Theodore G. Clark

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.

Functional identification of dendritic cells in the teleost model, rainbow trout (Oncorhynchus mykiss).

Dendritic cells are specialized antigen presenting cells that bridge innate and adaptive immunity in mammals. This link between the ancient innate immune system and the more evolutionarily recent adaptive immune system is of particular interest in fish, the oldest vertebrates to have both innate and adaptive immunity. It is unknown whether dendritic cells co-evolved with the adaptive response, or if the connection between innate and adaptive immunity relied on a fundamentally different cell type early in evolution. We approached this question using the teleost model organism, rainbow trout (Oncorhynchus mykiss), with the aim of identifying dendritic cells based on their ability to stimulate naïve T cells. Adapting mammalian protocols for the generation of dendritic cells, we established a method of culturing highly motile, non-adherent cells from trout hematopoietic tissue that had irregular membrane processes and expressed surface MHCII. When side-by-side mixed leukocyte reactions were performed, these cells stimulated greater proliferation than B cells or macrophages, demonstrating their specialized ability to present antigen and therefore their functional homology to mammalian dendritic cells. Trout dendritic cells were then further analyzed to determine if they exhibited other features of mammalian dendritic cells. Trout dendritic cells were found to have many of the hallmarks of mammalian DCs including tree-like morphology, the expression of dendritic cell markers, the ability to phagocytose small particles, activation by toll-like receptor-ligands, and the ability to migrate in vivo. As in mammals, trout dendritic cells could be isolated directly from the spleen, or larger numbers could be derived from hematopoietic tissue and peripheral blood mononuclear cells in vitro.

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.

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.

Avidin-Biotin Micropatterning Methods for Biosensor Applications

High-resolution patterning methods have been developed to immobilize functional proteins onto a silicon dioxide surface for biosensor applications. Antibody lines, as small as 5 μm in width, with intervening 5 μm spacings, were patterned on oxidized silicon wafers using avidin-biotin chemistry. The N-hydroxysuccinimide (NHS) ester of photoactivatable biotin was covalently bound to a self assembled monolayer (SAM) of 3-amino-propyltriethoxysilane (3-APTS) after irradiation by 350 nm ultraviolet (UV) light from a 25 W Hg arc lamp. The patterned layers were evaluated using fluorescent imaging of Alexa-488 conjugated avidin and two fluorescence-conjugated antibodies. This technique allows binding of any biotinylated compound without exposure to harmful UV light, extreme pH, toxic chemicals, or high salinity.

The I-antigens of Ichthyophthirius multifiliis are GPI-Anchored Proteins

Abstract The parasitic ciliate Ichthyophthirius multifiliis has abundant surface membrane proteins (i-antigens) that when clustered, trigger rapid, premature exit from the host. Similar antigens are present in free-living ciliates and are GPI-anchored in both Paramecium and Tetrahymena. Although transmembrane signalling through GPI-anchored proteins has been well-documented in metazoan cells, comparable phenomena have yet to be described in protists. Since premature exit of Ichthyophthirius is likely to involve a transmembrane signalling event, we sought to determine whether i-antigens are GPI-anchored in these cells as well. Based on their solubility properties in Triton X-114, the i-antigens of Ichthyophthirius are amphiphilic in nature and partition with the detergent phase. Nevertheless, following treatment of detergent lysates with phospholipase C, the same proteins become hydrophilic. Concomitantly, they are recognized by antibodies against a cross-reacting determinant exposed on virtually all GPI-anchored proteins following cleavage with phospholipase C. Finally, when expressed in recombinant form in Tetrahymena thermophila, full-length i-antigens are restricted to the membrane, while those lacking hydrophobic C-termini are secreted from the cell. Taken together, these observations argue strongly that the i-antigens of Ichthyophthirius multifiliis are, in fact, GPI-anchored proteins.

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.

Metallothionein Gene from Tetrahymena thermophila with a Copper-Inducible-Repressible Promoter

ABSTRACT We describe a novel metallothionein gene from Tetrahymena thermophila that has a strong copper-inducible promoter. This promoter can be turned on and off rapidly, making it a useful system for induction of ectopic gene expression in Tetrahymena and enhancing its applications in cell and molecular biology, as well as biotechnology.

Tetrahymena genome database Wiki: a community-maintained model organism database

When funding for Tetrahymena Genome Database (TGD) ended in 2006, no further updates were made to this important community resource and the main database was taken offline in 2008. We have restored and updated this important resource for use by the Tetrahymena research community. We have also retooled the TGD website (now TGD Wiki) to allow members of the community to directly update the information presented for each gene, including gene names, descriptions and Gene Ontology annotations, from a web browser. Maintenance of genome annotations by the authors generating and publishing primary data, rather than dedicated scientific curators, is a viable alternative for the upkeep of genomes, particularly for organisms with smaller research communities. By combining simple, intuitive displays with the powerful search functions made possible by its underlying relational database, TGD Wiki has been designed to maximize participation by bench scientists in the development of their community bioinformatics resource. Database URL: http://ciliate.org

Copper sulfate toxicity to two isolates of Ichthyophthirius multifiliis relative to alkalinity

Theronts from 2 different strains of Ichthyophthirius multifiliis (AR1 and AR5) were exposed to copper sulfate (CuSO4) in waters of different total alkalinities and observed for 4 h to determine relative toxicity and kinetics of parasite mortality. Consistent with the known solubility properties of the metal, Cu was significantly more toxic to cells maintained under low (48 mg l(-1)) compared with high (243 mg l(-1)) total alkaliniity conditions. This was reflected in both the median lethal concentration (LC50) values and rates of mortality for both parasite strains; strain differences were also observed. The AR1 strain was significantly more resistant to copper toxicity than the AR5 strain in both high and low alkalinity waters. In general, these strain differences were more evident under conditions of low stress (i.e. low CuSO4 concentration and high alkalinity), and suggest that genetic factors are overridden under high stress conditions. The present study establishes a role for alkalinity in the effectiveness of CuSO4 treatment of ichthyophthiriasis and reveals differences in the susceptibility of parasite populations that are clearly important for control programs.