Selected parasitosis in cultured and wild fish.

While intensive aquaculture has and will continue to supply the ever growing population with highly nutritious protein, it also comes with problems which include more frequent outbreaks of diseases in fish farms and transmission of diseases between farmed and wild fish. We have selected four Phyla of economically important fish parasites for our present discussion-a haemoflagellate (Cryptobia salmositica), a microsporidian, (Loma salmonae), a monogenean (Gyrodactylus salaries) and two copepods (Lepeophtheirus salmonis, Caligus rogercresseyi). This review consists of two parts with a brief description of each parasite and its biology related to transmission, followed by discussions on epizootic outbreaks in both wild and farmed fish, interactions between wild and farmed fish, and disease prevention and control.

Protective immunity in fish against protozoan diseases.

The demand for and costs of producing land-based animal protein continues to escalate as the world population increases. Fish is an excellent protein, but the catch-fishery is stagnant or in decline. Intensive cage culture of fish is a viable option especially in countries with lakes/rivers and/or a long coastline; however, disease outbreaks will likely occur more frequently with cage culture. Hence protective strategies are needed, and one approach is to exploit the piscine immune system. This discussion highlights immunity (innate/natural and adaptive/acquired) in fish against three pathogenic protozoa (Amyloodinium ocellatum, Ichthyophthirius multifiliis and Cryptobia salmositica). Histone-like proteins in the mucus and skin of naturally resistant fish kill trophonts of A. ocellatum, and also may cause abnormal development of tomonts. Breeding of Cryptobia-resistant brook charrs is possible as resistance is controlled by a dominant Mendelian locus, and the parasite is lysed via the Alternative Pathway of Complement Activation. Production of transgenic Cryptobia-tolerant salmon is an option. Recovered fish are protected from the three diseases (acquired immunity). Live I. multifiliis theronts injected intraperitoneally into fish elicit protection. Also, a recombinant immoblizing-antigen vaccine against ichthyophthirosis has been developed but further evaluations are necessary. The live Cryptobia vaccine protects salmonids from infections while the DNA-vaccine stimulates production of antibodies to neutralize the disease causing factor (metalloprotease) in cryptobiosis; hence infected fish recover more rapidly.

Humoral response and susceptibility of five full-sib families of Atlantic salmon, Salmo salar L., to the haemoflagellate, Cryptobia salmositica.

Susceptibility and antibody production against pathogenic and vaccine strains of the haemoflagellate, Cryptobia salmositica were investigated in five full-sib families (A-E) of Atlantic salmon, Salmo salar. Humoral response and susceptibility of families were compared within three treatments: infection, vaccination and vaccination followed by challenge. Parasitaemias caused by the vaccine strain of C. salmositica were considerably lower than those caused by the pathogenic strain. All vaccinated families were protected when challenged with the pathogenic strain. Family B had significantly lower parasitaemias (with both strains) than the other families. When naïve fish were infected with the pathogenic strain, this family had a significantly lower and earlier peak parasitaemia (4.3 +/-1.3 x 10(6) parasites mL(-1) blood at 3 weeks post-infection; w.p.i.) than the other families. Family C had the highest peak (11.1 +/- 1.2 x 10(6) parasites mL(-1) blood), which occurred at 4 w.p.i. Antibodies against C. salmositica were detected earlier in Family B (3 w.p.i.) than in Family C (5 w.p.i.). This demonstrates an association of increased susceptibility with a delayed antibody response. Western immunoblot identified antibodies against 112, 181 and 200 kDa antigens earlier in more resistant fish (Family B). Antigenic stimulation leading to a stronger antibody response was shown with the vaccine strain and in the later stages of infection.

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.

Cryptobia (Trypanoplasma) salmositica and salmonid cryptobiosis.

