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Arctic charr ( Salvelinus alpinus) are the northernmost distributed freshwater fish and can grow at water temperatures as low as 0.2 °C. Other teleost species have impaired immune function at temperatures that Arctic charr thrive in, and thus, charr may maintain immune function at these temperatures. In this study, a fibroblastic cell line, named ACBA, derived from the bulbus arteriosus ( BA) of Arctic charr was developed for use in immune studies at various temperatures. ACBA has undergone more than forty passages at 18 °C over 3 years, while showing no signs of senescence-associated β-galactosidase activity and producing nitric oxide. Remarkably, ACBA cells survived and maintained some mitotic activity even at 1 °C for over 3 months. At these low temperatures, ACBA also continued to produce MH class I proteins. After challenge with poly I:C, only antiviral Mx proteins were induced while MH proteins remained constant. When exposed to live viruses, ACBA was shown to permit viral infection and replication of IPNV, VHSV IVa and CSV at 14 °C. Yet at the preferred temperature of 4 °C, only VHSV IVa was shown to replicate within ACBA. This study provides evidence that Arctic charr cells can maintain immune function while also resisting infection with intracellular pathogens at low temperatures. [ABSTRACT FROM AUTHOR]

A cell line, WE-cfin11e, with an epithelial-like morphology was developed from a caudal fin of walleye, Sander vitreus ( Mitchill), characterized as distinct from the established walleye caudal fin fibroblast-like cell line, WE-cfin11f, and compared with WE-cfin11f for susceptibility to VHSV IVb. Immunocytochemistry and confocal microscopy were used to localize the intermediate filament protein, vimentin, the tight junction protein, zonula occludens-1 ( ZO-1), the extracellular matrix protein, collagen I, and the viral protein, G. Although both cell lines contained vimentin, only WE-cfin11e stained for ZO-1 and only WE-cfin11f stained for collagen I. Ascorbic acid increased the accumulation of collagen I and caused the appearance of collagen fibres only in WE-cfin11f cultures. At 14 °C, both cell lines produced VHSV IVb, but the infection developed more rapidly in WE-cfin11f. At 4 °C, both cell lines became infected with VHSV IVb as judged by the expression of viral proteins, N and G, but only WE-cfin11f produced virus. The results suggest that cold temperatures can modulate viral tropism. [ABSTRACT FROM AUTHOR]

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). [ABSTRACT FROM AUTHOR]

A cell line, WE-cfin11f, with a fibroblast-like morphology was developed from a walleye caudal fin and used to study the intersection of thermobiology of walleye, Sander vitreus ( Mitchill), with the thermal requirements for replication of viral haemorrhagic septicaemia virus ( VHSV) IVb. WE-cfin11f proliferated from 10 to 32 °C and endured as a monolayer for at least a week at 1-34 °C. WE-cfin11f adopted an epithelial shape and did not proliferate at 4 °C. Adding VHSV IVb to cultures at 4 and 14 °C but not 26 °C led to cytopathic effects ( CPE) and virus production. At 4 °C, virus production developed more slowly, but Western blotting showed more N protein accumulation. Infecting monolayer cultures at 4 °C for 7 days and then shifting them to 26 °C resulted in the monolayers being broken in small areas by CPE, but with time at 26 °C, the monolayers were restored. These results suggest that at 26 °C, the VHSV IVb life cycle stages responsible for CPE can be completed, but the production of virus and the initiation of infections cannot be accomplished. [ABSTRACT FROM AUTHOR]

A letter to the editor related to fomites for viral hemorrhagic septicemia virus, is presented.

