Studies of Atlantic salmon (Salmo salar L.) blood, spleen and head kidney leucocytes using specific monoclonal antibodies, immunohistochemistry and flow cytometry

Monoclonal antibodies (mabs) raised against Atlantic salmon serum IgM (C7G7 and G2H3) and isolated peripheral blood leucocytes (PBL) (E3D9, C4B6 and D8B3) were applied in this study. Using immunoenzymehistochemistry, immunofluorescence and flow cytometry, the distribution of mab+ cells in blood, spleen and head kidney from Atlantic salmon were studied. Immunostaining on cytospin preparations and flow cytometry of isolated PBL showed that the Ig+ cells recognised by C7G7 and G2H3 were mononuclear leucocytes (MNL). The cytospin preparations showed some Ig+ cells with strong cytoplasmic staining, most likely plasma cells. The salmon blood neutrophils were the only E3D9+ cells in cytospin preparations of PBL, and E3D9 recognised about 94% of the defined neutrophil fraction in flow cytometry. The reactivities of C4B6 and D8B3 were to a large degree similar in both immunoenzymehistochemistry and flow cytometry, recognising both MNL and blood neutrophils. Immunofluorescence double staining of PBL with C4B6 and D8B3 showed double staining of all mab+ cells and D8B3 was apparently not able to block the binding of C4B6 to PBL. Immunofluorescence double staining of PBL also revealed more E3D9+ than C4B6+ neutrophils. In immunostaining on cryostat sections of spleen and head kidney, staining of cells was observed with all the mabs, the head kidney generally containing more positive cells than the spleen. Some potential applications for immunological studies using these mabs are suggested.     

Studies of Atlantic salmon (Salmo salar L.) blood, spleen and head kidney leucocytes using specific monoclonal antibodies, immunohistochemistry and flow cytometry

Monoclonal antibodies (mabs) raised against Atlantic salmon serum IgM (C7G7 and G2H3) and isolated peripheral blood leucocytes (PBL) (E3D9, C4B6 and D8B3) were applied in this study. Using immunoenzymehistochemistry, immunofluorescence and flow cytometry, the distribution of mab+ cells in blood, spleen and head kidney from Atlantic salmon were studied. Immunostaining on cytospin preparations and flow cytometry of isolated PBL showed that the Ig+ cells recognised by C7G7 and G2H3 were mononuclear leucocytes (MNL). The cytospin preparations showed some Ig+ cells with strong cytoplasmic staining, most likely plasma cells. The salmon blood neutrophils were the only E3D9+ cells in cytospin preparations of PBL, and E3D9 recognised about 94% of the defined neutrophil fraction in flow cytometry. The reactivities of C4B6 and D8B3 were to a large degree similar in both immunoenzymehistochemistry and flow cytometry, recognising both MNL and blood neutrophils. Immunofluorescence double staining of PBL with C4B6 and D8B3 showed double staining of all mab+ cells and D8B3 was apparently not able to block the binding of C4B6 to PBL. Immunofluorescence double staining of PBL also revealed more E3D9+ than C4B6+ neutrophils. In immunostaining on cryostat sections of spleen and head kidney, staining of cells was observed with all the mabs, the head kidney generally containing more positive cells than the spleen. Some potential applications for immunological studies using these mabs are suggested.     

A monoclonal antibody recognising a surface marker on rainbow trout (Oncorhynchus mykiss) monocytes

