Biological characterization of a monoclonal antibody against a surface membrane antigen on Cryptobia salmositica Katz, 1951

A monoclonal antibody (IgG 1) (designated as MAb-001) was produced against the pathogenic haemoflagellate Cryptobia salmontica Katz. The antibody agglutinated live parasites under in vitro conditions. Live C. salmositica, incubated with MAb-001 at 10 °C, did not multiply and were dead within 4 weeks in culture. About 50% of juvenile rainbow trout, Oncorhynchus mykiss (Walbaum), inoculated intraperhoneally with C. salmositica, incubated in MAb-001 prior to inoculation, did not become infected, while in adult rainbow trout, the peak parasitaemia was reduced. These results indicate that MAb-001 is a protective monoclonal antibody and the antigen it recognizes is located on rhe surface membrane of C. salmositica. The antibody also inhibits multiplication and affects viability of the parasite under in vitro conditions.     

Inhibitory effects of a monoclonal antibody (MAb-001) on in vitro oxygen consumption and multiplication of the pathogenic haemoflagellate, Cryptobia salmositica Katz

A monoclonal antibody (MAb-001), against a surface glycoprotein on Cryptobia salmositica inhibited the multiplication and oxygen consumption of both virulent and avirulent strains of the parasite. The classical cysteine proteinase inhibitor (E-64) and a cysteine proteinase activator (EDTA) affected the in vitro multiplication of C. salmositica . Concentrations of E-64 higher than 10 μ M reduced the multiplication of C. salmositica while 5 m M of EDTA enhanced its multiplication. We propose that the cysteine proteinase is an important metabolic enzyme in C. salmositica and that binding of MAb-001 to the enzyme inhibited parasite multiplication and reduced oxygen consumption.     

The immune response of rainbow trout, Salmo gairdneri Richardson, to the haemoflagellate, Cryptobia salmositica Katz, 1951

Abstract. Rainbow trout that recovered from experimental Cryptobia salmositica infection 6 and 10 weeks earlier were protected against multiple intraperitoneal challenges of 50 000 and 10 000 parasites isolated from infected fish. The immunity was non-sterile; low parasitaemias were detected following a larger challenge (112 000 parasites). The indirect haemagglutination test was used to detect C. salmositica -specific agglutimns. Antibody titers increased during the first 18 weeks of infection. The infectivity of cultured C. salmositica was neutralized by incubation in heat-inactivated immune plasma. Infectivity of C, salmositica from infected fish was not neutralized by similar treatment. Complement fixing antibody was detected using the in vitro immune lysis test. Immune lysis occurred when cultured C. salmositica were used. Adoptive transfer of both leucocytes and plasma from immune fish conferred partial protection against the parasite in naive recipients. Complement fixing antibody may be important during early acute infection while phagocytosis may be important during the later chronic phase.     

Vaccination of rainbow trout, Oncorhynchus mykiss (Walbaum), against cryptobiosis: efficacy of the vaccine in fresh and sea water

Adult rainbow trout, Oncorhynchus mykiss (Walbaum), maintained in either fresh or sea water were vaccinated with a live Cryptobia salmositica vaccine. All vaccinated fish were protected 4 weeks later against the cryptobiosis, while unvaccinated rainbow trout developed the disease (e.g. high parasitaemia and severe anaemia) after challenge with virulent C. salmositica . There was also no disease in vaccinated fish when they were transferred from fresh to sea water immediately after vaccination. Complement fixing antibodies (CFAbs) were detected in vaccinated fish and the CFAbs lysed parasites under in vitro conditions. The antibody titres increased rapidly at one week post-challenge in vaccinated fish in fresh water and vaccinated fish transferred from fresh water to sea water after vaccination. However, the production of CFAbs was delayed by one week in vaccinated fish in sea water and the antibody titre was significantly lower than that in fish maintained in fresh water.     

An enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies against the pathogenic haemoflagellate, Cryptobia salmositica Katz, and protection against cryptobiosis in juvenile rainbow trout, Oncorhynchus mykiss (Walbaum), inoculated with a live vaccine

Abstract. An enzyme-linked immunosorbent assay using dried blood on filter paper, was developed for the detection of antibodies against the haemoflagellate Cryptobia salmositica in juvenile rainbow trout, Oncorhynchus mykiss (Walbaum). Each fish (average weight about 5g) in three experimental groups was either inoculated with 20000 attenuated live C. salmositica vaccine, or inoculated with 2000 or 20000 pathogenic parasites per fish. The vaccine was effective in protecting juvenile trout 4 weeks after vaccination and antibody titers were higher in vaccinated and challenged fish than in unvaccinated and infected ones. Specific antibodies were detected one week post-infection (w.p.i.) with the pathogen and declined to low levels at 6 w.p.i. The high-dose group (20000 per fish) had antibody titres comparable to those of the vaccinated and challenged fish.     

Characterization of a monoclonal antibody against the 47- kDa antigen of Cryptobia salmositica Katz and its use in an antigen-capture enzyme-linked immunosorbent assay for detection of parasite antigen in infected rainbow trout, Oncorhynchus mykiss (Walbaum)

A monoclonal antibody (designated MAb-007) was produced against the pathogenic haemoflagellate Cryptobia salmositica Katz. This IgG3 antibody recognized the 47-kDa antigenic polypeptide of C. salmositica (SDS-PAGE and Western immuno-blotting). The antibody did not agglutinate live parasites, and there was no change in the staining intensity of the 47-kDa band on Western immunoblots after immunoabsorption of MAb-007 with live intact parasites. The 47-kDa antigen recognized by MAb-007 was localized in the cytoplasm of the parasite (immunogold labelling and electron microscopy). The monoclonal antibody cross- reacted with the 47-kDa polypeptides of C. bullocki Srrout and C. catostomi Bower & Woo. It was used in an antigen-capture ELISA for the detection of parasite antigen in the plasma of rainbow trout inoculated with the parasite, or with an attenuated vaccine strain of C. salmositica. All pre-infection plasma were negative while all infected fish with detectable parasitaemias were positive for antigen at 1–9 weeks after infection. Parasite antigen was even detected in vaccinated fish that were negative for parasites using the wet mount microscopic technique. The antigen-capture ELISA detected C, salmositica antigen in whole cell lysate preparations at concentrations as low as 0.5 μg ml^-1. Fifty microlitres of fish plasma was required in the antigen-capture ELISA, and the use of a plate reader and 96-well plates facilitated rapid analysis of a large number of plasma samples. The sensitivity of the assay makes it a potentially useful tool for detection of Cryptobia infections.     

Immunosuppression in rainbow trout, Salmo gairdneri Richardson, caused by the haemoflagellate Cryptobia salmositica Katz, 1951

Abstract. An experimental Cryptobia salmositica infection in rainbow trout, Salmo gairdneri Richardson, produced suppression of the humoral response against sheep red blood cells as measured by direct haemagglutination. Two-month and 5-month infections produced equal suppression. The parasite also produced suppression of the humoral response against a bacterial pathogen, Yersinia ruckeri . Anti- Y. ritckeri titres were significantly lower in most fish infected with C. salmositica than in non-infected fish. Immunosuppression became evident when C. salmositica first appeared in the blood (first 2 weeks of infection), Immunosuppression was confirmed by challenge with Y. ruckeri . Mortality at challenge occurred in 64·3% to 83·3% of the fish already infected with C. salmositica at the time of initial Y. ruckeri exposure. There was no mortality at challenge if fish were not infected with C. salmositica at initial bacterial exposure, nor in those concurrently infected with both pathogens. Antigenic competition may have caused the immunosuppression.     

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.     

