Flumequine in Atlantic salmon Salmo salar: disposition in fish held in sea water versus fresh water

14C-labeled flumequine was administered as a single oral (5 mg kg(-1), 86 microCi kg(-1)) or intravenous (5 mg kg(-1), 82 microCi kg(-1)) dose to Atlantic salmon Salmo salar held in sea water or in fresh water. The absorption, tissue distribution and elimination were determined by means of liquid scintillation counting and whole-body autoradiography. The drug was rapidly absorbed and extensively distributed in all groups of fish. Radiolabeled compound was present in blood and muscle for more than 8 wk in the freshwater groups. In the seawater groups, however, no radioactivity was detected in the blood and muscle after 4 d and 2 wk, respectively. It was concluded that flumequine was eliminated at a substantially higher rate from Atlantic salmon in sea water than in fresh water.     

Resistance to reinfection in chinook salmon Oncorhynchus tshawytscha to Loma salmonae (Microsporidia).

Chinook salmon Oncorhynchus tshawytscha were experimentally infected per os with Loma salmonae and held in flow-through seawater tanks at 12 to 14 degrees C. The fish exhibited 100% infection when first examined at 7 wk post initial exposure (p.e.), and by 20 wk p.e. they had completely recovered from gill infections. The recovered fish were then re-exposed the following week. All of these fish showed strong protection to new L. salmonae infections, while na?ve fish exposed to the same inoculum developed the infection. Most of the re-exposed fish exhibited a few free spores or spores within phagocytes in the kidney interstitium at 20 to 29 wk p.e., but xenomas were not detected in either the gills or visceral organs. The kidney is the primary site of reticulo-endothelial activity, and thus these spores were likely deposited in the kidney by entrapment by fixed macrophages. It is possible that these spores provide immunologic stimuli to reinforce the resistance to new L. salmonae infections.     

Analysis of Kudoa thyrsites (Myxozoa: Myxosporea) spore antigens using monoclonal antibodies.

A method employing Percoll gradient centrifugation was developed to purify Kudoa thyrsites spores from somatic muscle tissue of Atlantic salmon Salmo salar. Highly purified spores were then used to immunize inbred BALB/c mice for derivation of hybridomas secreting Kudoa-specific monoclonal antibodies (mAbs). Analysis of mAbs by immunofluorescence microscopy and flow cytometry showed that several were specific for antigens on the surface of K. thyrsites spores whereas other mAbs reacted with polar capsules or with polar filaments of spores of K. thyrsites, K. paniformis and K. crumena. Immunoblots on spore lysates using the surface-binding mAbs showed a broad band of 46 to > 220 kDa, whereas mAbs specific for antigens of polar capsules and polar filaments detected sharper bands of various molecular masses, depending on the Kudoa species. The dominant epitope of the K. thyrsites spore surface antigen was shown to be carbohydrate as determined by its sensitivity to treatment with anhydrous trifluoromethane sulfonic acid and by its resistance to treatment with Proteinase K. Immunofluorescence microscopy using the K. thyrsites-specific mAbs on isolated, intact, permeabilized plasmodia and on thin sections of somatic muscle tissue containing plasmodia revealed intense labeling of spores both within the spore-producing plasmodia and in the flesh of infected Atlantic salmon. As few as 100 spores were detected by immunoblotting, indicating that these mAbs have potential for use in developing a field-based diagnostic test.     

Virus-like particles associated with heart and skeletal muscle inflammation (HSMI)

The first cases of heart and skeletal muscle inflammation (HSMI), in Atlantic salmon Salmo salar were registered in 1999 in the Hitra/Fr?ya area of Norway. The disease has since spread south to Rogaland, i.e. the southernmost county with salmon farming in Norway. The disease outbreaks usually start 5 to 9 mo after release into seawater but may occur as early as 2 wk after sea release. The present study focuses on possible pathogens associated with HSMI. It was not possible to find any parasites or bacteria that could explain HSMI, and none of the well-known viruses (infectious salmon anaemia virus, Norwegian salmonid alphavirus, infectious pancreatic necrosis virus, Atlantic salmonid paramyxovirus) were consistently present. Use of transmission electron microscopy showed the presence of epitheliocystis agent in 3 of 4 farms included in this study, and several virus-like particles. Type I and Type II virus particles, previously described for salmon suffering from haemorrhagic smolt syndrome (HSS), and erythrocytic inclusion body syndrome (EIBS) virus were consistently present in salmon suffering from HSMI in all 4 farms included in this study. The 2 HSS viruses (Type I and Type II) were also cultured in Atlantic salmon kidney (ASK) cells from salmon suffering from HSMI. However, a causal relationship between the observed virus particles and HSMI remains to be demonstrated.     

