Journal of Fish Diseases

Published by Wiley
Online ISSN: 1365-2761
Print ISSN: 0140-7775
Publications
The long and narrow Hardanger fjord in western Norway has a high density of salmon farms and has had severe salmon lice, Lepeophtheirus salmonis, problems. In the years 2004-06, salmon lice numbers were recorded in selected salmon farms in the fjord as part of a larger research project. Most farm sites participated in a strategic control programme and were deloused between November and January in each year. The aim of the programme was to achieve a mean abundance of <0.3 adult female lice at this time and to minimize the infection pressure on wild smolts in the spring. Dedicated teams carried out detailed counting of lice on farmed fish in April-September each year. Temperature conditions were fairly similar throughout the fjord and amongst years, but wide variations in salinities were observed. The two innermost zones, B and C, had the lowest lice mean abundances, whereas the outermost zones, D and E, consistently had more lice. General linear model analyses showed that differences in adult female lice abundance between the zones were associated with differing levels of salinity and emamectin benzoate treatments strategically administered. Mean fish weight was significantly positively correlated with mean abundance of adult female lice.
 
between dose and time of administration of dietary b 1,3 glucan on innate immune factors in Clarias batrachus 
This study investigated the effects of short and prolonged administration of a yeast beta-glucan on non-specific immune parameters, growth rate and the disease resistance of Asian catfish, Clarias batrachus. Fish fed with a basal diet (control) and test diet (basal diet supplemented with 0.1% glucan) for 1, 2 and 3 weeks were assayed for superoxide production, serum myeloperoxidase (MPO) content, natural haemagglutinin level, complement and lysozyme activities. Fish were weighed at weekly intervals and specific growth rate (SGR, % increase in body weight per day) was determined. After each week, fish were challenged with Aeromonas hydrophila to measure the level of protection. Results showed that glucan administration at 0.1% in feed, significantly (P<0.05) enhanced MPO and lysozyme levels, superoxide production, haemagglutination titre and level of protection against A. hydrophila challenge, irrespective of length of exposure. The alternative complement activity and SGR were not affected by the dietary supplementation of yeast glucan. As glucan feeding at 0.1% for 1 week is able to enhance the non-specific immunity and disease resistance of catfish efficiently, short-term feeding might be used in farmed catfish diets to enhance disease resistance.
 
The effects of six 1,3;1,6-β-D-glucooligo- and polysaccharides with different structures (ranging from 1 to 10 kDa in molecular mass and containing 10-25% of β-1,6-linked glucose residues) from brown algae, Saccharina cichorioides, on development of the chum salmon, Oncorhynchus keta (Walbaum), were evaluated. Exposure of chum salmon eggs to 1,3;1,6-β-D-glucans with a molecular mass of more than 2 kDa increased the survival of embryos and juveniles and their resistance to Saprolegnia infection by up to 2.5-fold, leading to a weight gain in juveniles of 40-55% compared with The control chum salmons. The 1,3;1,6-β-D-glucans with molecular mass of 6-8 kDa and used at a at concentration of 0.5 mg mL(-1) rendered the best stimulative effect.
 
Genetic variability in 16S rRNA gene sequences has been demonstrated among isolates of Flavobacterium columnare, and a restriction fragment length polymorphism (RFLP) assay is available for genetic typing of this important fish pathogen. Interpretation of restriction patterns can be difficult due to the lack of a formal description of the expected number and sizes of DNA fragments generated for each of the described genomovars. In this study, partial 16S rRNA gene sequences (ca. 1250-bp fragment) from isolates representing each described genomovar and isolates generating unique restriction patterns were cloned and sequenced. The results demonstrated that some isolates contained up to three different 16S rRNA genes whose sequences generate different RFLP patterns due to intragenomic heterogeneity within HaeIII restriction sites. The occurrence of HaeIII restriction sites within the portion of the 16S rRNA gene used for typing the F. columnare isolates and intragenomic heterogeneity within these sites explained the restriction patterns observed following RFLP analyses. This research provides a standard protocol for typing isolates of F. columnare by RFLP and a formal description of the expected restriction patterns for the previously described genomovars I, II, II-B and III. Additionally, we describe a new genomovar, I/II.
 
Lactococcus garvieae is recognized as an emerging pathogen in fish. Several PCR-based methods have been developed for the detection and identification of L. garvieae; however, the sensitivity of these methods is still in question regarding the discrimination of this organism from other closely related species. Two primers, ITSLg30F and ITSLg319R, were designed from the sequence in the 16S-23S internal transcribed spacer (ITS) region and used for the specific detection of L. garvieae. L. garvieae strains including fish isolates were positive by this method. In contrast, previously developed PCR methods showed false-positive results with non-L. garvieae species. Our results indicate that a PCR method using the newly designed ITS primer set provides a sensitive and efficient tool for the detection of L. garvieae in fish and aquaculture environments.
 
