that the interbranchial lymphoid tissue (ILT) is a distinct structure largely consisting of T cells embedded in a meshwork of epithelial cells, with no direct resemblance to previously described lymphoid tissues. In this study, we aim to focus on the T cell population and the possibility of the ILT being a thymus analog. By characterizing structural responsiveness to Ag challenge, the presence of recombination activating genes, and different T cell-related transcripts, we attempt to further approach the immunological function of the ILT in salmonid gills. In addition to eight healthy individuals, a group of eight infectious salmon anemia virus-challenged fish were included to observe T cell responses related to infection. The results showed reduced size of ILT in the infected group, no expression of RAG-1 and -2, and a high degree of T cell diversity within the ILT. Taking into account that the ILT can be regarded as a strategically located T cell reservoir and possibly an evolutionary forerunner of mammalian MALTs right at the border to the external environment, the alteration in transcription observed may likely represent a shift in the T cell population to optimize local gill defense mechanisms.
Journal of immunology (Baltimore, Md. : 1950). 08/2014;
[Show abstract][Hide abstract] ABSTRACT: The presence of melanin in muscle fillets of farmed salmon represents a considerable quality problem for the salmon industry with major economic concerns. In this study, we have examined the presence of abnormal pigmentation in vaccinated versus unvaccinated Atlantic salmon, Salmo salar L., and evaluated possible differences between diploid and triploid fish. Furthermore, the impact of the smolt production regime at ambient (4.5 °C) versus elevated temperature (16 °C) was investigated. Pigmented muscle spots were analysed for the expression of genes involved in melanization (tyrosinase gene family) and immune-related response in addition to morphological investigations. The proportion of fish with intramuscular melanin deposits was not significantly different between vaccinated and unvaccinated fish, regardless of ploidy. However, an interaction between vaccination and smolt regime was shown, where smoltification at elevated temperature after vaccination increased the number of affected individuals compared with vaccination followed by simulated natural smoltification. Furthermore, there were overall more fish with melanin spots amongst the triploids compared with their diploid counterparts. Transcription of the tyrosinase gene family confirmed an onsite melanogenesis in all pigment spots. The histological examination and the expression of the immune-related genes revealed a chronic polyphasic myopathy that was not affected by vaccination, ploidy or smolt production regime.
Journal of Fish Diseases 04/2014; 37(4):327-340. · 1.59 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Fish meal and fish oil are increasingly replaced by ingredients from terrestrial sources in the feeds for farmed salmonids due to expanding production and reduced availability of marine feed raw material. Fish oil that is rich in n-3 polyunsaturated fatty acids is considered beneficial to human health in general and to prevent intestinal inflammation and carcinogenesis in particular. In contrast, n-6 fatty acids that are present in many vegetable oils have been associated with increased risk of colitis and colon cancer in rodents and humans, as well as lowered transcription levels of certain stress and antioxidant-related genes in Atlantic salmon.The aim of the present study was to investigate the intestinal health in Atlantic salmon fed with different vegetable oils as partial substitutes of fish oil in the diet. A feed trial lasting for 28 weeks included one reference diet containing fish oil as the sole lipid source and three diets where 80% of the fish oil was replaced by a plant oil blend with either olive oil, rapeseed oil or soybean oil as the main lipid source. These plant oils have intermediate or low n-3/n-6-ratios compared to fish oil having a high n-3/n-6-ratio. The protein and carbohydrate fractions were identical in all the feeds.
Morphometric measurements showed significantly shorter folds in the mid intestine in all groups fed vegetable oils compared to the group fed fish oil. In the distal intestine, the complex folds were significantly shorter in the fish fed soybean oil compared to the fish fed rapeseed oil. Histological and immunohistochemical examination did not show clear difference in the degree of inflammation or proliferation of epithelial cells related to dietary groups, which was further confirmed by real-time RT-PCR which revealed only moderate alterations in the mRNA transcript levels of selected immune-related genes.
Shortened intestinal folds might be associated with reduced intestinal surface and impaired nutrient absorption and growth, but our results suggest that partial substitution of dietaryfish oil with vegetable oils does not have any major negative impact on the intestinal health of Atlantic salmon.
