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.     

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.     

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.     

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.     

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 × 10⁶ 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 × 10⁶ 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.     

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.     

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.     

In vitro secretion of metallo-protease (200 kDa) by the pathogenic piscine haemoflagellate,Cryptobia salmositica Katz,and stimulation of protease production by collagen

Cryptobia salmositica cultured in minimum essential medium secreted metallo-protease, and a significantly higher amount of the protease was found in media supplemented with either type I or type IV collagen. The enhancement of the protease secretion by type I collagen was dose-dependent. Using haemoglobin (substrate) SDS-PAGE, the secreted protease was detected as a single clear band (about 200 kDa). The 200 kDa metallo-protease was also detected on the C. salmositica surface membrane and the amount was increased by incubating the parasite with collagen. Collagen as a specific substrate may enhance the role of the C. salmositica metallo-protease in the disease process in infected fish.     

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.     

In vivo and in vitro cell-mediated immune responses of rainbow trout, Oncorhynchus mykiss (Walbaum), against Cryptobia salmositica Katz, 1951 (Sarcomastigophora: Kinetoplastida)

Abstract. Delayed-type hypersensitivity (DTH) reaction (an in vivo manifestation of cell-mediated immunity) was detected in Oncorhynchus mykiss maintained on a pantothenic-acid-supplemented diet 2 weeks after infection with Cryptobia salmositica. The reaction was similar to that in mammals with mononuclear cell infiltration into the dermis and muscle layers and the presence of oedema. DTH reaction was also displayed by fish on a pantothcnic-acid-supplemented diet that had recovered from the infection and were protected against further infection. The reaction was less marked in infected or protected fish on a pantothenie-acid-deficient diet. Inhibition of macrophage migration (an in vitro expression of cell-mediated immunity) was observed when head kidney cell suspensions from protected fish maintained on either pantothenic acid supplemented or deficient diets were incubated with Cryptobia antigen. No inhibition of migration was evident when head kidney cell suspensions from the above fish were incubated without antigen, nor was it evident when cells from uninfected fish were used. The occurrence of a typical DTH reaction in rainbow trout and the feasibility of assessing it by measuring the thickness of the induration provides a simple and practical method for assessing cell-mediated immunity in large scale vaccination programmes against pathogens.     

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.     

Dietary modulation of humoral immune response and anaemia in rainbow trout, Oncorhynchus mykiss (Walbaum), infected with Cryptobia salmositica Katz, 1951

Abstract. The humoral immune response in Cryptobia-salmositica -infected rainbow trout on pantothenic-acid-deficient/low protein (19%) diets was depressed. There was no significant difference in parasitaemia between fish on the pantothenic-acid-deficient diet and those on the supplemented diet; however, the parasitaemia in fish on 19% protein diet was significantly lower than that in fish on 38% protein diet. The low humoral responders, in contrast to the high responders (on 38 and 29% protein diets), had less severe anaemia and the packed cell volume at the end of the experiment was not significantly different from that at 2 weeks post-infection. Red cells from the low responders were Coombs' negative while those from the high responders were Coombs' positive from 4 to 6 weeks post-infection. The authors suggest that, in the low responders, red cell destruction is essentially due to the'lytic component' of the parasite antigen in contrast to the involvement of both the ^llytic' and the 'immune complex-forming' components in the high responders. The present study indicates that certain deficient diets are useful in modulating the parasitaemia and also in decreasing red cell destruction. Therefore, dietary modification may help in altering the course of such infections and the progression of the disease.     

Experimental Cryptobia salmositica (Kinetoplastida) infections in Atlantic salmon, Salmo salar L.: cell-mediated and humoral immune responses against the pathogenic and vaccine strains of the parasite

Hatchery-reared Atlantic salmon, Salmo salar L., were vaccinated intraperitoneally (i.p.) with a live attenuated Cryptobia salmositica vaccine (either 100 000 or 5000 parasites fish⁻¹) and 4 weeks later were challenged with the parasite (either 100 000 or 5000 parasites fish⁻¹). Unvaccinated, infected salmon had high parasitaemias and were anaemic. Fish given a high dose (100 000 parasites fish⁻¹) had higher parasitaemias than fish given the lower dose. Vaccinated fish had low parasitaemias and a mild anaemia, but recovered quickly after challenge. Complement-fixing antibody increased in vaccinated fish after challenge and was highest at 2 weeks post-challenge. The cell-mediated response (both T cells and B cells) was depressed in infected fish until 4 weeks after infection. In vaccinated fish, the humoral response (i.e. B-lymphocytes) was greater than the cell-mediated response (i.e. T-lymphocytes). In contrast, infected fish had a greater cell-mediated than humoral immune response.     