Flavobacterium columnare is the causative agent of columnaris disease in diverse fish species worldwide. Although columnaris is an important disease, the antimicrobial susceptibility pattern of F. columnare is not well studied. Thus, the purpose of this study was to test the in vitro antimicrobial susceptibility of 97 F. columnare isolates collected worldwide between 1987 and 2011 from 17 fish species. The broth microdilution technique was utilized for reliable testing of these fastidious organisms. None of the isolates displayed acquired resistance to florfenicol, gentamicin, ormetoprim-sulfadimethoxine and trimethoprim-sulfamethoxazole. Acquired resistance to chloramphenicol was detected in 1%, to nitrofuran in 5%, to oxytetracycline in 11% and to enrofloxacin, flumequine and oxolinic acid in 10%, 16% and 16% of the isolates, respectively, as reflected by a bimodal or trimodal distribution of their minimum inhibitory concentrations (MICs). One isolate showed acquired resistance towards several antimicrobial agents including erythromycin. Another isolate revealed acquired resistance towards - amongst others - ampicillin. The isolates displaying acquired resistance originated from ornamental fish species or Vietnamese catfish, except for two isolates coming from wild channel catfish in which acquired resistance was encountered towards oxytetracycline only. Fifty per cent of the resistant isolates from ornamental fish were shown to have acquired resistance against three classes of antimicrobial agents, assigning these isolates as multiple resistant. These data might indicate less prudent use of antimicrobials especially in ornamental fish species.
 
The stone loach, Barbatula barbatula (L.), is a typical and dominant intermediate host of Raphidascaris acus in lowland streams of Central Europe. The prevalence of infection of R. acus in B. barbatula from the River Haná ranged throughout the year from 73.3 to 100%. The abundance and the mean intensity of infection also varied throughout the year with a peak in September. Larvae were located mainly in the liver parenchyma. High numbers of larvae and their migration through the tissue caused cyst- or abscess-like formations in the host parenchyma. The severity of the disease condition ranged from mild to severe. We speculate that the infection of stone loach by R. acus larvae regulates the population density and abundance of the intermediate host in lowland streams where natural predators are absent.
 
The aim of this study was to induce Lactococcus garvieae infection in young and adult fish through different routes [intraperitoneal (IP) and immersion (IM)] and to investigate the pathogenesis and histopathological and immunohistochemical findings comparatively. For this purpose, a total of 180 rainbow trout (90 young, 20 ± 5 g and 90 adult, 80 ± 10 g) obtained from a commercial fish farm were used. The fish were divided into eight groups, four experimental groups (Young-Adult IP groups and Young-Adult IM groups, each contain 30 fish) and four control groups (Young-Adult IP Control groups and Young-Adult IM control groups, each contain 15 fishes). The experimental study was conducted using L. garvieae, and confirmatory identification was performed by PCR. The sequence result of the PCR amplicon of 16S rDNA from isolate L. garvieae LAC1 was determined and deposited in the GenBank database under accession number KC883976. Fish in the IP groups were intraperitoneally administered an inoculate containing 10(6 ) cfu mL(-1) bacteria 0.1 mL. In the IM groups, fish were kept in inoculated water containing 10(8 ) cfu mL(-1) bacteria for 20 min. Mortality as well as clinical and pathological findings was recorded daily, and significant differences in macroscopic and microscopic results were observed between the IP and IM administration groups. All tissue samples were immunohistochemically stained by the avidin-biotin-peroxidase complex and immunofluorescence (IF) methods using polyclonal antibody to detect L. garvieae antigens. In immunoperoxidase staining in the IP groups, positive reactions to bacterial antigens were most commonly seen in the spleen, kidney, heart, liver, peritoneum and swim bladder. In the IM groups, bacterial antigens were most commonly found in the eye, gill, spleen and kidney. In the IF method, the distribution of antigens in tissue and organs was similar to the reactions with immunoperoxidase staining. Finally, in this experimental study, an important correlation was seen between the distribution of L. garvieae antigens and lesions developing in many organ and tissues.
 
In response to pathogens, the higher vertebrate innate immune system activates pro-inflammatory caspase-1 which is responsible for the processing and secretion of several important cytokines involved in the host's defence against infection. To date, caspase-1 has been described in few teleost fish, and its activity has been demonstrated through substrate cleavage and inhibition by pharmacological agents. In this study, the detection of the active form of caspase-1 during the immune response in salmonid fish is described, where two antibodies were produced. These antibodies differentially recognize the structural epitopes of the inactive pro-caspase-1 and the processed active form of the caspase. Firstly, caspase-1 activation was demonstrated in vitro by ELISA, Western blotting and immunocytochemistry in rainbow trout macrophages exposed to different pathogen-associated molecular patterns plus the pathogen Aeromonas hydrophila. This activity was clearly abrogated by a caspase inhibitor and seems to be unrelated to IL-1β secretion. Caspase-1 activation was then validated in vivo in gill cells from fish challenged with Aeromonas salmonicida. These results represent the first demonstration of caspase-1 activation in salmonids, and the first evidence of the putative regulatory role which this protease plays in inflammatory response in this fish group, as described for some other teleosts and mammals. © 2014 John Wiley & Sons Ltd.
 