Cell membrane glycoconjugates on virulent and avirulent strains of the haemoflagellate Cryptobia salmositica (Kinetoplastida)

The glycoconjugates on the cell membranes of Cryptobia salmositica (virulent and avirulent strains) were analysed using 13 highly purified lectins. The virulent strain of C. salmositica was agglutinated by two of these lectins (Concanavalin A (Con A) Canavalia ensiformis , specific for α-mannose and α- D -glucosyl residues; Pisum sativau agglutinin (PSA), specific for N-acetyl α- D -galactosaminyl) but the avirulent strain of C. salmositica was agglutinated by 11 lectins. No agglutination of C. salmositica (virulent and avirulent strains) was observed with lectin Tetragonolobus purpureas agglutinin (TPA, specific for α- L -fucose). Glycoprotein analysis with digoxigenin or biotin labelled lectins showed strain specific staining patterns for C. salmositica; with the avirulent strain showing stronger reactions than the virulent strain. These results indicate that the surface carbohydrate residues changed with attenuation of the pathogen and this change may be related to its loss of virulence.     

Brook charr, Salvelinus fontinalis (Mitchill), and cryptobiosis: a potential salmonid reservoir host for Cryptobia salmositica Katz, 1951

Abstract. Laboratory-raised Cryptobia -susceptible brook charr, Salvelinus fontinalis (Mitchill), and rainbow trout, Oncorhynchus mykiss (Walbaum), were vaccinated intraperitoneally with a live Cryptobia salmositica vaccine (250000 parasites per fish), and 4 weeks later were challenged with the pathogen (250000 parasites per fish). Unvaccinated and infected brook charr had high parasitaemias but no clinical signs of disease, while unvaccinated and infected rainbow trout had anaemia and general oedema. Vaccinated and challenged fish had very low parasitaemias compared to unvaccinated and infected brook charr and rainbow trout. Complement fixing antibodies were detected in vaccinated and challenged fish 2 weeks after challenge. Unvaccinated and infected brook charr had consistently higher litres of complement fixing antibody than unvaccinated and infected rainbow trout. Parasitaemias were lower in all fish in which titres of complement fixing antibody were high. In a second experiment, brook charr inoculated intraperitoneally or intramuscularly with 100000 C. salmositica per fish had high parasitaemias but no anaemia or other clinical signs. The results show that susceptible brook charr do not suffer from cryptobiosis and may serve as reservoir hosts for C. salmositica in areas where the disease is prevalent. Vaccination to reduce the parasitaemia when fish become infected may be a control strategy in these areas.     

Characterization of a host response to the myxosporean parasite, Ceratomyxa shasta (Noble), by histology, scanning electron microscopy and immunological techniques

Abstract. The tissue response of Salmo gairdneri Richardson, against the myxosporean parasite. Ceratomyxa shasta (Noble), was investigated using histological techniques, scanning electron microscopy and immunological methods. The progress of infection in C. shasta -susceptible and resistant steelhead and rainbow trout was examined by standard histological techniques and by indirect fluorescent antibody methods using monoclonal antibodies directed against C. shasta antigens. Trophozoite stages were first observed in the posterior intestine and there was indication that resistance was due to the inability of the parasite to penetrate this tissue rather than to an inflammatory response. Examination of a severely infected intestine by scanning electron microscopy showed extensive destruction of the mucosal folds of the posterior intestine. Western blotting and indirect fluorescent antibody techniques were used to investigate the immunological component of the host response. No antibodies specific for C. shasta were detected by either method.     

Comparative in vitro studies on virulent and avirulent strains of Cryptobia salmositica (Sarcomastigophora: Kinetoplastida)

Abstract. The optimum temperature for in vitro multiplication of Cryptobia salmositica was 10°C. The avirulent strain multiplied more rapidly than the virulent strain. The haemolytic components, lytic component (LC) and immune complex-forming component (ICC), were secreted by the two strains into the culture medium and were detectable from one week post-inoculation. The haemolytic activity in the supernatant increased with increasing parasite numbers in both strains. Although cultures of the avirulent strain had higher parasite numbers than those of the virulent strain, the haemolytic activity was significantly lower than that of the virulent strain. Antiserum against ICC was produced in rabbit by immunization with ICC-coated rainbow trout red blood cells.     

