Cryptobia salmositica: susceptibility of infected rainbow trout, Salmo gairdneri, to environmental hypoxia

Using the sealed jar technique (also called residual oxygen bioassay), rainbow trout fry infected with Cryptobia salmositica were more susceptible than non-infected fish to environmental hypoxia. The Winkler technique (azide modification) was used to determine the residual dissolved oxygen in the water. Susceptibility of infected fish increased with 1) time after infection and was most evident in 3-7 wk infections, 2) the severity of anemia, and 3) increasing parasitemia. In prolonged infections, susceptibility was reduced when there were decreases in anemia and parasitemia; however, these infected fish were still more susceptible than non-infected fish. The increase in susceptibility of infected fish to hypoxia may be an important contributing factor to mortality of fish in hatcheries where there is inadequate water flow and overcrowding. The sealed jar technique is recommended in future studies on the pathogenesis of parasitic fish diseases, especially if the metabolic and/or respiratory systems are affected by the infection.     

The in vitro effects of isometamidium chloride (Samorin) on the piscine hemoflagellate Cryptobia salmositica (Kinetoplastida, Bodonina)

Isometamidium chloride (Samorin) is therapeutic in rainbow trout (Oncorhynchus mykiss) during preclinical and chronic cryptobiosis. However, the toxic mechanism of isometamidium on Cryptobia salmositica has not been elucidated. The objective of the present study was to examine the in vitro effects of isometamidium on C. salmositica. Under in vitro conditions, isometamidium chloride reduced the infectivity of C. salmositica suspended in whole fish blood. It accumulated rapidly in the kinetoplast (within 1 min) and caused disruption and decantenation of kinetoplast DNA. The in vitro cryptobiacidal activity of isometamidium was reduced when parasites were incubated in medium containing serum supplement, suggesting that isometamidium also binds to plasma proteins. Isometamidium altered glycoprotein receptors (epitopes) for antibodies on the surface of C. salmositica and thus protected some of the parasites from lysis by complement-fixing antibodies. In vitro oxygen consumption and carbon dioxide production decreased in drug-exposed C. salmositica, with increased products of glycolysis, i.e., lactate and pyruvate, after exposure to isometamidium. This suggests that some C. salmositica switched from aerobic respiration to glycolysis when the mitochondrion was damaged by isometamidium.     

Some observations of rainbow trout, Salmo gairdneri, Richardson, infected with Cryptobia salmositica

Rainbow trout ( Salmo gairdneri ) were infected with an inoculum of infected blood containing 10⁶ Cryptobia salmositica. Parasitaemia was cyclical with a major peak at 14–21 days and a smaller peak at 42 days. The first peak of parasitaemia was correlated with low glucose, plasma proteins, and haematocrit levels. During this time clinical symptoms (exophthalmia, ascites, pale liver and gills) of the pathogenic condition were observed. The partial disappearance of the parasites from the blood was corrrelated with their appearance in the peritoneal cavity. As plasma glucose, proteins, and haematocrit levels returned towards control values, the oedema and gill paleness declined. Examination of the liver and spleen showed an exudate of fibrous matrix on their exterior surfaces. In addition, the presence of 2–4×10³ mm⁻³ Cryptobia in asymptomatic surviving fish suggests that a chronic parasitaemia can be tolerated.     

In vitro attenuation of Cryptobia salmositica and its use as a live vaccine against cryptobiosis in Oncorhynchus mykiss

The present communication reports on the attenuation of a pathogenic hemoflagellate, Cryptobia salmositica Katz (Sarcomastigophora: Kinetoplastida) and its use as a live vaccine against cryptobiosis. The parasite was attenuated by continuous in vitro culture (at 10 C for 55 wk) in minimum essential medium. Attenuated (culture) forms are morphologically similar to virulent (blood) forms. They are however more slender and have a shorter anterior flagellum and a smaller nucleus and kinetoplast. The attenuated form returned to its normal size and multiplied when inoculated into naive Oncorhynchus mykiss. It produced a low parasitemia but did not cause disease (e.g., no exophthalmia or anemia) in fish. At four wk after infection, the vaccinated fish were challenged with the virulent parasite. They were protected from the disease, whereas the control (naive) fish, infected with only the virulent parasite, had the usual clinical signs (e.g., anemia, exophthalmia). No parasite was detected in any of 10 vaccinated fish at 22 wk after challenge with the virulent parasite. However, 5 of 9 fish infected with culture forms and 6 of 9 fish infected with blood forms still had detectable parasites at 26 and 22 wk after infection, respectively.     

