Immunity to Ichthyophthirius infections in fish: a synopsis.

Ichthyophthirius multifiliis is a ciliated protozoan parasite that infects freshwater fish. It has been the subject of both applied and basic research for over 100years, which can be attributed to its world-wide distribution and its significant economic impact on both food and aquarium fish production. I. multifiliis serves as a model for studies in fish on innate and acquired immunity, as well as on mucosal immunity. Although an obligate parasite, I. multifiliis is relatively easily passaged from infected to naïve fish in laboratory aquaria, and is easily observed and manipulated under laboratory conditions. It parasitizes the epithelia of the skin and gills, which facilitates in vivo experimentation and quantification of challenge. This review provides a description of both mucosal and systemic innate and adaptive immune responses to parasite infection, a synopsis of host-parasite immunobiology, vaccine research, and suggested areas for future research to address critical remaining questions. Studies in carp and rainbow trout have shown that extensive tissue damage occurs when the parasite invades the epithelia of the skin and gills and substantial focal and systemic inflammatory responses are elicited by the innate immune response. The adaptive immune response is initiated when phagocytic cells are activated by antigens released by the parasite. It is not known whether activated T and B cells proliferate locally in the skin and gills following infection or migrate to these sites from the spleen or anterior kidney. I. multifiliis infection elicits both mucosal and systemic antibody production. Fish that survive I. multifiliis infection acquire protective immunity. Memory B cells provide long-term humoral memory. This suggests that protective vaccines are theoretically possible, and substantial efforts have been made toward developing vaccines in various fish species. Exposure of fish to controlled surface infections or by intracoelomic injection of live theronts provides protection. Vaccination with purified immobilization antigens, which are GPI-anchored membrane proteins, also provides protection under laboratory conditions and immobilization antigens are currently the most promising candidates for subunit vaccines against I. multifiliis.

Endosymbiotic bacteria in the parasitic ciliate Ichthyophthirius multifiliis.

Endosymbiotic bacteria were identified in the parasitic ciliate Ichthyophthirius multifiliis, a common pathogen of freshwater fish. PCR amplification of DNA prepared from two isolates of I. multifiliis, using primers that bind conserved sequences in bacterial 16S rRNA genes, generated an approximately 1,460-bp DNA product, which was cloned and sequenced. Sequence analysis demonstrated that 16S rRNA gene sequences from three classes of bacteria were present in the PCR product. These included Alphaproteobacteria (Rickettsiales), Sphingobacteria, and Flavobacterium columnare. DAPI (4',6-diamidino-2-phenylindole) staining showed endosymbionts dispersed throughout the cytoplasm of trophonts and, in most, but not all theronts. Endosymbionts were observed by transmission electron microscopy in the cytoplasm, surrounded by a prominent, electron-translucent halo characteristic of Rickettsia. Fluorescence in situ hybridization demonstrated that bacteria from the Rickettsiales and Sphingobacteriales classes are endosymbionts of I. multifiliis, found in the cytoplasm, but not in the macronucleus or micronucleus. In contrast, F. columnare was not detected by fluorescence in situ hybridization. It likely adheres to I. multifiliis through association with cilia. The role that endosymbiotic bacteria play in the life history of I. multifiliis is not known.

Occurrence of Ichthyophthirius multifiliis within the peritoneal cavities of infected channel catfish Ictalurus punctatus.

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.

Surface display of a parasite antigen in the ciliate Tetrahymena thermophila.

The ciliated protozoan, Tetrahymena thermophila, offers an attractive medium for the expression of heterologous proteins and could prove particularly useful for the display of foreign proteins on the cell surface. Although progress has been made in transformation of Tetrahymena with heterologous DNA, methods that permit reliable expression of foreign genes have been lacking. Using a mutant strain of T. thermophila carrying a negatively selectable allele of a beta-tubulin gene, we have been able to direct foreign genes to this locus by homologous recombination. Transformed cell lines producing foreign proteins were readily identified and, in at least one case, targeting of proteins to the plasma membrane was accomplished.

