J. L. Fryer

THE IN VITRO CULTIVATION OF TISSUE AND CELLS OF PACIFIC SALMON AND STEELHEAD TROUT

Introduction The application of cell culture techniques has become a n invaluable tool for the study of viruses in both human and veterinary medicine. Wolf’ in America and Grutzner’ in Europe have demonstrated the usefulness of cell cultures for the diagnosis and study of fish viruses. The need for fur ther development of these techniques has gained added emphasis as a result of discovery and recognition of the importance of viral diseases in fish populations. Interest in fish cell culture a t this laboratory was stimulated by an epizootic caused by the Oregon sockeye virus in stocks of juvenile sockeye salmon (Oncorhynchus nerka ) . Work with this virus had been restricted to experimental infection of the intact animal until methods were developed which allowed the preparation of reliable cultures of salmon cells. These methods were based upon those originally developed for mammalian cells and were modified when necessary to fit the needs of the coldblooded vertebrate cells.

White Spot Syndrome Virus Infection of Cultured Shrimp in China

Abstract In 1993 and 1994, an epizootic of disease (white spot) occurred among cultured shrimp in China, resulting in mass mortality. During the periods of outbreak epizootiological surveys were undertaken. Based on these surveys it is known that the disease occurred among populations of cultured Penaeus chinensis, P. japonicus, and P. monodon, infecting shrimp from 2.4 cm to adult. The disease presumably spread among farms as a result of transport of contaminated shrimp seedlings and seawater. Water temperatures exceeding 25°C caused the disease to spread more rapidly. Challenge experiments showed the causative agent was highly virulent. Both naturally and experimentally infected shrimp exhibited white spots on the carapace. Moribund shrimp contained turbid hemolymph, hypertrophied lymphoid organ, and a necrotic hepatopancreas. By electron microscopy, viral particles were observed in gills, stomach, lymphoid organ, and hypodermal tissue of infected shrimp. The virions were slightly ovoid with an envelope...

A Fluorescent Antibody Test for Detection of the Rickettsia Causing Disease in Chilean Salmonids

Abstract An indirect fluorescent antibody test (IFAT) was developed for detection of the rickettsia that was causing epizootics among salmonids cultured in seawater net-pens in southern Chile. Antiserum against the rickettsial agent was produced in New Zealand white rabbits with a preparation grown in antibiotic-free chinook salmon embryo (CHSE-214) cell cultures and partially purified by a combination of filtration and centrifugation steps. The IFAT was effectively used on blood films, tissue sections, and smears. Two gram-negative and two gram-positive bacterial pathogens of salmonids did not react in this test. Detection of the rickettsial agent has previously been restricted to examination by light microscopy or isolation in salmonid cells. The IFAT provides a simple, rapid, sensitive method for detection of the agent and diagnosis of the disease. The rickettsia is thought to be a member of the tribe Ehrlichieae and was tested by IFAT with sera from animals infected with other rickettsial agents.

Relation of Water Temperature to Bacterial Cold-Water Disease in Coho Salmon, Chinook Salmon, and Rainbow Trout

Abstract The effect of water temperature on the progress of experimentally induced Cytophaga psychrophila infection was investigated in juveniles of coho salmon Oncorhynchus kisutch, chinook salmon O. tshawytscha, and rainbow trout O. mykiss (formerly Salmo gairdneri). A virulent strain of C. psychrophila was administered to fish by subcutaneous injection. Infected fish were held in tanks containing pathogen-free well water at temperatures ranging from 3 to 23°C. Mean times from infection to death of the fish were shortest at 12–15°C, which were the temperatures associated with the shortest time for doubling the population of this bacterium in vitro. Juvenile steelhead (anadromous rainbow trout) injected with viable C. psychrophila cells and held in 22°C water did not become diseased.

Epizootiology of Erythrocytic Inclusion Body Syndrome

Abstract Coho salmon Oncorhynchus kisutch were infected artificially with the virus that causes erythrocytic inclusion body syndrome (EIBS). A specific and consistent time course of infection was demonstrated for inclusion body number and hematocrit values. At 12°C, the inclusions appeared approximately 11 d postinjection and were most common near day 21. Hematocrit values were lowest at day 28. By day 30, inclusions disappeared, and hematocrits returned to normal by day 45. Fish that recovered from the disease were much less susceptible to reinfection, and their sera could be used successfully to passively immunize susceptible fish. The stages of the progression of EIBS were all discerned in infected fish held at temperatures of 6, 9, 12, 15, and 18°C, but as the temperature increased, the incubation time, time until maximum inclusions, duration of the inclusion stage, and the time until recovery decreased. Under laboratory conditions, the virus was transmitted horizontally, and rainbow trout Oncorhynchu...

