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.     

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.     

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.     

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.     

Trypanoplasma salmositica: experimental infections in rainbow trout, Salmo gairdneri.

Trypanoplasma salmositica was successfully cultured in Hanks' medium with 10% heated fetal calf serum. The culture forms were morphologically similar to blood forms and were infective to rainbow trout by inoculation. T. salmositica produced a disease in experimentally infected rainbow trout (Salmo gairdneri). The clinical signs were anemia, exophthalmia, abdominal distension with ascites, and splenomegaly. These clinical signs were observed in fish that were infected with three substrains (field substrain, cultured substrain, and cloned substrain) thus satisfying Koch's postulates. The anemia was microcytic and hypochromic and was coincident with increasing parasite number in the blood. The hemoglobin in the infected fish dropped from a normal of about 6 g% to about 1 g% in the first 10 weeks postinfection. Similarly, the hematocrit and red cell count declined as the infection progressed. Abdominal distension and exophthalmia was obvious 10 weeks postinfection. Up to 5 ml ascites fluid were recovered from each of three fish. The fluid contained large numbers of Trypanoplasma and macrophages. Some of the macrophages were engulfing the Trypanoplasma. At about this time the spleen in the infected fish was enlarged 5 to 10 times over that of control fish. The hematocrit centrifuge technique was less sensitive than wet mount examinations for the detection of the organism in blood. Fluctuations in parasite number during the course of infection may be due to antigenic change by the parasite to evade the host immune system.     

In vitro oxygen consumption and motility of Cryptobia salmositica, Cryptobia bullocki, and Cryptobia catostomi (Sarcomastigophora: Kinetoplastida).

Cryptobia salmositica (pathogenic and vaccine strains), Cryptobia bullocki (pathogenic), and Cryptobia catostomi (nonpathogenic) have similar oxygen consumption rates (0.17 +/- 0.01 nm O2/10(6) parasites). Incubation with sodium azide (5 microliters of a 1-M solution to 1 ml of parasite suspension, i.e., a 5-mM final concentration) reduced the oxygen consumption by approximately 4.5-fold. Motility of the parasites was also greatly reduced in sodium azide. The oxygen consumption and motility of the parasites returned to preazide treatment levels when the azide was removed even after 24 hr of incubation in sodium azide. The activities of hexokinase, pyruvate kinase, and cytochrome C oxidase were not detected in the 3 species of Cryptobia.     

Relations between histopathology and parasitaemias in Oncorhynchus mykiss infected with Cryptobia salmositica, a pathogenic haemoflagellate

Ichthyophthirius multifiliis is a ciliated protozoan parasite that infects the skin and gills of freshwater fish. This report describes the unusual finding of I. multifiliis within the peritoneal cavities of experimentally infected channel catfish Ictalurus punctatus. Twenty catfish fingerlings were exposed to I. multifiliis theronts using a standardized protocol. Five infected fish and 2 control fish were killed at various time points after infection and their tissues examined. Formalin-fixed, paraffin embedded sections were processed for light microscopy and immunohistochemical detection of I. multifiliis immobilization antigen. Trophonts were observed in skin and gill sections of all exposed fish. Parasites were associated with epithelial hyperplasia, focal areas of cellular disruption and necrosis. In addition to these usual sites of infection, individual trophonts were unexpectedly found within the peritoneal cavities of 4 fish. Staining for parasite antigen facilitated their detection within abdominal adipose tissue or adjacent to intestines. This discovery is interesting as it suggests I. multifiliis may be found in tissues other than the skin and gills during the course of a normal infection.     

Experimental infections of Atlantic salmon Salmo salar with Spironucleus barkhanus

Atlantic salmon Salmo salar L. (Salmonidae) were experimentally infected with Spironucleus barkhanus (Diplomonadida: Hexamitidae). Parasites were found in the blood 1 to 8 wk after infection, after which they disappeared from the blood and were found mainly in the internal organs (e.g. spleen and liver), eye socket or muscles. Mortality (38 out of 40 infected fish) occurred when fish had lesions in internal organs and/or on the body surface. Uninfected fish cohabiting with infected fish became infected after 4 wk, indicating direct transmission. There was no difference in susceptibility to spironucleosis between 3 different families of Atlantic salmon. All families developed the disease with a similar pattern of parasitaemia in the blood, similar clinical signs and gross pathology, and with very high mortality (29 out of 30). Clinical signs of systemic spironucleosis may include anemia, skin blisters, muscle ulcerations or unilateral exophthalmia. Gross pathologies include hemorrhaging of internal organs, splenomegaly or deformed (globulated) spleen, or granulomatous lesions in the spleen and liver.     

