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.     

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.     

鲑鱼隐鞭虫病及其防治策略

鲑鱼隐鞭虫病的病原为Cryptobiasalmositica。据报道,北美西海岸的所有大西洋鲑(Oncorhynchusspp.)均有该虫的寄生。该病除了通常情况下经河流中的水蛭传播外,在特定的条件下可直接经鱼类传播。鲑隐鞭虫病的临床症状包括贫血、食欲减退、脾脏肿大、全身性水肿、腹水性腹胀。被感染的鱼类需要付出巨大的能量代价,表现出明显的新陈代谢及游泳功能的衰退。鱼体对缺氧相当敏感,免疫系统也受到抑制。鱼类对隐鞭虫病的感染严重程度和死亡率可因鱼种而存在差异。预防感染的有效策略包括选育抗隐鞭虫的新品种,“抗隐鞭虫感染”在遗传上是孟德尔显性遗传。在抗隐鞭虫的鱼体内,虫体可被宿主免疫系统的“补体旁路途径”所裂解。在“隐鞭虫耐受性鱼”(处于感染状态但未发病)的血液中存在一种天然的抗蛋白酶———α2巨球蛋白,可以中和分泌的金属蛋白酶(毒力因子)。因此,繁殖转基因“隐鞭虫耐受性”鲑鱼是一种很好的选择,而无需通过疫苗和药物来治疗隐鞭虫病。康复后的鱼体具有对该病的免疫保护力,因而,利用这一特点采取疫苗免疫也是防治隐鞭虫病的另一种策略。我们所研制的致弱疫苗单一剂量免疫鱼体可获得至少两年的免疫保护。免疫保护机制包括产生“抗体结合补体”、强化吞噬作用及细胞介导的细胞毒作用。氯化氮氨菲啶是防治隐鞭虫病的有效药物,它通过溶解虫体而杀灭寄生虫,也可在线粒体中富集,显著影响有氧呼吸的顺利进行并造成线粒体的损伤。通过药物与抗体连接可以显著增强药效,这种利用“免疫化学疗法”控制鲑隐鞭虫病的策略,其优点在于减小了药物剂量,因而大大降低了化学治疗所引起的副作用。对于食用性鱼类而言,减少药物在鱼体的富集符合食品安全的需要     

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.     

Use of a live vaccine to modulate Cryptobia salmositica infections in Salmo salar

The vaccine strain of Cryptobia salmositica multiplies in Atlantic salmon Salmo salar and it can modulate the severity of the disease in Cryptobia-infected individuals. Fish injected with the vaccine 3 d post-infection with C. salmositica had lower peak parasitaemias and higher antibody titres than infected fish given the vaccine 7 d post-infection or those infected fish that were not given the vaccine.     

Protective immune response of fish to parasitic flagellates

The piscine immune system is normally quite efficient in protecting the host (innate and acquired immunity) from parasitic infections. Innate immunity may occur at two distinct levels — between host species and within a host species. If the resistance is at the host species (or a higher taxonomic group) level, then it is inter-host innate immunity. For example, Oncorhynchus mykiss can be infected with the pathogenic hemoflagellate, Cryptobia salmositica isolated from Oncorhynchus spp. but cannot be infected with Cryptobia catostomi from Catostomus commersoni. At the next level, there are individuals within a susceptible host species that are resistant to infection — this is intra-host innate immunity; e.g. some Salvelinus fontinalis are resistant to C. salmositica infection while others are not. This resistance to infection is not dependent on age or size of the fish; it is inherited and is controlled by a dominant gene. Protection at both levels of innate immunity is via the activation of the alternative pathway of complement activation to lyse the parasite. Also, S. fontinalis can be infected with the pathogenic C. salmositica have very high parasitaemias but they do not suffer from the disease as O. mykiss. This resistance to disease is related to high levels and rapid production of α₂-macroglobulin which is one of two natural antiproteases. The α₂-macroglobulin in the blood neutralises the metallo-protease secreted by the pathogenic C. salmositica. Acquire immunity was shown in fish that survived infections of pathogenic flagellates. Fish that have recovered from Amyloodinium ocellatum, C. salmositica, Cryptobia bullocki, and Trypanosoma danilewskyi are protected. This protection requires prior exposure to the pathogen and/or its antigens. Humoral (e.g. complement fixing antibodies to lyse the parasite) and cell-mediated (e.g. T-cell cytotoxicity, phagocytosis) are part of the protective mechanism in acquired immunity. Also, an attenuated live C. salmositica vaccine has been developed and it protects juvenile and adult salmonids from cryptobiosis for at least 2 years.     

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.     

Effects of the pathogenic haemoflagellate, <Emphasis Type="Italic">Cryptobia salmositica</Emphasis> on brood fish, <Emphasis Type="Italic">Oncorhynchus mykiss</Emphasis>

Sexually matured rainbow trout, Oncorhynchus mykiss, were experimentally infected with the pathogenic Cryptobia salmositica. Spawning female trout were more susceptible to cryptobiosis than sexually mature males. Most infected females (seven of nine) with eggs died before or shortly after spawning while all (nine) infected males survived the disease. Also, none of the uninfected females died. Males initially increased milt production and sperm concentration; however semen production declined as the disease progressed. Sperm from infected males fertilized more eggs than those from non-infected males. No differences in weight and survival were observed between progeny of infected and uninfected males.     

