Gill lesions and death of bluegill in an acid mine drainage mixing zone.

The toxicity of an acid mine drainage (AMD) mixing zone was investigated by placing bluegill (Lepomis macrochirus) at the confluence of a stream contaminated by AMD and a stream having neutral pH. A mixing channel receiving water from both streams was assembled in the field, during July and October 1996, to determine the toxicity of freshly mixed and aged water (2.9-7.5 min). The AMD stream had elevated concentrations of Al and Fe, which precipitated upon mixing, and of Mn, which did not precipitate in the mixing zone. Fish exposed to freshly mixed water had higher mortality than fish exposed to water after aging. Precipitating Al, but not Fe, accumulated on the gills of bluegill, and accumulation was more rapid early during the mixing process than after aging. Fish exposed for 3.5 h to freshly mixed water had hypertrophy and hyperplasia of gill filament and lamellar epithelial cells. Similar lesions were observed after 6.0 h in fish exposed to water aged after mixing. Results demonstrated that Al was the predominant metal accumulating on the gills of fish in this AMD mixing zone, and that mixing zones can be more toxic than AMD streams in equilibrium.

Detection of Yersinia ruckeri in rainbow trout blood by use of the polymerase chain reaction.

We evaluated a polymerase chain reaction (PCR) method for detecting Yersinia ruckeri, the bacterial pathogen causing enteric redmouth disease (ERM), in blood of rainbow trout Oncorhynchus mykiss. Identification of the PCR product was confirmed by Southern blot hybridization with a 32P-labeled oligonucleotide probe matching a sequence within the small subunit ribosomal RNA gene of Y. ruckeri. Following a 1 h immersion of rainbow trout in water with 4.5 x 10(6) colony-forming units of Y. ruckeri l(-1), the PCR was positive for all blood samples from 1 h (first sample) to 5 d and was negative from 9 to 30 d (last sample). Fish in this experiment did not show signs of disease, probably because they had been vaccinated against Y. ruckeri. To test this method with naturally infected fish, 42 rainbow trout from hatcheries were examined. Four of these fish had clinical signs of ERM and were infected with Y. ruckeri based on bacteriological culture. The PCR method detected Y. ruckeri in blood, intestine, liver, and trunk kidney from the 4 fish with ERM and from 5 additional rainbow trout that were bacteriologically negative for Y. ruckeri. Three of 5 rainbow trout from streams receiving effluent from hatcheries were positive for Y. ruckeri when tested with PCR, although there was no growth of Y. ruckeri on culture plates inoculated with the same samples. Samples were successfully stored for 1 wk in lysis buffer at 25 degrees C. This study demonstrated that a non-lethal blood sample can be used with PCR to detect Y. ruckeri.

Preliminary description of lesions in juvenile largemouth bass injected with largemouth bass virus.

Juvenile largemouth bass Micropterus salmoides were intraperitoneally injected with largemouth bass virus (LMBV), a member of the genus Ranavirus, family Iridoviridae. Moribund fish which had been injected with 10(6.2) tissue culture infectious doses, 50% endpoint (TCID50), were sampled 4 d after injection; other largemouth bass injected with this dose died between 3 and 5 d after injection. Fish injected with 10(2.8) TCID50 of LMBV were also examined after 4 d and had lesions similar to those of fish injected with the high dose. Clinical signs included darker pigmentation, inflammation and necrosis at the site of injection, distended abdomen, corkscrew swimming, and lateral recumbency. Internally, fish had focally pale livers, bright red spleens and reddened intestinal ceca. Histologically acute fibrinous peritonitis affected the surface of all organs in the peritoneal cavity, but deeper portions of organs appeared normal. There was also necrosis of the gastrointestinal mucosa. Except for the injection site, lesions were confined to the peritoneal cavity.

Effect of tumor necrosis factor-alpha on progesterone production by granulosa cells in laying hens of different genetic lines.