In vitro effects of fetal bovine serum and glucose on multiplication of Cryptobia salmositica

Cryptobia salmositica multiplied rapidly at 10 C in a minimum essential medium (containing 1.0 mg glucose/ml, Hanks' salts and L-glutamine) supplemented with heat-inactivated fetal bovine serum (FBS) and HEPES buffer (25 mM). The multiplication rate of C. salmositica was related to the amount of FBS; the peak number (approximately 9 x 10(6) parasites/ml) was attained in about 6 wk when the medium contained 25% final concentration of FBS. Glucose utilization was related to the number of parasites; the maximum utilization was reached before peak numbers. Formation of rosette colonies was correlated with multiplication rate and numbers of parasites in cultures. Degenerate round forms found in old cultures probably were caused by the accumulation of metabolic wastes in the medium.     

Further observations on Cryptobia dahli (Mastigophorea: Kinetoplastida) parasitizing marine fish.

Studies were conducted primarily to ascertain the mode of transmission of Cryptobia dahli parasitizing the digestive tract of lumpfish (Cyclopterus lumpus). Another flagellate, morphologically similar to C. dahli, was also observed in the gut of a deepsea fish (Macrourus berglax). Several invertebrates, which are food for lumpfish, were examined for flagellates, but were neither infected nor showed evidence of cystic stages. Parasites were more abundant in the stomach, especially at about pH 5, than in other areas of the digestive tract. Transmission was achieved by pipetting the parasites into the stomach of uninfected fish, by feeding food contaminated with flagellates, and also by holding infected and uninfected fish in the same aquarium. In nature, lumpfish probably acquire parasites during winter when they aggregate and regurgitate into seawater because parasites can survive for short periods outside their host.     

Further Observations on Cryptobia dahli (Mastigophorea: Kinetoplastida) Parasitizing Marine Fish

Studies were conducted primarily to ascertain the mode of transmission of Cryptobia dahli parasitizing the digestive tract of lumpfish ( Cyclopterus lumpus ). Another flagellate, morphologically similar to C. dahli , was also observed in the gut of a deepsea fish ( Macrourus berglax ). Several invertebrates, which are food for lumpfish, were examined for flagellates, but were neither infected nor showed evidence of cystic stages. Parasites were more abundant in the stomach, especially at about pH 5, than in other areas of the digestive tract. Transmission was achieved by pipetting the parasites into the stomach of uninfected fish, by feeding food contaminated with flagellates, and also by holding infected and uninfected fish in the same aquarium. In nature, lumpfish probably acquire parasites during winter when they aggregate and regurgitate into seawater because parasites can survive for short periods outside their host.     

Effect of Cryptobia salmositica-induced anorexia on feeding behavior and immune response in juvenile rainbow trout Oncorhynchus mykiss

At 10 degrees C, rainbow trout Oncorhynchus mykiss (n = 13 per group) infected with Cryptobia salmositica Katz, 1951 became anorexic at 3 wk post-infection (w.p.i.), with feed-intake decreasing significantly from 1.33 to 0.94% body weight (b.w.). Anorexia was most severe at 4 w.p.i. (0.80% b.w.), coinciding with peak parasitemia (9.2 x 10(6) parasites ml blood(-1)) and anemia. At 8 w.p.i., fish had recovered their appetite although they still had contained detectable parasites (6.8 x 10(5) parasites ml(-1)) and were anemic (pack cell volume, PCV, of 24.4%). However at 5 degrees C, anorexia occurred at 5 w.p.i. (0.81% b.w.), and was most severe at 7 w.p.i. (0.40% b.w.). At 8 w.p.i. (0.43% b.w.), fish displayed high parasitemia (4.6 x 10(6) parasites ml(-1)) and low PCV (10.8%). Fish at 5 degrees C had lower gastric evacuation (GE) rates (GE48h) than 10 degrees C fish, however there were no differences between infected and naive fish at both temperatures. Before anorexia, there was no significant correlation between mean share of meal (MSM, a measure of how food was partitioned within a group) and coefficient of variation in feeding but this became significant during anorexia (p = 0.02 and p = 0.0002 at 10 and 5 degrees C respectively). Significant correlations were detected between b.w. and MSM before onset of anorexia at 10 degrees C (p = 0.005) and 5 degrees C (p = 0.02); this was maintained at 10 degrees C (p = 0.001) but not at 5 degrees C (p = 0.98). Fish on an anorexic diet (0.93% b.w.) responded well at 10 degrees C to a live C. salmositica vaccine; this could partly be due to constant antigenic stimulation by the live vaccine.     