The gene for an abundant parasite coat protein predicts tandemly repetitive metal binding domains.

Immobilization antigens are highly abundant surface membrane proteins that coat the surface of hymenostomatid ciliates. While their function is unknown, recent studies with the common fish parasite, Ichthyophthirius multifiliis, suggest their involvement in a novel mechanism of humoral immunity involving an effect of antibody on parasite behavior. To gain further insight into the nature of these proteins, we have cloned a gene encoding the 48kDa i-antigen of I. multifiliis. Analysis of the gene (designated IAG48[G1]) reveals a single, uninterrupted reading frame that predicts a protein of 442 amino acids. Based on its deduced amino acid sequence, the protein contains hydrophobic amino acid domains at its N- and C-terminus that are characteristic of signal peptide and GPI-anchor addition sites, respectively. The most striking feature of the predicted protein, however, is a series of tandem repeats that spans most of its length. The repeats themselves are characterized by periodic cysteine residues that fall into register when the homologous segments are aligned. Interestingly, the spacing of cysteines (C-X2,3-C) within a framework of larger (C-X2-C-X20-C-X3-C-X20-C-X2-C) motifs is entirely consistent with the structure of known zinc-binding proteins. Finally, comparison of the coding sequence of the 48kDa i-antigen gene with a partial cDNA previously thought to encode this protein reveals nearly complete identity except at their 3' ends, suggesting that alternative forms of the antigen exist.

Antibody-mediated effects on parasite behavior: Evidence of a novel mechanism of immunity against a parasitic protist.

The parasitic ciliate Ichthyophthirius multifiliis is well known in commercial aquaculture as the etiological agent of 'white spot', a disease that afflicts a wide range of fresh-water fish. While Ichthyophthirius is highly pathogenic, animals exposed to controlled infections develop a strong acquired resistance to the parasite. Recent studies suggest host resistance involves a novel mechanism of humoral immunity affecting parasite behavior. Rather than being killed, parasites are forced to exit fish prematurely in response to antibody binding. The target antigens involved in this process are a class of highly abundant glycosylphosphatidyl-inositol-anchored coat proteins referred to as immobilization antigens, or i-antigens. Here, Theodore Clark and Harry Dickerson describe this phenomenon and offer a number of hypotheses that could account for the forced exit.

Analysis of plasmids cloned from a virulent avian Escherichia coli and transformed into Escherichia coli DH5 alpha.

Three of four plasmids from a virulent wild-type avian Escherichia coli were cloned or transformed into an avirulent laboratory recipient E. coli DH5 alpha and tested for the ability to confer a virulence phenotype. The three plasmids transformed into E. coli DH5 alpha were 5, 6, and 56 kb. A fourth plasmid of 64 kb was not successfully transformed. Parameters used to measure virulence included presence of type 1 pili and a smooth lipopolysaccharide (LPS) layer, motility, production of Colicin V, resistance to host complement, and embryo lethality. The 5-kb plasmid encoded for ampicillin resistance, whereas the 6-kb plasmid encoded for tetracycline resistance. The 56-kb plasmid encoded for streptomycin, sulfisoxazole, and tetracycline resistance. Twelve-day embryos inoculated with 467 colony-forming units of E. coli DH5 alpha containing the 56-kb plasmid had increased death rates (45%) in the embryo lethality assay and a decreased weight of surviving embryos with cranial hemorrhages as compared with embryos inoculated with similar amounts of E. coli DH5 alpha (0%) and phosphate-buffered saline (0%). Embryos inoculated with the wild-type virulent E. coli had 90% deaths. The 56-kb plasmid also had homology with a probe for Colicin V production (cvaC). No differences in LPS layer, complement resistance, motility, Colicin V activity, type 1 pili, cell-free supernatant proteins, or outer membrane proteins were observed in the transformants when compared with nontransformed E. coli DH5 alpha.

Surface antigen cross-linking triggers forced exit of a protozoan parasite from its host.