A novel fibroblast-like cell line RTHDF was established from hypodermal connective tissue of rainbow trout Oncorhynchus mykiss and telomerase activity was demonstrated early and late in cell line development. When RTHDF cells were exposed to bioenergetic stress, i.e. anoxia, activation of the stress activated member of the mitogen-activated protein kinase family, p38MAPK and induction of heat shock protein (Hsp70) were evident. The time-course of the p38MAPK activation and the induction of Hsp70 expression in RTHDF were studied in response to chemically induced anoxia. p38MAPK was activated rapidly, with maximal activity after 10 min of anoxia. Hsp70 was induced after 30 min of anoxia, followed by overnight recovery in growth medium at 21° C. Using the p38MAPK-specific inhibitor SB203580, the enhanced expression of Hsp70 occurred independently of p38MAPK activation in RTHDF. These data suggests that RTHDF can be useful in studying biochemical responses of teleost cells to environmental stress. [ABSTRACT FROM AUTHOR]

Collagenase was used to prepare primary cell cultures from rainbow trout, Oncorhynchus mykiss (Walbaum), gills. Although difficult to subcultivate, one primary culture led to the development of a cell, RTgil1-W1. RTgiIl-W1 grew in the basal medium, L-15, supplemented with foetal bovine serum at between 5 and 10%, but not in L-15 alone. The cells have been passaged approximately 50 times over a 4-year period. At confluency, the cell shape was predominantly polygonal or epithelial-like. RTgill-W1 cultures were demonstrated by DNA staining with H33258 and by growth on agar to be contaminated with mycoplasma, but this contaminant was eradicated by treatment with mycoplasma removal agent (MRA) and BM cyclin. [ABSTRACT FROM AUTHOR]

The influence of ultraviolet (UV) irradiation on creosote toxicity was investigated with the rainbow trout (Oncorhynchus mykiss) gill cell line, RTgill-W1, and two indicator dyes, alamar Blue(TM) and 5-carboxyfluorescein diacetate acetoxymethyl ester. These monitor redox potential and membrane integrity, respectively. After solubilization andchemical analysis, creosote was presented to cells in the dark to measure cytotoxicity or concurrently with UV irradiation to evaluate photocytotoxicity. Additionally, creosote was photomodified by 2 h of UV irradiation before presentation to cells in the dark or together with UV. Cytotoxicity was detected only at high nominal creosote concentrations, but photocytoxicity occurred at creosote concentrations 35-fold lower, All the aromatic hydrocarbons in creosote appeared to contribute to cytotoxicity, but photocytotoxicity was due only to the fluoranthene, pyrene, anthracene, and benzo[a]anthracene in the mixture. Photomodified creosote was much more cytotoxic than intact creosoteand this difference was most pronounced in the alamar Blue assay. Likely, this was due to photomodification products that impaired the mitochondrial electron transport chain. Photomodified creosote was slightly less photocytotoxic than intact creosote. Overall these results indicate that UV irradiation potentially enhances the toxicity of creosote in fish in several different but significant ways. [ABSTRACT FROM AUTHOR]

For livers of lake trout from three Great Lakes sites, the 2,3,7,9-tetrachlorodibenzo-p-dioxin (TCDD) equivalent concentrations (TEQs) were determined in four ways. Bioassay-derived (BD) TEQs were measured with rat (H4IIE) and rainbow trout (RTL-W1) cell lines, and chemistry-derived (CD) TEQs were calculated with TCDD equivalency factors (TEFs) derived with H4IIE and RTL-W1, Generally, BD-TEQs and CD-TEQs for individual samples did not differ significantly for either H4IIE or RTL-W1, indicating that all of the AhR-active compounds in a sample were accounted for by the congener analysis and that these compounds acted in an additive fashion in both mammalian and piscine systems. However contributions of individual chemicals to overall CD-TEQs differed with H4IIE and RTL-W1 TEFs, and for some individual samples, TEQs did depend on the method used. Hepatic TEQs and etlioxyresorufin-O-deethylase (EROD) levels differed significantly between sites. For TEQs,the order was Glenora > Jackfish Bay > Black Bay; for EROD activity,the order was Jackfish Bay > Glenora = Black Bay. No correlation wasfound between hepatic TEQs and EROD activity, which suggests that the two measurements are evaluating different but related consequences of contaminant exposure. [ABSTRACT FROM AUTHOR]