Zebrafish were exposed to the wood extractive betulinol (5 microg L(-1)) and to 17beta-oestradiol (E2, 0.27 microg L(-1)) for 8 weeks in an attempt to study the possible endocrine-disrupting activity of betulinol. Females exposed to betulinol showed increased spawning intensity, while males exposed to betulinol and E2 had increased incidences of structural alterations in the testes. However, histological examination of the fish revealed that they were infected by acid-fast bacteria suspected to be Mycobacterium sp. despite a careful examination of their health state prior to the onset of the experiment. Fish exposed to betulinol and E2 showed more serious consequences of the bacterial infection than control fish indicating that the test chemicals had weakened the immune defence of the fish. When the exposure was repeated with healthy fish, an increase in the proportion of spermatogonia was seen in the testes of betulinol-treated males. A similar alteration, although not statistically significant, was also seen in the first experiment. However, no increase in the incidences of structural alterations in the testes was seen in betulinol- and E2-treated fish in the second experiment. Our study indicates that betulinol might have an endocrine-disrupting effect in zebrafish, but the increase in incidences of structural alterations in the testes might have been caused by a synergistic action between the test compounds and the bacterial infection. Our study stresses the importance of carefully checking the health of experimental fish, not only prior to the onset of an experiment but also upon termination of the experiment, in order to avoid misinterpretation of the results.
 
Fish are becoming an increasingly important research species as investigators seek alternatives to mammalian models. Combined positron emission tomography/computed tomography with (18) F-fluorodeoxyglucose (FDG-PET/CT) is a powerful new technology that has been extensively applied for high-resolution imaging in mammals but not fish. CT scanning provides detailed anatomical three-dimensional imaging. PET scanning detects areas of cellular activity using radio-labelled molecular probes with specific uptake rates appropriate to the tissue involved. FDG-PET is used in oncology because tissues with high glucose uptake, such as neoplasms, are intensely radio-labelled. PET/CT combines the two technologies, so that images acquired from both devices are merged into one superimposed image, thus more precisely correlating metabolic activity with anatomical three-dimensional imaging. Our objective was to determine if fish can be viable replacement animals in cancer studies using this technique by analysing the similarities between fish and humans in glucose uptake in select organs across multiple fish species. Rapid, quantifiable glucose uptake was demonstrated, particularly in brain, kidneys and liver in all imaged fish species. Standard uptake values for glucose uptake in the major organ systems of fish were more similar to those of humans than mice or dogs, indicating that fish may serve as effective alternative animal models using this technology. Applications for this technique in fish may include oncogenesis and metabolism studies as well as screening for environmental carcinogenesis.
 
The pathological changes induced by an infection of Diphyllobothrium dendriticum (Nitzsch, 1824) plerocercoids in powan, Coregonus lavaretus (L.), from Loch Lomond, Scotland, were assessed using immunohistochemical and ultrastructural techniques. In a sample of 26 powan, the occurrence of encysted plerocercoids of D. dendriticum on the outer surface of the stomach was 38.5% (n = 10) with the number of cysts ranging from 4 to 15 and measuring 4.2 +/- 1.0 mm x 3.4 +/- 0.9 mm (mean +/- SD). Histological examination of intestinal samples also revealed plerocercoids (2-21) encapsulated within a proliferation of mesenteric fibrous tissues of the gastric wall and, occasionally, by the gut lamina propria-submucosa and lamina muscularis. In section, cysts were tri-layered and were formed from a series of concentric whorls of fibroblast and collagen fibre-based connective elements. The extent of necrosis within each muscle layer and the serosa of the stomach differed, notably within the latter that was marked by a chronic inflammatory reaction and fibrosis. Within the cyst and around it, a large number of degranulating mast cell/eosinophilic granule cells were seen, in addition to melano-macrophage centres. Immunohistochemical staining of sections of infected stomach revealed a high density of elements, in close proximity to plerocercoids, staining positive for serotonin, bombesin, substance P and galanin. Uninfected material did not present the same levels of activity. Sections through both infected and uninfected tissue were also tested for elements containing vasoactive intestinal peptide, met-enkephalin, calcitonin gene-related peptide, neuropeptide Y and nitric oxide synthase, but these were absent.
 
Infections by the gill fluke Zeuxapta seriolae are a serious concern for sea cage aquaculture of kingfish, Seriola lalandi. The present study aimed to determine the pathophysiological effects of a progressive infection with Z. seriolae and the effects of treatment with hydrogen peroxide. For the progression of infection study, infected fish were taken from a sea cage farm, treated to remove parasites and then infected by cohabitation with heavily infected fish. Samples were taken at 2-week intervals for 8 weeks. Infection intensity peaked at 4 weeks post-infection (mean intensity 565.9) and the number of mature worms (2 mm fixed length or larger) peaked at 6 weeks post-infection. Attachment of Z. seriolae appeared to cause little localized pathology; however, the occurrence of hyperplastic lamellae increased as the infection progressed. Haemoglobin concentrations were negatively correlated with Z. seriolae intensity and were lower than controls at 4 weeks (35.8% decrease) and 6 weeks (57.4% decrease) post-infection. Blood lactate concentration and plasma osmolality increased throughout the course of infection. For the effect of treatment experiment, groups of infected and non-infected fish were sampled either before or after treatment with hydrogen peroxide. Treated fish from both infected and uninfected groups had increased plasma lactate, osmolality and pH compared with pre-treatment groups. Treatment with hydrogen peroxide appeared to have acute effects on fish health but the magnitude (e.g. lactate, osmolality) and extent of the effects (e.g. haemoglobin) was much less than that caused by chronic infection with Z. seriolae.
 