Salmonid cryptobiosis is caused by Cryptobia (Trypanoplasma) salmositica. The haemoflagellate has been reported from all species of Pacific Oncorhynchus spp. on the west coast of North America. It is normally transmitted by the freshwater leech, Piscicola salmositica, in streams and rivers, and sculpins, Cottus spp., are considered important reservoir hosts. The pathogen can also survive on the body surface of fish because it has a contractile vacuole to osmoregulate when the fish is in fresh water. This allows for direct transmission between fish, especially in aquaculture facilities. The parasite divides rapidly by binary fission in the blood to cause disease, the severity of which is directly related to parasitaemia. Cryptobia salmositica has a mitochondrium and it normally undergoes aerobic respiration; however, if its mitochondrium is damaged it will switch to glycolysis. Its glycolytic enzymes and catalase are contained in glycosomes. Cysteine protease is a metabolic enzyme, and its neutralization inhibits oxygen consumption and multiplication of the parasite. An important virulent factor in cryptobiosis is a secretory metalloprotease. The protective mechanism involves production of complement fixing antibodies, phagocytosis by macrophages, and cell-mediated cytotoxicity. Recovered fish are protected, probably for life as the immunity is non-sterile. Clinical signs of the disease include anaemia, anorexia, splenomegaly, general oedema and abdominal distension with ascites. The metabolism and swimming performance of infected fish are significantly reduced and the bioenergetic cost of the disease is very considerable. Fish are susceptible to hypoxia and their immune system is depressed during acute cryptobiosis. Severity of the disease and mortality rates vary significantly between species and stocks of salmon. Protective strategies include selective breeding of Cryptobia-resistant fish. This is innate resistance to infection and it is controlled by a dominant Mendelian locus. In these fish the parasite is lysed via the alternative pathway of complement activation. In Cryptobia-tolerant fish (infected with the pathogen but which do not suffer from disease) the metalloprotease secreted by the parasite is neutralized by alpha2 macroglobulin. Hence, the production of a transgenic Cryptobia-tolerant salmon is an option. This strategy has the advantage in that human intervention (e.g. vaccination, chemotherapy) is not required once the transgenic fish is produced. Acquired immunity is another option; a single dose of the attenuated live vaccine protects fish for at least 2 years. The protective mechanism in vaccinated fish is similar to that in recovered fish. The trypanocidal drug, isometamidium chloride, is an effective therapeutic and prophylactic agent. It accumulates in the mitochondrium of the parasite and significantly disrupts aerobic respiration by causing lesions in the organelle. Efficacy of the drug is significantly increased after its conjugation to antibodies. This immuno-chemotherapeutic strategy has the advantage in that it will lower the drug dosage and hence side-effects of chemotherapy. It will probably reduce the accumulation of the drug in fish, an important consideration in food fish.

In vitro secretion of metabolic end-products by piscine haemoflagellates Cryptobia salmositica and C. bullocki (Kinetoplastida: Bodonidae) and the relationship of these products to the pH in the medium.

Pathogenic and nonpathogenic strains of Cryptobia salmositica Katz, 1951 and C. bullocki Strout, 1965 produced hydrogen peroxide, pyruvate and lactate under in vitro conditions in Minimum Essential Medium (MEM). As parasite number increased, the phenol red in the medium changed from red to yellow. This change was not associated with a decrease in pH, or an increase in pyruvate or lactate, but was correlated with an increased secretion of hydrogen peroxide. Parasites incubated at 10 degrees C in medium at pH 6.0, 6.5, 7.0 and 7.3 were active for about I week with decreasing activity in the absence of serum. Parasites in saline (pH 6.0, 6.5, 7.0 and 7.3) were nonmotile within 24 h and were dead in about 1 week. This suggests that these Cryptobia spp. are sensitive to changes in pH and require medium which is buffered, either with serum or Hepes.

Cryptobiosis and its control in North American fishes.

