The ciliated protozoan Ichthyophthirius multifiliis (Ich) is an obligate parasite of freshwater fish. The trophont -stage- resides beneath epithelia of the skin and gills. After 5 to 7 days, the parasite leaves the fish and multiplies into 200-1000 free-swimming infective tomites. A severe initial infection is lethal. However, fish which survive a mild infection become resistant to even severe subsequent infections. Our work during the past 12 months of this project has contributed significantly towards the understanding of which antigens might be involved in stimulating this immunity. Furthermore, we have demonstrated that fish can acquire protective immunity to Ich by vaccination as well as by natural infection. Preliminary studies indicate that the highest concentration of antibodies directed against protozoan antigens occurs 21 days following a single intraperitoneal (i.p.) injection. Based on this fact, channel catfish immunized with various Ich antigens were challenged with the live parasite 21 days after i.p. injection. Fish immunized with tomites killed by fixation in 1% formalin or by freeze-thawing were not protected from lethal infection. However, fish injected with formalin-fixed trophonts had significantly lowered mortality rates (20-50%) when compared to controls (100%). From these findings it appears that antigens expressed differentially during the feeding trophont stage of the parasite stimulate protective immunity. Hyperimmune fish and rabbit polyclonal antibodies have been produced against trophont whole cells as well as cilia and surface membrane proteins. Monoclonal antibodies have been produced from mice immunized with cilia and formalin-fixed trophont cells. These antibodies are being used to identify immunogenic and protective proteins. Genes coding for these proteins will be selected from a cDNA library constructed from trophont poly A message RNA.