指状拟舟虫诱导牙鲆抗血清免疫球蛋白分析

对引起牙鲆体表溃烂的原纤毛虫－指状舟虫诱导牙鲆免疫反应产生的免疫球蛋白ＩｇＭ进行分析,结果表明,病原纤毛虫免疫注射牙鲆 ,可诱导牙鲆发生特异性免疫反应,产生抗体型上鲆抗指状拟舟虫血清的凝集试验,发现纤毛虫停止游动并发虫体聚集;抗血清经与标准分子量和鼠ＩｇＭ单克隆抗体以及对照血清的ＳＤＳ－ＰＡＧＥ电泳比较分析证明,牙鲆抗指状拟舟虫血清免疫球蛋白ＩｇＭ重链分子量为７１０００,轻链分子量为２３０００;Ｉ     

The in vitro inhibition of proteases from Cryptobia salmositica Katz by a monoclonal antibody (MAb-001) against a glycoprotein on the pathogenic haemoflagellate

The monoclonal antibody (MAb-001), which was produced against a surface membrane glycoprotein on C. salmositica , significantly inhibited the activities of the intracellular proteases of the parasite. The total activity in the partially purified metallo-protease, and about 80% of activity in the partially purified cysteine protease, were inhibited by the antibody (at 10 μg protein ml^–1). The inhibitory effects of the antibody on the proteases were also demonstrated using haemoglobin (substrate)-SDS-PAGE. The activities of the metallo-protease band (200 kDa) and the three cysteine protease bands (66, 70 and 97 kDa) were inhibited by MAb-001, but the activity of the fourth cysteine protease band (49 kDa) was not affected. Similar inhibitory effects of the antibody were also found in the crude protease extract (parasite lysate), except that more antibody was required to obtain the same level of inhibition. The metallo-protease band was more sensitive than the cysteine protease bands to the antibody.     

Development of monoclonal antibodies to PK'X', the causative agent of proliferative kidney disease

Abstract. Two monoclonal antibody probes were produced against PK'X' cells. The parasites were partially purified by filtration and centrifugation of kidney tissue from rainbow trout with proliferative kidney disease and used as antigen for immunization of mice. The resulting monoclonal antibodies reacted with PK'X' cell antigens in enzyme-linked immunosorbent assay and immunohistochemistry tests. One antibody (Mab 12) appeared to be specific for PK'X'in kidney tissue, while the other (Mab 18) cross-reacted with host cell antigens in the kidney tubules. These probes are invaluable tools for the investigation of parasite surface antigens and life cycle studies.     

Impact of a pathogenic haemoflagellate, Cryptobia salmositica Katz, on the metabolism and swimming performance of rainbow trout, Oncorhynchus mykiss (Walbaum)

Abstract. Oxygen consumption of juvenile rainbow trout (5 g at 13°C) at moderate swimming speeds did not change significantly when infected with Cryptobia salmositica. However, significant reductions of as much as 44% of the maximum aerobic scope for activity and 24% of the critical swimming speed were observed when the parasitaemia reached a maximum of 57.6 × 10⁶ ml⁻¹ fish blood at 3 weeks post- infection. Blood haematocrit was significantly reduced from the initial 34.1 to 19.7% at 4 weeks post- infection, probably as a result of haemolysis by the parasite. The destruction of red blood cells clearly led to lower oxygen carrying capacity, and reduced respiratory and swimming performance.     

Experimental evidence for direct in situ binding of IgM and IgT to early trophonts of Ichthyophthirius multifiliis (Fouquet) in the gills of rainbow trout, Oncorhynchus mykiss (Walbaum)

Freshwater fish are able to mount a protective immune response against the parasite Ichthyophthirius multifiliis (Ich) following a non‐lethal exposure. Factors involved in immunity comprise cellular and humoral factors, but antibodies have been suggested to play a prominent role in protection. However, host antibodies have not yet been demonstrated to bind to the parasite in situ. By the use of immunohistochemical techniques, this study demonstrated that IgT and IgM bind to surface structures, including cilia, on the early feeding stage of the parasite in the gills of immune rainbow trout, Oncorhynchus mykiss, shortly (2 h) after invasion. No binding of IgT and no or only a weak binding of IgM was observed on the parasites in the gills of similarly exposed but naïve rainbow trout. This study indicates that antibodies play an important part in the protection of immune fish against Ich although additional humoral and cellular factors may contribute to this reaction.     