The characterisation of a monoclonal antibody (mab 45) reacting with phagocytic leucocytes isolated from blood and spleen of rainbow trout (Oncorhynchus mykiss, Walbaum) is described. The surface marker labelled by this mab is expressed at relative low levels on the membrane of large, nearly nongranulated trout leucocytes, and having the typical morphology of monocytes in flow cytometry (Kfoury et al., 1999, Fish Pathology, 34, 1-6). No reaction of mab 45 with granulocytes, lymphocytes or thrombocytes was detected. In spleen and head kidney, large, polymorphonuclear leucocytes were immunostained. The mab most strongly recognised an antigen of 48 kDa prepared from trout leucocytes of different organs, but not in trout plasma. In an in vitro phagocytosis assay trout monocytes were stained with mab 45 after phagocytosis of Aeromonas salmonicida labelled with the lipophilic fluorescent cell surface linker PKH26. However, previous binding of mab 45 on trout leucocytes did not inhibit the phagocytosis of A. salmonicida particles. Using mab 45, the dynamics of monocytes in blood, spleen and peritoneal cavity could be demonstrated after intraperitoneal injection of trout with inactivated A. salmonicida. The described mab serves as a useful tool to investigate the involvement of monocytes/macrophages in immune reactions of trout to a variety of pathogens.     

A monoclonal antibody recognizing a complex tubular structure in rainbow trout pillar cells

Pillar cells are important to maintain the structural integrity of the fish gills. This study describes a new monoclonal antibody (mab 25) which detects a complex tubular structure of rainbow trout Oncorhynchus mykiss gill pillar cells. This mab was selected and established after immunization of mice with density gradient separated trout mononuclear cells. In primary screening using a cell enzyme-linked immunoabsorbant assay (ELISA) the corresponding hybridoma supernatant reacted with viable and fixed trout leukocytes. In immunofluorescence analysis mab 25 showed a distinct reaction with a prominent snow chain like tubular structure in pillar cells. It consists of seven to 11 concave turned tubuli of about 5 μm length forming a circle of about 7 μm. Reactivity of mab 25 with fibrous structures was observed in cryostat sections of gill, pseudobranchia, spleen, striated muscle and heart muscle. mab 25 recognized a protein with a molecular weight of about 58 kDa as determined by radioimmunoprecipitation. This structure appears to be related to elements which have been described as membrane enclosed collagen columns by electronmicroscopic analysis and is reminiscent of patterns labelled with anti-human cytokeratin antibodies. mab 25 may be an important tool to study the impact of environmental and physiological conditions on the structural integrity of this complex structure in trout gill pillar cells.     

Immunogenicity of a recombinant infectious hematopoietic necrosis virus glycoprotein produced in insect cells.

A recombinant infectious hematopoietic necrosis virus (IHNV) glycoprotein (G protein), produced in Spodoptera frugiperda (Sf9) cells following infection with a baculovirus vector containing the full-length (1.6 kb) glycoprotein gene, provided very limited protection in rainbow trout Oncorhynchus mykiss challenged with IHNV. Fish were injected intraperitoneally (i.p.) with Sf9 cells grown at 20 degrees C (RecGlow) or 27 degrees C (RecGhigh) expressing the glycoprotein gene. Various antigen (Ag) preparations were administered to adult rainbow trout or rainbow trout fry. Sera collected from adult fish were evaluated for IHNV neutralization activity by a complement-dependent neutralization assay. Anti-IHNV neutralizing activity was observed in sera, but the percent of fish responding was significantly lower (p < 0.05) in comparison to fish immunized with a low virulence strain of IHNV (LV-IHNV). A small number of fish immunized with RecGlow or RecGhigh possessed IHNV G protein specific antibodies (Abs) in their serum. Cumulative mortality (CM) of rainbow trout fry (mean weight, 1 g) vaccinated by i.p. injection of freeze/thawed Sf9 cells producing RecGlow was 18% in initial trials following IHNV challenge. This level of protection was significant (p < 0.05) but was not long lasting, and neutralizing Abs were not detected in pooled serum samples. When trout fry (mean weight, 0.6 g) were vaccinated with supernatant collected from sonicated Sf9 cells, Sf9 cells producing RecGlow, or Sf9 cells producing RecGhigh, CM averaged 46%. Protection was enhanced over negative controls, but not the positive controls (2% CM), suggesting that in the first trial soluble cellular proteins may have provided some level of non-specific protection, regardless of recombinant protein expression. Although some immunity was elicited in fish, and RecGlow provided short-term protection from IHNV, Ab-mediated protection could not be demonstrated. The results suggest that recombinant G proteins produced in insect cells lack the immunogenicity associated with vaccination of fish with an attenuated strain of IHNV.     