The effects of hydrogen peroxide administered at a concentration of 1500 ppm for 20 min at 7.5 °C on different life-cycle stages of Lepeophtheirus salmonis were examined experimentally. The mobile adult and pre-adult stages of L. salmonis readily reattached to Atlantic salmon after hydrogen peroxide treatment. Adult female lice, but not adult males or pre-adults, reattached in significantly lower numbers than untreated controls. Survival of early chalimus stages (I and II) was not affected by hydrogen peroxide treatment, but their subsequent development to chalimus III and IV was delayed compared with untreated controls. Nauplii and copepodid larvae of L. salmonis were almost all dead by 1 hour post-treatment. A very few copepodids survived 24 h post-treatment. Egg strings of L. salmonis at an early stage of development failed to hatch after hydrogen peroxide treatment, but those with pigmented eggs did hatch, although in significantly reduced numbers. Treated egg strings did produce viable copepodids, although in significantly reduced numbers compared with controls.
 
A sensitive and specific immunohistochemical technique was developed to improve the diagnosis of tenacibaculosis and to better understand its pathogenesis. Senegalese sole Solea senegalensis Kaup, 1858 were inoculated subcutaneously with a bacterial suspension of Tenacibaculum maritimum, and samples were taken at different hours post-inoculation. Sections from different organs were used as positive controls. In addition, a total of 128 field samples from different organs collected from tenacibaculosis outbreaks were used. Tenacibaculum maritimum antigens were detected in several organs of experimentally infected Senegalese sole and in at least one of the tissues from fish suffering from natural tenacibaculosis previously confirmed by culture and PCR-based methods. In fish collected during outbreaks, a strong positive reaction was detected in ulcerative skin areas. Moreover, bacterial antigen was identified inside scale pockets and in sites of the skin with mild lesion. In kidney and spleen, evident immunostaining of bacterial antigen was detected in both naturally and experimentally infected fish. Besides, the presence of T. maritimum in the intestinal tract without associated histological changes suggests that this organ may act as a reservoir for T. maritimum. The results of this study confirm the usefulness of IHC for the diagnosis of tenacibaculosis in paraffin-embedded tissues.
 
Confirmation of positive clones by RE digestion. Lane 1: 100-bp marker; lane 2: plasmid (extra small virus antisense (XSVAS) before RE digestion); lane 3: plasmid (XSVAS after RE digestion).
Different treatments in experimental design
Physicochemical characteristics of chitosan and chitosan-conjugated DNA
Survival rate for different treatments.
The protective efficacy of a DNA construct containing extra small virus antisense (XSVAS) gene of nodavirus encapsulated with chitosan nanoparticles (NPs) was investigated in giant freshwater prawn Macrobrachium rosenbergii (De Man, 1879). The delivery was carried out using oral and immersion methods. A plasmid concentration of 100 ng μL(-1) when conjugated with chitosan NPs was found to be more effective in increasing the survivability of the infected prawn. The particle mean size, zeta potential and loading efficiency percentage were 297 nm, 27 mV and 85%, respectively. The ability of the chitosan to form a complex with the plasmid was studied by agarose gel electrophoresis. The NPs were characterized by atomic force microscopy (AFM). Persistence study showed the presence of the DNA construct up to 30th day post-treatment. The oral treatment was found to be better than the immersion treatment for delivery of the chitosan-conjugated DNA construct. This is probably the first report on the delivery of nanoconjugated DNA construct in M. rosenbergii, against nodavirus.
 
Microscopical observation of the ciliate parasite found in the eye's orbital cavity of a moribund H. hippocampus. Scale bar: 50 μm.
Maximum likelihood tree showing the phylogenetic position of the ciliates found in the eye of cultured seahorses (in bold) compared against scuticociliate SSU rRNA sequences available in GenBank. Bootstrap probabilities higher than 60 after 1000 replications are shown.
Even though some of the 33 recognized species are reared successfully, a sharp decrease on the survival rate occurred during early development stages of the cultured seahorses Hippocampus hippocampus. Moribund seahorse juveniles showing symptoms of lethargy, anorexia, rubbing, dark body pigmentation, lateral and water surface swimming were observed. During culturing of H. hippocampus mass mortality events were recorded. A ciliate parasite infestation was detected in the ascitic fluid and orbital cavities of seahorses. By amplification of the genomic small-subunit 18S rDNA the ciliate sequences showed a 99% of similarity with the 18S rRNA of Porpostoma notata (HM236335). The systematic position of the ciliate was determined among thirty-eight scuticociliatia sequences available in GenBank and integrated in the phylogenetic analysis. The histophagous parasite Porpostoma notata has been never described before on seahorses and only isolated from seawater samples and dead cultured crabs. Further studies would be necessary to determine if P. notata is a primary causative agent of mortality in seahorses and to define its pathogenicity. Nucleotide sequence data reported in this study are available in the EMBL database under the accession number HG326610.
 