BMC Veterinary Research 03/2014; 10(1):60. · 1.86 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Previously, it has been assumed that fish lack organized mucosal-associated lymphoid structures. Recently, an interbranchial lymphoid tissue (ILT) was described in salmonid gills at a site with substantial exposure to antigen. In this study, immune responses were examined in gills, mid-kidney and the laser-dissected ILT of Atlantic salmon (Salmo salar L) infected with infectious salmon anemia virus (ISAV). A strong innate response was observed in gills and mid-kidney and even in the laser-dissected ILT, despite the fact that no virus could be traced in this tissue. A small delayed increase in IgT transcripts, exclusively in the ILT, could indicate that this tissue has a role as a secondary lymphoid organ with clonal expansion of IgT expressing B-cells. Compared to the other examined tissues, gills displayed the earliest replication of the virus, further supporting this tissue as the main entry route for infection with ISAV.
Developmental and comparative immunology 02/2014; · 3.29 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Heart morphology is particularly plastic in teleosts and differs between farmed and wild Atlantic salmon. However, little is known about how different culture practices and sex affect heart morphology. This study investigated how vaccination, triploidy and sex affected heart size and heart morphology (ventricle shape, angle of the bulbus arteriosus) in farmed Atlantic salmon for 18 months following vaccination (from c. 50-3000 g body weight). In addition, hearts were examined histologically after 7 months in sea water. All fish sampled were sexually immature. Vaccinated fish had significantly heavier hearts relative to body weight and a more triangular ventricle than unvaccinated fish, suggesting a greater cardiac workload. Irrespective of time, triploids had significantly heavier hearts relative to body weight, a more acute angle of the bulbus arteriosus and less fat deposition in the epicardium than diploids. The ventricle was also more triangular in triploids than diploids at seawater transfer. Sex had transient effects on the angle of the bulbus arteriosus, but no effect on relative heart weight or ventricle shape. From a morphological perspective, the results indicate that vaccination and triploidy increase cardiac workload in farmed Atlantic salmon.
Journal of Fish Diseases 01/2014; · 1.59 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Atlantic salmon were fed a standard extruded dry feed (Control) or the same feed supplemented with 1.5% l-glutamate (Glu) (triplicate net pens per diet) from May 2009 (body weight = 105 g) to May 2010 (body weight = 3.1 kg). No significant differences were observed in growth (TGC = 3.1) or feed conversion ratio (1.0) between the dietary treatments. Instrumental texture analyses showed that Glu supplementation resulted in significantly (P ≤ 0.05) firmer fillets after ice storage (10.1 vs. 9.1 N) and after frozen storage (8.7 vs. 6.3 N). Additionally the Glu group had less organ adhesions (score = 0.5 vs. 1.1), lower hepato-somatic index (0.91 vs. 0.99%) and less fat accumulated in the livers (1.8 vs. 2.1 g). The condition factor, carcass and fillet yield, and cardio- and spleen-somatic indices were unaffected by dietary treatment. Hepatocellular vacuolization, intestinal inflammation and muscle degeneration were observed in the Control (50, 13, and 5%, respectively) and the Glu group (40, 7, and 25%, respectively). No abnormal observations were found in the spleen or kidney. Plasma analyses revealed significantly lower activity of creatine kinase (3.3 vs. 5.8 U/mL) and alanine aminotransferase (5.8 vs. 7.5 U/L) in the Glu fed group. Muscle pH was significantly higher in the Glu group (6.22 vs. 6.19), but the fat-, protein-, amino acid-, and collagen contents were similar. The Glu group had significantly higher concentrations of saturated fatty acids and docosapentaenoic acid (1.9 vs. 1.7 and 17.4 vs. 17.1% of fatty acids, respectively), and n− 3 fatty acids and n− 3/n− 6 ratio in the muscle tended to be higher (P < 0.09). Collagen properties determined as degree of glycation, solubility, thermal behaviour, pyridinoline bonds and structure were similar for both dietary groups. Compared with the Control, the skeletal muscle of salmon fed the Glu supplemented diet showed up-regulation of genes involved in stress response, mitochondrial functions, and amino acid and lipid metabolism, whereas several genes involved in cytoskeletal structure were down-regulated. Glu supplementation resulted in firmer fillets, coinciding with altered energy metabolism and improved health related parameters. It is suggested that optimal dietary amino acid levels for growth may differ from optimal levels for good fish health and flesh quality.