In vitro progesterone production by granulosa cells in the presence or absence of human recombinant tumor necrosis factor-alpha (hrTNF-alpha) was measured at 10, 20, and 30 wk of egg production in White Leghorn hens selected for high (HA)- or low-antibody (LA) response to sheep red blood cell challenge. Isolated granulosa cells from the three largest preovulatory follicles (F1-F3) were incubated with 5 or 250 ng/ml hrTNF-alpha, and progesterone production was determined by the use of a validated radioimmunoassay. F1, F2 and F3 granulosa cells from HA hens produced more (P < or = 0.05) progesterone (140.8, 107.2, and 49.7 ng/ml) than LA hens (109.4, 78.9, and 26.9 ng/ml). The treatment of granulosa cells with hrTNF-alpha consistently inhibited (P < or = 0.05) progesterone secretion by all follicles among HA and LA hens, but not always at both doses. Generally, 5 ng/ml hrTNF-alpha was the maximum inhibitory dose. In the laying hen, a decrease in steroid production in response to cytokines may upset the steroid balance created by follicular hierarchy and inhibit or delay ovulation.

Potassium flux in juvenile striped bass (Morone saxatilis): influence of external concentrations of sodium chloride and calcium.

Whole-body influx and efflux of K(+) were determined for 25-day-old striped bass,Morone saxatilis, in conditions that simulated harvesting fish from ponds. During the first 5h in fresh water with combined high NaCl (80 mM) and low Ca(2+) (0.12 mM) concentrations, a combination that is acutely lethal to this age of striped bass, K(+) influx for fish in 0.07 mM K(+) was 21±1.7 (SEM) compared to 3.4±0.33 nmol g(-1) h(-1) for fish in water with low Na(+) (0.25 mM) or high Ca(2+) (2.5 mM) concentrations. Influx of K(+) was inhibited during the first few hours after fish were placed in flux chambers. Potassium efflux as percentage of(42)K lost per hour was two-fold higher from fish in the high Na-low Ca treatment compared to fish in low concentrations of Na(+) or high concentrations of Ca(2+). Potassium efflux was probably much greater than influx, but exact values for efflux could not be calculated from the data available. Survival of fish in water with high Na-low Ca was not increased by addition of KCl to the water, indicating that the net loss of K(+) was probably not the cause of death.

Follicular development and superovulation response in cows administered multiple FSH injections early in the estrous cycle.

To determine whether follicular development, superovulation and embryo production were affected by the absence or presence of a dominant follicle, cows were administered injections of FSH twice daily in the early (Days 2 to 6, estrus=Day 0) or middle stage (beginning on Day 10 or 11) of the estrous cycle. Treatment with FSH early in the cycle stimulated follicular development in 83 to 100% of all cows from 4 groups evaluated at different times after PGF2alpha treatment on Days 6 and 7. However, the proportion of cows with >2 ovulations varied from 31 to 62.5%, indicating that induction of follicular development may occur in the absence of superovulation. When compared with cows treated in the middle of the cycle, no differences were observed in the proportion of cows with >2 ovulations (31 vs 20%), ovulation rate. (26.0+/-6.3 vs 49.6+/-25.8), production of ova/embryos (13.3+/-3.2 vs 14.4+/-3.4), or the number of transferable embryos (8.0+/-3.6 vs 5.4+/-1.5; early vs middle, respectively). The proportion of the total number of embryos collected that were suitable for transfer was greater (P<0.01) in cows treated early in the cycle (60%) than at midcycle (37.5%). The diameter of the largest follicle observed by ultra-sound prior to initiation of FSH treatment in the early stage of the cycle (10.0+/-2.0 mm) was smaller (P<0.05) than at midcycle (16.8+/-1.3 mm). These results demonstrate that superinduction of follicular development is highly consistent after FSH treatment at Days 2 to 6 of the cycle and that superovulation and embryo production are not less variable than when FSH is administered during the middle of the cycle. However, superovulation in the early stage of the cycle may increase the proportion of embryos suitable for transfer.