Map of southern Norway showing the localization of farms A–D along the West coast.
Summary of epidemiological characteristics for Atlantic salmon seawater farms with outbreaks of amoebic gill disease (AGD)
Gill pathology of Atlantic salmon with amoebic gill disease. (a) Patchy white changes along filaments (arrow) and excessive mucus (arrowhead). Photo by L.B. Rønneberg. (b–d) H&E stained histological sections. (b) Filaments with segmental changes: adhesions between lamellae, especially apically, and epithelial hyperplasia. There is a distinct transition (arrowhead) to a segment with no apparent changes. Many amoebae are between filaments and lamellae (bar = 200 μm). (c) Extensive adhesions and hyperplasia and amoebae (arrow) within an enclosed interlammellar space (bar = 25 μm). (d) Enclosed interlamellar space with amoeba (arrow) and hypertrophied superficial epithelial cells (arrowhead; bar = 10 μm).
Amoebae at gill surface of Atlantic salmon with amoebic gill disease. H&E stained histological sections in different planes through amoebae showing nuclei (arrow) and parasomes (arrowhead). Bar = 10 μm in all micrographs. (a) Thicker section showing nucleus and parasome with basophilic material. (b) Parasomes appeared as basophilic opposed crescent-shaped structures. Nucleus is partly out of focus. (c) Details of nucleus with an amphiphilic core surrounded by an irregular basophilic ring and an outermost amphiphilic zone.
Cladogram showing the 18S rRNA gene inferred phylogeny of Neoparamoeba spp. and position of the Norwegian strains of N. perurans with related marine amoebae Vexilifera armata, Korotnevella hemistylolepis and Vannella aberdonia as the outgroup. All sequences are presented with their GenBank database accession numbers. The topology was inferred using maximum likelihood analysis incorporating the GTR + G + I model and values at nodes represent bootstrap support by fast stepwise-addition analysis with 100 replicates and resampling of all characters using the PAUP software. Main nodes where the bootstrapping failed to provide good support are marked with an asterisk. Details beyond species level are not shown as bootstrapping generally did not support such high resolution. All branches are to scale and the bar represents 0.01 substitutions per site. 146 × 204 mm (600 × 600 DPI).
Amoebic gill disease (AGD) was observed in seawater farmed Atlantic salmon at four geographically distant locations on the western coast of Norway. To the best of our knowledge, these are the first detected AGD outbreaks in Norway. The outbreaks lasted for 7-12 weeks in late autumn 2006 and were for the most part concurrent. The crude, cumulative mortality was in the range of 12-20% at three farms and 82% at a fourth. The histopathology showed uniform parasomal amoebae in lesions characteristic for AGD. Another gill disease, proliferative gill inflammation (PGI), was also present to a variable degree and the distinction between the two gill problems is discussed. Seawater temperatures were 3.5 degrees C higher than average before disease outbreaks, which subsided in early winter. The geographical and time pattern of these outbreaks strongly indicates simultaneous infection from the marine environment. Two contiguous 18S cDNA sequences, obtained by reverse transcriptase PCR from gill tissue with AGD-related lesions, showed highest similarity (99.2%) to a newly recognized species designated Neoparamoeba perurans and maximum likelihood analysis demonstrates that they represent Norwegian strains of this Neoparamoeba lineage.
 
The life cycle of the histozoic myxozoan parasite Henneguya nuesslini was investigated in two salmonid host species. Naive brown trout, Salmo trutta, and brook trout, Salvelinus fontinalis, were experimentally infected in two trials by triactinomyxon type actinospores from naturally infected Tubifex tubifex. In exposed common carp, Cyprinus carpio, no myxospore production was detected. The parasite formed cysts with mature myxospores in the connective tissue of the fish 102 days post-exposure. The morphology of both actinosporean and myxosporean stages was described by light microscopy and a 1417-bp fragment of the 18S rDNA gene was sequenced. Sequence analysis confirmed the absolute congruence of the two developmental stages and assisted in determining species identity. Host range, tissue specificity and myxospore measurements provided sufficiently distinctive features to confirm species validity and were thus crucial for identification. The triactinomyxon spores had 16 secondary germ cells, unique dimensions, a very opaque sporoplasm matrix and three conspicuously protruding, pyriform polar capsules. This is the first record of a Henneguya sp. life cycle with a triactinomyxon-type actinospore, which suggests a close relationship with the Myxobolus group and a polyphyletic origin of the genus Henneguya.
 
Elongate plasmodia with myxosporean spores belonging to the genus Unicapsula, Davis, 1924 were found in the skeletal muscle of the striped seabream, Lithognathus mormyrus (L.), a candidate for the mediterranean aquaculture. The only species of Unicapsula described from the Mediterranean is Unicapsula pflugfelderi Schubert et al. 1975, which occurs in the picarel, Spicara smaris (L.). For morphological and molecular comparison of U. pflugfelderi from S. smaris with Unicapsula sp. from L. mormyrus measurements of plasmodia and spores, ultrastructural details and 18S and 28S rDNA sequences were analysed. Whereas plasmodia were 2-3 times larger in S. smaris than in L. mormyrus (length 2.47-0.81 mm; width 0.22-0.09 mm; P = 0.000), spore morphology showed minor differences and both 18S and 28S rDNA sequences were 100% identical identifying the myxozoan as U. pflugfelderi. Scanning electron microscopy of the spores revealed a different shell valve distribution than the one used for the diagnosis of the genus Unicapsula. This resulted in a review of the genus Unicapsula dividing it into two morphological groups of different spore valve arrangement. TEM revealed the presence of a yet undescribed crystalline structure in the sporoplasm of the spores.
 