We used the common fish pathogen Ichthyophthirius multifiliis as a model for studying interactions between parasitic ciliates and their vertebrate hosts. Although highly pathogenic, Ichthyophthirius can elicit a strong protective immune response in fish after exposure to controlled infections. To investigate the mechanisms underlying host resistance, a series of passive immunization experiments were carried out using mouse monoclonal antibodies against a class of surface membrane proteins, known as immobilization antigens (or i-antigens), thought to play a role in the protective response. Such antibodies bind to cilia and immobilize I. multifiliis in vitro. Surprisingly, we found that passive antibody transfer in vivo caused rapid exit of parasites from the host. The effect was highly specific for a given I. multifiliis serotype. F(ab)2 subfragments had the same effect as intact antibody, whereas monovalent Fab fragments failed to protect. The activity of Fab could, nevertheless, be restored after subsequent i.p. injection of bivalent goat anti-mouse IgG. Parasites that exit the host had detectable antibody on their surface and appeared viable in all respects. These findings represent a novel instance among protists in which protective immunity (and evasion of the host response) result from an effect of antibody on parasite behavior.

Sustained growth of Ichthyophthirius multifiliis at low temperature in the laboratory.

Applied and basic research on the ciliate Ichthyophthirius multifiliis, an obligate parasite of freshwater fishes, requires passage on fish hosts to maintain laboratory stocks. However, continual repeated passage results in senescence of parasite clones over time. Because growth and development are directly correlated to water temperature, our objective was to grow the parasite at low temperature in order to extend the period that the organism remains on the fish, thus reducing: (1) the number of passages and (2) the number of fish required to maintain the parasite over time. Lowering of water temperature from 25 C to 9 C resulted in significant slowing of growth on channel catfish (parasites remained on fish for 20.4 days at 9 C, as compared to 5-6 days at 25 C), with no discernible changes in viability, antigenicity, or infectivity. Low-temperature growth is proposed as an improved method for stable maintenance of I. multifiliis cultures in the laboratory.

Serotypic variation among isolates of Ichthyophthirius multifiliis based on immobilization.

Efforts have been made to determine whether surface antigens could be used as biochemical markers to define strain differences in the parasitic ciliate Ichthyophthirius multifiliis. In previous studies, a wild-type isolate designated G1 was found to have surface proteins analogous to the immobilization antigens of Paramecium and Tetrahymena; rabbit antiserum against this strain immobilizes homologous cells in vitro. It has now been shown for two additional Ichthyophthirius isolates (designated G1.1 and G2) that immobilization antigens are both present and serologically distinct. Proteins of similar size, which cross-react in Western blots with rabbit antisera against immobilization antigens of the G1 strain, are nevertheless found in the G1.1 and G2 isolates. As shown by Southern blotting analysis, the G1.1 and G2 strains also contain genomic DNA sequences which hybridize with an immobilization antigen cDNA from G1 when probed under conditions of reduced stringency. The serotypic differences in immobilization between I. multifiliis isolates appear to be stable over time and provide a means of discriminating strains. In addition to providing a basis for comparative studies, the work described here has implications for the development of vaccines against this important fish parasite.

Developmental expression of surface antigen genes in the parasitic ciliate Ichthyophthirius multifiliis.

A 1.2-kilobase (kb) cDNA encoding a major surface antigen of the holotrich ciliate Ichthyophthirius multifiliis (an obligate parasite of fish) has been isolated and used as a probe to examine the expression of immobilization antigen (i-antigen) genes in this system. The cDNA encodes a predicted protein of 394 amino acids with a tandemly repeated structure characteristic of the i-antigens of the related free-living ciliates Paramecium and Tetrahymena. As shown by Northern hybridization analysis with both total and poly(A)+ RNAs, the 1.2-kb cDNA recognizes distinct transcripts of 1.6 and 1.9 kb which are differentially expressed through the parasite life cycle. During the transition from the host-associated trophozoite stage to the infective tomite stage, steady-state levels of the 1.9-kb RNA undergo a marked increase of greater than or equal to 50-fold, while the 1.6-kb transcript increases only slightly. The absolute amounts of RNA encoding the i-antigen have been quantitated and were found to reach extremely high levels equivalent to approximately 6% of the poly(A)+ RNA of I. multifiliis tomites. Southern hybridization analysis with I. multifiliis genomic DNA suggests that at least two genes encode the i-antigen transcripts. In experiments to examine the effects of temperature on the expression of I. multifiliis i-antigen genes, levels of the 1.6- and 1.9-kb transcripts were found to remain relatively constant in cells maintained at different temperature extremes. These studies indicate that genes encoding i-antigens of I. multifiliis are developmentally regulated, and they suggest the existence of alternative mechanisms for the control of surface antigen expression in ciliates.

