metallothionein and Fish-Diseases

Globally, parasites are sensitive toward environmental changes, and, in some cases, they are even more sensitive than their hosts. However, there is limited knowledge on the physiological responses of parasites and their effects on their hosts in relation to environmental degradation. In this study, metallothioneins (MTs) were isolated and compared between the ectoparasite Lamproglena clariae and its host fish Clarias gariepinus. Differences in the levels of MTs in the parasite and host were compared to physicochemical water quality variables and metals to determine if MT expression was linked with changes in water quality. Clarias gariepinus individuals were sampled from 2 sites of differing water quality along the Vaal River using gill nets and assessed for L. clariae. Gill, muscle, and liver tissue of the host and L. clariae were collected and stored in liquid nitrogen for analysis of MT. Water and sediment samples were collected for metal analysis by inductively coupled plasma-optical emission spectrometry and inductively coupled plasma-mass spectrometry. Nutrient levels and water hardness in water samples were assessed using spectrophotometry. MTs were quantified using spectrophotometry and size exclusion chromatography in the host and parasite, respectively. Infections by L. clariae differed between sites, with higher parasite intensity at the unpolluted Vaal Dam site. Concentrations of MT in host tissues and L. clariae were significantly higher at the polluted site, below the Vaal River Barrage, compared to the Vaal Dam site. Parasite MT concentrations were significantly lower compared to concentrations in the liver and gill tissue of C. gariepinus individuals. In conclusion, differences in the concentrations of MT and infection biology of L. clariae reflected the state of the environment and support the usefulness of this parasite and other Lamproglena spp. as bioindicators.

Topics: Animals; Catfishes; Chromatography, Gel; Copepoda; Ectoparasitic Infestations; Environmental Biomarkers; Fish Diseases; Gills; Liver; Metallothionein; Muscles; Rabbits; Water Quality

In the present study, the importance of considering fish intestinal parasites i.e. the acanthocephalans in metal exposure assessment was estimated under low metal contamination conditions. Two acanthocephalan species, Pomphorhynchus laevis and Acanthocephalus anguillae were examined in 59 specimens of European chub (Squalius cephalus L.) sampled at 5 locations along the Sava River, Croatia. Concentrations of essential (Cu, Mn) and non-essential (Ag, Cd, Pb) metals were higher in intestinal parasites than chub gastrointestinal tissue, but levels of essential metals Fe and Zn were comparable or lower in parasites, respectively. The highest accumulation in both acanthocephalan species was found for non-essential metals and followed the order: Ag>Pb>Cd. Higher infection intensity with P. laevis allowed us to present their spatial metal distribution and evaluate the influence of P. laevis on metal levels and sub-cellular biological responses (total protein and metallothionein levels) in the host infected with P. laevis. Even in the river section with low metal contamination, parasitism affected metal levels, resulting in lower Cu, Cd and Pb concentrations in chub infected with P. laevis than in uninfected chub. Although total protein and metallothionein levels remained constant in infected and uninfected chub, acanthocephalans should be considered a potential confounding factor in metal exposure assessments. Moreover, P. laevis-chub system can be suggested as an appropriate tool in biomonitoring, since in both species increased Cu and Cd concentrations towards the downstream locations were found. Higher Cu and Cd levels in P. laevis suggest acanthocephalans to be sensitive bioindicators if low metal levels have to be detected.

Topics: Acanthocephala; Animals; Cadmium; Carps; Copper; Croatia; Fish Diseases; Gastrointestinal Tract; Helminthiasis, Animal; Humans; Iron; Lead; Manganese; Metallothionein; Metals; Rivers; Silver; Water Pollutants, Chemical; Zinc

The aim of the present study was to assess the prevalence of the flavobacteria within farmed trout and to quantify their bacterial burden. A total of 61 fish were sampled from seven farms, and were distributed in two groups: (1) visibly diseased fish suffering from the rainbow trout fry syndrome or the bacterial cold water disease caused by the bacteria Flavobacterium psychrophilum and (2) normally appearing fish. F. psychrophilum cells were titered by qPCR, targeting a specific area of the 16S rRNA gene in skin, muscle, gills, liver, spleen and kidney from all fish. The pathogen was detected in these organs whatever the health status, with titers ranging from 10(4) to 6 × 10(7)bacteria/g of tissue in normally appearing fish, thus showing they were bacterial carriers. Two organs allowed differentiation between diseased and normally appearing fish: spleen and kidney, with titers ranging from 10(6) to 10(7)bacteria/g of tissue in normally appearing fish vs 10(11) to 10(12)bacteria/g of tissue in diseased fish. No relationship was found between immunoglobulin M-like titer in plasma and health status. Gene expression analysis in fish organs revealed two genes that were markers of the bacterial infection: mt-a and il-1β genes encoding the metallothionein A and the interleukin1-β, respectively. These genes were both over-expressed in gills, liver, spleen and kidney of diseased fish. Four genes encoding immunity markers were down-regulated in spleen (a key organ implicated in immunity) of diseased fish: tgf-β, cd8-α, mhc2-β and igt, suggesting a weakened immune system in diseased fish.