Epitopes associated with mature spores not recognized on Kudoa thyrsites from recently infected Atlantic salmon smolts

Atlantic salmon Salmo salar skeletal muscle was examined for Kudoa thyrsites by polymerase chain reaction (PCR) and positive fish were further examined by in situ hybridization (ISH) and immunohistochemistry (IHC). The infection was detected in 42% of salmon by PCR following a 60 d exposure to infective seawater at a temperature of 10 degrees C (= 600 degree-days, degreeD). The parasite was detected by ISH in skeletal and cardiac muscle but not in gill, kidney, spleen, liver, stomach, intestine, pyloric caeca and skin. None of 4 monoclonal antibodies (2F4, 4H2, 1H2, 3E8) raised against mature K. thyrsites spores reacted with the stages identified by ISH following a 600 degreeD exposure, but they did react with ISH-identified stages following a 1600 degreeD exposure. In contrast, a polyclonal antibody reacted with K. thyrsites stages in salmon with both 600 and 1600 degreeD exposures, suggesting that the parasite observed in 600 degreeD infections represents an antigenically distinct developmental stage of K. thyrsites.     

Arsenobetaine in Atlantic salmon (Salmo salar L.): influence of seawater adaptation

Glycine betaine has been suggested to improve the maintenance of ionic and osmotic homeostasis during seawater adaptation in teleost fish. Arsenobetaine may also behave as an osmolyte, due to its structural similarity to glycine betaine. The influence of seawater adaptation on intestinal uptake and muscle accumulation of arsenobetaine in the teleost Atlantic salmon (Salmo salar L.) was investigated. Atlantic salmon (freshwater and seawater adapted) were given a single oral dose of arsenobetaine, which was absorbed over the intestine within 6 h after exposure. Seawater adapted Atlantic salmon had significantly higher levels of accumulated arsenobetaine in blood compared to the freshwater adapted salmon. However, seawater adaptation had no effect on the levels of accumulated arsenobetaine in muscle tissue. Similar retention of the administered dose was found in muscle tissue in both freshwater and seawater adapted salmon, with 49+/-6% and 50+/-10% retention after 144 h, respectively. Results indicate that muscle retention was not influenced by salinity in seawater adapting teleosts.     

First detection of piscine reovirus (PRV) in marine fish species

Heart and skeletal muscle inflammation (HSMI) is a disease that affects farmed Atlantic salmon Salmo salar L. several months after the fish have been transferred to seawater. Recently, a new virus called piscine reovirus (PRV) was identified in Atlantic salmon from an outbreak of HSMI and in experimentally challenged fish. PRV is associated with the development of HSMI, and has until now only been detected in Atlantic salmon. This study investigates whether the virus is also present in wild fish populations that may serve as vectors for the virus. The virus was found in few of the analyzed samples so there is probably a more complex relationship that involves several carriers and virus -reservoirs.     

Evidence for a carrier state of infectious hematopoietic necrosis virus in chinook salmon Oncorhynchus tshawytscha.

In British Columbia, Canada, infectious hematopoietic necrosis virus (IHNV) is prevalent in wild sockeye salmon Oncorhynchus nerka and has caused disease in seawater net-pen reared Atlantic salmon Salmo salar. In this study, chinook salmon Oncorhynchus tshawytscha experimentally exposed to an isolate of IHNV found in British Columbia became carriers of the virus. When Atlantic salmon were cohabited with these virus-exposed chinook salmon, IHNV was isolated from the Atlantic salmon. Identification of chinook salmon populations that have been exposed to IHNV may be difficult, as virus isolation was successful only in fish that were concurrently infected with either Renibacterium salmoninarum or Piscirickettisia salmonis. Also, IHNV-specific antibodies were detected in only 2 of the 70 fish experimentally exposed to the virus. Two samples collected from chinook salmon exposed to IHNV while at a salt water net-pen site had a seroprevalence of 19 and 22%; however, the inconsistencies between our laboratory and field data suggest that further research is required before we can rely on serological analysis for identifying potential carrier populations. Because of the difficulty in determining the exposure status of populations of chinook salmon, especially if there is no concurrent disease, it may be prudent not to cohabit Atlantic salmon with chinook salmon on a farm if there is any possibility that the latter have been exposed to the virus.     

