Temporary protection of rainbow trout gill epithelial cells from infection with viral haemorrhagic septicaemia virus IVb.

The branchial epithelium is not only a primary route of entry for viral pathogens, but is also a site of viral replication and subsequent shedding may also occur from the gill epithelium. This study investigated the potential of agents known to stimulate innate immunity to protect rainbow trout epithelial cells (RTgill-W1) from infection with VHSV IVb. RTgill-W1 cells were pretreated with poly I:C, FuGENE(®) HD + poly I:C, lipopolysaccharide (LPS), LPS + poly I:C or heat-killed VHSV IVb and then infected with VHSV IVb 4 days later. Cytopathic effect (CPE) was determined at 2, 3, 4, 7 and 11 days post-infection. Virus in cells and supernatant was detected using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). All of the treatments delayed the onset of CPE (per cent of monolayer destruction), compared with untreated controls; however, killed VHSV or poly I:C combined with LPS was the most effective. Similarly, the detection of viral RNA in the supernatant was delayed, and the quantity was significantly (P < 0.05) reduced by all treatments with the exception of LPS alone (4 days). Unlike many of the other treatments, pretreatment of RTgill-W1 with heat-killed VHSV did not upregulate interferon 1, 2 or MX 1 gene expression.

Pathogenesis of spring viremia of carp virus in emerald shiner Notropis atherinoides Rafinesque, fathead minnow Pimephales promelas Rafinesque and white sucker Catostomus commersonii (Lacepede).

Spring viremia of carp (SVC) is a reportable disease to the World Organization of Animal Health (OIE) as it is known to cause significant international economic impact. In Canada, the first and only isolation of SVC virus (SVCV) was in 2006, from common carp Cyprinus carpio L., at Hamilton Harbour, Lake Ontario. The susceptibility of fathead minnow Pimephales promelas Rafinesque, emerald shiner Notropis atherinoides Rafinesque and white sucker Catostomus commersonii (Lacepede) to intraperitoneal injection of the Canadian isolate (HHOcarp06) was evaluated using experimental infection, virus isolation, quantitative reverse transcription polymerase chain reaction (qRT-PCR), histopathology and immunohistochemistry (IHC). Emerald shiner and fathead minnow were most susceptible with 43 and 53% cumulative mortality, respectively, compared with koi at 33%. Quantitative RT-PCR demonstrated that koi had high viral loads throughout the experiment. At 34 days post-infection, SVCV was detected from sampled emerald shiner and white sucker in very low titre and was not detected from fathead minnow. Koi, fathead minnow and emerald shiner had gross lesions typical of SVC disease. The histopathological picture was mostly dominated by necrotic changes in kidney, spleen, liver, pancreas and intestine. IHC further confirmed SVCV infection, and staining was largely correlated with histological lesions.

Walleye Sander vitreus (Mitchill) are relatively resistant to experimental infection with VHSV IVb and extant walleye strains vary in susceptibility.

Compared to fathead minnow, walleye demonstrate low susceptibility to experimental infection with VHSV IVb, regardless of route of exposure or water temperature at time of infection. In triplicate and duplicate groups, walleye were intraperitoneally (i.p.) injected (102 -108  pfu/fish) or waterborne-exposed (w; 1.4 × 107  pfu mL-1 ) with VHSV IVb. High cumulative mortality (64-100%) and severe gross lesions associated with VHSV IVb infection were evident only in fish i.p. injected with 108  pfu at 12 °C. These fish had multifocal necrosis of several tissues including the gill and heart. There was no difference in mortality between walleye infected (w or i.p.) at 12 °C (spring stocking) compared with a declining temperature profile from 18 to 12 °C (fall stocking). There were significant differences (P < 0.05) in mortality between four extant walleye strains following i.p. infection, indicating that the choice of walleye strain for stocking might be an important consideration. Viral antigen was found in both i.p. and w-exposed walleye using immunohistochemistry, mostly within the gill and skin of w-exposed fish and most prominently in dermal fibrocytes. VHSV IVb was detected in multiple tissues from 6 to 21 days post-infection using reverse transcriptase quantitative polymerase chain reaction (RT-qPCR).

Amyloid associated with neoplasia in two captive tricolour sharkminnows Balantiocheilus melanopterus Bleeker.

Amyloid associated with pancreatic adenocarcinoma was discovered in two captive adult tricolour sharkminnows Balantiocheilus melanopterus Bleeker found dead in a freshwater display. Enlarged abdomens expanded by bloody ascitic fluid and grossly visible masses of abnormal tissue were present surrounding sections of the stomach and intestine. Histologically, the masses were composed of areas of well-organized exocrine pancreatic acini interspersed with cords of poorly differentiated, spindle-shaped cells that compressed and effaced normal parenchyma. These cells possessed small numbers of cytoplasmic zymogen granules; the exocrine nature of these cells was confirmed using transmission electron microscopy (TEM). Fibrovascular connective tissue of the hepatopancreas and mesenteries was expanded by lightly eosinophilic, hyaline, homogeneous acellular material. Similar material greatly expanded the tunica media of large blood vessels in the hepatopancreas. After staining with Congo red or thioflavin T, this material exhibited red-green dichroism under polarized light or bright green fluorescence under ultraviolet light (255 nm), respectively. The non-branching fibrils, of indeterminate length, had an approximate diameter of 10-20 nm using TEM. Although exocrine pancreatic neoplasia is relatively common in fish, the presence of amyloid is not. To our current knowledge, the latter has not yet been described in association with a neoplastic lesion in fish.