Topics: Animals; Aquaculture; DNA Primers; Down-Regulation; Female; Fish Diseases; Flavobacteriaceae Infections; Flavobacterium; Gene Expression; Immunoglobulin M; Interleukin-1beta; Kidney; Metallothionein; Oncorhynchus mykiss; Prevalence; RNA, Ribosomal, 16S; Spleen; Transforming Growth Factor beta

We developed a suicidal DNA vaccine (pIRF1A-G-pMT-M) for salmonid fish susceptible to Infectious Hematopoietic Necrosis Virus (IHNV). The suicidal vaccine consists of two operons: i) an inducible fish promoter, the interferon regulatory factor 1A promoter (pIRF1A), driving the expression of the IHNV viral glycoprotein (G) gene that induces protection, and ii) a ZnCl(2) inducible fish promoter, the metallothionein promoter (pMT), driving the expression of the IHNV matrix (M) protein that induces apoptosis. The vaccine induces an immune response to the G protein and then induces the cell to undergo apoptosis to eliminate the DNA vaccine-containing cell. Also developed is another suicidal construct (pCMV-luc-pMT-M) for monitoring the persistence of luciferase (luc) expression after induction of apoptosis. In this study, we evaluated the inducibility of the MT promoter with ZnCl(2) and the capacity of cells transfected with the suicidal vector pCMV-luc-pMT-M to undergo apoptosis after ZnCl(2) addition. We also demonstrated the protective immunity elicited by the suicidal DNA vaccine pIRF1A-G-pMT-M, the survival of fish after treatment with ZnCl(2), and the elimination of the suicidal vector in fish after ZnCl(2) treatment.

Topics: Animals; Apoptosis; Cell Line; Chlorides; Fish Diseases; Gene Expression Regulation; Infectious hematopoietic necrosis virus; Luciferases; Metallothionein; Oncorhynchus mykiss; Promoter Regions, Genetic; Rhabdoviridae Infections; Survival Analysis; Transfection; Vaccines, DNA; Zinc Compounds

We analyzed the relationships between the macroparasite community of the European eel and the expression of genes involved in the host physiology during its continental life. The genes studied are implicated in (1) host response to environmental stress, i.e., heat shock protein 70 (HSP70) and metallothionein (MT); (2) osmoregulation, i.e., beta thyroid hormone receptor (betaTHR) and Na+/K+ATPase; and (3) silvering, i.e., betaTHR, freshwater rod opsin (FWO), and deep-sea rod opsin (DSO). All were enumerated by quantitative reverse-transcription polymerase chain reaction. The epizootiological results for 93 yellow eels caught in the Salses-Leucate Lagoon (France) included 11 species: 1 nematode, 2 acanthocephalans, 1 monogenean, and 7 digeneans. The molecular results revealed (1) a significant negative relationship between digenean abundance and the expression level of all the tested genes, except FWO; (2) a significant negative relationship between the abundance of the nematode Anguillicola crassus and the expression level of the Na+/K+ATPase gene; and (3) a significant positive relationship between the A. crassus abundance and the expression level of the MT gene. Eels infected with digeneans had, on average, a lower level of expressed genes. We hypothesize that the parasites may disturb the eel's ability to withstand environmental stress and delay their migration to the Sargasso Sea because of degeneration of the gut. We further propose that the effect of the invasive species, A. crassus, on the gene expression was mainly linked to an increased trophic activity of infected eels. Moreover, it is possible that the parasite may have an effect on the fish's migratory behavior, which is tied to reproductive purposes. Additional work, including an experimental approach, is required to confirm our hypotheses.

Topics: Anguilla; Animals; Eye; Fish Diseases; Gene Expression; Gene Expression Profiling; Gills; HSP70 Heat-Shock Proteins; Liver; Metallothionein; Parasitic Diseases, Animal; Prevalence; Reverse Transcriptase Polymerase Chain Reaction; Rod Opsins; Sodium-Potassium-Exchanging ATPase; Stress, Physiological; Thyroid Hormone Receptors beta; Water-Electrolyte Balance

Cultured and wild sea bass (Dicentrarchus labrax) from the Arade Estuary were sampled in summer and winter and the degree of exposure to metals and polycyclic aromatic hydrocarbons (PAHs) assessed, together with some biochemical responses against those and other pollutants. The highest levels of copper (up to 997 microg g-1 dry weight) and cadmium (up to 4.22 microg g-1 dry weight) were detected in the liver and kidney of cultured specimens, whereas the highest exposure to PAHs was observed in wild fish. Significant alterations in some biochemical markers were detected and associated to pollutant exposure. Thus, metallothionein concentrations were higher in the tissues of cultured fish and positively correlated with metal residues. The activity 7-ethoxyresorufin O-deethylase ranged from 28 pmol/min/mg protein in cultured fish to 83 pmol/min/mg protein in wild fish collected near a marina area. Cultured fish and wild fish from the marina area had depressed acetylcholinesterase in muscle tissue and a parasitic infection in the gonads. The obtained results support the usefulness of the combined use of chemical and biochemical markers to assess the impact of anthropogenic pollutants in both wild and cultured fish.