The stress-response of rainbow trout to experimental infection with the blood parasite Cryptobia salmositica Katz, 1951

Physiological responses of rainbow trout, Salmo gairdneri Richardson, to experimental infection with the blood haemoflagellate Cryptobia salmositica were monitored for 7 weeks to determine whether the parasitaemia elicited a classical stress-response as evidenced by elevated plasma cortisol and glucose levels. There was a progressive anaemia, hepatomegaly, splenomegaly, and depressed plasma T3, T4, protein and glucose concentration, and lowered liver glycogen content in the parasitized fish throughout the study, which clearly indicated that the animals were under physiological duress. Moreover, these hormonal and metabolic indicators showed no indication of recovery with the decline in blood parasite numbers. However, there were no differences in plasma cortisol or glucose levels between infected and control fish throughout the study, suggesting that the parasitaemia did not activate the pituitary-interrenal axis.     

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.     

Detection of infection and susceptibility of different Pacific salmon stocks (Oncorhynchus spp.) to the haemoflagellate Cryptobia salmositica

The haematocrit centrifugation technique, modified by keeping the haematocrit tubes cold (between 1 and 10 C), was sensitive for detecting light infections of Cryptobia salmositica (as few as 75 flagellates per ml of blood). In wet mount preparations, infections lighter than 7.5 X 10(3) flagellates per ml of blood could not be detected consistently. Different Pacific salmon stocks from British Columbia demonstrated differences in susceptibility to C. salmositica in experimental studies using laboratory reared juvenile fish. Oncorhynchus keta and Oncorhynchus tshawytscha from the Big Qualicum River stocks (Vancouver Island), and Oncorhynchus nerka from the Fulton River stock (Skeena River system), were all equally susceptible and suffered high mortalities at low exposures (100 flagellates in 0.1 ml physiological saline inoculated intraperitoneally per fish). Oncorhynchus nerka from the Weaver Creek stock (Fraser River system) was the most resistant with no mortalities even at exposures of 10(6) flagellates (in 0.1 ml physiological saline) per fish. Oncorhynchus kisutch seemed to be slightly less resistant than the Weaver Creek O. nerka, but fewer than 16% of the inoculated fish died. Oncorhynchus kisutch from the Big Qualicum River seemed to be slightly more resistant than O. kisutch from the Capilano River stock (a coastal river near Vancouver), with fewer mortalities and lighter infections when the experiments were terminated. Differences in susceptibility are believed to be associated with innate, genetically transmitted resistance.     

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.     

The chromosomes of Salmo salar

A new chromosome number, 2n=58, is reported for Salmo solar, the Atlantic salmon. The complement comprises 8 pairs of metacentrics, 21 pairs of acrocentrics. The question of variation in chromosome numbers for different salmon stocks is very briefly discussed.     

Increased susceptibility of Atlantic salmon Salmo salar to infections with Gyrodactylus derjavini induced by dexamethasone bath treatment

Dexamethasone, a known immunosuppressant, was administered by bath or injection to Atlantic salmon Salmo salar (Conon stock) to study if this treatment could affect the susceptibility of fish to infections with a Danish strain of Gyrodactylus derjavini (Monogenea). Three groups of S. salar (Conon stock) were immersion treated either with 10, 60 or 240 microg dexamethasone l-1 water, respectively. In addition, one group (positive control) was treated intraperitoneally with 200 microg dexamethasone per fish and one negative control group was kept untreated. A single G. derjavini parasite was placed on the anal fin of each fish and the infection was subsequently monitored weekly for 6 weeks. An increase in parasite populations on the salmon was positively correlated with the amount of immunosuppressant used. Infection levels in the group immersion treated with dexamethasone (240 microg l-1 water) and in the i.p. treated positive control group were significantly higher compared to the untreated control group.     