Gastric dilation and air sacculitis in Chinook salmon, Oncorhynchus tshawytscha (Walbaum); correlation of macroscopic and microscopic lesions, and relationship of the syndrome to glomerulonephritis and serum biochemistry.

Macroscopic and microscopic assessment procedures were developed to evaluate the severity and enable diagnosis from histological samples, of gastric dilation and air sacculitis (GDAS) in Chinook salmon, Oncorhynchus tshawytscha. Stomachs and swim bladders were examined from young fish with experimentally induced GDAS and from larger fish with the syndrome held in commercial saltwater net-pens. Fish fed a diet previously known to induce GDAS had significantly wider stomachs with decreased prominence of longitudinal stomach folds that contained larger amounts of fluid (P < 0.001), and thinner stomach walls with greater inter-nerve distances (P < 0.001), than fish fed an alternative commercial diet not associated with the syndrome. These fish also had swim bladders that were more likely to be opaque and contain more fluid (P < 0.001). These observations correlated well with selected criteria for stomach tissue (P < 0.002) and swim bladder (P < 0.04) that could be evaluated microscopically. Four stomach measurements, primarily measurements of wall or partial wall thickness and inter-nerve distances, were suitable for discriminating between affected and non-affected fish. A stomach width ratio, that was independent of fish weight and highly correlated with macroscopic stomach measurements (P < 0.0001), was particularly useful; this ratio was derived from the distances between both the outer border of the muscularis mucosa and mesothelium of the serosal surface to the stratum compactum. Serum biochemistry parameters (osmolality, calcium and magnesium) did not differ between fish fed different diets, but serum creatinine concentration was correlated with the microscopic thickness of the muscularis externa of the stomach wall and the total stomach thickness (P < 0.001 and P < 0.003, respectively). A glomerulonephritis was also noted in these fish. The severity of the lesion was not significantly related to GDAS nor to any serum biochemistry parameter assayed however.

Nodular gill disease causing proliferative branchitis and mortality in Chinook salmon (Oncorhynchus tshawytscha).

CASE HISTORY: A period of escalating mortality occurred among Chinook salmon (1-5 g) held in freshwater raceways at a commercial culture facility. The mortality rate peaked at 1.5% of the population per day, water temperature was 9-10 degrees C, and water quality had recently deteriorated due to an influx of suspended solids. The affected fish did not respond to several chloramine-T bath treatments (10 ppm for 1 hour). CLINICAL FINDINGS: The dead fish did not exhibit flared opercula typical of infection with bacterial gill disease (BGD) nor did the remaining fish show obvious signs of respiratory distress. Clinical lesions were limited to the gills of the fish, and were consistent with a proliferative branchitis, including substantial hyperplasia and fusion of lamellae. DIAGNOSIS: Numerous angular amoebic organisms (approximately 10 x 8 microm), with a central, large vesicular nucleus and faintly acidophilic cytoplasm, visible on H&E-stained sections, were located on the hyperplastic epithelia of the lamellae, filaments, and remaining lining of the branchial cavity. Morphologically, the amoebae were similar to representatives of the genus Cochliopodia spp. described from North American salmonids, that presented similar lesions and clinical signs; consequently, the disease was diagnosed as nodular gill disease (NGD). CLINICAL RELEVANCE: Grossly, NGD can be mistaken for BGD; the distinction is important because chloramine-T, routinely used in the treatment of BGD, is ineffective for the treatment of NGD. Static formalin baths of 170 ppm (37% formaldehyde) for 1 hour, supplemented with oxygen, may reduce amoebic infections but should be used cautiously as treatment can precipitate mortality in fish with gill disease. Cues for the onset of NGD are not completely understood, although poor water quality and elevations in temperature are likely to exacerbate infections. Similarly, the prevalence of this disease is not accurately known, but it is possible that it may be more common among salmonids in New Zealand than is presently recognised.

Metabolism of praziquantel in kingfish Seriola lalandi.

Investigations into the metabolism of drugs used in aquatic animal therapy are useful for understanding the mechanisms of xenobiotic transformation systems and can aid the development of dosing regimens. This study investigated the metabolism of the synthetic anthelmintic praziquantel, which has application in helminthiasis treatment for several fish species including kingfish Seriola lalandi, a commercial aquaculture finfish species. At least 7 mono- or dihydroxylated derivatives of the parent compound were identified in kingfish after administration of a 150 mg kg(-1) oral praziquantel dose, paralleling findings in mammals. The structure of one representative mono-hydroxylated species that was prominent in the skin, muscle, liver, kidney and plasma of kingfish was investigated using fragmentation experiments; this revealed that hydroxylation of the parent molecule occurred in the tetrahydroisoquinoline region of praziquantel, analogous with mammalian metabolites, but different to that of the active mammalian metabolite (trans-4-OH-praziquantel). The implications of these findings with regard to biotransformation systems for this drug in mammals and fish are discussed.