Topics: Acetylcholinesterase; Animals; Animals, Wild; Bass; Cytochrome P-450 CYP1A1; Fish Diseases; Fusobacterium Infections; Male; Metallothionein; Metals, Heavy; Organ Specificity; Polycyclic Aromatic Hydrocarbons; Portugal; Seasons; Testis; Tissue Distribution; Water Pollutants, Chemical

Biomarkers of exposure and effect of pollutants were analyzed in croakers Micropogonias furnieri (Teleostei: Sciaenidae) captured in winter and summer in a polluted and in a non-polluted site at the Patos Lagoon estuary (Southern Brazil). Catalase and glutathione S-transferase activities (exposure biomarkers) and lipid peroxidation (effect biomarker) were analyzed in liver samples. Other two effect biomarkers were also studied: blood cells DNA damage (through comet assay and micronucleus test) and respiratory burst measurements. In a broad view, results point to an important seasonal variation of the biochemical biomarkers analyzed. However, data obtained clearly indicate that croakers collected in winter at the polluted site were subjected to a level of clastogenic agents sufficient to generate irreversible genetic damages (mutations) and impair the fish immune system.

Topics: Animals; Biomarkers; Blood Cells; Brazil; Catalase; DNA Damage; Environmental Exposure; Environmental Monitoring; Fish Diseases; Glutathione Transferase; Immune System Diseases; Lipid Peroxidation; Metallothionein; Perciformes; Respiratory Burst; Seasons; Seawater; Water Pollution

Cadmium concentrations, (Cd,Zn)-metallothionein (MT) concentrations, MT synthesis and the relative amounts of cadmium bound to (Cd,Zn)-MTs were determined in gills, liver and kidney of common carp Cyprinus carpio exposed to 0, 0.5 microM (0.06 mg.l(-1)), 2.5 microM (0.28 mg.l(-1)) and 7 microM (0.79 mg.l(-1)) Cd for up to 29 days. Cadmium accumulation was in the order kidney > liver > gills. Control levels of hepatic (Cd,Zn)-MT were four times higher compared to those of gills and kidney. No increases in (Cd,Zn)-MT concentrations were observed in liver during the exposure period. In comparison with control carp, (Cd,Zn)-MT concentrations increased up to 4.5 times in kidney and two times in gills. In both these organs, (Cd,Zn)-MT concentrations were linearly related with cadmium tissue levels and with the de novo synthesis of MTs. Hepatic cadmium was almost completely bound to (Cd,Zn)-MT, while percentages of non-MT-bound cadmium were at least 40% in gills and 25% in kidney. This corresponded with a total saturation of (Cd,Zn)-MT by cadmium in kidney and a saturation of approximately 50 and 60% in gills and liver, respectively. The final order of non-MT-bound cadmium was kidney > gills > liver. Our results indicate that cadmium exposure causes toxic effects, which cannot be correlated with the accumulated levels of the metal in tissues. Although cadmium clearly leads to the de novo synthesis of MT and higher (Cd,Zn)-MT concentrations, the role of this protein in the detoxification process is clearly organ-specific and its synthesis does not keep track with cadmium accumulation.

Topics: Animals; Cadmium Poisoning; Carps; Fish Diseases; Gills; Kidney; Liver; Metallothionein; Time Factors; Tissue Distribution

Topics: Animals; Cadmium Poisoning; Cell Line; Cloning, Molecular; Fish Diseases; Fishes; Gene Deletion; Metallothionein; Metals; Transfection; Water Pollutants, Chemical

Juvenile coho salmon (Oncorhynchus kisutch) 3-8 g body weight were exposed to sublethal concentrations of copper acetate in water of hardness 280 mg/l (as CaCO3) using flow-through conditions. Concentrations of copper were approximately 1, 50, 100 or 150 micrograms Cu/l. Uptake of copper into blood, gills, kidney and liver was measured using 67Cu and into hepatic metallothionein. Chromatography on Sephadex G-75 was used to separate the protein from the cytosol. Copper was determined using atomic absorption spectrophotometry. The ratio of rates of uptake of copper and synthesis of metallothionein was consistent with published values for the copper content of copper-thionein, i.e. 7 to 1. After 4 weeks of continuous exposure the concentrations of copper and hepatic metallothionein levelled off at concentrations that were a function of the copper concentration in the water. After the plateau or steady-state had been reached, the rate of loss of metallothionein was measured by determining the change in specific-activity of isotope with time after incorporation of 35S-L-cysteine or 3H-L-lysine into the protein. The half-life for loss of metallothionein was a function of the exposure to copper, being about 13 days for control fish and 30 days for fish exposed to the greatest concentration of copper. Salmon that had been exposed to copper for 8 weeks neither gained hepatic metallothionein in a further 4 week exposure to copper, nor lost it when transferred for 4 weeks to clean water. Tolerance to copper exhibited by fish that continued to be exposed to it was partially lost after 4 weeks depuration.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Copper; Fish Diseases; Kinetics; Liver; Metallothionein; Salmon; Water Pollution, Chemical