[Show abstract][Hide abstract] ABSTRACT: Melanised focal changes (black spots) are common findings in the white skeletal muscle of seawater-farmed Atlantic salmon (Salmo salar). Fillets with melanised focal changes are considered as lower quality and cause large economic losses. It has been suggested that red focal changes (red spots) precede the melanised focal changes. In the present work, we examined different populations of captive and wild salmon for the occurrence of both types of changes, which were investigated for the presence of different viruses by immunohistochemistry and RT-qPCR. The occurrence of red or melanised foci varied significantly between the populations, from none in wild fish control group, low prevalence of small foci in fish kept in in-house tanks, to high prevalence of large foci in farm-raised salmon. Large amounts of Piscine orthoreovirus (PRV) antigen were detected in all foci. No other viruses were detected. Red focal changes contained significantly higher levels of PRV RNA than apparently non-affected areas in white muscle of the same individuals. Some changes displayed a transient form between a red and melanised pathotype, indicating a progression from an acute to a chronic manifestation. We conclude that PRV is associated with the focal pathological changes in the white muscle of farmed Atlantic salmon and is a premise for the development of focal melanised changes.
Veterinary Research 09/2015; 46(1):89. DOI:10.1186/s13567-015-0244-6 · 2.82 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Yersinia ruckeri is the causative agent of enteric redmouth disease (ERM) in rainbow trout, and the first commercially available fish vaccine was an immersion vaccine against ERM consisting of Y. ruckeri bacterin. The ERM immersion vaccine has been successfully used in aquaculture farming of salmonids for more than 35 years. The gills and the gastrointestinal (GI) tract are believed to be the portals of antigen uptake during waterborne vaccination against ERM; however, the actual sites of bacterin uptake are only partly understood. In order to obtain insight into bacterin uptake during waterborne vaccination, optical projection tomography (OPT) together with immunohistochemistry (IHC) was applied to visualize bacterin uptake and processing in whole rainbow trout fry. Visualization by OPT revealed that the bacterin was initially taken up via gill lamellae from within 30 seconds post vaccination. Later, bacterin uptake was detected on other mucosal surfaces such as skin and olfactory bulb from 5 to 30 minutes post vaccination. The GI tract was found to be filled with a complex of bacterin and mucus at 3 hours post vaccination and the bacterin remained in the GI tract for at least 24 hours. Large amounts of bacterin were present in the blood, and an accumulation of bacterin was found in filtering lymphoid organs such as spleen and trunk kidney where the bacterin accumulates 24 hours post vaccination as demonstrated by OPT and IHC. These results suggest that bacterin is taken up via the gill epithelium in the earliest phases of the bath exposure and from the GI tract in the later phase. The bacterin then enters the blood circulatory system, after which it is filtered by spleen and trunk kidney, before finally accumulating in lymphoid organs where adaptive immunity against ERM is likely to develop.
PLoS ONE 02/2015; 10(2):e0117263. DOI:10.1371/journal.pone.0117263 · 3.23 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 02/2015; 38(2):151-160. DOI:10.1111/jfd.12216 · 2.06 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Infection of fish with the facultative intracellular bacterium Francisella noatunensis remains an unresolved problem for aquaculture industry worldwide as it is difficult to vaccinate against without using live attenuated vaccines. Outer membrane vesicles (OMVs) are biological structures shed by Gramnegative bacteria in response to various environmental stimuli. OMVs have successfully been used to vaccinate against both intracellular and extracellular pathogens, due to an ability to stimulate innate, cell-mediated and humoral immune responses. We show by using atomic force and electron microscopy that the fish pathogenic bacterium E noatunensis subspecies noatunensis (F.n.n.) shed OMVs both in vitro into culture medium and in vivo in a zebrafish infection model. The main protein constituents of the OMV are IgIC, PdpD and PdpA, all known Francisella virulence factors, in addition to the outer membrane protein FopA and the chaperonin GroEL, as analyzed by mass spectrometry. The vesicles, when used as a vaccine, reduced proliferation of the bacterium and protected zebrafish when subsequently challenged with a high dose of F.n.n. without causing adverse effects for the host. Also granulomatous responses were reduced in F.n.n.-challenged zebrafish after OMV vaccination. Taken together, the data support the possible use of OMVs as vaccines against francisellosis in fish. (C) 2014 The Authors. Published by Elsevier Ltd.