Bioavailability and pharmacokinetics of a praziquantel bolus in kingfish Seriola lalandi.

Oral praziquantel (PZQ) preparations have recently been investigated for the treatment of monogeneans that infect the skin and gills of kingfish Seriola lalandi cultured in sea-cages. To evaluate an oral PZQ dosing strategy, the pharmacokinetics of a dissolved and in feed oral PZQ preparation (40 mg kg(-1) body weight) were compared with an intravenous bolus in kingfish plasma and skin using HPLC. Compared with intravenous administration, PZQ bioavailability (area under curve, AUC0-24h) was slightly improved when the drug was administered with food in both kingfish plasma (56.8% in feed vs. 50.8% in solution) and skin (55.5% in feed vs. 50.3% in solution). After oral dosing, maximum drug concentrations in skin were approximately one-third of those achieved in plasma and higher when the drug was administered in solution (5.26 microg ml(-1)) than in feed (3.96 microg ml(-1)); additionally, the time to achieve maximum PZQ concentration was similar in plasma and skin, although markedly reduced when the drug was administered in solution (1 h) than in feed (6 h). However, clearance of the drug was delayed in skin; administered as an oral formulation, PZQ concentrations in the systemic circulation fell below the limit of quantification after 24 h, but remained quantifiable (0.3 microg g(-1)) in skin at this time. These initial studies indicate that a daily treatment interval will lead to the exposure of parasites to highly variable anthelmintic concentrations, which may be sub-optimal for the treatment of monogeneans in this finfish species.

Effects of temperature on fecundity in vitro, egg hatching and reproductive development of Benedenia seriolae and Zeuxapta seriolae (Monogenea) parasitic on yellowtail kingfish Seriola lalandi.

Globally, aquaculture industries involved with commercial culture of kingfish (Seriola spp.) experience outbreaks of monogenean parasites, which can cause heavy stock losses. In Australia and New Zealand, aquaculturists of kingfish Seriola lalandi incur financial losses caused by two monogenean species: Benedenia seriolae and Zeuxapta seriolae which parasitise the skin and gills, respectively. This study provides information on some basic temperature-dependent life-cycle parameters of these problematic monogeneans on S. lalandi. Hatching times and age at maturity were inversely related to water temperature within the range experienced by wild kingfish in New Zealand (13-21 degrees C). Mature B. seriolae in vitro laid on average 37 eggs/day that hatched over approximately 4 days; peak hatching occurred 9, 11 and 22 days post-deposition at temperatures of 21, 17.5 and 13+/-1.0 degrees C, respectively. Z. seriolae in vitro laid on average 246 eggs/day that hatched over 2 days; peak hatching occurred 7, 9 and 15 days post-deposition at these respective temperatures. B. seriolae matured within 20, 25 and 48 days p.i. at 21, 18 and 13 degrees C. Z. seriolae matured within 25, 37 and >52 days p.i. at the same temperatures. This research describes stages in the reproductive development of B. seriolae and Z. seriolae and discusses the inclusion of basic parasitic life-cycle parameters into management strategies designed to maximise treatment efficacy and limit monogenean epizootics in sea-cage kingfish culture.

The dopamine D3 receptor antagonist nafadotride inhibits development of locomotor sensitization to amphetamine.

Behavioral sensitization is a well-studied model of behavioral plasticity mediated at least in part by dopaminergic systems believed to play an important role in several psychiatric conditions. In the rodent, locomotion is regulated by the opposing balance of D3 and D2 receptors, with D2 activation increasing and D3 stimulation inhibiting locomotion. However, receptor occupancy of D3 dopamine receptors is far greater than D2 or D1 occupancy at typical post-stimulant dopamine concentrations. We therefore hypothesized that tolerance of D3 receptor inhibition of locomotion contributes to the development of sensitization. To test this hypothesis, we examined the effect of the D3 receptor antagonist nafadotride on sensitization. As predicted, nafadotride inhibits augmentation of the locomotion response to repetitive amphetamine. This finding is consistent with the proposed model of adaptive down-regulation of D3 dopamine receptor function contributing to the development of behavioral sensitization.

Foreign-gas broadening of nitrous oxide absorption lines.

We have measured the foreign-gas broadening coefficients for collisional broadening of lines in the v(3) fundamental of N(2)O by He, Ne, Ar, Kr, Xe, H(2), D(2), and CH(4). These coefficients, which give the ratio of the line-broadening ability of these gases to the line-broadening ability of N(2), can be used with recent measurements and calculations of N(2) broadening to obtain optical collision cross sections.