Comparison of damage to live v. euthanized Atlantic salmon Salmo salar smolts from passage through an Archimedean screw turbine

This study assessed the usefulness of passing euthanized Atlantic salmon Salmo salar smolts through an Archimedean screw turbine to test for external damage, as compared with live, actively swimming smolts. Scale loss was the only observed effect. Severe scale loss was 5·9 times more prevalent in euthanized turbine‐passed fish (45%) than the live fish (7·6%). Additionally, distinctive patterns of scale loss, consistent with grinding between the turbine helices and housing trough, were observed in 35% of euthanized turbine‐passed smolts. This distinctive pattern of scale loss was not seen in live turbine‐passed smolts, nor in control groups (live and euthanized smolts released downstream of the turbine), which suggests that the altered behaviour of dead fish in turbine flows generates biased injury outcomes.     

Overlooked aspects of the Salmo salar and Salmo trutta lifecycles

Reviews in Fish Biology and Fisheries - The salmonid lifecycle has been studied for over a 100 years. Our literature search indicated that the Atlantic salmon (Salmo salar) and brown trout...     

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.     

Diseases of farmed Atlantic salmon Salmo salar associated with infections by the microsporidian Paranucleospora theridion

The microsporidian Paranucleospora theridion was discovered in Atlantic salmon Salmo salar suffering from proliferative gill disease in a marine farm in western Norway in 2008. The parasite develops in cells of the reticuloendothelial system, cells important for normal immune function. The aim of this study was to see if P. theridion could play a part in some of the diseases with unclear causes in salmon production in Norway, i.e. proliferative gill disease (PGI), pancreas disease (PD), heart and skeletal muscle inflammation (HSMI) and cardiomyopathy syndrome (CMS). P. theridion was present in all areas with salmon farming in Norway, but high prevalence and densities of the parasite in salmon and salmon lice were only seen in southern Norway. This region is also the main area for PGI and PD in Norway. Quantification of pathogens associated with PGI, PD, HSMI and CMS diagnoses showed that P. theridion levels are high in southern Norway, and may therefore play a role in susceptibility and disease development. However, among the different diagnoses, fish with PGI are particularly heavily infected with P. theridion. Therefore, P. theridion appears as a possible primary agent in cases with high mortality in connection with PGI in western Norway.     

Characterization of a New HpaI Centromeric Satellite DNA in Salmo salar

A highly repeated HpaI DNA family was revealed in Atlantic salmon (Salmo salar) and analyzed by Southern blotting and fluorescence in situ hybridization (FISH). In this report, we describe the nucleotide sequence, genomic structure and chromosomal localization of this HpaI repeat. This novel satellite appeared tandemly arrayed and located at centromeric areas of three acrocentric chromosome pairs as evidenced by FISH. The sequence was characterized by a high AT content (63%), a short consensus motif (A/T)(G/C)AAA(T/C) similar to other centromeric satellites motifs, and by short AT enriched stretches. The presence of this sequence in other salmonid species was also tested by Southern blot hybridization and used to analyze its evolution within this group.     

Phylogenetic position of a paramyxovirus from Atlantic salmon Salmo salar

A paramyxovirus has been isolated from Atlantic salmon Salmo salar suffering from epitheliocystis. This virus does not cause any mortality when used to challenge disease-free salmon, but has been associated with 2 cases of mortality in salmon farms in Norway. Atlantic salmon paramyxovirus (ASPV) has been suggested as a name for the virus. The ASP virus is a slow-growing virus in cell cultures (rainbow trout gill cells: RTgill-W1). Little is known about its importance and its phylogenetic position is uncertain. Hence, the need for a fast and sensitive diagnostic method for studying the prevalence of this virus in salmon farms and for more basic knowledge about its identity were the motivation for this study. A partial nucleotide sequence (816 bp) from the large protein (L protein) gene of the ASP virus has been sequenced from 2 different isolates. The putative amino acid sequence has been compared with the L protein of other paramyxoviruses. This sequence gives strong support to a relationship between the ASP virus and members of the subfamily Paramyxovirinae, genus Respirovirus.