Atlantic salmon, Salmo salar L. parr (age 1+), infected by the monogenean ectoparasite Gyrodactylus salaris (Malmberg, 1957), were exposed to chlorine (Cl)-enriched water at three different concentrations: Cllow (0-5 μg Cl L(-1) ), Clmedium (18 μg Cl L(-1) ) and Clhigh (50 μg Cl L(-1) ). There was a negative correlation between G. salaris infections and the hypochlorite concentrations added. The parasite infection was eliminated by day 6-8 and day 2-4 in the groups Clmedium and Clhigh , respectively, while inhibition of G. salaris population growth was observed in the Cllow group. An important note to this matter, however, is that the G. salaris specimens observed at day 6 in Clmedium and at day 2 in Clhigh were all considered dead by subjective judgement. No mortality in the salmon parr was observed during the first 8 days of the experiment, demonstrating that Cl has a stronger effect on G. salaris than on the salmonid host. The differences in sensitivity between the parasite and the Atlantic salmon indicate that hypochlorite has a potential use as a parasiticide with a therapeutic margin. The low-dose sensitivity may imply that Cl pollution in urban areas may pose a greater risk towards biodiversity than previously assumed.
 
Amoebic gill disease (AGD) has been attributed to infection by Neoparamoeba sp. The causal mechanisms for AGD lesion development and the primary pathogenic role of Neoparamoeba sp. require elucidation. Three groups of Atlantic salmon were exposed to viable gill isolated amoebae, to sonicated amoebae, or to sea water containing viable amoebae without direct contact with gill epithelia. Fish were removed 8 days post-exposure and the gills assessed histologically for AGD. AGD occurred only when fish were exposed to viable trophozoites. Consequently, in an accompanying experiment, infection was evaluated histologically at 12, 24 and 48 h post-exposure in three groups of salmon, one group being mechanically injured 12 h prior to exposure. A progressive host response and significant increase (P < 0.001) in the numbers of attached amoebae was apparent over the 48-h duration in undamaged hemibranchs in both treatment groups. There were no significant differences to mucous cell populations. Attachment of Neoparamoeba sp. to damaged gill filaments was significantly reduced (P < 0.05) by 48 h post-exposure. These data further confirm and describe the primary pathogenic role of Neoparamoeba sp. and the early host response in AGD. Preliminary evidence suggests that lesions resulting from physical gill damage are not preferentially colonized by Neoparamoeba sp.
 
A total of 18 Neoparamoeba strains were characterized both morphologically and using the SSU rRNA gene sequences as molecular markers. Nine were isolated from gills of farmed Atlantic salmon, Salmo salar L., six from sediments sampled in areas of sea-cage farms and three from net material of sea-cages. The newly obtained sequences extended substantially the dataset of Neoparamoeba strains available for phylogenetic analyses, which were used to infer taxonomic relatedness among 32 strains morphologically assigned to this genus. In addition to the N. pemaquidensis and N. aestuarina clades, phylogenetic analyses clearly distinguished a third clade with sequences from six strains. Members of this clade are characterized as representatives of a new species, N. branchiphila n. sp. The diagnostic primers for the identification of this species are introduced.
 
Prevalence of infectious pancreatic necrosis virus (IPNV) in Atlantic salmon broodstock sites in Scotland from 1990 to 2002. The black line shows the estimate of the mean level of IPNV with 95% pointwise confidence intervals shown as grey shading. Figure 3 Predicted effect of sex on the prevalence of infectious pancreatic necrosis virus (IPNV) in Atlantic salmon broodstock sites in Scotland in 1996. For interpretability, the effects are shown for IPNV prevalence in 1996. The vertical bars give approximate 95% confidence intervals for the levels of the predicted effect. A Wald test of significance for sex gave P = 0.24.
Predicted effect of age on the prevalence of infectious pancreatic necrosis virus (IPNV) in Atlantic salmon broodstock sites in Scotland in 1996. For interpretability, the effects are shown for IPNV prevalence in 1996. The vertical bars give approximate 95% confidence intervals for the levels of the predicted effect. A Wald test of significance for age gave P = 0.75.
Predicted effect of geographical area on the prevalence of infectious pancreatic necrosis virus (IPNV) in Atlantic salmon broodstock sites in Scotland in 1996. For interpretability, the effects are shown for IPNV prevalence in 1996. The vertical bars give approximate 95% confidence intervals for the levels of the predicted effect. A Wald test of significance for geographical area gave P = 0.62.
Throughout this study period the prevalence of infectious pancreatic necrosis virus (IPNV) in Scottish farmed Atlantic salmon was high in the marine environment but relatively low in fresh water. In order to minimize the risk of vertical transmission of infection from parent to progeny, all IPNV infected broodstock populations had to undergo testing of all fish for the virus at the time of stripping and eggs from positive parents were destroyed. Between 1990 and 2002 over 68 000 Atlantic salmon broodfish were individually screened for IPNV by cell culture isolation and enzyme linked immunosorbent assay. Generalized linear mixed models were used to assess the influence of geographical region, age, sex and year on IPNV prevalence in Atlantic salmon broodstock. This analysis determined that the age and sex of the broodfish and the geographical region of the broodstock stripping site did not have a statistically significant influence on IPNV prevalence within the broodstock parental population. However, there was a statistically significant temporal increase in IPNV prevalence from 1990 to 2002.
 