Mortality in Atlantic salmon (Salmo salar) associated with trichodinid ciliates

A protozoan infection (Trichodina truttae) was identified in captive Atlantic salmon (Salmo salar) kelts that died in spring of 1988 and 1989. Fish with intense infections showed signs of listlessness, erratic swimming and inappetence. The infection induced excessive mucus secretion, epithelial sloughing and lesions that probably permitted entry of opportunistic bacteria which eventually caused ulcers and death. A seawater bath for 30 min each week for 4 wk effectively controlled the parasite.     

Enzyme profiles of single muscle fibers never exposed to normal neuromuscular activity.

Do muscle fiber properties commonly associated with fiber types in adult animals and the population distribution of these properties require normal activation patterns to develop? To address this issue, the activity of an oxidative [succinic dehydrogenase (SDH)] and a glycolytic [alpha-glycerophosphate dehydrogenase (GPD)] marker enzyme, the characteristics of myosin adenosinetriphosphatase (myosin ATPase, alkaline preincubation), and the cross-sectional area of single fibers were studied. The soleus and medial gastrocnemius of normal adult cats were compared with cats that 6 mo earlier had been spinally transected at T12-T13 at 2 wk of age. In control cats, SDH activity was higher in dark than light ATPase fibers in the soleus and higher in light than dark ATPase fibers in the medial gastrocnemius. After transection, SDH activity was similar to control in both muscles. GPD activity appeared to be elevated in some fibers in each fiber type in both muscles after transection. The cross-sectional areas most affected by spinal transection were light ATPase fibers of the soleus and dark ATPase fibers of the medial gastrocnemius, the predominant fiber type in each muscle. These data demonstrate that although the muscle fibers of cats spinalized at 2 wk of age presumably were never exposed to normal levels of activation, the activity of an oxidative marker enzyme was maintained or elevated 6 mo after spinal transection. Furthermore, although the absolute enzyme activities in some fibers were elevated by transection, three functional protein systems commonly associated with fiber types, i.e., hydrolysis of ATP by myosin ATPase and glycolytic (GPD) and oxidative (SHD) metabolism, developed in a coordinated manner typical of normal adult muscles.     

Cultured gill-derived Neoparamoeba pemaquidensis fails to elicit amoebic gill disease (AGD) in Atlantic salmon Salmo salar

Amoebic gill disease (AGD) affects the culture of Atlantic salmon Salmo salar in the southeast of Tasmania. The disease is characterised by the presence of epizoic Neoparamoeba spp. in association with hyperplastic gill tissue. Gill-associated amoebae trophozoites were positively selected by plastic adherence for culture in seawater, where they proliferated using heat-killed E. coli as a nutrient source. One isolate of gill-harvested amoebae designated NP251002 was morphologically consistent to N. pemaquidensis under light, fluorescence and transmission electron microscopy. Rabbit anti-N. pemaquidensis antiserum bound to NP251002, and N. pemaquidensis small subunit (SSU) ribosomal DNA (18S rDNA) was detected in NP251002 genomic DNA preparations using PCR. A high degree of similarity in the alignment of the NP251002 18S rDNA PCR amplicon sequence with reference isolates of N. pemaquidensis suggested conspecificity. While short-term culture (72 h) of gill-harvested amoebae does not affect the capacity of amoebae to induce AGD, Atlantic salmon challenged with NP251002 after the trophozoites had been 34 and 98 d in culture exhibited neither gross nor histological evidence of AGD. It is not known if NP251002 were avirulent at the time of isolation, had down-regulated putative virulence factors or virulence was inhibited by the culture conditions. Therefore, the time in culture could be a limiting factor in maintaining virulence using the culture technique described here.     

Randomized clinical trial to investigate the effectiveness of teflubenzuron for treating sea lice on Atlantic salmon

A double-blind, randomized control clinical trial was performed to investigate the effectiveness of teflubenzuron in controlling sea lice Lepeophtheirus salmonis on farmed Atlantic salmon Salmo salar. A total of 40 sea cages from 3 commercial cage sites in Atlantic Canada were used in this Good Clinical Practice (GCP) trial. The teflubenzuron was administered in the feed at a dosage of 10 mg kg(-1) biomass d(-1) for 7 d. Medicated and control cages were matched by site, cage size, and pre-treatment mean lice counts using cages as the unit of concern. Post-treatment lice counts and staging of developmental stages were performed at 1 and 2 wk after the end of treatment. Chalimus stages in medicated cages were significantly lower than in control cages at 1 wk (79% reduction in mean lice counts, p < 0.001), and at 2 wk (53% reduction, p < 0.001). Mobile (pre-adult and adult) stages were also significantly reduced in medicated cages at 1 wk (69% reduction, p < 0.01), and at 2 wk (40% reduction, p < 0.01) post-treatment, respectively. Teflubenzuron was proven effective for reducing lice burdens on salmon despite the low parasite levels experienced during the trial and the recruitment of lice from the untreated cages. The use of cage as the unit of concern was an important design component of this trial.     