Dietary vitamin E and rainbow trout (Oncorhynchus mykiss) phagocyte functions: effect on gut and on head kidney leucocytes

The effects of vitamin E (deficiency or supplementation) on the non-specific immune system in rainbow trout, Oncorhynchus mykiss, were evaluated. Rainbow trout were fed daily a semi-purified diet supplemented with vitamin E at 0, 28 and 295 mg x kg(-1) of diet. After 80 days of experimental feeding, the phagocytic function (respiratory burst evaluated by the CL response, phagocytosis) from gut leucocytes and head kidney enriched macrophages was measured; head kidney cell pinocytosis and serum lysozyme activity were also analysed. The results showed that some phagocyte functions were influenced by dietary vitamin E. When fish were fed the high dietary dose of vitamin E an enhancement of phagocytosis was found, but only significantly for the leucocytes isolated from the gut of rainbow trout; moreover, an impaired response was also observed in the fish fed no vitamin E for 80 days. However, no significant differences were noticed on the oxidative burst (CL) response of both gut and head kidney cells according to the dietary dose of vitamin E. Pinocytosis evaluated on head kidney cells was not influenced by dietary vitamin E. Fish fed vitamin E at 295 mg x kg(-1) had a lower serum lysozyme activity than those fed with vitamin E at 28 mg x kg(-1) and the fish fed no vitamin E for 80 days had an impaired activity. Thus, the present results demonstrate that altered dietary levels of vitamin E modulates the phagocytic functions of gut leucocytes in rainbow trout; moreover, the vitamin E diet effect seems to be greater on the local intestinal response as compared to systemic (head kidney). Taken together, this study confirms the crucial role of gut phagocytes in mucosal non-lymphoid defences in fish.     

The rainbow trout classical MHC class I molecule Onmy-UBA*501 is expressed in similar cell types as mammalian classical MHC class I molecules

Onmy-UBA is a polymorphic classical major histocompatibility (MHC) class I locus in rainbow trout (Oncorhynchus mykiss). A common allomorph is Onmy-UBA*501, which has been detected in several wildtype strains, in the clonal homozygous rainbow trout C25 and, in the current study, in the rainbow trout gonad cell line RTG-2. The extracellular domain of this allomorph was expressed in E. coli and a murine monoclonal antibody designated H9 was generated against the recombinant protein. In Western blot analysis Mab H9 specifically recognised an n-glycosylated protein of 45 kDa in leucocytes and erythrocytes of C25 fish and in RTG-2 cells. The level of Onmy-UBA*501 expression in erythrocytes was very low. Immunocytochemistry of isolated cells indicated expression in lymphocytes, macrophages, neutrophils, erythrocytes, RTG-2 cells and Onmy-UBA *501 transfected CHO cells, but not in untransfected CHO cells. Immunohistochemistry using frozen sections of C25 fish indicated that Onmy-UBA*501 expression is strong in the lymphoid organs (thymus, head kidney and spleen) and in the epithelia and endothelia of several organs. No significant expression was observed in muscle fibres, hepatocytes or neurons. These observations demonstrate that in jawed fish, the lowest phylogenetic group possessing an MHC system, the classical MHC class I molecules are expressed in similar cell types as in higher vertebrates.     

Isolation and characterization of Edwardsiella tarda from fall chinook salmon (Oncorhynchus tshawytscha).

