Mortality and kidney histopathology of chinook salmon Oncorhynchus tshawytscha exposed to virulent and attenuated Renibacterium salmoninarum strains.

An isolate of Renibacterium salmoninarum (strain MT 239) exhibiting reduced virulence in rainbow trout Oncorhynchus mykiss was tested for its ability to cause bacterial kidney disease (BKD) in chinook salmon Oncorhynchus tshawytscha, a salmonid species more susceptible to BKD. Juvenile chinook salmon were exposed to either 33209, the American Type Culture Collection type strain of R. salmoninarum, or to MT 239, by an intraperitoneal injection of 1 x 10(3) or 1 x 10(6) bacteria fish(-1), or by a 24 h immersion in 1 x 10(5) or 1 x 10(7) bacteria ml(-1). For 22 wk fish were held in 12 degrees C water and monitored for mortality. Fish were sampled periodically for histological examination of kidney tissues. In contrast to fish exposed to the high dose of strain 33209 by either injection or immersion, none of the fish exposed to strain MT 239 by either route exhibited gross clinical signs or histopathological changes indicative of BKD. However, the MT 239 strain was detected by the direct fluorescent antibody technique in 4 fish that died up to 11 wk after the injection challenge and in 5 fish that died up to 20 wk after the immersion challenge. Viable MT 239 was isolated in culture from 3 fish that died up to 13 wk after the immersion challenge. Total mortality in groups injected with the high dose of strain MT 239 (12%) was also significantly lower (p < 0.05) than mortality in groups injected with strain 33209 (73 %). These data indicate that the attenuated virulence observed with MT 239 in rainbow trout also occurs in a salmonid species highly susceptible to BKD. The reasons for the attenuated virulence of MT 239 were not determined but may be related to the reduced levels of the putative virulence protein p57 associated with this strain.

Route of vaccine administration: effects on the specific humoral response in rainbow trout Oncorhynchus mykiss.

The specific humoral response of teleost fish to extracellular bacteria was examined using a rainbow trout-Vibrio anguillarum model. Treatment groups were immunized by oral, immersion, and injection routes. All 3 delivery methods conferred full protection in controlled laboratory challenges (p < 0.01). Prior to boosting, serum antibody titers did not correlate with protection in the orally and immersion-vaccinated groups, but, contrary to previous studies, titers measured 10 and 17 d after boosting correlated positively with protection in all 3 vaccinated groups. The route of administration strongly affected the magnitude of the antibody response as measured by enzyme-linked immunosorbent assay (ELISA) and Western blots; however, the antigenic epitopes recognized were not substantially altered by delivery method as evidenced in immunoblot patterns. Given that the primary and booster vaccination protocols were identical, the data suggest that all 3 vaccinated groups may have had a specific humoral response following initial immunization but that specific serum antibody levels before boosting were too low to be detected by ELISA in fish vaccinated by oral and immersion routes. An anamnestic response was evident in all 3 groups. The data support the possibility that teleosts, like higher vertebrates, have a protective immune response to extracellular bacteria that is predominantly humoral. Route of delivery may primarily affect the efficiency with which the immunogenic constituents of the vaccine are presented to the relevant recognition and effector components of the immune system.

Purification of Piscirickettsia salmonis and partial characterization of antigens.

Piscirickettsia salmonis is the etiological agent of salmonid rickettsial septicemia, an economically significant disease affecting the salmon aquaculture industry. As with other rickettsial pathogens, antigenic analysis of P. salmonis has been limited by the inherent difficulties of purifying an intracellular organism away from host cell material. In this report, we describe the use of diatrizoate meglumine and diatrizoate sodium (DMDS) density gradient centrifugation to purify P. salmonis grown in chinook salmonis was consistently concentrated in a visible band within the DMDS density gradient at density of 1.15 to 1.16 g ml(-1). Recovery of purified, viable organisms from DMDS density gradients varied from 0.6 to 3%. Preparations of uninfected CHSE-214 cells, CHSE-214 cells infected with P. salmonis, and gradient-purified P. salmonis were compared using sodium dodecyl sulfate polyacrylamide gel electrophoresis to assess the degree of purification and to identify P. salmonis-specific proteins. Although gradient-purified P. salmonis preparations were not completely free of host cell material, 8 bacterial proteins were identified. Polyclonal rabbit antiserum was used in an immunoblot of proteins from purified P. salmonis to identify 3 major and 5 minor antigens. The major antigens of 56, 30 and 20 kDa were potential candidates for experimental vaccines and development of novel diagnostic assays.