Oncogene expression in hepatocytes of the fish Rivulus ocellatus marmoratus during the necrotic and regenerative phases of diethylnitrosamine toxicity.

Livers of mangrove rivulus (Rivulus ocellatus marmoratus) were examined after an acutely necrogenic dose of diethyl-nitrosamine (DEN). Immunohistochemical detection of oncoproteins and bromodeoxyuridine (BrdU), enzyme histochemical detection of gamma-glutamyltranspeptidase, and histological stains were used in an attempt to separate changes in protooncogene expression related to hepatic regeneration from those changes that were putatively preneoplastic. Perivenous hepatocytes were rounded and shrunken within 3 days of the beginning of DEN exposure, and widespread necrosis and hepatocyte proliferation occurred by 21 days (the last day of DEN exposure). Twenty-four days after the end of DEN exposure, livers were primarily composed of nodules of regenerated hepatocytes. Epidermal growth factor receptor expression in hepatocytes increased in inflamed areas and then returned to control levels as inflammation subsided. Increased expression of Fos, Ras and Myc occurred prior to necrosis in a zonal and chronological progression consistent with regeneration of hepatocytes. Fos, Ras, Myc and p53 expression persisted in scattered cells and foci for 24 days after the end of DEN exposure, and this expression was at levels higher than during normal cell-cycle progression. The spatial pattern and persistence of cells expressing Fos, Ras, Myc and p53 at high levels may have represented preneoplastic changes.

Oncogene expression in hepatic and biliary neoplasms of the fish Rivulus ocellatus marmoratus: correlation with histologic changes.

One day old mangrove rivulus (Rivulus ocellatus marmoratus) were exposed to 9 mg/l diethylnitrosamine (DEN) for 6 weeks, kept in water without DEN for an additional 18-20 months, then necropsied. Oncogene expression was detected by immunohistochemical staining of freeze-dried cryofixed livers. Positive controls for immunohistochemistry included tumors grown by injecting athymic nude mice with cell lines having known oncogene expression. Livers from 15 DEN-exposed fish contained 178 altered foci and neoplasms; 48% of these lesions over-expressed Ras, Myc, Fos, p53 or epidermal growth factor receptor (EGFR). Raf overexpression was not detected. Myc overexpression was positively correlated (P < 0.05) with smaller hepatocyte size in both hepatocellular neoplasms and in altered foci. Increased EGFR expression occurred primarily in inflamed lesions. Increased Ras expression in hepatocellular neoplasms was correlated with anaplasia, gamma-glutamyltranspeptidase activity and lesions that contained mixed acinar and trabecular profiles. Accumulation of p53 occurred more often in neoplasms than in altered foci and correlated with unusual cytoplasmic vacuoles. In hepatocellular neoplasms, Fos overexpression was correlated with increased cell diameter, nuclear pleomorphism, and enlarged nucleoli. Only 1/14 biliary neoplasms overexpressed an oncoprotein (Myc). None of the changes in oncoprotein expression were correlated with cell proliferation (bromodeoxyuridine staining). Although several of the correlations found in mangrove rivulus also occur in mammals, the general relevance of some of our findings can be determined only after they are confirmed in other species.

Endogenous enzymes cause structural and chemical artifacts in methacrylate- and celloidin-embedded sections of unfixed freeze-dried tissues.

Bovine exocrine pancreas and fish (Rivulus ocellatus marmoratus) liver containing pancreatic acini were cryofixed, freeze-dried, and embedded in methacrylate or double-embedded in celloidin and paraffin. In chemically unfixed sections incubated in aqueous solutions, dissolution of zymogen granules was coincident with loss of tissue structure and antigenicity. Type II-S soybean protease inhibitor at 150 mg/liter during section flotation and in aqueous reagents used for immunohistochemistry prevented these artifacts and allowed the use of more dilute antibody solutions. Loss of glycogen from fish hepatocytes was most rapid in areas adjacent to pancreatic acini. Rapid loss of glycogen was attributed to amylase and was prevented by using poly-L-lysine instead of 3-aminopropyltriethoxysilane slide adhesive and by using alcoholic solutions during PAS staining. Inhibition of endogenous enzymes is an important consideration in the development of histological protocols with freeze-dried tissue sections.