A new bacterial pathogen of chinook salmon (oncorhynchus tshawytscha) was isolated from fish in Oregon's Rogue River. The bacteria are biochemically and serologically related to strains of Edwardsiella tarda. Initially isolated from chinook salmon, the bacteria were also pathogenic for steelhead and rainbow trout (Salmo gairdneri), and channel catfish (Ictalurus punctatus). The 50% lethal doses for chinook salmon, steelhead trout, and channel catfish injected intraperitoneally and maintained in 18 degrees C water were 4.1 x 10(6), 5.6 x 10(6), and 4.0 x 10(5) respectively. When chinook salmon and rainbow trout were injected intraperitoneally and held in 12 degrees C water, the mean lethal doses were 6.4 x 10(7) and 1.7 x 10(6), respectively. The invasiveness of the organism was low in steelhead trout exposed to the bacteria by the waterborne route. The optimum growth temperature of the bacteria in brain heart infusion broth was approximately 35 degrees C. The guanine plus cytosine content of DNA obtained from E. tarda isolated from salmon was 59 mol%.     

Calcein AM release-based cytotoxic cell assay for fish leucocytes

A non-specific cytotoxic cell assay for fish is presented that is based on the release of the activated fluorochrome calcein AM from lysed carp epithelioma papulosum cyprini (EPC) cells. To establish the suitability of treating EPC cells with calcein AM the uptake and spontaneous release of the calcein AM by the EPC cells was evaluated. Incubation of 5 microM calcein AM in culture medium with 1x10(5)EPC cells well(-1)for a minimum of 3 h provided sufficient labelling. Spontaneous release of fluorescence from the labelled EPC cells during 10 h of post labelling incubation ranged from 30 to 39% of the total observed fluorescence. Cytotoxic activity of trout leucocytes was evaluated at three leucocyte to target cell ratios (10:1, 2:1 and 1:1) following incubation (4, 6, 8, and 10 h) with calcein AM-labelled EPC cells at 15 degrees C. In some instances, the monoclonal antibody specific for the NCC surface receptor NCCRP-1 (MAb5C.6) was included in the cultures. The activity of NCC cells was significantly inhibited in the presence of 0.25 microg well(-1)of MAb5C.6 relative to no antibody (P相似文献   

A function-associated molecule on rat natural killer cells identified by anti-idiotypic monoclonal antibodies.

Monoclonal antibodies were generated against idiotopes on an NK target antigen-specific IgM monoclonal antibody (mab). This mab (18C2) was originally produced against (NC-37) human EBV-transformed B cells. The 18C2 mab inhibits natural killer cell lysis of NC-37 and other target cells by preventing conjugate formation. Anti-18C2(id) mabs were tested for binding to effector cells and screened by ELISA, flow cytometry, and by inhibition of NK cytotoxicity. Two of the anti-18C2(id) (anti-id) mabs (12H1.C5 and 6D9.B11) were chosen for further study. The idiotypic specificity of these anti-id mabs was confirmed by testing their binding to 18C2 hybridoma cells in the presence of homologous and heterologous "cold" inhibitor mabs. Experiments were also conducted to determine the functional properties of these mabs. Anti-18C2(id) mab 12H1.C5 inhibited the cytotoxic activity of rat splenic NK (nylon wool nonadherent cells, NWNA) and rat ALAK cells. Flow cytometric (FCM) analysis of the binding of the anti-18C2(id) mabs demonstrated that mab 12H1.C5 bound 75.43% rat NWNA spleen cells, 43.74% rat ALAK cells, and 74.33% rat CRC- cells. Anti-id mab 6D9.B11 bound 45.20% NWNA cells, 70.45% rat ALAK cells, and 55.86% CRC- cells. Two-color FCM analysis demonstrated that the anti-id mabs not only bound to the same molecule on NK cells, but also these mabs bound to the same molecule as 5C6, an anti-NK cell mab. Biochemical analysis of the antigen recognized by mab 12H1.C5 was determined by Western blotting. The determinant on NWNA cells recognized by mab 12H1.C5 had an M(r) of 40 kDa and appeared to be identical to that recognized by mab 5C6. The same experiment using a transformed rat RNK-16 (CRC-) cell extract and Western blot analysis, demonstrated an M(r) of 42 and 48 kDa in the presence of mabs 5C6 and 12H1.C5. Monoclonal antibody 5C6 was previously shown to recognize a vimentin-like function-associated molecule on NK cell membranes. The anti-id mabs were also shown to have cross-reactivity with the intermediate filament vimentin as determined by Western blot analysis.     