Cryptobiosis is caused by the haemoflagellates Cryptobia bullocki and Cryptobia salmositica. These parasites infect food fishes (e.g. flounders, salmon) on both the Atlantic and Pacific coasts of North America and clinical signs of the disease include anaemia, and abdominal distention with ascites. The virulent factor in salmonid cryptobiosis, caused by C. salmositica, is a secretory metalloprotease (200 kDa). Fish mortality may be up to 100% in the absence of treatment, consequently strategies have been developed to protect them from disease/mortality. A single dose of a live vaccine protects fish for at least 2 years, and it is via the production of complement-fixing antibodies, enhanced phagocytosis and cell-mediated cytotoxicity. Inhibition of the parasite's cysteine protease by a monoclonal antibody reduces multiplication, infectivity and survival of the parasite. Consequently, the recombinant cysteine protease (49 kDa) of the parasite will be tested as a potential vaccine. The trypanocidal drug, isometamidium chloride (1.0 mg/kg), is effective (therapeutic and prophylactic) against C. salmositica in chinook salmon. Its efficacy is significantly enhanced if it is conjugated either to a monoclonal antibody or to polyclonal antibodies from immune fish. Selective breeding of Cryptobia-resistant brook charr (innate resistance to infection) is possible, and the resistant factor(s) is controlled by a dominant Mendelian locus. In these resistant charr the parasite is lysed via the alternate pathway of complement activation (innate immunity to infection). There are also Cryptobia-tolerant charr, fish that are susceptible to infection but have no clinical disease (innate resistance to disease). In these fish, one of the natural anti-proteases, alpha2-macroglobulin, neutralises the metalloprotease secreted by C. salmositica. Production of transgenic Cryptobia-tolerant salmon is an option to vaccination and or chemotherapy. Also, transgenic pathogen-tolerant animals may be an alternate strategy against other pathogens where the disease mechanism is similar to cryptobiosis.

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.

Therapeutic and prophylactic effects of isometamidium chloride (Samorin) against the hemoflagellate Cryptobia salmositica in chinook salmon (Oncorhynchus tshawytscha) and the effects of the drug on uninfected rainbow trout (O. mykiss).

A series of compounds (triphenylmethanes, thiazines, xanthenes, benzidines, phenanthridiniums, napthalamines, and diamidines) were screened for in vitro toxicity against Cryptobia salmositica. Isometamidium chloride (Samorin) was cryptobiacidal at low concentrations and was examined for therapeutic and prophylactic activities against C. salmositica in chinook salmon (Oncorhynchus tshawytscha). An intramuscular dose (1.0 mg/kg) of Samorin 3 weeks post-infection significantly reduced the parasitemia in adult chinook. A higher dose (2.5 mg/kg) eliminated the infection in 30% of adult fish and parasitemias were significantly reduced in the remaining infected fish. Juvenile chinook treated with 1.0 mg Samorin/kg at 2-3 weeks post-infection survived, while 100% of untreated control fish died from cryptobiosis. The high dose (2.5 mg/kg) was lethal to small fish (98.93 +/- 12.09 g) and 50% died within 24 h of treatment, while all large fish (168.38 +/- 13.87 g) survived. Samorin (1.0 mg/kg) did not affect growth, food consumption, complement, or hematocrit values in uninfected rainbow trout (O. mykiss).

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.

An antigen-capture enzyme-linked immunosorbent assay (ELISA) to detect isometamidium chloride in Oncorhynchus spp.

An antigen-capture enzyme-linked immunosorbent assay (ELISA) was developed to detect and measure isometamidium chloride in the plasma of Oncorhynchus tshawytscha and O. mykiss. Isometamidium-ovalbumin conjugate and anti-isometamidium antibodies were used to coat polystyrene plates. The peroxidase saturation technique was used to optimize the coating antigen concentration; it demonstrated low affinity of the isometamidium-ovalbumin conjugate but high affinity of the anti-isometamidium antibodies for polystyrene surface sites. The optimal conditions of antiisometamidium antibodies to coat plates was at pH 7.3 and a 1:1000 dilution (0.0012 mg ml(-1) protein). The ELISA was sensitive as it detected 0.0006 mg ml(-1) of isometamidium in fish plasma. Isometamidium diluted with saline could not be detected at concentrations less than 0.05 mg ml(-1). The results indicate that this ELISA is much more sensitive when isometamidium is bound to plasma than unbound isometamidium in saline.

The therapeutic use of isometamidium chloride against Cryptobia salmositica in rainbow trout Oncorhynchus mykiss.