Monoclonal antibody-based immunoassay of vitellogenin in the blood of male channel catfish (Ictalurus punctatus).

1. A monoclonal antibody to vitellogenin of channel catfish (Ictalurus punctatus) was made, and its specificity was demonstrated using Western blots of serum from female fish, estradiol-treated male fish, untreated male fish, vitellogenin purified by three different methods and egg extracts. 2. An enzyme-linked immunosorbent assay (ELISA), using this monoclonal antibody, detected vitellogenin in the plasma of 59 out of 60 untreated 17-24-month-old male channel catfish with a mean concentration of 338 micrograms/ml and a maximum concentration of 4240 micrograms/ml. 3. Vitellogenin levels in male channel catfish were unrelated to testicular stage, gonadosomatic index and month.

Aging and restriction of dietary calories increases insulin receptor mRNA, and aging increases glucocorticoid receptor mRNA in the liver of female C3B10RF1 mice.

We investigated the influence of age and a 20% or 52% reduction in dietary calories (caloric restriction) on expression of mRNA for a number of transcription factors and signal-transducing proteins using 4, 16, and 30-month-old female mice of the long-lived C3B10RF1 strain. In all age groups, 52% caloric restriction, which extends maximum life span by approximately 33%, increased insulin receptor mRNA by 15% to 25% over the levels in animals fed ad libitum. Aging increased insulin receptor mRNA and glucocorticoid-receptor mRNA in all dietary groups. A similar increase in glucocorticoid receptor mRNA was not observed for male mice of three other strains, suggesting the change is sex- or strain-specific and not a general feature of aging. These changes appear to be specific. Neither caloric restriction nor age had an effect on the level of mRNA for insulin-like growth factor-I, RNA polymerase II elongation-factor S-II, or transcription factors Sp1, CCAAT and enhancer binding protein, or proto-oncogene c-jun.

Influence of age and caloric restriction on expression of hepatic genes for xenobiotic and oxygen metabolizing enzymes in the mouse.

The influence of age and life-span-prolonging caloric restriction on the expression of hepatic genes for xenobiotic and activated oxygen metabolism was investigated in female C3B10RF1 mice, a long-lived hybrid strain. Animals were fed either ad libitum, or diets reduced 20% or 52% in total calories but approximately unchanged in total protein, vitamins, and minerals. Cytochrome P1- and P3-450 (cyp1A1 and cyp1A2, respectively) mRNA levels decreased approximately 40% between age 4-5 months (young) and 30-31 months (old) in ad libitum fed animals (p less than or equal to .05). Caloric restriction eliminated this decrease. Manganese-superoxide dismutase mRNA decreased significantly in old ad libitum fed mice, and caloric restriction eliminated this decrease. No change in manganese-superoxide dismutase activity was detected, probably due to its low level and the large variability inherent in the assay. Catalase mRNA increased with age, but was not affected by diet. Catalase activity increased significantly with caloric restriction in young and old mice, in the absence of an increase in catalase mRNA, suggesting translational or posttranslational effects. CuZn-superoxide dismutase, glutathione peroxidase and epoxide hydrolase mRNA, and the ratio of ribosomal to total mRNA did not change with age or diet.

Granulomatous inflammation and monstrous giant cells in response to intraperitoneal hormone implants in channel catfish (Ictalurus punctatus).