Occurrence of Flavobacterium psychrophilum in fish-farming environments

Occurrence of Flavobacterium psychrophilum in fish farms and fish-farming environments was studied using agar plate cultivation, the immunoflourescence antibody technique (IFAT) and nested PCR. Characteristics of 64 F. psychrophilum isolates from rainbow trout Oncorhynchus mykiss, fish farm rearing water, ovarian fluid and wild fish were serotyped, ribotyped and compared biochemically. Virulence of F. psychrophilum isolates from different sources was compared by injection into rainbow trout. Additionally, the number of F. psychrophilum cells shed by naturally infected rainbow trout was determined. F. psychrophilum was detected and isolated from skin mucus, skin lesions and internal organs of diseased rainbow trout and from fish without clinical disease. The pathogen was also present in wild perch Perca fluviatilis, roach Rutilus rutilus, and ovarian fluids of farmed rainbow trout brood fish. Isolates were biochemically homogenous, excluding the capability to degrade elastin. Five different agglutination patterns with different antisera against F. psychrophilum were found among the isolates studied. Although several different ribopatterns were found (ClaI: 12 ribopatterns and HaeIII: 9 ribopatterns), ribotype A was the most dominant. Farmed rainbow trout brood fish carried a broad-spectrum of serologically and genetically different F. psychrophilum in ovarian fluids. Virulence of the tested isolates in rainbow trout varied and naturally infected rainbow trout shed 10(4) to 10(8) cells fish(-1) h(-1) of F. psychrophilum into the surrounding water.     

Tissue astaxanthin and canthaxanthin distribution in rainbow trout (Oncorhynchus mykiss) and Atlantic salmon (Salmo salar)

A comparative investigation of tissue carotenoid distribution between rainbow trout, Oncorhynchus mykiss, and Atlantic salmon, Salmo salar, was undertaken to identify the relative efficiency of utilization of astaxanthin and canthaxanthin. Higher apparent digestibility coefficients (ADCs) (96% in trout vs. 28-31% in salmon; P<0.05), and pigment retention efficiencies (11.5-12.5% in trout vs. 5.5% in salmon; P<0.05), for both astaxanthin and canthaxanthin, were observed for rainbow trout. Astaxanthin deposition was higher than canthaxanthin in rainbow trout, while the reverse was true for Atlantic salmon, suggesting species-specificity in carotenoid utilization. The white muscle (95% in trout vs. 93% in salmon) and kidneys (0.5% in trout vs. 0.2% in salmon) represented higher proportions of the total body carotenoid pool in rainbow trout than in Atlantic salmon (P<0.05), whereas the liver was a more important storage organ in Atlantic salmon (2-6% in salmon vs. 0.2% in trout; P<0.05). The liver and kidney appeared to be important sites of carotenoid catabolism based on the relative proportion of the peak chromatogram of the fed carotenoid in both species, with the pyloric caecae and hind gut being more important in Atlantic salmon than in the rainbow trout. Liver catabolism is suspected to be a critical determinant in carotenoid clearance, with higher catabolism expected in Atlantic salmon than in rainbow trout.     

Flow cytometric analysis of the neutrophil respiratory burst of ayu,Plecoglossus altivelis: comparison with other fresh water fish

Neutrophils of vertebrates undergo respiratory burst activity (RBA) as a defense mechanism against bacterial infections. We report here that ayu (Plecoglossus altivelis) have unusually high RBAs even when they are in a healthy condition. Kidney and blood leukocytes were obtained from ayu, rainbow trout (Oncorhynchus mykiss), carp (Cyprinus carpio), eel (Anguilla japonica), and pond smelt (Hypomesus nipponensis). Neutrophil RBA was measured by flow cytometry using dihydrorhodamine after stimulation with phorbol myristate acetate. The amount of RBA of neutrophils from both blood and kidney was significantly higher in ayu than in the other species (e.g. the fluorescence intensity of ayu blood neutrophils was about 3-7 times higher than that from trout and carp, and that of ayu kidney neutrophils was 2-19 times higher than that of rainbow trout, carp, eel, and pond smelt). This unique character of ayu neutrophils was invariable even at different ages, locations, and sex-maturation stages.     