[Show abstract][Hide abstract] ABSTRACT: Atlantic salmon (Salmo salar L.) with soft fillets are not suited for manufacturing high quality products. Therefore fillets with insufficient firmness are downgraded, leading to severe economic losses to the farming and processing industries. In the current study, morphological characteristics of salmon fillets ranging from soft to hard were analysed. Different microscopic techniques were applied, including novel methods in this field of research: morphometric image analysis, periodic acid Schiff staining, immunofluorescence microscopy, transmission electron microscopy and fourier transform infrared microscopy. The results showed that the myocytes of soft muscle had detached cells with mitochondrial dysfunctions, large glycogen aggregates and enlarged inter cellular areas, void of extracellular matrix proteins, including lower amounts of sulfated glycoproteins. Myofibre-myofibre detachment and disappearance of the endomysium in soft muscles coincided with deterioration of important connective tissue constituents such as Collagen type I (Col I), Perlecan and Aggrecan. In summary our investigations show for the first time an association between soft flesh of Atlantic salmon and massive intracellular glycogen accumulation coinciding with degenerated mitochondria, myocyte detachment and altered extracellular matrix protein distribution. The results are important for further understanding the etiology of soft salmon.
PLoS ONE 01/2014; 9(1):e85551. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: It is widely accepted that melanin formation may play an immunologic role in invertebrates and ectothermic vertebrates. In farmed Atlantic salmon, cardiomyopathy syndrome (CMS) is a common viral disease associated with severe cardiac inflammation that may be accompanied by heavy melanisation of the heart. By the use of histology, laser capture microdissection and transcription analysis of tyrosinase genes, we here show that this melanisation is linked to de novo melanogenesis by melanomacrophages, suggesting an active part in the inflammatory reaction. No general systemic activation of the extracutaneous pigmentary system in response to viral infections with affinity to the heart was observed.
Veterinary Research 11/2013; 44(1):107. · 3.43 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cytotoxic T-cells (CTLs) play a pivotal role in eliminating viruses in mammalian adaptive immune system. Many recent studies on T-cell immunity of fish have suggested that teleost CTLs are also important for antiviral immunity. Cellular functional studies using clonal ginbuan crucian carp and rainbow trout have provided in vivo and in vitro evidence that in many respects, virus-specific CTLs of fish have functions similar to those of mammalian CTLs. In addition, mRNA expression profiles of CTL-related molecules, such as CD8, TCR and MHC class I, have shown that in a wide range of fish species, CTLs are involved in antiviral adaptive immunity. These findings are a basis to formulate possible vaccination strategies to trigger effective antiviral CTL responses in teleost fish. This review describes recent advances in our understanding of antiviral CTL functions in teleost fish and discusses vaccination strategies for efficiently inducing CTL activities.
Developmental and comparative immunology 08/2013; · 3.29 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Aeromonas salmonicida subsp. achromogenes, the causative agent of atypical furunculosis in many fish species, secretes the toxic metalloendopeptidase AsaP1. This study aimed to analyze innate and adaptive immune parameters induced in Arctic charr (Salvelinus alpinus, L.) infected with wild type (wt) A. salmonicida subsp. achromogenes and its isogenic asaP1 deletion mutant (AsaP1-deficient). Head-kidney, liver and spleen were obtained from i.p. infected charr (wt, AsaP1-deficient), during a time schedule of 7 d post infection. Reverse transcription quantitative real-time PCR (RT-qPCR) was applied to study the expression of immune parameters: pro-inflammatory cytokines IL-1β and TNF-α; anti-inflammatory cytokine IL-10; chemokines CXCL-8 (IL-8) and CC-chemokine; the cytokines IFN-γ and IL-4/13A as tracers for Th1 and Th2 immune responses, respectively; and the cell markers CD8α and CD83. In addition, lymphoid organs were histopathologically examined at days 3 and 7 post infection, including B (IgM) and T (CD3ε) cell staining. The detected immune responses were initially driven by innate mechanisms represented by the up-regulation of pro-inflammatory cytokines and chemokines and later on by adaptive Th2 related responses cumulating in B cell recruitment as shown by regulation of immune parameters in spleen and head-kidney, with significant differences between mutant and wt infected fish. Histological sections revealed IgM-positive cells around ellipsoid arterioles in spleen, while CD3ε positive cells were found in clusters scattered all over the section. However, histopathological differences were only detected between infected and non-infected fish, but not between AsaP1-deficient mutant and wt infected fish. This work represents the first study on innate and adaptive immune responses of Arctic charr induced by a bacterial infection.