The genetic diversity and epizootiology of infectious hematopoietic necrosis virus.

Infectious hematopoietic necrosis virus (IHNV) is a rhabdovirus which causes a serious disease in salmonid fish. The T1 ribonuclease fingerprinting method was used to compare the RNA genomes of 26 isolates of IHNV recovered from sockeye salmon (Oncorhynchus nerka), chinook salmon (O. tshawytscha), and steelhead trout (O. mykiss) throughout the enzootic portion of western North America. Most of the isolates analyzed in this study were from a single year (1987) to limit time of isolation as a source of genetic variation. In addition, isolates from different years collected at three sites were analyzed to investigate genetic drift or evolution of IHNV within specific locations. All of the isolates examined by T1 fingerprint analysis contained less than a 50% variation in spot location and were represented by a single fingerprint group. The observed variation was estimated to correspond to less than 5% variation in the nucleic acid sequence. However, sufficient variation was detected to separate the isolates into four subgroups which appeared to correlate to different geographic regions. Host species appeared not to be a significant source of variation. The evolutionary and epizootiologic significance of these findings and their relationship to other evidence of genetic variation in IHNV isolates are discussed.

Molecular cloning and sequence analysis of the gene coding for the 57-kDa major soluble antigen of the salmonid fish pathogen Renibacterium salmoninarum.

The complete sequence coding for the 57-kDa major soluble antigen of the salmonid fish pathogen, Renibacterium salmoninarum, was determined. The gene contained an opening reading frame of 1671 nucleotides coding for a protein of 557 amino acids with a calculated M(r) value of 57,190. The first 26 amino acids constituted a signal peptide. The deduced sequence for amino acid residues 27-61 was in agreement with the 35 N-terminal amino acid residues determined by microsequencing, suggesting the protein is synthesized as a 557-amino acid precursor and processed to produce a mature protein of M(r) 54,505. Two regions of the protein contained imperfect direct repeats. The first region contained two copies of an 81-residue repeat, the second contained five copies of an unrelated 25-residue repeat. Also, a perfect inverted repeat (including three in-frame UAA stop codons) was observed at the carboxyl-terminus of the gene.

Inactivation of infectious hematopoietic necrosis virus by low levels of iodine.

The fish rhabdovirus infectious hematopoietic necrosis virus (IHNV) was rapidly inactivated by extremely low concentrations of iodine in water. A 99.9% virus reduction was obtained in 7.5 s when virus (10 PFU/ml) and iodine (0.1 mg/liter, final concentration) were combined in distilled-deionized or hatchery water. Iodine efficacy decreased at pHs greater than 7.5 or when proteinaceous material was added to the water. Bovine serum albumin blocked iodine inactivation of the virus more effectively than did equal concentrations of fetal bovine serum or river sediment. Sodium thiosulfate effectively neutralized free iodine. Powder, iodophor, and crystalline iodine solutions inactivated IHNV equally. Iodine rapidly inactivated IHNV isolates representing each of the five electropherotypes. Under the conditions used in this study, inactivation was not affected by temperature, salinity, or water hardness. When Dworshak National Fish Hatchery water was continuously treated to provide a free iodine concentration of 0.14 mg/liter, a 7.5-s exposure to iodine was sufficient to inactivate 99.9% of the IHNV. Iodine added to water that contained IHNV prevented infection of rainbow trout (Oncorhynchus mykiss) fry. These results suggest that the waterborne route of IHNV transmission can be blocked by adding low iodine concentrations to the water supplies of hatcheries.

Advantages of using aquatic animals for biomedical research on reproductive toxicology.

Major advantages of the use of aquatic animals, such as trout, English sole, or sea urchins, for studying the mechanisms of reproductive toxicology are discussed. The remarkable synchrony of differentiation of gametes in large quantities for detailed morphologic and biochemical measurements enables research not readily done on mammalian nonseasonal breeders. Structural differences such as the absence of a fibrous sheath in the more simple structure of fish and sea urchin sperm flagella facilitates comparative study of the mechanism of action of microtubules in flagella movement and the coupling of mitochondrial energy production to microtubules movement.