Rainbow trout Oncorhynchus mykiss injected intramuscularly with isometamidium chloride (0.01 or 0.1 mg kg-1) at 3 wk post-infection and given a booster 2 wk later had significantly lower parasitaemias than infected controls. Packed cell volume increased after treatment and remained higher than in infected controls. The concentration of isometamidium in plasma was highest at 2 wk after injection and then declined. An intramuscular dose of 1.0 mg kg-1 of isometamidium chloride at 1, 2 and 3 wk postinfection (preclinical) significantly reduced the parasitaemia in rainbow trout 2 wk after treatment. A booster at 9 wk postinfection (chronic disease phase) reduced the parasitaemia further in all fish. The packed cell volume in these fish was higher than in infected controls. Treatment at 5, 6, and 7 wk postinfection (acute disease) had no effects and parasitaemias in treated fish were higher than in infected controls; also, anti-Cryptobia salmositica antibodies and titres of complement-fixing antibody were higher in these than in infected controls. Incubation of immune plasma or complement with isometamidium for 3 h did not affect the lytic titres of complement-fixing antibodies nor rainbow trout complement.

The in vitro effects of crystal violet on the pathogenic haemoflagellate Cryptobia salmositica Katz, 1951 (Sarcomastigophora: Kinetoplastida).

Crystal violet does not inhibit in vitro multiplication of a nonpathogenic strain of Cryptobia salmositica at low concentrations (0.01 microM and 0.001 microM) but multiplication is inhibited at higher concentrations (> or = 0.05 microM). In contrast, the pathogenic strain of C. salmositica does not multiply in vitro when incubated with crystal violet (0.001 microM, 0.01 microM and 0.05 microM). The infectivity of the pathogenic strain is significantly reduced after in vitro exposure to crystal violet. Crystal violet lyses C. salmositica (100.0 microM) and causes lesions on mitochondrial and nuclear membranes of the parasite. Pathogenic strains of Cryptobia salmositica and C. bullocki are more susceptible to lysis after in vitro exposure to crystal violet than are nonpathogenic strains of Cryptobia salmositica and C. catostomi.

Identification of carbohydrates on the surface membrane of pathogenic and nonpathogenic piscine haemoflagellates, Cryptobia salmositica, C. bullocki and C. catostomi (Kinetoplastida).

Carbohydrates and protein glycoconjugates on the cell membranes of Cryptobia salmositica, C. bullocki and C. catostomi were analyzed using 13 highly purified lectins (unlabelled or digoxigenin/biotin labelled). No agglutinations were observed with C. salmositica, C. bullocki and C. catostomi using lectin TPA (Tetragonolobus purpureas agglutinin, for alpha-L-fucose). C. salmositica was agglutinated by 3 of 12 lectins [Con A, for alpha-man and alpha-D-glc; PSA, for alpha-man; PWM, for (glcNAc)3], while C. bullocki was agglutinated by 8 lectins and C. catostomi was agglutinated by 10 lectins. Glycoconjugate analysis with digoxigenin or biotin labelled lectins showed a species-specific staining pattern in pathogenic and nonpathogenic Cryptobia spp. The nonpathogenic C. catostomi had the strongest reaction. These results indicate that the surface carbohydrate residues and glycoconjugate compositions on Cryptobia spp. are different between species they may be related to the virulence of the parasite.

Characterization of a 200 kDa glycoprotein (Cs-gp200) on the pathogenic piscine haemoflagellate Cryptobia salmositica.

The 200 kDa antigenic doublet of the pathogenic haemoflagellate Cryptobia salmositica Katz, 1951, was detected using a monoclonal antibody (MAb-001) in 1-dimensional SDS-PAGE. This antigenic doublet is a glycoprotein since it was susceptible to both protease K and to sodium m-periodate oxidation and is designated Cs-gp200. It has a PI (isoelectric point) value of about 5.5 (using 2-dimensional gel electrophoresis). It migrated faster under reducing conditions than under nonreducing conditions and was partitioned into the aqueous phase in Triton X-114 phase separation. It is a secretory-excretory product since it was detected in a non-protein culture medium with C. salmositica. These results suggest that the Cs-gp200 is a glycoprotein consisting of carbohydrate determinants and conformational polypeptide with internal disulphide bonds. It is a hydrophilic antigen, is a secretory product of the parasite, and plays a important role in antibody elicitation in immunized fish.

Characterization of purified metallo- and cysteine proteases from the pathogenic haemoflagellate Cryptobia salmositica Katz 1951.