Purification and partial characterization of immobilization antigens from Ichthyophthirius multifiliis.

Ichthyophthirius multifiliis, a parasitic ciliate of freshwater fishes, was found to have surface antigens (Ag) which elicited immobilizing antibodies (Ab) when injected into rabbits. An effort was made to purify and characterize these Ag (referred to as immobilization Ag) because of their potential role in protective immunity in fishes. Mice immunized with theront cilia were used for production of immobilizing monoclonal antibodies (MAb). Hybridomas were screened by indirect immunofluorescent light microscopy and immobilization of live parasites. Six hybridomas producing immobilizing MAb were cloned. Immobilizing MAb were used to affinity purify Ag solubilized with Triton X-114 and Na deoxycholate. Two membrane protein Ag of approximately 48 and 60 kDa were identified. Immobilizing MAb failed to react with these Ag on Western blots and, conversely, MAb that reacted with the Ag on Western blots did not immobilize live organisms. These results suggest that immobilization required native conformational epitopes which were altered by Western blotting procedures. Individual MAb reactive on Western blots recognized both the 48- and 60-kDa proteins indicating the presence of common epitopes. Affinity purified Ag elicited immobilizing antisera when injected into rabbits, mice, and channel catfish.

Comparison of a complement resistance test, a chicken embryo lethality test, and the chicken lethality test for determining virulence of avian Escherichia coli.

Results with four pathogenic avian Escherichia coli isolates and one avirulent isolate in a complement resistance test, a chicken lethality test, and a chicken embryo lethality test were compared. Results of the complement resistance test with these isolates were highly correlated to results of the chicken lethality test of virulence. The chicken embryo test yielded results that were of a medium positive correlation with the chicken lethality results. The results of the complement resistance and chicken embryo lethality tests were highly correlated.

Influence of calcium on secretion and activity of the cytolysins of Actinobacillus pleuropneumoniae.

In vitro production of a secreted hemolytic cytolysin of Actinobacillus pleuropneumoniae has been reported to be dependent on the presence of calcium in culture media. This is not the case with Escherichia coli hemolysins, however, where calcium has been shown to be required only for activation and binding to target cells. Because the cytolysins of A. pleuropneumoniae have structural and functional similarities to those of hemolytic E. coli, we sought to reexamine the role that calcium plays in the secretion and activity of A. pleuropneumoniae cytolysins. A. pleuropneumoniae hemolytic strain S4074 secreted two major proteins into culture supernatants independent of the presence of calcium in growth medium. These proteins were identified with murine monoclonal antibodies as the 105-kDa cytolysin I and the 103-kDa cytolysin II. It was found that both cytolysins required calcium for binding to erythrocyte membranes. Culture fluids from bacteria grown with calcium lysed porcine erythrocytes even after free calcium in the fluid was removed prior to the hemolytic assay. When bacteria were grown in medium depleted of calcium, no lysis of erythrocytes was detected unless calcium was added to assay buffers. Culture supernatants from A. pleuropneumoniae nonhemolytic strain 1421 grown with or without calcium contained two predominant proteins, which were identified with mouse monoclonal antibodies as the 103-kDa cytolysin II and the 120-kDa cytolysin III. Binding to erythrocytes (without hemolysis) by cytolysin II was dependent on calcium. Cytolysin III did not bind to erythrocytes. These results indicate that the ability of strain S4074 to lyse swine erythrocytes (and the inability of strain 1421 to do so) was directly correlated with the presence of cytolysin I. Cytolysins I, II, and III bound to swine neutrophils and purified neutrophil membranes only in the presence of calcium. When calcium was depleted, cytolysin I of strain S4074 had a reduced binding and cytolysis II and III of strain 1421 did not bind at all. The data suggest that regardless of the target cell involved, calcium plays an integral role in the function but not the production of A. pleuropneumoniae cytolysins.