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.     

A new type of cysteine proteinase inhibitor--the salarin gene from Atlantic salmon (Salmo salar L.) and Arctic charr (Salvelinus alpinus)

Salarin is a 43 kDa glycoprotein which is found so far only in salmonid fish species. It is a strong inhibitor of cysteine proteinases. Here we characterised the salarin gene from Atlantic salmon and cDNA from Arctic charr. The salarin gene has 13 exons and 1026 bp long coding sequence. The translated amino acid sequence has four similar domains. The sequence resembles the proregion of cathepsins, known to inhibit cysteine proteinases. Salarin can be a new type of cysteine proteinase inhibitor.     

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.     

Anorexia reduces the severity of cryptobiosis in Oncorhynchus mykiss

Cryptobia salmositica multiplied more rapidly and caused a more severe disease with a higher mortality (65%) in rainbow trout (Oncorhynchus mykiss) fed a 52% protein diet than in fish fed 37 or 22% protein diets (25 and 30% mortalities, respectively). Also, plasma protein was significantly higher in fish fed a 52% protein diet than in fish fed 37 or 22% protein diets. Anorexia in infected trout was related positively to parasitemia and was most significant at 4 wk postinfection. During the chronic phase of the infection, food consumption increased with declining parasitemia. It is hypothesized that anorexia lowers plasma protein level, which reduces the multiplication rate of parasites, thus decreasing the severity of the disease.     

MISET: an immunological technique for the serodiagnosis of Cryptobia salmositica (Sarcomastigophora: Kinetoplastida) infection in Oncorhynchus mykiss

A serodiagnostic technique, microscopic immuno-substrate-enzyme technique (MISET), is described using the Cryptobia-Oncorhynchus mykiss system. The reactions of specific antibodies, phosphatase-labeled antibody, and substrate with subsequent color development on the parasite (cell membrane, flagella, kinetoplast, and nucleus) are observed using light microscopy. Using this technique, humoral response to Cryptobia salmositica was detected in 2 of 10 O. mykiss 7 days after infection. Subsequently, antibodies were detected in all 10 infected fish. Parasites cultured in minimum essential medium or from experimentally infected pink salmon, Oncorhynchus gobuscha, worked equally well. Antibodies eluted from antisera or whole blood dried on filter paper (stored at -20 C) gave similar reactions as those from antisera stored in containers. MISET is at least as sensitive as the indirect fluorescent antibody technique. When MISET is modified and adapted for other parasitic diseases (e.g., for those of medical and or of economic importance), it may be useful especially in smaller centers or in developing countries where equipment cost, maintenance of equipment, operating costs, and well trained personnel are important considerations. Because MISET worked with dried whole blood or serum on filter paper it may also be a useful field technique for large scale seroepidemiological surveys.     

Identification of genetic markers associated with Gyrodactylus salaris resistance in Atlantic salmon Salmo salar

Gyrodactylus salaris Malmberg, 1957 is a freshwater monogenean ectoparasite of salmonids, first recorded in Norway in 1975 and responsible for extensive epizootics in wild Atlantic salmon Salmo salar L. The susceptibility of different populations of Atlantic salmon to G. salaris infection differs markedly, with fish from the Baltic being characterised as relatively resistant whereas those from Norway or Scotland are known to be (extremely) susceptible. Resistance to Gyrodactylus infection in salmonids has been found to be heritable and a polygenic mechanism of control has been hypothesised. The current study utilises a 'Quantitative trait loci' (QTL) screening approach in order to identify molecular markers linked to QTL influencing G. salaris resistance in B1 backcrosses of Baltic and Scottish salmon. Infection patterns in these fish exhibited 3 distinct types; susceptible (exponential parasite growth), responding (parasite load builds before dropping) and resistant (parasite load never increases). B1 backcross fish were screened at 39 microsatellite markers and single marker-trait associations were examined using general linear modelling. We identified 10 genomic regions associated with heterogeneity in both innate and acquired resistance, explaining up to 27.3% of the total variation in parasite loads. We found that both innate and acquired parasite resistance in Atlantic salmon are under polygenic control, and that salmon would be well suited to a selection programme designed to quickly increase resistance to G. salaris in wild or farmed stocks.     

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.     

