Surface Immobilization Antigen of the Parasitic Ciliate Ichthyophthirius multifiliis Elicits Protective Immunity in Channel Catfish (Ictalurus punctatus) (original) (raw)
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Infection and Immunity, 1996
Fish acquire immunity against the ciliated protozoan parasite Ichthyophthirius multifiliis following sublethal infection. The immune response includes the elaboration of humoral antibodies against a class of abundant surface membrane proteins referred to as immobilization antigens (i-antigens). Antibodies against these proteins immobilize the parasite in vitro, suggesting a potential role for the i-antigens in protective immunity. To test this hypothesis, passive immunization experiments were carried out with naive channel catfish, Ictalurus punctatus, using immobilizing murine monoclonal antibodies (MAbs). Fish were completely protected against lethal challenge following intraperitoneal injection of 20 to 200 micrograms of MAb. Although fish succumbed to infection at lower doses, palliative effects were observed with as little as 2 micrograms of antibody. In experiments in which animals were challenged at various times following inoculation, an inverse relationship between parasite...
Surface immobilization antigens of Ichthyophthirius multifiliis: Their role in protective immunity
Annual Review of Fish Diseases, 1995
In response to infection with the pathogenic ciliate Ichthyophthirius multifiliis, fish produce serum and mucus antibodies that immobilize the parasite in vitro. The antigens responsible for this phenomenon (referred to as immobilization antigens, or i-antigens) are thought to be involved in protective immunity and are being studied in connection with efforts to develop subunit vaccines. Using mammalian antibodies, the i-antigens of Ichthyophthirius have been identified as a family of related surface proteins with Mr's in the 40–60 kDA range. The amino acid sequence deduced from a 1.2 kb cDNA encoding a member of this family predicts a protein with a highly periodic structure characteristic of the i-antigens of the free-living ciliates, Paramecium and Tetrahymena. To date, four distinct immobilization serotypes of I. multifiliis have been identified. Northern hybridization studies indicate that i-antigen genes of Ichthyophthirius are developmentally regulated during the parasite life cycle and are expressed at extraordinarily high levels in the infective stage. Synthesis of i-antigen mRNA transcripts may also be accompanied by novel RNA processing events. A role for the i-antigens in protective immunity is strongly suggested by the results of passive immunization studies with immobilizing monoclonal antibodies (i-mAbs). Following intraperitoneal injection of naïve channel catfish, i-mAbs confer complete protection against an otherwise lethal parasite challenge. In conjunction with ELISA and in vitro immobilization assays, passive immunization experiments indicate that protection requires the presence of antibody at the site of infection (that is, at the surface of fish). The results of these studies are discussed in the light of current knowledge about mechanisms of protection against I. multifiliis, and a model of surface immunity is presented.
Immune response of channel catfish to ciliary antigens of Ichthyophthirius multifiliis
Developmental and Comparative Immunology, 1988
Channel catfish were rendered immune to the protozoan pathogen, Ichthyophthirius multifiliis, by exposure to sublethal infections. Sera from test animals were then screened for antibodies against the parasite using enzyme-linked immunoassays. Ichthyophthirius cilia were blotted onto nitrocellulose filters and reacted with catfish sera, followed by rabbit anti-catfish Ig antibodies coupled to horseradish peroxidase. Subsequent color development revealed the presence of anti-ciliary antibodies in a number of fish tested. Reactions appeared to be highly specific; little cross-reactivity was seen in equivalent assays with heterologous cilia from Tetrahymena. Ciliary antigens were associated predominantly with a membrane polypeptide fraction isolated from intact cilia by phase separation in solutions of the nonionic detergent, Triton X-114. The relative levels of anti-ciliary antibodies in sera from individual fish were quantitated by photometric scanning of immunoblot assays. A strong correlation (P<.03) was found between antibody levels and the ability of sera to agglutinate live parasites in vitro.