Sister chromatid exchange analysis in cultured peripheral blood leukocytes of the coldwater marine fish, Pacific staghorn sculpin (Leptocottus armatus): a feasible system for assessing genotoxic marine pollutants.

The genotoxicity of environmental contaminants and test compounds to aquatic and marine fish has primarily been assessed by in vivo techniques that require sacrifice of the test organism for analysis. The major objective of this research was to develop an in vitro sister chromatid exchange (SCE) assay which would utilize cultured peripheral blood leukocytes (PBLs) of a coldwater marine fish species. Use of PBLs in cytogenetic genotoxicity tests has several advantages, the major one being that the experimental fish need not be sacrificed for sample collection. In addition, this nondestructive method of tissue collection permits the investigator to take multiple samples from a single individual and thereby allows the use of an individual as its own control and to monitor its SCE frequency over time. A suitable in vitro culture method for fish PBLs was a prerequisite for cytogenetic analysis of this tissue. The in vitro culture conditions necessary to provide a sufficient number of dividing cells for performance of the SCE assay were established in our laboratory for the PBLs of the Pacific staghorn sculpin (Leptocottus armatus), a common bottom-dwelling Puget Sound fish. The major components of this culture system are heparinized whole blood, fetal bovine serum-supplemented enriched tissue culture medium (RPMI 1640), purified protein derivative of tuberculin as a mitogen, and an incubation temperature of 13.5 degrees C. This in vitro PBL culture system is unique because it involves cultured blood cells from a coldwater marine fish species. Using this culture method, SCE induction was investigated in Pacific staghorn sculpin PBLs which had been exposed in vitro to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), a known direct-acting inducer of SCEs. Cultured cells exposed in vitro responded to MNNG in a dose-related manner in regard to SCE induction, and the frequency of "outlier" cells increased at the higher concentrations of MNNG. With further development, this technique may be adaptable for use with in vivo genotoxicity studies and provide information concerning the induction and persistence of chemically induced SCEs in fish. This PBL/SCE assay may also be a feasible assessment tool for detecting exposure of marine fish to genotoxic environmental contaminants in laboratory and field situations.

Sequestration and release of polycyclic aromatic hydrocarbons by vertebrate cells in vitro.

Vertebrate fibroblasts grown in vitro and exposed to various concentrations of the mutagen/carcinogen benzo(a)pyrene (B(a)P) internalized the compound and recrystallized it in lysosomes by 6-18 h postexposure. This phenomenon occurred when B(a)P at or above 10 micrograms/ml was introduced to the culture medium in a solvent such as DMSO or acetone but not when introduced dissolved in serum. Likewise, high fetal bovine serum concentrations in the culture medium (greater than 20%) as well as human serum (10%) inhibited crystal formation, presumably owing to lipid competition for the compound. Electron-microscopic observations of the cells during the uptake and crystal forming periods revealed that the cell membrane became altered within 3 h of exposure to B(a)P. This was followed by a return to normal of the membrane and the appearance of vesicles within the cell by 6-8 h. The vesicles then became filled with crystals which continued to grow while in the presence of B(a)P and disappeared when the cells were exposed to B(a)P-free culture medium or serum lipids. Introduction of B(a)P into culture medium containing delipidated serum resulted in crystal formation indistinguishable from that which occurred when whole serum was present. Crystals were not formed when total lipoprotein and the lipoprotein components VLDL, LDL, HDL2, and HDL3 were used as solvents for B(a)P. The process of crystal formation was inhibited by the addition of 10(-3) M KCN, and removal of the crystals was dependent on the concentration of lipoprotein in the culture medium.

Effects of benzo(a)pyrene on early development of flatfish.

The ontogenetic effects of the environmental carcinogen benzo(a)pyrene (BAP) on three species of larval flatfish were investigated using concentrations (from 0.10 to 4.2 ppb) which were comparable to levels found in polluted harbors. BAP-treated sand sole (Psettichthys melanostichus) eggs displayed a significant decline in hatching success and a significantly higher incidence of developmental anomalies than did control eggs. Flathead sole (Hippoglossoides elassodon) eggs exposed to a single dose of a water-soluble BAP-bovine serum albumin complex demonstrated evidence of toxic injury with pycnotic nuclei present in the integument and, more commonly, in ocular and neural tissues. An increased incidence of morphological anomalies in English sole (Parophyrs vetulus) eggs and larvae exposed to BAP was not detected.