Comparative susceptibility of rainbow trout Oncorhynchus mykiss and brown trout Salmo trutta to Myxobolus cerebralis, the cause of salmonid whirling disease.

The susceptibility of rainbow trout Oncorhynchus mykiss and brown trout Salmo trutta to Myxobolus cerebralis, the cause of salmonid whirling disease, was assessed following dosed exposures to the infectious stages (triactinomyxons). Parallel groups of age-matched brown trout and rainbow trout were exposed to 10, 100, 1000 or 10,000 triactinomyxons per fish for 2 h and then placed in aquaria receiving single pass 15 degrees C well water. Severity of infection was evaluated by presence of clinical signs (whirling and/or black tail), prevalence of infection, severity of microscopic lesions, and spore counts 5 mo after exposure. Clinical signs of whirling disease, including a darkened caudal region (black tail) and radical tail chasing swimming (whirling), occurred first among rainbow trout at the highest dose at 6 to 7 wk post exposure. Black tail and whirling occurred among rainbow trout receiving 1000 and 100 triactinomyxons per fish at 8 to 9 wk post exposure. Only 1 of 20 fish had a black tail among rainbow trout receiving 10 triactinomyxons per fish, although 30% of the fish were infected at 5 mo post exposure. Black tails were observed in brown trout at 1000 and 10,000 triactinomyxons per fish beginning at 11 and 7 wk post exposure, respectively. There was no evidence of the tail chasing swimming (whirling) in any group of brown trout. The prevalence of infection, spore numbers, and severity of microscopic lesions due to M. cerebralis among brown trout were less at each exposure dose when compared to rainbow trout. Infections were found among rainbow trout at all doses of exposure but only among brown trout exposed to doses of 100 triactinomyxons per fish or greater. Risk of infection analyses showed that rainbow trout were more apt to be infected at each exposure dose than brown trout. Spore counts reached 1.7 x 10(6) per head among rainbow trout at the highest dose of exposure compared to 1.7 x 10(4) at the same exposure dose among brown trout. Spore numbers increased with dose of exposure in rainbow trout but not in brown trout. As microscopic lesion scores increased from mild to moderate, spore numbers increased in rainbow trout but not brown trout. The mechanisms by which brown trout resist infections with M. cerebralis were not determined. Cellular immune functions, including those of eosinophilic granular leukocytes that were more prominent in brown trout than rainbow trout, may be involved.     

Flow-cytometric isolation of testicular germ cells from rainbow trout (Oncorhynchus mykiss) carrying the green fluorescent protein gene driven by trout vasa regulatory regions

There is a need to isolate different populations of spermatogenic cells to investigate the molecular events that occur during spermatogenesis. Here we developed a new method to identify and purify testicular germ cells from rainbow trout (Oncorhynchus mykiss) carrying the green fluorescent protein gene driven by trout vasa regulatory regions (pvasa-GFP) at various stages of spermatogenesis. Rainbow trout piwi-like (rtili), rainbow trout scp3 (rt-scp3), and rainbow trout shippo1 (rt-shippo1) were identified as molecular markers for spermatogonia, spermatocytes, and spermatids, respectively. The testicular cells were separated into five fractions (A-E) by flow cytometry (FCM) according to their GFP intensities. Based on the molecular markers, fractions A and B were found to contain spermatogonia, while fractions C and D contained spermatocytes, and fraction E contained spermatids. We also classified the spermatogonia into type A, which contained spermatogonial stem cells (SSCs), and type B, which did not. As none of the molecular markers tested could distinguish between the two types of spermatogonia, we subjected them to a transplantation assay. The results indicated that cells with strong GFP fluorescence (fraction A) colonized the recipient gonads, while cells with weaker GFP fluorescence (fraction B) did not. As only SSCs could colonize the recipient gonads, this indicated that fraction A and fraction B contained mainly type A and type B spermatogonia, respectively. These findings confirmed that our system could identify and isolate various populations of testicular cells from rainbow trout using a combination of GFP-dependent FCM and a transplantation assay.     

