Harry W. Dickerson

Ichthyophthirius multifiliis: a model of cutaneous infection and immunity in fishes

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.

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.

Systemic and Cutaneous Mucus Antibody Responses of Channel Catfish Immunized against the Protozoan Parasite Ichthyophthirius multifiliis

ABSTRACT 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 μg/fish) in Freunds incomplete adjuvant. At 5 weeks, infected and inoculated fish had a mean serum (1:80 dilution) antibody absorbance (A405) value of 0.54 ± 0.17 and 0.35 ± 0.03, respectively, which were significantly higher (α = 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. I. multifiliis infection induced a transient mucosal antibody response that coincided with the resolution of infection. Whether elicited by infection or intraperitoneal injection of i-antigen, the serum and mucus antibody responses of channel catfish immunized against I. multifiliis did not occur synchronously.

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 β-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.

Surface Immobilization Antigen of the Parasitic Ciliate Ichthyophthirius multifiliis Elicits Protective Immunity in Channel Catfish (Ictalurus punctatus)

ABSTRACT Channel catfish (Ictalurus punctatus) that survive infection with the parasitic ciliate Ichthyophthirius multifiliis acquire immunity to subsequent challenge and produce specific antibodies in serum that immobilize the parasite in vitro. Cellular surface protein antigens targeted by these antibodies are referred to as immobilization antigens (i-antigens). By using an immobilizing mouse monoclonal antibody as a ligand, the i-antigen of I. multifiliis isolate G5 was purified to homogeneity by immunoaffinity chromatography, and its immunogenicity was confirmed by inoculating rabbit and channel catfish to produce immobilizing antisera. To test the purified i-antigen as a subunit vaccine, channel catfish fingerlings were injected intraperitoneally (i.p.) with purified i-antigen at a dose of 10 μg/fish in complete Freund’s adjuvant on day 1, followed by a second i.p. injection of the same amount of i-antigen in incomplete Freund’s adjuvant on day 15. Negative control fish were immunized similarly with either bovine serum albumin (BSA) or an immobilization-irrelevant I. multifiliis protein. On day 84, the fish were challenged with live I. multifiliis G5 theronts at a dose of 15,000 cells per fish. Seventy-two percent of the fish immunized with i-antigen survived the challenge. All negative control fish died within 16 days of exposure. There was a significant difference in the median days to death between the negative control fish injected with BSA and the fish that died following vaccination with i-antigen. Fish injected with i-antigen developed high immobilizing antibody titers in serum. This is the first demonstration of a direct role for i-antigens in the elicitation of protective immunity, suggesting that these proteins by themselves serve as effective subunit vaccines against I. multifiliis.

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.

Immune response of fishes to ciliates

Ciliates are highly evolved protists comprising a phylum of diverse species, many of which are opportunistic or obligate parasites. Ciliates parasitic to fish consist of salt and freshwater forms with endo- or ectoparasitic modes of infection. Some of the more commonly encountered genera include Chilodonella, Brooklynella, Ophryoglenina, Ichthyophthirius, Cryptocaryon, Uronema, Tetrahymena, Epistylus, and Trichodina. Species range from obligate parasites and commensals to opportunistic, facultative forms. Some parasitic ciliates are highly pathogenic and fishes in closed environments such as aquaria and farm ponds are particularly susceptible to high mortalities. Nevertheless, fish have evolved an immune system capable of mounting an effective protective response against parasite challenge. Much of the experimental research on immunity against ciliates has been carried out with Ichthyophthirius multifiliis, on obligate parasite that invades surface epithelia of virtually all freshwater fish species. Interest in the immune response against I. multifiliis stems from the fact that convalescent fish become resistant to subsequent challenge (suggesting the possibility of immunoprophylaxis), and the need to curtail severe losses caused by this parasite in intensively farmed fishes. Furthermore, I. multifiliis has proven to be a useful experimental model because it is amenable to study under laboratory conditions. In this review cellular and humoral factors involved in both innate and acquired immunity against ciliates are covered and include natural killer cells, phagocytic cells, and antibody responses. Current ideas on the mechanisms of antibody-mediated cutaneous immunity against I. multifiliis are discussed and approaches toward the development of vaccines against this and other ciliate parasites are presented.

Endosymbiotic Bacteria in the Parasitic Ciliate Ichthyophthirius multifiliis

ABSTRACT 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 ∼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.

Surface immobilization antigens of Ichthyophthirius multifiliis: Their role in protective immunity

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 Mrs 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 naive 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.

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.