Enzyme treatment of Trypanosoma danilewskyi (Laveran and Mesnil) increases its susceptibility to lysis by the alternative complement pathway of goldfish, Carassius auratus (L.)

This study examined whether in vitro-cultured Trypanosoma danilewskyi were susceptible to lysis in the presence or absence of anti-parasite antibodies and complement. Cultured trypanosomes were resistant to lysis by either immune or non-immune goldfish serum. However, trypanosomes treated with the proteolytic enzyme trypsin, which destroys surface proteins of the parasites, became susceptible to lysis when exposed to either immune or non-immune goldfish serum. The lysis by goldfish serum was dependent on the presence of heat-labile factors and occurred at 4 and 20 degrees C. The lysis was also dependent on the presence of Mg(2+) ions but not Ca(2+) ions. Furthermore, treatment of the parasites with different sialidases did not enhance their susceptibility to lysis by goldfish serum. Trypsinized parasites regained resistance to lysis after at least 6-h cultivation in the absence of trypsin and the restoration of full resistance was observed after 24-h cultivation. The resistance to lysis was abrogated when the protein synthesis inhibitor, puromycin, was added to the cultures. These results suggest that trypsinized trypanosomes were susceptible to lysis by goldfish complement (alternative pathway) and that protective surface proteins of the parasite were required for the resistance of normal trypanosomes to lysis.     

Preliminary investigation into the killing effect of kingfish (Seriola lalandi) serum and mucus against the monogenean parasites Benedenia seriolae and Zeuxapta seriolae

The survival of two economically important monogenean parasites, Benedenia seriolae and Zeuxapta seriolae, exposed to either naïve or infected yellowtail kingfish Seriola lalandi serum and mucus was observed over an 8-h period. Infection status of the host had no effect on survival of monogeneans in either serum or mucus therefore data were grouped according to each parasite. Serum exposure at the highest concentration of 1:5 did not have any effect on Z. seriolae, however, 50% killing activity against B. seriolae was observed within 3 h at concentrations greater than 1:100. This killing activity was completely abolished following heat treatment and inactivation of the serum. Prior incubation of parasites with heat-treated serum for 2 h in order to potentially coat the parasites with antibodies did not enhance the activity of fresh serum against either species. Addition of 5 mM EDTA, but not 5 mM EGTA-Mg, inhibited the killing ability of fresh serum against B. seriolae, suggesting that previously observed activity against this parasite was most likely mediated via the alternative complement pathway. Cutaneous mucus was not found to have any effect on B. seriolae or Z. seriolae survival. Overall these results suggest that the blood-feeding gill parasite Z. seriolae is considerably more resistant to host immune responses compared with skin- and mucus-dwelling B. seriolae.     

Cryptobia salmositica: cortisol increases the susceptibility of Salmo gairdneri Richardson to experimental cryptobiosis

Abstract. Intraperitoneal implants of cortisol (cortisol suspended in hydrogenated coconut oil) were used to induce a graded hypercortisolism in rainbow trout, Salmo gairdneri Richardson. There was no obvious reduction in circulating lymphocytes in cortisol-implanted rainbow trout (70, 140 or 210μg/g body weight). Cortisol-implanted fish infected with Cryptobia salmositica had significantly higher parasitaemia and lower antibody litres compared with controls infected with haemonagellate but given coconut oil implants. These confirm the immunodepressive effects of the steroid. The parasite was also more readily detected at the early stage of the infection (shorter prepatent period, more infected fish and higher parasitaemia) in cortisol-implanted fish (140 and 210 μg/g body weight) than in controls. The mortality of the infected cortisol-implanted fish was higher than that of the infected fish implanted with only coconut oil, or the cortisol-implanted but non-infected fish. This in vivo study suggests that protective immunity against C. salmositica is, in part, due to a humoral response.