Fish & Shellfish Immunology 06/2013; · 2.96 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The main function of the immune system is to maintain the organism's homeostasis when invaded by foreign material or organisms. Prior to successful elimination of the invader it is crucial to distinguish self from non-self. Most pathogens and altered cells can be recognized by immune cells through expressed pathogen- or danger-associated molecular patterns (PAMPS or DAMPS, respectively), through non-self (e.g. allogenic or xenogenic cells) or missing major histocompatibility (MHC) class I molecules (some virus-infected target cells), and by presenting foreign non-self peptides of intracellular (through MHC class I - e.g. virus infected target cells) or extracellular (through MHC class II - e.g. from bacteria) origin. In order to eliminate invaders directly or by destroying their ability to replicate (e.g. virus-infected cells) specialized immune cells of the innate and adaptive responses appeared during evolution. The first line of defence is represented by the evolutionarily ancient macrophages and natural killer (NK) cells. These innate mechanisms are well developed in bony fish. Two types of NK cell homologues have been described in fish: non-specific cytotoxic cells and NK-like cells. Adaptive cell-mediated cytotoxicity (CMC) requires key molecules expressed on cytotoxic T lymphocytes (CTLs) and target cells. CTLs kill host cells harbouring intracellular pathogens by binding of their T cell receptor (TCR) and its co-receptor CD8 to a complex of MHC class I and bound peptide on the infected host cell. Alternatively, extracellular antigens are taken up by professional antigen-presenting cells such as macrophages, dendritic cells and B cells to process those antigens and present the resulting peptides in association with MHC class II to CD4(+) T helper cells. During recent years, genes encoding MHC class I and II, TCR and its co-receptors CD8 and CD4 have been cloned in several fish species and antibodies have been developed to study protein expression in morphological and functional contexts. Functional assays for innate and adaptive lymphocyte responses have been developed in only a few fish species. This review summarizes and discusses recent results and developments in the field of T and NK cell responses with focus on economically important and experimental model fish species in the context of vaccination.
Fish & Shellfish Immunology 05/2013; · 2.96 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The present study was performed to address putative links between the immune and pigmentary systems. A pigment-producing leukocyte-like cell-line (SHK-1 cells) of Atlantic salmon (Salmo salar L.) was exposed to different temperatures, poly I:C, bacterin or infected with virus (infectious pancreatic necrosis virus or infectious salmon anaemia virus). The effect of this stimulation regarding the transcription-pattern of the tyrosinase gene family (melanin genes) and the immune-related genes MHC class II and IFN-1 was analysed using real-time RT-qPCR. At 10°C cultivation, tyrosinase and dopachrome tautomerase remained unregulated. At 15°C, a moderate up-regulation was induced, while at 20°C, these genes were up-regulated in an exponential manner over time. Temperature did not affect the transcription of the immune-related genes. Virus infections, poly I:C or bacterin had no influence on the transcription of the melanogenesis-related genes, but triggered the immune-related genes. Our findings revealed no connections between the pigmentary and immune systems, but demonstrated a hereto undiscovered temperature-effect on the tyrosinase gene family.