Enzymes released from Lepeophtheirus salmonis in response to mucus from different salmonids

Adult and mobile preadult sea lice Lepophtheirus salmonis were incubated with mucus samples from rainbow trout (Oncorhynchus mykiss), coho salmon (O. kisutch), Atlantic salmon (Salmo salar), and winter flounder (Pseudopleuronectes americanus) to determine the response of L. salmonis to fish skin mucus as assessed by the release of proteases and alkaline phosphatase. There was variation in the release of respective enzymes by sea lice in response to different fish. As well, sealice collected from British Columbia responded differently than New Brunswick sea lice to coho salmon mucus. Fish mucus and seawater samples were also analyzed using protease gel zymography to observe changes in the presence of low molecular weight (LMW) proteases after L. salmonis incubation. Significantly higher proportions of sea lice secreted multiple bands of L. salmonis-derived LMW proteases after incubation with rainbow trout or Atlantic salmon mucus in comparison with seawater, coho salmon, or winter flounder mucus. Susceptibility to L. salmonis infections may be related to the stimulation of LMW proteases from L. salmonis by fish mucus. The resistance of coho salmon to L. salmonis infection may be due to agents in their mucus that block the secretion of these LMW proteases or factors may exist in the mucus of susceptible species that stimulate their release.     

Identification, characterization and deduced amino acid sequence of the dominant protease from Kudoa paniformis and K. thyrsites: a unique cytoplasmic cysteine protease

Kudoa paniformis and Kudoa thyrsites (Myxozoa: Myxosporea) infections are associated with severe proteolysis of host muscle tissue post-mortem. The present study was undertaken to identify and characterize the protease responsible for myoliquefaction and determine mechanisms controlling protease function in vivo. N-terminal sequence analysis of partially purified protease from hake muscle infected with K. paniformis and K. thyrsites revealed a 23 amino acid sequence that aligned with cysteine proteases. Enzyme inhibition assays confirmed the presence of an essential active site cysteine residue. Using the above K. paniformis amino acid sequence data, a corresponding cDNA sequence from K. thyrsites plasmodia was elucidated revealing a cathepsin L proenzyme (Kth-CL). The translated amino acid sequence lacked a signal sequence characteristic of lysosomal and secreted proteins suggesting a unique cytoplasmic location. Only the proenzyme form of Kth-CL was present in Atlantic salmon muscle anti-mortem but this form became processed in vivo when infected muscle was stored at 4 degrees C. The proenzyme of Kth-CL showed uninhibited activity at pH 6.0, negligible activity at pH 6.5 and no measurable activity at pH 7.0 whilst the processed protease showed stability and function over a broad pH range (pH 4.5-8.8). The pH dependent processing and function of Kth-CL was consistent with histidine residues in the proregion playing a critical role in the regulation of Kth-CL.     

Infectious pancreatic necrosis virus infection in Atlantic salmon Salmo salar post-smolts affects the outcome of secondary infections with infectious salmon anaemia virus or Vibrio salmonicida.

Infectious pancreatic necrosis (IPN) virus (IPNV) infection in Atlantic salmon Salmo salar L. post-smolts and its influence on the outcome of secondary infections with infectious salmon anaemia (ISA) virus (ISAV) or Vibrio salmonicida were studied. The infections with ISAV or V salmonicida were performed both in a period of acute IPN and in the following IPNV carrier stage, 3 and 6 to 8 wk after experimental IPNV challenge, respectively. An IPNV carrier condition at low virus titre did not influence the mortality rates after secondary infections. Neither the ISAV infection nor the V. salmonicida infection in experimentally induced IPNV carriers resulted in mortalities different from those observed after challenge of IPNV-free fish. At higher IPNV titres in Atlantic salmon with acute IPN, the outcome of secondary infections was quite different from that observed in IPNV-free fish and in IPNV carriers. In 2 different experiments significantly more fish died when fish with acute IPN were infected with V salmonicida than when fish were infected with V salmonicida alone. Mortality also started earlier in the double-infected group than in the group challenged with V. salmonicida alone, 3 to 4 and 8 d after V salmonicida infection, respectively. Similar results were observed independent of whether mortalities due to IPN alone were registered in the experiments. When Atlantic salmon with acute IPN were infected with ISAV, significantly fewer fish died than when fish were infected with ISAV alone. The ongoing IPNV infection seemed to provide some protection against development of ISA.     