[Show abstract][Hide abstract] ABSTRACT: Consumption of fatty acids from fishes is widely regarded as beneficial for preventing cardiovascular disorders. Nevertheless, salmonids themselves are victims of vascular diseases. As the pathogenesis and nature of these changes are elusive, they are here addressed using novel morphological and transcriptional approaches. Coronary arteries of wild Atlantic salmon Salmo salar L., (n = 12) were investigated using histological and immunohistochemical techniques, and RT-qPCR was employed to investigate expression of stretch-induced genes. In an experimental trial, fish were fed diets with different fatty acids composition, and histological features of the coronary arteries (n = 36) were investigated. In addition, the heart fatty acid profile (n = 60) was analysed. There were no differences in morphological or immunological features between wild fish and groups of experimental fish. Arteriosclerotic lesions consisted of smooth muscle cells in dissimilar differential stages embedded in considerable amounts of extracellular matrix in a similar fashion to what is seen in early stages of human atherosclerosis. No fat accumulations were observed, and very few inflammatory cells were present. In affected arteries, there was an induction of stretch-related genes, pointing to a stress-related response. We suggest that salmon may have a natural resistance to developing atherosclerosis, which corresponds well with their high investment in lipid metabolism.
Journal of Fish Diseases 10/2014; DOI:10.1111/jfd.12321 · 2.06 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Prophylactic measures against Aeromonas salmonicida subsp. salmonicida, the causative agent of furunculosis, have been an active field of research for decades, with studies mainly focused on Atlantic salmon (Salmo salar). In the present study we have examined the protective and adverse effects of mineral oil-adjuvanted injection vaccines on rainbow trout (Oncorhynchus mykiss). A commercial vaccine and an experimental auto vaccine, as well as their respective adjuvant formulations alone were used to evaluate their individual effects, both prior to and during an experimental waterborne infection challenge. Macro- and microscopic examination revealed signs of vaccine-induced adverse effects from 10 weeks to 14 months post vaccination. Both vaccines induced statistically significant protection during the experimental challenge (P=0.018 for both vaccines), as well as significantly elevated levels of specific circulating antibodies prior to and during the experimental challenge when compared to an unvaccinated control group. During the early, critical time points of the infection, both vaccines appeared to protect against pathological changes to the liver and spleen, which provides a probable explanation for the reduced mortality seen in the vaccinated groups. A significant correlation was found between the level of A. salmonicida-specific antibodies measured prior to challenge and the endpoint survival of each group after the experimental infection, and furthermore, the levels of these antibodies remained elevated for at least 14 months post vaccination.
[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.
The Journal of Immunology 08/2014; DOI:10.4049/jimmunol.1400797 · 4.92 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We isolated the infectious viral agent from infected Asian seabass, Lates calcarifer (Bloch, 1790) using SSN-1 cells. The isolated virus was cultivated in indigenous marine fish cell lines developed from ornamental sea anemone fish Amphiprion sebae and from Asian seabass. Reverse transcriptase PCR using betanodavirus-specific primers gave positive results for the RNA from virus-infected cells (hereinafter referred to as L. calcarifer nervous necrosis virus Indian strain – LCNNV-In01). Serum neutralization test indicated partial homogeneity between Atlantic halibut nervous necrosis virus (AHNNV 692/9/98) and LCNNV-In01. Transmission electron micrographs of the virus-infected cell culture showed large numbers of 25–30-nm particles in cellular vacuoles with features similar to betanodaviruses. Pathogenicity study in seabass fingerlings using cell culture grown virus revealed nervous necrosis in retinal cells following a 21-day challenge trial. The nodavirus was reisolated from the infected fish using clownfish and seabass cell lines. Sequence analysis of the coat protein gene showed that the virus belonged to RGNNV group (GenBank Acc. No. FR669249). The isolation of the nodavirus from seabass in India and its potential to grow in non-homologous cell lines indicate the serious threat of the Indian strain of betanodavirus (LCNNV-In01) to cultured and ornamental marine fish that have their hatchery production successfully standardized in India.
