Research Program: Dr. Ted Clark, Associate Professor, Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University.
Channel catfish with severe Ich infection
We are interested in several broad areas related to fish disease including host-parasite interactions, mechanisms of cutaneous immunity, development of recombinant vaccines, and genetic factors underlying resistance/susceptibility to infectious agents. Our principal model for work in these areas is Ichthyophthirius multifiliis, a parasitic ciliate that infects virtually all species of freshwater fish. Ichthyophthirius is pathogenic to a number of important farm-raised species (including channel catfish, rainbow trout, carp, eels and tilapia), and has a major impact on commercial aquaculture worldwide. Although the parasite is quite lethal, animals that survive epizootics develop a strong acquired resistance, and we can routinely immunize fish in the laboratory by exposing them to sublethal infections followed by chemical treatment of the water to eliminate free-swimming stages that would eventually kill fish. Resistance is seen within 3-4 weeks of initial exposure and involves, at least in part, a cutaneous (mucosal) antibody response to a defined class of parasite membrane proteins known as immobilization antigens, or i-antigens (Clark et al., 1995; Dickerson and Clark, 1998).
I-antigens are highly abundant GPI-anchored membrane proteins present on the surface of a variety of ciliates including the free-living species, Paramecium and Tetrahymena. As likely vaccine candidates, a large part of our work has been directed toward the isolation and characterization of the antigens themselves along with their corresponding genes. We have identified serotypic variants of I. multifiliis based on these proteins and, to date, have cloned and sequenced the genes encoding i-antigens from two such variants (serotypes A and D). These genes specify proteins with tandemly repetitive amino acid sequence domains (~80 residues each) that have the potential to bind zinc, and other metal ions (Clark et al., 1999). A comparison of the deduced amino acid sequence of the two proteins has revealed regions of conservation between i-antigen alleles and allowed the development of PCR primers that are now being used for diagnostic purposes (Clark, unpublished). From the standpoint of vaccine development, isolation of i-antigen genes will permit large-scale expression of recombinant proteins in heterologous systems, as well as the construction of DNA vaccines that can be used for genetic immunization. In this regard, novel adjuvants are being designed for use in vaccine formulations. Finally, comparisons of the i-antigen genes of different serotypes may eventually lead to the identification of epitopes responsible for protective immunity, as well as the development of peptide vaccines.
On a more basic level, recent studies have indicated that a novel mechanism of immunity is at play against Ichthyophthirius involving an effect of antibody on parasite behavior. Rather than being killed on the host, a majority of parasites are forced to exit fish prematurely in response to antibody binding (for review, see Clark and Dickerson, 1997). We know from passive immunization studies that antibody-binding alone is not sufficient to induce the effect; antigen cross-linking is also required (Clark et al, 1996). Based on these observations, we believe that forced exit is triggered by a signal transduction cascade leading either to 1) an avoidance response, 2) a change in developmental state, or 3) a short-circuiting of the normally metabolic pathway used by parasites to exit the host. A number of avenues are currently being explored to address these possibilities and to examine the signal transduction pathways leading to premature exit. Although the mechanism(s) responsible for forced exit has yet to be elucidated, Ichthyophthirius can be easily visualized within host tissues using simple light microscopic techniques. Indeed, the ability to observe parasites in vivo make this an extremely powerful system for studying host-parasite interactions in general.
Recently, we have begun to explore the use of zebrafish molecular genetics to elucidate host factors responsible for susceptibility and resistance to infectious disease. Unlike channel catfish, Danio rerio appears highly resistant to parasite infection (Clark, unpublished) suggesting a role for innate mechanisms of protection that have yet to be identified.
Clark, T.G., Lin, T. L., Jackwood, D. A., Sherrill, J., Lin, Y. and H. W. Dickerson (1999). The gene for an abundant parasite coat protein predicts tandemly repetitive metal binding domains. Gene (In Press).
Dickerson, H.W. and T.G. Clark (1998). Ichthyophthirius multifiliis: a model of cutaneous infection and immunity in fishes. Immunol. Rev. 166, 377-384.
Clark, T. G. and H. W. Dickerson (1997). Antibody-mediated effects on parasite behavior: evidence of a novel mechanism of immunity against a parasitic protist. Parasitology Today 13, 477-480.
Clark, T.G., Lin, T.L. and H.W. Dickerson (1996). Surface antigen cross-linking triggers forced exit of a protozoan pathogen from its host. Proc. Nat. Acad. Sci. (USA) 93, 6825-6829.
Clark, T.G., Lin, T.-L. and H.W. Dickerson (1995). Surface immobilization antigens of Ichthyophthirius multifiliis: their role in protective immunity. Ann. Rev. Fish Dis. 5, 113-131.
Cross, M. L. and R. A. Matthews (1992). Ichthyophthiriasis in carp, Cyprinus carpio L.: Fate of parasites in immunized fish. J. Fish Dis. 15, 497-505.