In vitro and in vivo effects of rabbit anti-thymocyte serum on circulating leucocytes and production of complement fixing antibodies in thymectomized Oncorhynchus mykiss (Walbaum) infected with Cryptobia salmositica Katz 1951

Rabbit anti-thymocyte serum (RATS) against thymocytes of rainbow trout was toxic to leucocytes from intact and thymectomized rainbow trout at 10 °C under in vitro conditions. The total number of leucocytes decreased significantly in 24 h after RATS was injected intraperitoneally into intact rainbow trout, but the number returned to pre-injection level within 1 week. RATS destroyed a lower percentage of leucocytes in thymectomized fish than in intact fish under both in vitro and in vivo conditions and the recovery in the number of leucocytes was slower in thymectomized fish. The parasitaemia, packed cell volume and production of complement fixing antibody in thymectomized and intact fish (injected with RATS before Cryptobia salmositica infection) were not significantly different from control fish (not injected with RATS), and they both acquired protective immunity against cryptobiosis on recovery. This indicates that RATS is not cytotoxic to B-like cells in the lymphoid tissue which produce complement fixing antibody against C. salmositica and that the protective antigen in C. salmositica seems to be thymus-independent.     

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.     

Cryptobia salmositica: an in vitro and in vivo study on the mechanism of anaemia in infected rainbow trout, Salmo gairdneri Richardson

Abstract. There are two basic antigenic components in Cryptobia that are responsible for the anaemia in infected rainbow trout. A 'lytic component', which is dosage-dependent, causes lysis of red blood cells independent of antibody or complement. The second, an 'immune-complex-forming component', attaches to red blood cells, forms immune complexes with specific antibody and activates complement resulting in haemolysis. These two antigenic components, from both live and lysed Cryptobia , were present in the serum of infected fish. When sonicated antigen or heat-inactivated antiserum (from infected fish) was incubated with red cells from uninfected fish, a portion of the red cells was lysed and a positive Coombs' reaction was observed with the remaining intact red cells. The positive Coombs' reaction was due to immune complexes adsorbed onto the red cells and these lysed when incubated with complement. Antibody by itself did not adsorb onto the red cells. From the fourth week post-infection, a positive Coombs' reaction was observed in all infected fish and haemolysis occurred with complement. The authors suggest that, in infected fish, one or more components of the complement cascade is depleted continually during infection and that the anaemia is due to the lytic action of the antigen and immune complex formation on red cells. These lead to intra-vascular haemolysis as well as erythrophagocytosis. In general, the mechanism of anaemia in cryptobiosis appears similar to that in African trypanosomiasis.     

Development of monoclonal antibodies against VP28 of WSSV and its application to detect WSSV using immunocomb

White spot disease (WSD) is an important viral disease of penaeid shrimp caused by white spot syndrome virus (WSSV). WSSV isolated from WSD outbreaks in commercial shrimp (Penaeus monodon) farms in India were propagated in the laboratory in healthy shrimp. The virus was purified from the infected tissues by sucrose gradient centrifugation. The VP28 was electroeluted from SDS-PAGE gels and was used to immunize Balb/c mice to produce hybridomas secreting monoclonal antibodies (MAb) against WSSV. A total of five hybridoma clones secreting MAbs to VP28 were produced. The MAbs were of the isotypes IgG1, IgG2b and IgM. The MAbs reacted with VP28 of WSSV and not with any other viral or shrimp protein in western blot. The MAbs were used to develop dot immunoblot assay using an immunocomb to detect WSSV from field samples. The test developed had an analytical sensitivity of 625 pg and a diagnostic sensitivity of 100% compared to single step polymerase chain reaction (PCR). The test can be used as an alternate for first step PCR to detect WSSV from field samples.     

福建省夏季网箱养殖鱼类主要病害情况及对策

研究了6种中草药水提取物对刺激隐核虫(Crytocaryon irritans)的体外杀灭效果,发现用7.14 mg·mL^–1的乌梅(Fructus mume)、槟榔(Areca catechu)、贯众(Dryopteris setosa)和石榴皮(Punica granatum)的水提取物分别处理幼虫5 min,幼虫死亡率达100%,用28.57 mg·mL^–1处理包囊4 h,包囊死亡率达84.1%以上。为评估喂服乌梅水提物对珍珠石斑鱼(Epinephelus lanceolatus♀×E.fuscoguttatus♂)预防刺激隐核虫病的效果,投喂第14天后对石斑鱼的增重、溶菌酶活性、补体旁路途径溶血活性和鱼体滋养体数量及死亡率进行测试和评估。结果显示,药物组石斑鱼的增重和旁路补体溶血活性与对照组没有显著性差异(P>0.05),溶菌酶活性均显著高于对照组(P<0.05),感染刺激隐核虫后滋养体数量均显著低于对照组(P<0.05);致死剂量攻毒后,药物组(11.4 g·kg^–1、6.84 g·kg^–1、3.42 g·kg^–1)和对照组鱼的成活率分别为40%、20%、46.67%和0%。结果表明乌梅对预防刺激隐核虫病具有一定的开发前景。