Cryptobia cataractae from the blood of Semotilus atromaculatus: structure and division in the fish

A cryptobiid was found in the blood of 2 of 9 Semotilus atromaculatus from a tributary of the Saugeen River in Ontario, Canada. Blood inoculation produced an infection in 2 uninfected S. atromaculatus but not in any Oncorhynchus mykiss, Catostomus commersoni, or Carassius auratus. The flagellate was identified as Cryptobia cataractae, based on host restriction. Cryptobia cataractae occurred as slender and broad forms (body width 3.0-8.7 microns). The length of the anterior flagellum was equal to body length, whereas that of the free recurrent flagellum was half body length. Cryptobia cataractae divided by equal binary fission that produced elongate, slender daughter cells.     

Direct Transmission of a Hemoflagellate,Cryptobia salmositica (Kinetoplastida: Bodonina) Between Rainbow Trout Under Laboratory Conditions1

Transmission of Cryptobia salmositica occurred when infected and uninfected rainbow trout were held in the same tank. In tanks where infected and uninfected fish were allowed to mix freely, 67–80% of the uninfected fish acquired detectable infections by the 27th week. None of the control fish in another tank was infected. In another tank where the infected and uninfected fish were separated by a wire screen, 9 of 20 uninfected fish acquired detectable infections by the 22nd week. This is the first demonstration of direct transmission of a hemoflagellate via the water medium in aquatic vertebrates. Cryptobia salmositica was found in the mucus on the body surface of 9 of 10 fish examined 6 weeks after infection. These parasites were infective and some of them were morphologically similar to those in the blood or peritoneal fluid. It is suggested that the vascular species of Cryptobia were originally ectoparasitic on fishes and that these ectoparasitic species were descended from the free-living Procryptobia.     

Fatty acid beta-oxidation system in microbodies of n-alkane-grown Candida tropicalis.

Localization of fatty acid beta-oxidation system in microbodies of Candida tropicalis cells growing on n-alkanes was studied. Microbodies isolated from the yeast cells showed palmitate-dependent activities of NAD reduction, acetyl-CoA formation and oxygen consumption. When sodium azide, an inhibitor of catalase, was added to the system, palmitate-dependent formation of hydrogen peroxide was observed. Stoichiometric study revealed that two moles of NAD were reduced per one mole of oxygen consumed in the absence of sodium azide and the presence of the inhibitor doubled the oxygen consumption by microbodies without an appreciable change in NAD reduction. These results indicate that the yeast microbodies contain beta-oxidation system of fatty acid, and that catalase located in the organelles participates in the degradation of hydrogen peroxide to be formed at the step of dehydrogenation of acyl-CoA.     

Glucocorticoid receptors on and in a unicellular organism,Cryptobia salmositica

This is the first report to our knowledge that demonstrates a functional steroid hormone receptor in a protozoon. The study used Cryptobia salmositica, a pathogenic haemoflagellate found in salmonid fishes. It has been previously shown that cortisol and dexamethasone (a synthetic glucocorticoid) enhanced the multiplication of C. salmositica under in vitro conditions indicating the presence of glucocorticoid receptors on/in the parasite. Also, the glucocorticoid receptor antagonist, mifepristone (RU486), inhibited the stimulatory effect of the two glucocorticoids on parasite multiplication. In the present study, we used an antibody (produced in a rabbit against glucocorticoid receptor protein) agglutination test and confocal microscopy with immunohistofluorescence staining to demonstrate cortisol-glucocorticoid receptor-like protein receptors on the plasma membrane and in the cytoplasm of the parasite. In two in vitro studies, the addition of 50 ng ml⁻¹ of RU486 was more effective in inhibiting parasite replication in cultures with 7,000 parasites ml⁻¹ than in cultures with 14,000 parasites ml⁻¹. Also, 100 ng ml⁻¹ of RU486/ml was more effective than 50 ng ml⁻¹ in inhibiting parasite multiplication in the 14,000 parasites ml^-1 cultures. These in vitro studies indicate that the number of binding sites on/in the parasite is finite. The findings may be important in future studies especially on steroid receptor signalling pathways and dissection of ligand–receptor interactions, and for evaluating the adaptations that develop in pathogens as part of the host–parasite interaction.     