SSU rRNA gene sequence reveals two genotypes of Spironucleus barkhanus (Diplomonadida) from farmed and wild Arctic charr Salvelinus alpinus

Spironucleus barkhanus isolated from the blood of Arctic charr Salvelinus alpinus from a marine fish farm were genetically compared with S. barkhanus isolated from the gall bladder of wild Arctic charr. The wild Arctic charr were caught in the lake used as the water source for the hatchery from which the farmed fish originated. Sequencing of the small subunit ribosomal RNA gene (SSU rDNA) from these 2 populations showed that the isolates obtained from farmed and wild Arctic charr were only 92.7 % similar. Based on the sequence differences between these isolates, it is concluded that the parasites isolated from the farmed fish have not been transmitted from wild Arctic charr in the hatchery's fresh water source. It is therefore most likely that the farmed fish were infected by S. barkhanus after they were transferred to seawater. S. barkhanus isolated from diseased farmed Arctic charr were 99.7% similar to the isolates obtained from diseased farmed Chinook (Canada) and Atlantic salmon (Norway). The high degree of sequence similarity between S. barkhanus from farmed Arctic charr, Chinook and Atlantic salmon indicates that systemic spironucleosis may be caused by specific strains/variants of this parasite. The genetic differences between the isolates of farmed and wild fish are of such magnitude that their conspecificity should be questioned.     

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.     

Isolation of a novel fish thymidylate kinase gene, upregulated in Atlantic salmon (Salmo salar L.) following infection with the monogenean parasite Gyrodactylus salaris

Analysis of differential gene expression in salmon (Salmo salar) blood following infection with the monogenean parasite Gyrodactylus salaris, resulted in the isolation of a thymidylate kinase gene not previously described from fish and which showed similarity to an LPS-inducible thymidylate kinase gene isolated from mouse macrophages. This salmon TYKi-like gene may play a role in an innate generalised response to pathogen infection as it was upregulated in salmon following infection with the parasite, and also in response to injection with the immunostimulants LPS and Poly I:C, used to emulate bacterial and viral infections, respectively. The possible role of this gene in the biosynthesis of mitochondrial DNA in activated macrophages, in response to G. salaris infection is discussed.     

Cloning of a metalloprotease gene involved in the virulence mechanism of Vibrio anguillarum.

Genetic evidence has previously suggested that a zinc metalloprotease is involved in the invasive mechanism of the fish pathogen Vibrio anguillarum NB10. In this study, the metalloprotease gene was cloned and sequenced. The sequence encodes a polypeptide (611 amino acids) that contains a putative signal sequence followed by a large leader sequence and the mature protein (44.6 kDa). Since the purified protein has a molecular mass of 36 kDa instead of the predicted 44.6 kDa, the mature protein is most likely processed a third time. Comparative analyses of the protein sequence showed high homologies to other bacterial metalloproteases within the zinc-binding and active-site regions. The Vibrio cholerae hemagglutinin/protease and the Pseudomonas aeruginosa elastase were exceptions in that the homology extended throughout the entire putative preproprotein. A chromosomal metalloprotease mutant was made via the integration of foreign DNA into the protease gene. This mutant did not secrete the metalloprotease, as determined by sodium dodecyl sulfate (SDS)-polyacrylamide protein analysis and by growth on gelatin agar. Transcomplementation of the chromosomal mutation revived the secretion of the metalloprotease and its activity on gelatin agar. Interestingly, when supernatant proteins were analyzed by gelatin-SDS-polyacrylamide electrophoresis, two different proteases (75 and 30 kDa) were detected in the mutant strain but not in the transcomplemented strain or the wild-type strain. Moreover, fish infection studies were done, and implications for the role of the metalloprotease in the virulence mechanism of V. anguillarum are discussed.     

Fish microsporidia: immune response, immunomodulation and vaccination

Immune response to fish microsporidia is still unknown and there are current research trying to elucidate the events involved in the immune response to this parasite. There is evidence suggesting the role of innate immune response and it is clear that adaptive immunity plays an essential part for eliminating and then mounting a solid resistance against subsequent microsporidian infections. This review article discusses the main mechanisms of resistance to fish microsporidia, which are considered under four main headings. 1) Innate immunity: the inflammatory tissue reaction associated with fish microsporidiosis has been studied at the ultrastructural level, providing identification of many of the inflammatory cells and molecules that are actively participating in the spore elimination, such as macrophages, neutrophils, eosinophilic granular cells, soluble factors and MHC molecules. 2) Adaptive immunity: the study of the humoral response is relatively new and controversial. In some cases, the antibody response is well established and it has a protective role, while in other situations, the immune response is not protective or it is depressed. Study of the cellular response against fish microsporidia is still in its infancy. Although the nature of the microsporidian infection suggests participation of cellular mechanisms, few studies have focused on the cellular immune response of infected fish. 3) Immunomodulation: glucans are compounds that can modulate the immune system and potentiate resistance to microorganisms. These compounds have been proposed that can interact with receptors on the surface of leukocytes that result in the stimulation on non-specific immune responses. 4) Vaccination: little is known about a biological product that could be used as a vaccine for preventing this infection in fish. In the Loma salmonae experience, one of the arguments that favor the production of a vaccine is the development in fish of resistance, associated to a cellular immune response. A recently proved spore-based vaccine to prevent microsporidial gill disease in salmon has recently shown its efficacy by considerably reducing the incidence of infection. This recent discovery would be first anti-microsporidian vaccine that is effective against this elusive parasite.