Clone D of allogynogenetic gibel carp heavily infected with Myxobolus wulii in the hepatopancreas. (a) External appearance of infected fish showing swollen abdomen (bar = 2 cm). (b) Internal appearance of infected fish showing enlarged hepatopancreas, occupying most of the abdominal cavity and other internal organs (bar = 3 cm). (c) Liquefaction of hepatopancreas tissue immediately after death of host fish (bar = 3 cm).
Morphological comparison between Myxobolus wulii and its related species
Histological sections of the hepatopancreas of allogynogenetic gibel carp infected with Myxobolus wulii. Note the honeycomb-like structure of plasmodia (bar = 50 μm).
Goldfish infected with Myxobolus wulli in the hepatopancreas (a and b) and gills (c and d). Note swollen hepatopancreas in the abdomen (a), a bunch of grape-like plasmodia (b), and gills covered by numerous cysts (c and d) (bars for b and d are 50 μm and 3 mm, respectively).
Schematic drawings of a Myxobolus wulii spore. (a) Frontal and (b) sutural view (bar = 5 μm).
Myxobolus wulii (=Myxosoma magna) was first described from the gills of goldfish, Carassius auratus auratus, in China. Subsequently, a myxosporean infecting the hepatopancreas of allogynogenetic gibel carp, C. auratus gibelio, was designated as a different species, Myxobolus guanqiaoensis, although the morphological features were almost identical to those of M. wulii. In Japan, an unidentified Myxobolus sp. was found in the gills and hepatopancreas of goldfish. Morphological and molecular analyses in the present study identified these myxosporeans as M. wulii, which was thus shown to use different habitats in the host fish. Phylogenetic analyses of small subunit ribosomal RNA gene sequences showed that M. wulii is closely related to two gill-infecting Myxobolus species, M. ampullicapsulatus and M. longisporus. Fish infected with M. wulii in the hepatopancreas exhibit swollen abdomens and chronic mortality. Hepatopancreas tissues are virtually destroyed and replaced with plasmodia of M. wulii. A remarkable difference in susceptibility to M. wulii between two clones of allogynogenetic gibel carp was observed, suggesting that resistance to the myxosporean infection was established in a clone of fish bred by allogynogenesis.
 
Propagating epizootics due to Pilchard herpesvirus (PHV) occurred in the Australian population of pilchard, Sardinops sagax neopilchardus (Steindachner) (Clupeidae), in 1995 and 1998-99, with up to 60% losses. No mortality events have been evident in the ensuing 7 years, one reason for which could be that PHV is now endemic. During 2004, a survey was conducted to establish if PHV was present in pilchards in Australia. The pilchard is a highly active, pelagic schooling fish which is found in subpopulations, creating difficulties for the conduct of surveys. It occurs in Australian coastal waters and embayments below about 25 degrees S latitude, feeds on plankton and is predated by birds, mammals and larger fish. It reaches sexual maturity at 2 years of age, spawns at sea, enters embayments when about 5 months old and returns to sea when about 1 year old. It may live for 6-9 years, reaching a maximum length of 200 mm. It forms schools and may travel up to 30 km per day. Pilchards aggregate in mobile shoals of fish containing large highly mobile schools, which interact randomly and exchange individuals. Four subpopulations were defined for the purposes of this survey based on differences in biological characteristics: south-eastern Queensland/northern New South Wales (NSW), Victoria/South Australia (SA), south coast Western Australia (SWA) and west coast Western Australia (WWA). Specimens were obtained from the catch of commercial fishermen using random sampling where possible. Polymerase chain reaction (PCR) for the detection of PHV was performed after appraising the suitability of all available tests according to their impact on sample size requirements, total survey costs and logistical constraints. In the analysis, estimates of true prevalence (TP) of infection and 95% confidence limits were adjusted from the apparent prevalence estimates provided by PCR results. Percentage TP of PHV and corresponding 95% confidence intervals for the four subpopulations: NSW, SA, SWA and WWA were thus estimated as 0 (0-1.5), 31 (22-43), 42 (31-55) and 29 (20-41), respectively. PHV is now endemic in Australian populations of pilchard. Implications of the findings for fisheries management are discussed.
 
Analyses of a unique database containing sea lice records over an 11 year period provide evidence of changing infestation patterns in Scotland. The data, collected from more than 50 commercial Atlantic salmon farms, indicate that both species of sea lice commonly found in Scotland, Lepeophtheirus salmonis and Caligus elongatus, have declined on farms over the past decade. Reductions for both species have been particularly marked since 2001 when more effective veterinary medicines became available. Treatment data were also available in the database and these show a growing trend towards the use of the in-feed medication emamectin benzoate (Slice), particularly in the first year of the salmon production cycle. However, this trend towards single product use has not been sustained in 2006, the latest year for which data are available. There is some evidence of region to region variation within Scotland with the Western Isles experiencing higher levels of infestation. However, compared to the levels observed between 1996 and 2000, all regions have benefited from reduced lice infestation, with the overall pattern showing a particular reduction in the second and third quarters of the second year of production.
 