Histopathological changes due to infections with Cryptobia iubilans Nohýnková 1984, in two cichlid fishes

Histopathological study of infections with Cryptobia iubilans Nohýnková 1984 in two species of freshwater aquarium cichlids (Herichthys cyanoguttatum and Cichlasoma meeki) revealed severe tissue changes which were not limited to the digestive tract but also affected other principal body organs. The lesions were extensive enough to cause mortalities C. iubilans is thus far the only clearly pathogenic species among cryptobias infecting fish.     

Farnesol-induced generation of reactive oxygen species dependent on mitochondrial transmembrane potential hyperpolarization mediated by F(0)F(1)-ATPase in yeast

An isoprenoid farnesol (FOH) inhibited cellular oxygen consumption and induced mitochondrial generation of reactive oxygen species (ROS) in cells of Saccharomyces cerevisiae in correlation with hyperpolarization of the mitochondrial transmembrane potential (mtDeltaPsi). The FOH-induced events were coordinately abolished with the F(1)-ATPase inhibitor sodium azide as well as the F(0)F(1)-ATPase inhibitor oligomycin, suggesting the dependence of ROS generation on mtDeltaPsi hyperpolarization mediated by the proton pumping function of F(0)F(1)-ATPase as a result of ATP hydrolysis. The role of F(1)-ATPase activity in mtDeltaPsi hyperpolarization was supported by the intracellular depletion of ATP in FOH-treated cells and its protection with sodium azide. An indirect mechanism was suggested to exist in the regulation of F(0)F(1)-ATPase by FOH to accelerate its ATP-hydrolyzing activity.     

In vitro nutritional requirements and metabolic products of pathogenic and nonpathogenic strains of Cryptobia salmositica: essential carbohydrates and amino acids

Pathogenic and nonpathogenic strains of Cryptobia salmositica cultured in minimum essential medium (MEM) with several monosaccharides, disaccharides and amino acids were observed for differences in multiplication and motility. Metabolic end products (i.e. alanine, aspartate, carbon dioxide, lactate and pyruvate) were measured for logarithmically growing cells under aerobic conditions. The pathogenic strain of C. salmositica multiplied more readily in MEM supplemented with D(-)ribose, D(+)xylose, D(+)galactose, D(+)glucose, D(+)mannose and D(-)fructose. However, there were no significant differences in multiplication when the strains were cultured with the monosaccharide D(-)arabinose. The nonpathogenic strain multiplied significantly better than the pathogenic strain in the presence of the disaccharides alpha-lactose, maltose and sucrose. It also multiplied more readily when the amino acids L-glutamine and D(-)proline were added to MEM. The end products of carbohydrate catabolism under aerobic conditions were alanine, aspartate, carbon dioxide, lactate and pyruvate.     

Identification of glycosomes and metabolic end products in pathogenic and nonpathogenic strains of Cryptobia salmositica (Kinetoplastida: Bodonidae)

Whole cell lysates of pathogenic and nonpathogenic strains of Cryptobia salmositica were subjected to subcellular fractionation using differential and isopycnic centrifugation in sucrose. The glycolytic enzymes hexokinase, fructose-1,6-biphosphate aldolase, triosephosphate isomerase, glucosephosphate isomerase and glyceraldehyde-3-phosphate-dehydrogenase and the peroxisomal enzyme catalase were associated with a microbody that had a buoyant density in sucrose of 1.21 g cm-3. Lactate dehydrogenase was detected in whole cell lysates, but not in purified organelles. A microbody with a positive reaction for catalase was detected in electron microscope sections of the pathogenic and nonpathogenic strains. These catalase-containing microbodies fused with lipid bodies and vacuoles, arose by division from pre-existing microbodies and expelled their contents into the cytoplasm of the cell. Both strains also modified the catalase content in their microbodies. Under aerobic conditions, they metabolized glucose to pyruvate and lactate. We conclude that part of the glycolytic pathway in C. salmositica is compartmentalized in a microbody called the glycosome.     

Morphology and Ultrastructure of Cryptobia eilatica n. sp. (Bodonidae: Kinetoplastida), an Ectoparasite from the Gills of Marine Fish

ABSTRACT. A marine kinetoplastid flagellate, Cryptobia eilatica n. sp., is described from the gills of cultured gilt-head sea bream Sparus aurata L. and wild black-spot sea bream Diplodus noct (Valenciennes) in the Red Sea. The trophozoite is elongated and lacks a contractile vacuole and undulating membrane. The body averages 13.5 × 4.1 μm, anterior flagellum 9.7 μm, and free portion of recurrent flagellum 15.2 μm. The ultrastructural features of the species exhibit great similarity to various previously studied Cryptobiids. Cryptobia eilatica trophozoites feed on bacteria, show a preference for the branc hial interlamellar crypts, and attach to the host epithelium by means of the recurrent flagellum. Neither penetration into the epithelial cells, nor any direct damage to host tissue was observed. Cryptobia eilatica inhabits a purely marine habitat, but its trophozoite tolerates salinities as low as 10 ppt.     