Host responses against the fish parasitizing ciliate Ichthyophthirius multifiliis
Veterinary Parasitology, 2001
Recent studies have shown that fish are able to mount protective immune responses against various parasites. One of the best characterized parasite-host system in this context is the ciliate Ichthyophthirius multifiliis (Ich) parasitizing a range of freshwater fishes. Both specific and non-specific host defence mechanisms are responsible for the protection of fish against challenge infections with this ciliate. The specific humoral components comprise at least specific antibodies. The non-specific humoral elements included are the alternative complement pathway and probably lectins. Cellular factors involved in the specific response are B-cells and putative T-cells. The non-specific effector cells recognized are various leukocytes. In addition, goblet-cells and mast cells (EGC-cells) may have a function. The NCC-cell (suggested analogue to NK-cells in mammals) seems to play a role in the non-specific response. This well documented protective response in freshwater fishes against Ich has urged the development of anti-parasitic vaccines. Indeed, such products based on formalin killed parasites have been developed and found to offer the vaccinated host a satisfactory protection. However, the collection of parasites for vaccine production is extremely laborious. It involves keeping infected fish due to the fact that in vitro propagation of the parasite is still insufficiently developed. Gaining knowledge of amino acid sequences and its encoding DNA-sequences for the protective antigens (i-antigens) in the parasite was a major breakthrough. That achievement made it possible to produce a recombinant protein in E. coli and preliminary results indicated a certain protection of fish vaccinated with this product. Recent work has shown that the free-living and easily cultivated ciliate Tetrahymena can be transformed and express the i-antigen. This path seems to be promising for future development of vaccines against Ich. A novel approach in fish is the development of DNA-vaccines. Successful DNA-vaccination trials have been conducted in fish against viral infections and the technology also makes it possible to develop a DNA-vaccine against Ich. Other approaches to immuno-protection against Ich have been the use of heterologous vaccines. Thus, both bath and injection vaccination using live or killed (un-transformed) Tetrahymena has been reported to offer treated fish a certain level of protection. Such protection could be explained
Ichthyophthirius multifiliis Has Membrane-Associated Immobilization Antigens
Journal of Eukaryotic Microbiology, 1989
Sera from fish that survive infections with the ciliated protozoon, Ichthyophthirius multifiliis, immobilize the parasite in vitro. In order to identify ceil surface antigens involved in the immobilization response, integral membrane proteins were extracted from tomites with Triton X-l14 and used to immunize rabbits. The rabbit antisera immobilized the parasite in vitro and antigens were localized to cell and ciliary plasma membranes by indirect immunofluorescent microscopy. The membrane protein fractions from both whole cells and tomite cilia were characterized by 1 - and 2-dimensiona! SDS-PAGE. A 43,000-dalton (D) glycoprotein with an isoelectric point of 7.0 is the predominant protein in these fractions, comprising 12% and 60% of the total protein of whole cell and ciliary membranes, respectively. Western blot analysis of ciliary proteins with immune rabbit sera indicated that the 43,000-D glycoprotein is the principal antigen.
Fish & Shellfish Immunology, 2007
Vaccination of channel catfish with either of two serotypes of the parasitic ciliate Ichthyophthirius multifiliis conferred protection against challenge infection by either serotype. Fish were vaccinated by intracoelomic injection with live theronts of isolate G5 (serotype D) or isolate G12 (a new serotype), which express different surface immobilisation antigens. Vaccination with live G12 theronts conferred complete protection against subsequent challenge by both serotypes while vaccination with G5 theronts elicited only partial protection against both serotypes. Vaccination with trophont lysates did not protect against challenge infection. Sera from vaccinated fish were tested in immobilisation assays, ELISAs, and Western blots. Serum antibodies recognised only immobilisation antigens of the serotype used for vaccination in immobilisation assays or on Western blots. No antigens common to both serotypes were identified by Western blots. In contrast, serum antibodies bound antigens in cell lysates from both serotypes by ELISA, demonstrating that antibodies recognising both serotypes are produced in response to infection, which presumably confer observed cross-serotype protection.