Plastic implants (2.7 mm maximum dimension) of an ethyl vinyl acetate copolymer (EVAc) matrix, containing inulin, bovine serum albumin (BSA) and luteinizing hormone releasing hormone (LHRH), were covered with impervious EVAc and then surgically placed into the peritoneal cavity of 1-year-old channel catfish, Ictalurus punctatus. In fish kept in cold water (13 degrees C), 10 per cent of the implants per month were encapsulated by granulation tissue. In fish kept in warm water (27 degrees C), 20 per cent of the implants per month were encapsulated, with a total of 86 per cent encapsulated at 5 months. In addition to fibroblasts and capillaries, the granulation tissue included macrophages, neutrophils, lymphocytes, plasma cells, multinucleated giant cells and a matrix of collagen fibres. The density of the fibrous capsule increased with time. In a separate investigation, it was found that the thickness of the capsule was directly proportional to the degree of exposure of the EVAc matrix to the fish (exposure influenced by the rate of dissolution of the capsule content). Monstrous giant cells with up to 600 nuclei per 5 microns thick section were seen in capsules around implants. On intraperitoneally implanted cover glasses, whole giant cells contained up to 6000 nuclei and were interconnected by cytoplasmic bridges. Signs of neoplasia, implant expulsion or massive adhesions were not seen.

Aging alters hepatic expression of insulin receptor and c-jun mRNA in the mouse.

The clear association between species and life span suggests that aging, like development, is genetically orchestrated. To explore this hypothesis, the expression of mRNA for a number of transcription regulatory and signal transduction proteins was investigated during aging of B10.RIII, C57BL/10 and B10.BR mice. mRNA for glucocorticoid receptor, CCAAT and enhancer binding protein, transcription factor Sp1 and RNA polymerase II elongation factor S-II were unchanged between 4 and 24 months of age in these mice. These factors are required for the normal transcription of many genes, perhaps explaining their steady rates of expression throughout life. Insulin-like growth factor I mRNA also remained unchanged. By contrast, mRNA for the insulin receptor and transcription factor c-jun changed significantly during aging. c-Jun mRNA decreased approximately 55% between 4 and 12 months of age and then increased by 24-25 months of age to levels approximately equal to those found in young mice. Insulin receptor mRNA increased approximately 30% by 24-25 months of age in all strains of mice. These results suggest that factors determining the steady state level of these mRNAs are altered in level or activity during aging. Assessing the causes and significance of these changes will require further study. However, our results demonstrate that alterations in the expression of specific regulatory genes occur during aging.

Dietary energy restriction in mice reduces hepatic expression of glucose-regulated protein 78 (BiP) and 94 mRNA.

The influence of life span-prolonging dietary energy restriction on hepatic expression of glucose-regulated protein 78 and 94 (GRP78 and GRP94) RNA was investigated in female C3B10RF1 mice. Mice were either fed ad libitum or fed diets reduced 20 or 40% in energy but containing approximately equivalent amounts of protein, fats, vitamins and minerals. Aging produced no changes in GRP mRNA. However, GRP78 and GRP94 mRNA levels were reduced approximately 50 and 40%, respectively, by 40% energy restriction. This level of energy restriction produced a 43% reduction in the mean plasma glucose levels of young and old mice. The changes in GRP mRNA expression appear to be specific, because the levels of these RNAs were normalized to the level of polyadenylated RNA, and no changes were detected in the levels of a number of other mRNAs. Although extreme glucose deprivation increases GRP mRNA levels in cultured cell lines, physiologically relevant reductions in blood glucose had the opposite effect in the liver, in vivo. The regulatory pathway responsible for these effects is not known. GRP mRNA levels are elevated by agents that increase the level of malfolded proteins in the endoplasmic reticulum. Thus, energy restriction may act to reduce malfolded proteins in the endoplasmic reticulum of hepatic cells.

Age-related down regulation of hepatic cytochrome P1-450, P3-450, catalase and CuZn-superoxide dismutase RNA.