Virological, serological and histopathological evaluation of fish strain susceptibility to experimental infection with salmonid alphavirus

Pancreas disease (PD) of farmed Atlantic salmon Salmo salar L., which is caused by an alphavirus known as salmon pancreas disease virus (SPDV), can have serious economic consequences. An epidemiological survey carried out in Ireland in 2003 indicated that within individual farms there were significant differences in the susceptibility of different strains of farmed Atlantic salmon to infection with SPDV, as measured by levels of clinical disease and mortality. The aim of this preliminary study was to investigate this field observation by comparing lesion development, viraemia and serological responses of 3 commercial strains of Atlantic salmon (A, B and C) experimentally infected with SPDV. Highly significant differences in the severity of lesions in the pancreas at Day 21 post-infection (pi) were detected (p < 0.01), with Group B being more severely affected. There were also significant differences in the prevalence and severity of lesions in heart and skeletal muscle at Day 21 and 35 pi respectively, with Group B results again significantly higher than those from both Groups A and C (p < 0.05). There was no overlap between viraemia and the presence of specific SPDV antibody. Some fish in all groups had no viraemia, lesions or evidence of seroconversion. There were no significant differences seen between the challenged groups in relation to the percentage of viraemic fish at each time point. Viral loads were not determined. Differences between the number of antibody-positive fish in each challenge group were found at Days 28 and 35 pi (p < 0.1). Highly significant differences (p < 0.01) in the geometric mean titres of seropositive fish were detected at Day 28. These results, obtained using a challenge model, confirm that there are strain differences in the susceptibility to experimental SPDV infection in commercial farmed Atlantic salmon.     

Seasonal changes in hypoosmoregulatory ability in landlocked and anadromous populations of Arctic charr,Salvelinus alpinus,and Atlantic salmon,Salmo salar

Synopsis Seasonal changes in hypoosmoregulatory ability were compared in landlocked and anadromous strains of Arctic charr and Atlantic salmon. Seawater adaptability was assessed using periodic 48 h seawater challenge tests with 25. seawater. The landlocked strains of Arctic charr, two from northern Sweden and one from Southern Norway, displayed similar seasonal changes in seawater adaptability as the anadromous strain. Seawater tolerance increased during spring and remained high until the end of July — early August after which it declined. The two strains of Atlantic salmon displayed different seasonal patterns in hypoosmoregulatory ability. The anadromous strain showed a pronounced seasonal pattern with maximal seawater adaptability in early June. In contrast, seawater tolerance in the landlocked strain improved steadily during spring and remained high until late autumn. During the period of enhanced seawater tolerance, hypoosmoregulatory ability increased significantly with body size in both Arctic charr and anadromous Atlantic salmon. The minimum size at which fish were able to regulate plasma sodium following seawater transfer at a level comparable to freshwater levels (<170 mmol I^–1) differed significantly between anadromous Atlantic salmon (ca. 14 cm) and Arctic charr (ca. 22 cm). The results show that seasonal changes in hypoosmoregulatory ability are present in both Atlantic salmon and Arctic charr, and that these physiological traits are retained in the corresponding landlocked strains. However, the seasonal pattern of seawater adaptability as well as the minimum size at which seawater tolerance occurs differs between the two species.     

Major quantitative trait loci affect resistance to infectious pancreatic necrosis in Atlantic salmon (Salmo salar)

Infectious pancreatic necrosis (IPN) is a viral disease currently presenting a major problem in the production of Atlantic salmon (Salmon salar). IPN can cause significant mortality to salmon fry within freshwater hatcheries and to smolts following transfer to seawater, although challenged populations show clear genetic variation in resistance. To determine whether this genetic variation includes loci of major effect, a genomewide quantitative trait loci (QTL) scan was performed within 10 full-sib families that had received a natural seawater IPN challenge. To utilize the large difference between Atlantic salmon male and female recombination rates, a two-stage mapping strategy was employed. Initially, a sire-based QTL analysis was used to detect linkage groups with significant effects on IPN resistance, using two to three microsatellite markers per linkage group. A dam-based analysis with additional markers was then used to confirm and position any detected QTL. Two genomewide significant QTL and one suggestive QTL were detected in the genome scan. The most significant QTL was mapped to linkage group 21 and was significant at the genomewide level in both the sire and the dam-based analyses. The identified QTL can be applied in marker-assisted selection programs to improve the resistance of salmon to IPN and reduce disease-related mortality.