Immune response of channel catfish to ciliary antigens of
Developmental and Comparative Immunology, 1988
Channel catfish were rendered immune to the protozoan pathogen, , by exposure to sublethal infections. Sera from test animals were then screened for antibodies against the parasite using enzyme-linked immunoassays. cilia were blotted onto nitrocellulose filters and reacted with catfish sera, followed by rabbit anti-catfish lg antibodies coupled to horseradish peroxidase. Subsequent color development revealed the presence of anti-ciliary antibodies in a number of fish tested. Reactions appeared to be highly specific; little cross-reactivity was seen in equivalent assays with heterologous cilia from . Ciliary antigens were associated predominantly with a membrane polypeptide fraction isolated from intact cilia by phase separation in solutions of the nonionic detergent, Triton X-114. The relative levels of anti-ciliary antibodies in sera from individual fish were quantitated by photometric scanning of immunoblot assays. A strong correlation (P<.03) was found between antibody levels and the ability of sera to agglutinate live parasites .
Clinical and Vaccine Immunology, 2003
Fish acquire protective immunity against the ciliated protozoan parasite Ichthyophthirius multifiliis following sublethal infection or inoculation with I. multifiliis immobilization antigens (i-antigens). In both cases, parasite-immobilizing antibodies have been identified in sera and mucosal secretions. To investigate the kinetics of this immune response, antibody levels were determined by enzyme-linked immunosorbent assay (ELISA) in the sera and cutaneous mucus of channel catfish (Ictalurus punctatus) that were either infected with parasites or given a single injection of purified i-antigen (5.0 g/fish) in Freund's incomplete adjuvant. At 5 weeks, infected and inoculated fish had a mean serum (1:80 dilution) antibody absorbance (A 405 ) value of 0.54 ؎ 0.17 and 0.35 ؎ 0.03, respectively, which were significantly higher (␣ ؍ 0.05) than the pretreatment serum (1:80 dilution) antibody absorbance value of 0.24 ؎ 0.05. At 14 weeks, mean serum (1:80 dilution) ELISA absorbance values in the teo groups of fish increased to 0.79 ؎ 0.30 and 0.71 ؎ 0.24, respectively. In both groups of fish, antibody levels in cutaneous mucus (undiluted) were much lower than those in sera. Infected fish had detectable mucus (undiluted) antibody levels from 3 to 9 weeks, with the highest mean value (0.30 ؎ 0.07) occurring at 7 weeks. Although individual inoculated fish produced serum antibody absorbance values comparable to those seen in infected fish, the mean mucus antibody values in this group did not rise above pretreatment levels.
Ichthyophthirius multifiliis: a model of cutaneous infection and immunity in fishes
Immunological Reviews, 1998
Summary: The parasitic ciliate Ichthyophthirius multifilus offers a useful system for the study of cutaneous immunity against an infectious microorganism. Naive fish usually die following infection, but animals surviving sublethal parasite exposure become resistant to subsequent challenge. This resistance correlates with the presence of humoral antibodies in the sera and cutaneous mucus of immune fish. A mechanism of immunity has recently been elucidated that involves and body binding to surface proteins (referred to as immobilization antigens or i-antigens) located on the parasite cell and ciliary membranes. Antibody-mediated cross-linking of i-antigens triggers a response by the parasite resulting in its exit from the host. These effects can be observed directly on the surface of live fish. In addition to allowing the observation of effector responses in vivo, Ichthyophthirius also provides a means to study the ontogeny of the mucosal immune response. The sites of antigen capture and presentation, and the sites of antibody production, are unknown with regard to cutaneous immunity. Because the external epithelial surfaces of fish are often the points of pathogen entry, a basic understanding of the inductive immune mechanisms and immune cell interactions in the skin and gills is extremely important with regard to vaccine development. The development of Ichthyophthirius as an experimental system and how it might be used to address these issues are discussed in this review.