The influence of age on liver gene expression was investigated in two strains of H-2 congenic mice. In B10.RIII mice (H-2r), basal P1- and P3-450 RNA levels progressively decreased 65 and 95%, respectively, between 4 and 28 months of age (P less than or equal to 0.05). Polyaromatic hydrocarbon (PAH) induced P1- and P3-450 RNA levels decreased about 50% during this time (P less than or equal to 0.05). In contrast, in C57BL/10 mice (H-2b) little or no change was detected in basal or induced P1- or P3-450 RNA levels. CuZn-superoxide dismutase RNA decreased 80 to 90% between 4 and 9 months of age in B10.RIII mice, while a quantitatively smaller decrease of 50 to 65% was found in C57BL/10 mice (P less than or equal to 0.05). Catalase RNA decreased approximately 80% between 4 and 9 months of age in B10.RIII mice, and a similar decrease was found in C57BL/10 mice. Down regulation of these genes may explain the reduced activities of the cognate hepatic enzymes, and reduced xenobiotic metabolism found in older animals.

Vitellogenin induction by estradiol in channel catfish, Ictalurus punctatus.

In oviparous vertebrates estrogens induce hepatic synthesis of vitellogenin (VG), a blood protein sequestered in vitellogenic oocytes and from which lipovitellin (LV) and phosvitin are derived. Our objective was to identify VG in the channel catfish, Ictalurus punctatus. An intraperitoneal injection of estradiol-17 beta into adult male fish induced a dose-dependent accumulation of a 150 kDa protein (EP) in the plasma. EP was detectable in Coomassie blue-stained polyacrylamide gels within 24 hr after injection of 2 mg hormone/100 g body weight. During the next 4 days, EP increased from 5 to about 25% of the total plasma protein. Electrophoretic mobility, peptide mapping, and immunological crossreactivity showed EP to be indistinguishable from a plasma protein in adult females with vitellogenic ovaries. Two major yolk polypeptides, YP1 (120 kDa) and YP2 (29.6 kDa), were precipitated by (NH4)2SO4 from a yolk protein extract. YP1 but not YP2 reacted with an anti-EP polyclonal antiserum in Western blots. Peptide mapping after proteolysis with trypsin showed YPs 1 and 2 to be unique and revealed structural homologies between YP1 and EP. Liver but not pancreatic explants from an estradiol-treated male synthesized and secreted a [35S]methionine-labeled, 150 kDa protein beginning about 2 hr after initial exposure to the label. We tentatively conclude that EP and YP1 represent VG and LV, respectively. YP2 remains unidentified.

Molecular cloning of fragments of the channel catfish virus (Herpesviridae) genome and expression of the encoded mRNA during infection.

Eleven EcoRI DNA fragments from the genome of an isolate of channel catfish virus (CCV) were cloned into the bacterial vector pUC19. The cloned DNA fragments ranged in size from approximately 200 base pairs to greater than 5,400 base pairs and accounted for about 13.5% of the 130,000-base pair CCV genome. Nine of these CCV DNA fragments encoded sequences that were expressed during late CCV infection. Channel catfish (total length, 4 cm) injected with CCV expressed CCV mRNA at detectable amounts in greater than or equal to 1 tissues. Uninjected control fish failed to express CCV-specific mRNA or expressed CCV-specific mRNA at lower amounts because of the presence of endogenous CCV. Tissue samples from clinically normal channel catfish fingerlings from 2 other farms as well as from adult brood stock also expressed CCV-specific mRNA. The results suggest that CCV can persist in a dormant or transcriptionally active state without causing clinical disease.

Uptake of channel catfish virus from water by channel catfish and bluegills.

Radiolabeled channel catfish virus entered the gills of juvenile channel catfish (Ictalurus punctatus) and was then concentrated in the gut and the liver over 48 hours. Diminution of radioactivity was not detected in these tissues over the course of the experiment. Bluegills (Lepomis macrochirus) were capable of clearing the virus during a period of 48 hours. [3H]Thymidine alone had a different distribution in the channel catfish than did labeled virus.