Anaphase aberrations: a measure of genotoxicity in mutagen-treated fish cells.

Rainbow trout gonad cells (RTG-2) were cultured for various lengths of time in the presence of several classes of known mutagenic chemicals and several related compounds that possessed no known mutagenic/carcinogenic activity. During the course of exposure the cells were examined for the presence of abnormalities in the chromosome arrangement of anaphase figures during mitosis. Untreated and solvent-treated (dimethylsulfoxide-treated) cells exhibited a background abnormality rate of 12% with only minor chromosomal defects being observed. This was also true for those cells exposed to naphthol and anthracene, two chemicals with no proven mutagenic or carcinogenic activity. Conversely, significant increases in the frequency of anaphase aberrations were produced in cells treated with N-methyl-N'-nitro-N-nitrosoguanidine, benzo(a)pyrene, 9-aminoacridine and mitomycin-C. These abnormalities were also far more complex and extensive than those observed in the control and nonmutagen-treated cells. Many species of fish have extremely small and numerous chromosomes, making resolution of chromosome defects such as sister chromatid exchange and deletions more difficult than in most mammalian diploid cells, which generally have larger and fewer chromosomes. Examination of cells during anaphase eliminates the need to observe each chromosome separately as well as the need to produce well-spread metaphase chromosomes. Since the sensitivity of anaphase aberrations to known mutagenic/carcinogenic compounds appears to be quite high in trout cells and since hundreds of suitable cells are available for analysis, this may be an appropriate alternative or addition to some of the more standard chromosome macrolesion tests developed in mammalian systems.

In vitro effect of some mutagens/carcinogens on cultured fish cells.

Fibroblast cells derived from juvenile bluegill sunfish were tested for their response the effects of known mutagens/carcinogens. The cells grew well and cloned in Eagle's minimal essential medium which contained fetal bovine serum. Growth rate was temperature dependent and increased steadily as temperature was raised from 15 to 33 degrees C. Direct mutagens and promutagens were toxic to the cells, and fluorescence spectroscopy revealed the capability of metabolizing at least one promutagen, benzo(a)pyrene, to water-soluble intermediates. Ouabain resistant mutants were produced by exposing the cells to N-methyl-N-nitro-nitrosoguanidine (MNNG) or to benzo(a)pyrene (B(a)P). The mutant clones were resistant to 100-fold increases in concentration of the selective agent ouabain, over that which was lethal to wild type cells. Spontaneous mutation frequency to ouabain in mass cultures averaged 1.2 X 10(-6) for all experiments. A fluctuation test confirmed an expected random occurrence of spontaneous mutations. Visible crystals formed within the cells when high concentrations of B(a)P (greater than 5 micrograms/ml were added to the tissue culture medium. Crystal formation resulted in no apparent cell damage but did reduce the growth rate of the cultures. The crystals were rhombic, resembling those described for pure native B(a)P, and gradually disappeared when the cells were exposed to mutagen-free medium which contained serum.

Uptake of benzo[a]pyrene by gonadal tissue of flatfish (family Pleuronectidae) and its effects on subsequent egg development.

Accumulation of benzo[a]pyrene (BaP) by sexually mature flatfish gonad, its transfer to developing gametes, and its subsequent effects on developing embryos were studied. Thin-layer chromatography revealed both unmetabolized BaP and polar metabolites in the ovary, wolffian ducts, oocytes, and semen of English sole 24 h after ip injection with 200 microCi [3H]BaP. Concentrations of BaP and its metabolites were 3-11 times higher in oocytes and semen than in gonadal tissue. Fertilized eggs from flathead sole that had been fed 4.0 mg BaP 5 h before spawning demonstrated a significantly lower (p less than 0.001) hatching success (11.9%) than eggs from control fish (56.6%). Morphological abnormalities were found in only 1.6% of control embryos but in 5.6% of embryos from treated females.

Variation in response of channel catfish to Henneguya sp. infections (Protozoa: Myxosporidea).

Infections in channel catfish (Ictalurus punctatus, Rafinesque) induced by the sporozoan Henneguya (Protozoa: Myxosporidea) result in seven known and diverse disease manifestations. Most outstanding is an interlamellar branchial form responsible for significant losses among immature catfish, and a unique papillomatous form. The question of whether or not the species of Henneguya involved in these cases is H. exilis remains to be resolved.