The impact of salmon lice on the survival of migrating Atlantic salmon smolts was studied by comparing the adult returns of sea-ranched smolts treated for sea lice using emamectin benzoate or substance EX with untreated control groups in the River Dale in western Norway. A total of 143 500 smolts were released in 35 release groups in freshwater from 1997 to 2009 and in the fjord system from 2007 to 2009. The adult recaptures declined gradually with release year and reached minimum levels in 2007. This development corresponded with poor marine growth and increased age at maturity of ranched salmon and in three monitored salmon populations and indicated unfavourable conditions in the Norwegian Sea. The recapture rate of treated smolts was significantly higher than the controls in three of the releases performed: the only release in 1997, one of three in 2002 and the only group released in sea water in 2007. The effect of treating the smolts against salmon lice was smaller than the variability in return rates between release groups, and much smaller that variability between release years, but its overall contribution was still significant (P < 0.05) and equivalent to an odds ratio of the probability of being recaptured of 1.17 in favour of the treated smolts. Control fish also tended to be smaller as grilse (P = 0.057), possibly due to a sublethal effect of salmon lice.
 
A cross-sectional survey of Renibacterium salmoninarum infection in farmed rainbow trout (RBT) and wild fish populations was carried out in 10 farms and six river catchments, respectively, in England and Wales. The majority of the wild fish were sampled in 1998 and the farmed fish in 2000. Grayling, Thymallus thymallus, and brown trout, Salmo trutta, were the main wild species sampled. Two fish, one grayling and one salmon, Salmo salar, were R. salmoninarum culture-positive, compared with 40 confirmed polymerase chain reaction-positive wild fish. The highest prevalence of R. salmoninarum infection was found in grayling in rivers with RBT farms with a history of R. salmoninarum infection. One hundred and fifty fish were sampled from each RBT farm, but none of the fish was found to be R. salmoninarum-positive. Evidence was found, for the first time, for the presence of R. salmoninarum in an eel, Anguilla anguilla.
 
Vibriosis caused by the pathogenic bacterium Vibrio (Listonella) anguillarum leads to serious losses in European sea bass, Dicentrarchus labrax. Because of its pleiotropic activity in controlling immune and inflammatory responses against various pathogens, interleukin-1beta (IL-1beta) is an attractive candidate for resistance to bacterial vibriosis. Four polymorphisms c.76 + 52C>T, c.76 + 157A>G, c.76 + 215A>and c76 + 310A>G of IL1B were genotyped in progeny of four families of wild sea bass captured in geographically distinct regions of the Black Sea and Sea of Azov and challenged with V. anguillarum. In the transmission disequilibrium test, the TGGG haplotype of IL1B showed significant overtransmission from parents to surviving progeny, thereby suggesting an association with higher resistance to V. anguillarum infection (Odds Ratio 0.38, P < 10(-7)). Using a luciferase reporter assay, we found a 1.4-fold increase in transcription activity of the protective IL1B TGGG variant compared to the susceptible CAAA variant of IL1B. The higher transcriptional activity of IL1B TGGG may arise from the functional effects of c.76 + 157A>G and c.76 + 215A>G polymorphisms disrupting potential binding sites for glucocorticoid receptor and YY1, both are negative transcription regulators.
 
Location of research stations at the East Frisian Coast (German Bight-North Sea).
Mortality rates (%) of C. gigas at sampling stations at the East Frisian Coast during summer 2006
Abundance of C.gigas at various stations in Norderney, Norddeich and at the Lütetsburger Plate. Living oysters are represented in black, moribund in black-white, and dead in white columns.
Abundance of C.gigas in various size classes (2-18). Living oysters are represented in black, moribund in black-white, and dead in white columns.
Mortality rate of C.gigas in various size classes (2-17). Living oysters are represented in black, moribund in black-white, and dead in white columns.
In 2005, Pacific oysters, Crassostrea gigas, were collected from May to September along the East Frisian coast and processed for histology. Because of mass mortalities in September, additional samples of moribund oysters and apparently healthy blue mussels, Mytilus edulis, were subjected to virological and ultrastructural investigation. The oysters displayed a variety of pathological conditions including viral gametocytic hypertrophy which is reported here for the first time from the German coast. Haemocyte aggregations in the digestive tract, in the intestinal mucosa and submucosa, in the mid-gut gland and in the ventricle of the heart were commonly observed at some stations. In association with mass mortalities, severe gill necrosis occurred which may have contributed to the high mortality rates. Total mortality rates of up to approximately 60% were seen. All size classes and thus age classes of oysters were affected, with highest mortality rates within the youngest age classes which had just reached sexual maturity (shell lengths <40 mm). The smallest dead oysters had shell lengths of 10 mm. The phenomenon was mainly restricted to C. gigas stocks in harbours, probably because of favourable conditions for infection, i.e. limited water exchange, less food availability, reduced oxygen content and higher pollution levels.
 
Top-cited authors
Brian Austin
  • University of Stirling
Randolph H Richards
  • University of Stirling
R. J. Roberts
  • University of Stirling
Thomas Wahli
  • Universität Bern
Heike Schmidt-Posthaus
  • Institute for Fish and Wildlife Health