Normal Trout Serum Research Articles

Interaction between eicosanoids and the complement system in salmonid fish

Both eicosanoid generation and the complement system have long evolutionary histories predating the emergence of the vertebrates over 500 myr ago. This study investigated the interplay between these two systems in an example of a bony fish, the rainbow trout ( Oncorhynchus mykiss). Specifically, it examined whether purified complement fragments including C3a-1 and zymosan-activated serum, stimulate the biosynthesis of any of these eicosanoids by trout macrophages. Incubation of macrophages with zymosan pre-incubated with normal trout serum resulted in the phagocytosis of such particles and the generation of both intra- and extra-cellularly located lipoxygenase and cyclooxygenase products. Both eicosanoid generation and phagocytosis levels were significantly elevated following incubation of zymosan in trout serum in comparison with heat-inactivated (60 °C for 30 min) trout serum and saline alone. A combined mass spectrometry/high performance liquid chromatography approach was employed to conclusively demonstrate the presence of the cyclooxygenase product, prostaglandin E (PGE) in the culture supernatants of ionophore-challenged macrophages. Incubation of trout macrophages with zymosan-activated trout serum (i.e. no zymosan present) failed to stimulate PGE generation. Similarly, incubation of these cells for up to 60 min with C3a-1 (4 or 50 nM) failed to generate significant amounts of PGE or lipoxygenase products such as leukotriene B 4/5 or lipoxin A 4/5. Longer term (6 & 24 h) incubation of macrophages with C3a-1 (4 nM) resulted in a time dependent increase in the generation of PGE but not leukotriene B in culture supernatants. No conclusive evidence that the increase in PGE generation was caused by changes in the expression of either cyclooxygenase-1 or -2 was found.

Streptococcus iniae expresses a cell surface non-immune trout immunoglobulin-binding factor when grown in normal trout serum

Three capsulated isolates of S. iniae representing serotype I and II and being arginine dihydrolase positive, negative or variable (AD+ve, AD−ve, AD+−ve) were investigated for their ability to bind rainbow trout serum immunoglobulin by the Fc region. Using a coagglutination assay with bacteria grown in Todd–Hewitt broth (THB), no evidence of non-specific Fc-binding of trout immunoglobulin (Ig) was obtained. However, when grown in normal trout serum, all isolates produced similar protein patterns in SDS–PAGE, but they were markedly different from the patterns of the bacteria grown in THB. Some bands with MW 70 kDa and over 100 kDa were very intense in the profiles of the serum-grown isolates. In Western blots, these bands of all isolates were immunostained with the conjugated goat antiserum to trout Ig, after blocking with normal goat serum, demonstrating that the bacteria had bound the trout Ig during growth in the serum. When the isolates were grown overnight in trout antiserum against Lactococcus garvieae they coagglutinated with L. garvieae cells but S. iniae isolates grown in normal trout serum did not. These data indicate that S. iniae grown in serum express surface factors which can bind trout Ig by the Fc-region.

Comparsion of selected strains of infectious hematopoietic necrosis virus (IHNV) using neutralizing trout antisera

Comparsion of selected strains of infectious hematopoietic necrosis virus (IHNV) using neutralizing trout antisera

Protection of rainbow trout, Oncorhynchus mykiss (Richardson), against Diplostomum spathaceum (Digenea): the role of specific antibody and activated macrophages

Abstract. The degree of protection in immunized rainbow trout against the infective larval stages of Diplostomum spathaceum was investigated. Immunized rainbow trout demonstrated a 70–80% reduction in the number of metacercariae establishing in their eye‐lenses following challenge with live cercariae compared with unimmunized fish. Both normal trout serum and anti–diplostomule serum weTC cytotoxic to diplostomules in vitro. However, there was no correlation between the degree of protection and the level of serum antibody within individual fish. Reduced establishment of migrating diplostomules, indicative of in vivo killing, occurred in fish possessing activated macrophages and challenged with cercariae opsonized with specific antibody. An antibody–dependent cell–mediated cytotoxic (ADCC) mechanism is proposed, conferring protection in immunized fish. This is supported by the finding that cercarial and diplostomule antigens are capable of activating macrophages in immunized rainbow trout. The role of macrophage activation and specific antibody in conferring protection in immunized fish is discussed.

Tn5-induced protease-deficient strains of Aeromonas hydrophila with reduced virulence for fish.