Extracellular tyrosinase from the fungus Trichoderma reesei shows product inhibition and different inhibition mechanism from the intracellular tyrosinase from Agaricus bisporus

Tyrosinase (EC 1.14.18.1) is a widely distributed type 3 copper enzyme participating in essential biological functions. Tyrosinases are potential biotools as biosensors or protein crosslinkers. Understanding the reaction mechanism of tyrosinases is fundamental for developing tyrosinase-based applications. The reaction mechanisms of tyrosinases from Trichoderma reesei (TrT) and Agaricus bisporus (AbT) were analyzed using three diphenolic substrates: caffeic acid, L-DOPA (3,4-dihydroxy-l-phenylalanine), and catechol. With caffeic acid the oxidation rates of TrT and AbT were comparable; whereas with L-DOPA or catechol a fast decrease in the oxidation rates was observed in the TrT-catalyzed reactions only, suggesting end product inhibition of TrT. Dopachrome was the only reaction end product formed by TrT- or AbT-catalyzed oxidation of L-DOPA. We produced dopachrome by AbT-catalyzed oxidation of L-DOPA and analyzed the TrT end product (i.e. dopachrome) inhibition by oxygen consumption measurement. In the presence of 1.5mM dopachrome the oxygen consumption rate of TrT on 8mM L-DOPA was halved. The type of inhibition of potential inhibitors for TrT was studied using p-coumaric acid (monophenol) and caffeic acid (diphenol) as substrates. The strongest inhibitors were potassium cyanide for the TrT-monophenolase activity, and kojic acid for the TrT-diphenolase activity. The lag period related to the TrT-catalyzed oxidation of monophenol was prolonged by kojic acid, sodium azide and arbutin; contrary it was reduced by potassium cyanide. Furthermore, sodium azide slowed down the initial oxidation rate of TrT- and AbT-catalyzed oxidation of L-DOPA or catechol, but it also formed adducts with the reaction end products, i.e., dopachrome and o-benzoquinone.     

The Effect of Respiratory Inhibitors and Chelating Agents on the Frequencies of Chromosomal Aberrations Produced by X-Rays in Vicia

Nitrosophenylhydroxylamine-ammonium (cupferron), potassium cyanide, sodium azide, ethylenediaminetetraacetate (EDTA), α,α'-dipyridyl, and o-phenanthroline were tested (1) for their ability to enhance the frequencies of chromosomal aberrations produced by x-rays in the root tip cells of the broad bean, Vicia faba, and (2) for their ability to inhibit oxygen consumption of excised roots of the same plant. In all cases a close correlation was found between the inhibitory effect on respiration and the enhancement of the sensitivity to x-rays at low oxygen pressures. EDTA, dipyridyl, and o-phenanthroline did not affect respiration to any greater extent, and they were without influence on the radiosensitivity. Cyanide, azide, and cupferron, which strongly inhibited respiration, also increased the frequencies of chromosome aberrations produced by x-rays at low oxygen pressures. The relation between oxygen concentration and radiosensitivity was determined both in the presence and the absence of the respiratory inhibitor cupferron. When cupferron was present, the radiosensitivity was influenced by oxygen concentrations 30 times lower than those effective in the absence of the inhibitor. In an atmosphere of pure oxygen, an increase of radiosensitivity of about 20 per cent was obtained with cupferron, EDTA, and potassium cyanide.     

Measurements of oxygen consumption in Mytilus edulis during exposure to, and recovery from, high sublethal concentrations of formaldehyde, benzene and phenol.

1. The rate of oxygen consumption has been monitored continuously in M. edulis during acute exposure to high sublethal concentrations of formaldehyde, phenol and benzene and subsequent recovery periods of 96 hr. 2. The results are discussed in relation to changes in the electrochemical potential difference of sodium, the content of ATP and the tissue concentration of strombine. 3. After exposure to benzene and phenol, an increase in the rate of oxygen consumption that could not be explained by oxygen debt from the exposure period was observed. 4. Depression of the rate of oxygen consumption after exposure to formaldehyde may be explained by a reduced ability to extract oxygen from the water. 5. The pattern of oxygen consumption and behavioural responses, as well as the combined changes in the biochemical markers, were distinctly different in the three cases.