Fluorescent Antibody Test for the Rapid Diagnosis of Infectious Hematopoietic Necrosis

Abstract A fluorescent antibody test (FAT) was developed for the rapid detection of infectious hematopoietic necrosis virus (IHNV). Both polyclonal and monoclonal antisera prepared against IHNV were evaluated. Test variables investigated included type of fixative, dilution rate of antibody reagents, staining time, and type of fluorescent conjugate that would be optimal for detection of IHNV. Specificity tests of the FAT indicated no cross-reactivity of the two antisera with other viruses or with cell lines of salmonid and nonsalmonid origin. All strains of IHNV tested, which included different electropherotypes, those isolated from selected salmonids at different life stages, and those from different geographic regions, reacted with both antisera. The FAT has been used for the detection of IHNV in blood smears and organ imprints from clinically infected juveniles, and in IHNV-infected cells in ovarian fluid from adult carriers. With this FAT, IHNV was detected after 48 h in cell lines inoculated with infe...

Production of Monoclonal Antibodies (MAbs) against White Spot Syndrome Virus (WSSV)

Abstract Nine hybridoma clones secreting monoclonal antibodies (MAbs) were produced from mouse myeloma and spleen cells immunized with white spot syndrome virus (WSSV), an important pathogen of cultured penaeid shrimp in Asia since 1993. An indirect immunofluorescence assay was standardized to evaluate the usefulness of the MAbs for rapid diagnostic techniques to identify WSSV and for further study of this virus. Isotyping revealed that the MAbs were of immunoglobulin G (IgG) and immunoglobulin M (IgM) class, all with kappa light chains. Four MAbs were examined by using immune electron microscopy and colloidal gold as a marker. Three MAbs recognized epitopes on the envelope of the virus and one MAb recognized an epitope on the capsid. The specificity of the MAbs to WSSV was determined by a lack of reactivity with tissue containing a second penaeid shrimp agent, the baculoviral mid-gut gland necrosis virus.

Size-Related Susceptibility of Salmonids to Two Strains of Infectious Hematopoietic Necrosis Virus

Abstract Infectious hematopoietic necrosis is generally considered a disease of salmonid alevins, fry, and early juveniles. It has been suggested that this disease kills only young salmonids and that susceptibility decreases with increased size and age of the fish. To determine if decreased susceptibility occurs with increasing size, we exposed four different sizes of rainbow trout Oncorhynchus mykiss (mean weight, 0.2–13.1 g) and kokanee (lacustrine sockeye salmon) Oncorhynchus nerka (0.2–7.2 g) to two waterborne strains of infectious hematopoietic necrosis virus. Both species were exposed to four concentrations of a type-1 virus strain, which was isolated from fish in Oregon, and a type-2 virus strain obtained from fish in Idaho. Neither strain of virus was consistently more virulent over all exposure concentrations for kokanee at the 0.2-g fish size, At all other sizes, kokanee were consistently more susceptible to the Oregon strain than to the Idaho strain (sign test, P < 0.002). Conversely, all sizes...

Isolation, purification, and partial characterization of a lectin from chinook salmon ova☆

Abstract Chinook salmon ( Oncorhynchus tshawytscha ) ova contain a lectin that agglutinated human type B and rabbit erythrocytes and was specifically inhibited by the monosaccharides d -galactose and l -rhamnose. The lectin purified from homogenates of the ova by affinity chromatography on agarose possessed a p I of 4.5 in isoelectric focusing studies. The purified lectin inhibited the growth of four bacterial fish pathogens.

Binding affinity of tetrameric coho salmon Ig anti-hapten antibodies

Tetrameric Ig anti-fluorescyl antibody was purified from sera of immunized coho salmon by immunoadsorption and the ligand binding properties studied from a series of bleedings over a 120-day period. Purified antibody was assayed by fluorescence quenching and equilibrium dialysis for determination of the average intrinsic association constant (Ka) and heterogeneity index (a). All antibody preparations showed an equilibrium constant of ~4–5 × 105 M−1 for the fluorescyl ligand, regardless of the time of bleeding after primary or secondary immunizations. Heterogeneity indices, derived from Sips plots, indicated relatively restricted heterogeneity in all purified antibody populations. The ligand binding results are discussed in terms of apparent restricted regulatory functions in salmon with respect to affinity maturation.