Influence of antibody treatment of Campylobacter jejuni on the dose required to colonize chicks.

This study was designed to clarify the role of antibodies in controlling chicken colonization by Campylobacter jejuni. Cecal colonization by C. jejuni was compared after the organism was exposed either to phosphate-buffered saline, normal rabbit serum, rabbit hyperimmune anti-C. jejuni serum, or anti-C. jejuni antibodies extracted from chicken bile. Antibodies from chicken bile were extracted by affinity absorption against outer-membrane proteins from the challenge organism. Sera were heated 1 hour at 56 C to destroy complement activity. Bacterial inoculum levels were enumerated after 1 hour exposure at 4 C to the various treatments. The heated sera and the bile antibodies were not bactericidal, and bacterial agglutination was not evident. Serial dilutions of the antibody-treated C. jejuni were given by gavage into 1-day-old chicks. Six days later, the ceca were removed from the chicks, and samples were cultured on Campylobacter-charcoal differential agar. The colonization dose-50% was increased by twofold to 160-fold when the organism was preincubated with hyperimmune antiserum or the bile antibodies as compared with preincubation with phosphate-buffered saline. We conclude that antibodies inhibit chicken cecal colonization by C. jejuni.

Icthyophthirius multifiliis has membrane-associated immobilization antigens.

Sera from fish that survive infections with the ciliated protozoon, Ichthyophthirius multifiliis, immobilize the parasite in vitro. In order to identify cell surface antigens involved in the immobilization response, integral membrane proteins were extracted from tomites with Triton X-114 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-dimensional 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.

Immune response of channel catfish to ciliary antigens of Ichthyophthirius multifiliis.

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 less than .03) was found between antibody levels and the ability of sera to agglutinate live parasites in vitro.

Production and preliminary characterization of murine monoclonal antibodies to Ichthyophthirius multifiliis, a protozoan parasite of fish.

An initial panel of 34 hybridomas, each secreting antibodies reactive with an infective theront stage of an Ichthyophthirius multifiliis isolate, was produced. Three of these cell lines, each producing immunoglobulin M class antibodies, were cloned by limiting dilution and were expanded as ascites-producing tumors in syngeneic mice. Three monoclonal antibodies (MAB) reacted with intact whole theronts in an enzyme-linked immunosorbent assay to dilutions of 1:10,000 in ascitic fluids and had a similar pattern of surface and cytoplasmic staining in indirect immunofluorescent tests. Only antigen specified by MAB E6 could be characterized by acrylamide gel electrophoresis and immunoblotting; initial data indicated a molecular weight of 200,000. Physicochemical properties of the determinants recognized by the 3 MAB were tested by pronase digestion and periodate oxidation. Seemingly, a protein, glycoprotein, and carbohydrate were recognized by MAB E6, FE10, and AC8, respectively.

Effect of EDTA-tris on an Escherichia coli isolate containing R plasmids.

Solutions of ethylenediaminetetraacetate (EDTA)-tris combined with antibiotics have been shown to be effective in treating selected cases of persistent bacterial infections. Basic techniques in microbial genetics, including mating frequencies, chemical elimination of R plasmids, isolation of plasmid DNA and agarose gel electrophoresis, were used to determine if EDTA-tris has a curing effect on an R plasmid as part of its clinical action. Results of this study indicated that EDTA-tris by itself eliminated an antibiotic resistance marker from a clinical isolate of Escherichia coli and when combined with another chemical curing agent altered the isolate's mating frequency.