Protease-deficient strains of Aeromonas hydrophila TF7 were induced by transposon Tn5 mutagenesis, with Escherichia coli 1830(pJB4JI) as the Tn5 donor. The parent strain has the cell surface characteristics associated with virulence for fish, and as it produces a single metalloprotease, mutants could be distinguished by direct plating on brain-heart infusion skim milk agar. Mutants Pd-7 and Pd-10 still produced metalloprotease, but at reduced levels and only after prolonged incubation. The activities of other exoenzymes and hemolysin were unaffected, and the mutants autoagglutinated in broth, indicating that the cell surface characteristics of A. hydrophila TF7 had been retained. Unlike the parent strain, the mutants did not produce lesions or mortalities in rainbow trout (Salmo gairneri) when 5 X 10(6) CFU were injected intramuscularly. The bacterial cells were completely cleared from the site of the injection and the organs within 7 days. For 60-g rainbow trout held at 10 degrees C, the 50% lethal dose of Pd-10 was greater than 10(7) CFU, compared with 8.1 X 10(5) CFU for the parent strain. The mutants were significantly more susceptible than the parent strain to the bactericidal effect of fresh normal trout serum in vitro. Mutants Pd-7 and Pd-10 grew as well as the parent on M9 salts-glucose medium but more slowly on heat-inactivated fish serum. Thus, protease appears to be able to contribute to the establishment of A. hydrophila infection in fish both by overcoming initial host defenses and by providing nutrients for cell proliferation.

Inhibition of infectious pancreatic necrosis virus by serum from normal rainbow trout (Salmo gairdneri) in Canadian hatcheries.

Disease-free rainbow trout (Salmo gairdneri) in Europe have a natural (6 S) serum protein capable of inactivating a tissue culture-adapted isolate of infectious pancreatic necrosis virus, IPNV strain Sp (M. DORSON et al., C.R. Acad. Sci., 281, 1435-1438, 1975). We examined five distinct stocks of rainbow trout (from virus-free Canadian facilities) and all expressed a similar inhibitor that neutralized IPNV Sp in fathead minnow (FHM) cells at maximum (reciprocal) mean serum titers ranging from 128 to 512 (80 % plaque reduction, 0.1 ml inocula). Tests with different laboratory strains of virus, including Jasper, Buhl, Reno, Powder Mill, VR 299, OV, and Tellina virus (TV), indicated that greatest activity was directed against TV but Jasper and VR 299 were inactivated nearly as well by a typical serum preparation. Viral inhibition was slightly greater in the cyprinid (FHM) cell line than in the salmonid (CHSE-214) line. However, preparations of viruses that did not produce significant cytopathic effects (CPE) in FHM cells were comparatively insensitive to serum inhibition as determined in CHSE-214 cells. Buhl and Powder Mill were refractory to neutralization, as were VR 299 preparations without FHM-IPNV variants. Oyster virus (OV) was inhibited by normal trout serum, but to a considerably decreased extent compared to the Sp FHM-IPNV strain or to the serum-sensitive North American IPNV strains.

Infectious pancreatic necrosis virus of salmonids: biological and antigenic features of a pathogenic strain and of a non‐pathogenic variant selected in RTG‐2 cells

Abstract. A non‐pathogenic cell culture adapted variant was obtained from a normally pathogenic strain of IPN virus (Sp type) after several passages in RTG‐2 cells. This cell culture modified (CCA) strain was compared with the original wild (W) strain in various tests. Cross neutralization tests showed no obvious antigenic difference between the two. However the CCA strain was neutralized by a 1:5000 dilution of normal trout serum whereas the W strain was not. In RTG‐2 cells, CCA strain gave both large and small plaques, whereas the W strain gave only small ones. Both virus strains were heat sensitive and labile to cyclic freezing and thawing. The CCA virus was more stable in storage at 4°C under different pH conditions and its growth in RTG‐2 cells was more rapid. The rt (supraoptimal temperature at which viral yield is depressed by 90%) appeared to be 19‐20°C for the CCA strain and 18‐19°C for the W strain. Pre‐treatment of RTG‐2 cells with ultraviolet inactivated CCA virus could inhibit growth of W virus, but had no effect on replication of homologous virus.

Isolation and characterization of infectious pancreatic necrosis virus from pike (Esox lucius).

A nonenveloped icosahedral virus measuring 60 (+/-5)nm in diameter has been isolated from pike fry (Esox lucius). The isolate is stable to heat, lipid solvent and acid. Iododeoxyuridine has no effect on its replication. Serologically, the virus related to the major European strains of infectious pancreatic necrosis virus (IPN) Sp and Ab. The isolate is neutralized by normal trout serum and is not pathogenic for trout fry or for young pike. A line of CHSE-214 cells persistently infected with the isolate was established and has been passed 30 times.