Developmental and comparative immunology 04/2013; · 3.29 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Granulomatous peritonitis is often induced by intraperitoneal vaccination in fish. Peritonitis is a very painful condition in mammals, but little is known about how fish experience this condition. In a previous experiment we found increased latency to eat and a tendency to decreased swimming during feeding in vaccinated salmon housed in groups of three. These changes in behaviour correlated with the severity of the peritonitis. However, dominance relationships may influence the degree of painrelated changes in behaviour and physiology shown by group-housed fish, and we therefore conducted the present study using singly-housed fish. The aim was to describe changes in latency to eat, hiding,
swimming and bottom behaviour after vaccination, to test whether morphine would alleviate changes in behaviour, and finally to test whether vaccination and/or morphine would influence the response of the fish in the novel object test. In addition, we looked for microscopic changes of peritonitis two days after vaccination to test whether we would be able to detect inflammation at an early stage. Four treatment groups were used: VS (injected with vaccine intraperitoneally (ip) and saline intramuscularly (im)), VM (injected with vaccine ip and 300 mg kg-1 morphine im), SS (saline ip, saline im) and SM (saline ip, morphine 300 mg kg-1 im). Swimming during feeding decreased in the VS fish 2 days after treatment, both compared to baseline (p = 0.031) and to the SS group (p = 0.023).
The latency to eat increased for all groups from baseline to the four post-treatment times (F4, 88 = 18.7, p < 0.0001), and the VS group showed significantly higher latency to eat compared with the saline group 6.5 h and 2 days after treatment (p = 0.012 and 0.018, respectively). Contrary to predictions, we did not find an increase in bottom behaviour after vaccination. We could not detect any microscopic signs of peritonitis 48 h after vaccination. Morphine did not seem to have an analgesic effect in that there were no differences between the VM and VS groups. However, the SM fish spent significantly more time in the open during the novel object test compared with the SS fish ( χ2=6.97, df=1, p=0.0083) one day after injection, indicating an anxiolytic effect of morphine at that time-point. It is
important that the lack of analgesic effect is not taken to indicate lack of pain perception in salmon. Inter species differences in analgesic efficacy is well known in veterinary medicine, and the results from this paper indicate that we need to look at alternatives to morphine to find effective analgesics for Atlantic salmon.
[Show abstract][Hide abstract] ABSTRACT: Endothelial cells (ECs) line the luminal surfaces of the cardiovascular system and play an important role in cardiovascular functions such as regulation of haemostasis and vasomotor tone. A number of fish and mammalian viruses target these cells in the course of their infection. Infectious salmon anaemia virus (ISAV) attacks ECs and red blood cells (RBCs) of farmed Atlantic salmon (Salmo salar L.), producing the severe disease of infectious salmon anaemia (ISA). The investigation of ISA has up to now been hampered by the lack of a functional marker for ECs in Atlantic salmon in situ. In this study, we report the characterisation and use of a novel monoclonal antibody (MAb) detecting Atlantic salmon ECs (e.g. vessel endothelium, endocardial cells and scavenger ECs) and RBCs. The antibody can be used with immunohistochemistry, IFAT and on Western blots. It appears that the epitope recognised by the antibody is associated with the ISAV cellular receptor. Besides being a tool to identify ECs in situ, it could be useful in further studies of the pathogenicity of ISA. Finally, the detection of an epitope shared by ECs and RBCs agrees with recent findings that these cells share a common origin, thus the MAb can potentially be used to study the ontogeny of these cells in Atlantic salmon.
[Show abstract][Hide abstract] ABSTRACT: Infectious salmon anaemia virus (ISAV), a member of the Orthomyxoviridae family, infects and causes disease in farmed Atlantic salmon (Salmo salar L.). Previous studies have shown Atlantic salmon endothelial cells to be the primary targets of ISAV infection. However, it is not known if cells other than endothelial cells play a role in ISAV tropism. To further assess cell tropism, we examined ISAV infection of Atlantic salmon gill epithelial cells in vivo and in vitro. We demonstrated the susceptibility of epithelial cells to ISAV infection. On comparison of primary gill epithelial cell cultures with ISAV permissive fish cell cultures, we found the virus yield in primary gill epithelial cells to be comparable with that of salmon head kidney (SHK)-1 cells, but lower than TO or Atlantic salmon kidney (ASK)-II cells. Light and transmission electron microscopy (TEM) revealed that the primary gill cells possessed characteristics consistent with epithelial cells. Virus histochemistry showed that gill epithelial cells expressed 4-O-acetylated sialic acid which is recognized as the ISAV receptor. To the best of our knowledge, this is the first demonstration of ISAV infection in Atlantic salmon primary gill epithelial cells. This study thus broadens our understanding of cell tropism and transmission of ISAV in Atlantic salmon.