Aquaculture Research 08/2014; 45(9). DOI:10.1111/are.12095 · 1.38 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The intestinal mucosa is an important port of entry for many pathogens. Information of antigenic uptake mechanisms is essential to understand and to possibly prevent infections. In teleosts, several studies have aimed at investigating particulate uptake in the gastrointestinal system that seems to vary dependent on species and antigen. In the present study, particulate uptake in the Atlantic salmon intestine by anal intubation of yeast cells has been investigated. In the anal intubated fish, yeast were found in the epithelium close to nuclei of macrophage-like cells and inside large mononuclear cells in the intestinal lumen, indicating uptake and possible transport of large antigenic particles over the epithelium by macrophage-like cells.
[Show abstract][Hide abstract] ABSTRACT: Forming a barrier to the outside world, the gut mucosa faces the challenge of absorbing nutrients and fluids while initiating immune reactions towards potential pathogens. As a continuation to our previous publication focusing on the regional intestinal morphology in wild caught post smolt and spawning Atlantic salmon, we here investigate selected immune parameters and compare wild, reared unvaccinated and vaccinated post smolts. We observed highest transcript levels for most immune related genes in vaccinated post smolts followed by reared unvaccinated and finally wild post smolts, indicating that farming conditions like commercial feed and vaccination might contribute to more alerted immune system in the gut. In all groups, higher levels of immune transcripts were observed in the second segment of mid-intestine and in the posterior segment. In the life stages and conditions investigated here, we found no indication of a previously suggested population of intestinal T cells expressing MHC class II nor RAG1 expression.
[Show abstract][Hide abstract] ABSTRACT: Sialic acids are located at the terminal branches of the cell glycocalyx and secreted glycan molecules. O-Acetylation is an important modification of the sialic acids, however very few studies have demonstrated the in situ distribution of the O-Acetylated sialic acids. Here the distribution of glycoprotein bound 4-O-Acetylated sialic acids (4-O-Ac sias) in vertebrates was determined using a novel virus histochemistry assay. The 4-O-Ac sias were found in the circulatory system, i.e. on the surface of endothelial cells and RBCs, of several vertebrate species, though most frequently in the cartilaginous fish (class Chondrichthyes) and the bony fish (class Osteichthyes). The O-Acetylated sialic acid was detected in 64 % of the examined fish species. Even though the sialic acid was found less commonly in higher vertebrates, it was found at the same location in the positive species. The general significance of this endothelial labelling pattern distribution is discussed. The seemingly conserved local position through the evolution of the vertebrates, suggests an evolutionary advantage of this sialic acid modification.
[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. DOI:10.1111/jfd.12106 · 2.06 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.
Aquaculture 04/2014; s 426–427:288–295. DOI:10.1016/j.aquaculture.2014.01.034 · 1.88 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. DOI:10.1186/1746-6148-10-60 · 1.78 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Vaccination interferes with biological functions of the fish and may therefore cause some side-effects with consequences for economy and fish welfare. Although all delivery methods may cause side-effects, most of them are of minor significance. Most administration forms may cause acute or prolonged side-effects, but those caused by intraperitoneal injection of oil-adjuvanted vaccines in salmon are by far the most severe and will therefore constitute the main bulk of information in this chapter. The acute side-effects may grossly be divided into those resulting from poor handling, anesthesia, contamination of the vaccine, and genuine side-effects caused by the vaccine itself. Also, several of the more diffuse side-effects observed in vaccinated fish may be related to the induction of systemic autoimmunity. The chapter also discusses injection site reactions, abdominal lesions, and skeletal lesions.
Fish Vaccination, 03/2014: pages 153-161; , ISBN: 9780470674550
[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.
[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. DOI:10.1371/journal.pone.0085551 · 3.23 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. DOI:10.1186/1297-9716-44-107 · 2.82 Impact Factor