Age- and weight-dependent susceptibility of rainbow trout Oncorhynchus mykiss to isolates of infectious haematopoietic necrosis virus (IHNV) of varying virulence

The virulence of 5 European and 1 North American isolate of infectious haematopoietic necrosis virus (IHNV) was compared by infecting female sibling rainbow trout ('Isle of Man' strain) of different weights and ages (2, 20 and 50 g). The fish were exposed to 10(4) TCID50 IHNV per ml of water by immersion, and the mortality was recorded for 28 d. Two new IHNV isolates from Germany were included in the investigation. One was isolated from European eels kept at 23 degrees C (+/- 2 degrees C) and the other was not detectable by immunofluorescence with commercially available monoclonal antibodies recognising the viral G protein. The results showed that IHNV isolates of high or low virulence persisted in rainbow trout of all ages/weights for 28 d, with the exception of fish over 15 g in the eel IHNV (DF [diagnostic fish] 13/98)-infected groups from which the virus could not be reisolated on Day 28. The smallest fish were most susceptible to an infection with any of the IHNV isolates. The lowest cumulative mortality (18%) was observed in fingerlings infected with the North American isolate HAG (obtained from Hagerman Valley), and the highest mortality (100%) in DF 04/99 infected fish. The DF 04/99 and O-13/95 viruses caused mortality in fish independent of their weight or age. The isolates FR-32/87 and I-4008 were virulent in fish up to a weight of 20 g and caused no mortality in larger fish. In the IHNV HAG- and DF 13/98 (eel)-infected rainbow trout, no signs of disease were observed in fish weighing between 15 and 50 g. An age/weight related susceptibility of rainbow trout was demonstrated under the defined conditions for all IHNV isolates tested.     

DNA repair synthesis in cultured fish and human cells exposed to fish S9-activated aromatic hydrocarbons

Unscheduled DNA repair synthesis was measured autoradiographically in cultured rainbow trout gonad (RTG) and human fibroblast (HF) cells following exposure to aflatoxin B1 (AFB1), 3,4-benzopyrene (BP), 1,2,5,6-dibenzanthracene (DBA), 1,2-benzanthracene (BA) and pyrene (PY) activated with S9 prepared from rainbow trout liver. S9 from rainbow trout injected with Arochlor 1254 or an oil extract was compared with S9 from Fischer rats injected with Arochlor 1254 for the ability to activate AFB1 and cause DNA repair in RTG and HF cells. All three types of S9 activated AFB1, but the measured DNA repair response was greater in the HF cells. A significant grain count response was found following exposure of HF cells to fish S9-activated BP. Using assay conditions which enhance fish cell grain counts, a significant level of DNA repair was also found in RTG cells exposed to fish S9-activated BP. Marginal but statistically significant amounts of DNA repair were elicited in HF and RTG cells exposed to rainbow trout S9-activated BA and DBA, but no response was detected following PY exposure. Fish S9 was found to be able to activate a series of polycyclic aromatic hydrocarbons (PAH) and cause DNA repair synthesis in both fish and mammalian cells. The magnitude of the repair response roughly parallels the carcinogenic potential of the PAHs. These results elicit trans species and phyla comparisons which help to validate fish as models for aquatic carcinogenesis research, and also demonstrate PAH DNA-damaging effects on fish DNA, adding further credence for studying the effects of these chemicals on aquatic organisms.