Experimental evidence for direct in situ binding of IgM and IgT to early trophonts of Ichthyophthirius multifiliis (Fouquet) in the gills of rainbow trout, Oncorhynchus mykiss (Walbaum) L von Gersdorff Jørgensen 1 , R D Heinecke 1 , K Skjødt 2 , K J Rasmussen 2 and K Buchmann 1 1 Laboratory of Aquatic Pathobiology, Section of Biomedicine, Faculty of Life Sciences, University of Copenhagen, Frederiksberg C, Denmark 2 Department of Cancer and Inflammation, Institute of Molecular Medicine, University of Southern Denmark, Odense, Denmark Abstract Freshwater fish are able to mount a protective immune response against the parasite Ichthyoph- thirius multifiliis (Ich) following a non-lethal exposure. Factors involved in immunity comprise cellular and humoral factors, but antibodies have been suggested to play a prominent role in pro- tection. However, host antibodies have not yet been demonstrated to bind to the parasite in situ. By the use of immunohistochemical techniques, this study demonstrated that IgT and IgM bind to surface structures, including cilia, on the early feeding stage of the parasite in the gills of im- mune rainbow trout, Oncorhynchus mykiss, shortly (2 h) after invasion. No binding of IgT and no or only a weak binding of IgM was observed on the parasites in the gills of similarly exposed but naı ¨ve rainbow trout. This study indicates that antibod- ies play an important part in the protection of immune fish against Ich although additional humoral and cellular factors may contribute to this reaction. Keywords: binding, Ichthyophthirius multifiliis, IgM, IgT, rainbow trout, trophonts. Introduction The ciliate Ichthyophthirius multifiliis (Ich) is a protozoan parasite that infects freshwater fish worldwide causing considerable economic losses in aquaculture enterprises (Matthews 2005). It has a direct lifecycle comprising the infective theront, the feeding trophont and the external tomocyst stage producing numerous free-swimming infective ther- onts (Wagner 1960; Matthews 2005). Following a non-lethal infection with Ich fish can acquire immunity (Buschkiel 1910; Bauer 1953; Hines & Spira 1974), and several studies have indicated that both cellular and humoral factors are involved in the protective response (Cross & Matthews 1992; Dickerson & Clark 1996; Ewing, Kocan & Ewing 1985; Hines & Spira 1974; Olsen, Kania, Hei- necke, Skjoedt, Rasmussen & Buchmann 2011). Specific antibodies in serum and mucus from immune fish bind in vitro to specific parasite antigens called immobilization antigens (i-ags) (Clark, Dickerson & Findly 1988; Dickerson & Clark 1996; Cross & Matthews 1993; Lin & Dickerson 1992; Xu, Klesius & Shelby 2002; Maki & Dickerson 2003). It has been shown that theronts penetrate immune and naı ¨ve fish to the same extent but they leave immune hosts within 2 h (Cross & Matthews 1992; Wahli & Matthews 1999). It has been suggested that cross-binding by antibodies of i-ags is the stimulus triggering Journal of Fish Diseases 2011, 34, 749–755 doi:10.1111/j.1365-2761.2011.01291.x Correspondence L von Gersdorff Jørgensen, Laboratory of Aquatic Pathobiology, Section of Biomedicine, Faculty of Life Sciences, University of Copenhagen, Stigbøjlen 7, 1870 Frederiksberg C, Denmark (e-mail: lgj@life.ku.dk) 749 Ó 2011 Blackwell Publishing Ltd