The purified metalloprotease and the partially purified cysteine protease from pathogenic Cryptobia salmositica were characterized. Using haemoglobin gel electrophoresis, we detected five enzymatic bands in crude parasite lysate; one protease (200 kDa) yielded a metalloprotease band and other four, cysteine protease bands (97, 70, 66 and 49 kDa). Both the metalloprotease and the cysteine protease had high levels of proteolytic activity against azocasein, haemoglobin and fibrinogen. The metalloprotease had high levels of activity against azocoll and gelatin but a low degree of activity against albumin. In contrast, the cysteine protease had extensive activity against albumin but low levels of activity against azocoll and gelatin. The metallo- and cysteine proteases had no activity against Pz-peptide, a specific substrate for bacterial collagenase. The optimal pH for the metalloprotease and the cysteine protease was 7.0 and 5.0, respectively. The metalloprotease was inhibited by metal-chelating agents and excess of zinc ions but was activated by calcium ions. The cysteine protease was inhibited by thiol-blocking agents. The natural antiprotease alpha2-macroglobulin, but not alpha1-protease inhibitor, inhibited the activity of both proteases from C. salmositica. The optimal in vitro temperature for the purified metalloprotease was 30 degrees C.

Biochemical characterisation of an epitope on the surface membrane antigen (Cs-gp200) of the pathogenic piscine haemoflagellate Cryptobia salmositica Katz 1951.

A protective surface antigen (200 kDa) on C. salmositica was detected using a monoclonal antibody (mAb-001). Enzymatic studies on the epitope indicated that it was sensitive to nonspecific protease K and to site-specific trypsin and protease V8 but not to alpha-chymotrypsin. The reactivity of the epitope with mAb-001 was not affected when the antigen was denatured with 8 M urea; however, reduction of the antigen with dithiothreitol destroyed the epitope. The epitope was susceptible to sodium m-periodate oxidation and N-glycosidase F, but not to O-glycosidase or neuraminidase. It was also sensitive to mild potassium hydrochloride hydrolysis and to phospholipase C, which is specific for phosphatidylinositol. These results suggest that the epitope consists of a polypeptide, a carbohydrate, and probably a phospholipid. The asparagine-bound N-glycosidically linked hybrid-type carbohydrate chain has the minimum length of a chitobiose core unit. There is probably a phosphatidylinositol residue which anchors the polypeptide to the surface membrane. The antigen is extensively posttranslationally modified.

Improved culture media for piscine hemoflagellates, Cryptobia and Trypanosoma (Kinetoplastida).

Increasing the Hepes buffer in minimum essential medium from 25 mM to 100 mM yielded a significantly larger number of Cryptobia salmositica. Cryptobia salmositica (pathogenic and nonpathogenic strains), Cryptobia bullocki, and Trypanosoma danilewskyi did not multiply either in heat-inactivated trout plasma (< or =25%) or in less than 10% fresh trout plasma. Both strains of C. salmositica multiplied better in 10% fresh trout plasma than in 25% heat-inactivated fetal bovine serum. In contrast, C. bullocki and T. danilewskyi multiplied better in 25% fetal bovine serum; 10% fetal bovine serum did not significantly reduce multiplication of C. bullocki. The nonpathogenic vaccine strain of C. salmositica cultured in 10% fresh trout plasma still protected rainbow trout from high parasitemia when challenged with the pathogen.

Protection against Cryptobia (Trypanoplasma) salmositica and Salmonid Cryptobiosis.

Cryptobia (Trypanoplasma) salmositica is a haemoflagellate that causes morbidity and mortality in salmon, Oncorhynchus spp, on the Pacific coast of North America. In this review, Patrick Woo briefly describes the pathogen, its transmissions (either indirectly via its leech vector, Piscicola salmositica, or directly between fish) and the clinical signs of the disease. He then outlines strategies that have been developed to protect fish against the pathogen, and the mechanism of innate resistance to disease in Cryptobia-tolerant fish. Protective strategies include the breeding of fish that are resistant to infection, and the use of an attenuated C. salmositica strain to protect susceptible fish from disease for at least two years. He ends the review with suggestions for further research that include the use of the leech vector to deliver the vaccine and the development of more novel protective strategies (eg. immunochemotherapy, anti-idiotype vaccine) against cryptobiosis.