Immune response to Ichthyophthirius multifiliis and role of IgT

Abstract The parasitic ciliate Ichthyophthirius multifiliis causes white spot disease in freshwater fish worldwide. The theront penetrates external surfaces of the naïve fish where it develops into the feeding trophont stage and elicits a protective immune response both at the affected site as well as at the systemic level. The present work compiles data and presents an overall model of the protective reactions induced. A wide spectrum of inflammatory reactions are established upon invasion but the specific protection is provided by adaptive factors. Immunoglobulin IgT is involved in protection of surfaces in several fish species and is thereby one of the first adaptive immune molecules reacting with the penetrating theront. IgT producing lymphocytes occur in epithelia, dispersed or associated with lymphoid cell aggregations (skin epidermis, fins, gills, nostrils and buccal cavities) but they are also present in central immune organs such as the head kidney, spleen and liver. When theronts invade immunized fish skin, they are encountered by host factors which opsonize the parasite and may result in complement activation, phagocytosis or cell‐mediated killing. However, antibody (IgT, IgM and IgD) binding to parasite cilia has been suggested to alter parasite behaviour and induce an escape reaction, whereby specific IgT (or other classes of immunoglobulin in fish surfaces) takes a central role in protection against the parasite.


| INTRODUC TI ON
The parasitic ciliate Ichthyophthirius multifiliis is known to infect a wide range of freshwater teleosts worldwide and elicit the disease ichthyophthiriosis. 1 The pathognomonic white spots in the skin of the infected fish, which is the basis for the vernacular name of the disease, white spot disease WSD, are caused by the feeding stage of the parasite, the trophont, which induces proliferation of the epidermal cells enclosing the parasite. The continuously rotating ciliate in its epidermal enclosure appears as a light reflecting blister on the fish surface visible to the naked eye as a white spot (Figure 1). Heavy infections may be lethal but fish surviving an infection were already a century ago reported to be protected against reinfection. 2 The protection is correlated to the severity of the primary infection, 3 but the immunological mechanisms associated with the protection were largely unknown until it was demonstrated that immune carp produced substances in skin and plasma which were able to immobilize the infective stages (theronts) of the parasite. 4

Subsequent investigations have
shown that specific immunoglobulins may explain the immobilizing ability through cross-linking i-antigens on the parasite surface. 5,6 This stimulus may alter the behaviour of the ciliate, and induce an escape reaction. 7 However, it is evident that various host cells (comprising lymphocytes and granulocytes) are involved in the immunization process. [8][9][10][11][12][13][14] In addition, recent transcriptomic studies have demonstrated that a wide range of other immune factors are trum of inflammatory reactions are established upon invasion but the specific protection is provided by adaptive factors. Immunoglobulin IgT is involved in protection of surfaces in several fish species and is thereby one of the first adaptive immune molecules reacting with the penetrating theront. IgT producing lymphocytes occur in epithelia, dispersed or associated with lymphoid cell aggregations (skin epidermis, fins, gills, nostrils and buccal cavities) but they are also present in central immune organs such as the head kidney, spleen and liver. When theronts invade immunized fish skin, they are encountered by host factors which opsonize the parasite and may result in complement activation, phagocytosis or cell-mediated killing. However, antibody (IgT, IgM and IgD) binding to parasite cilia has been suggested to alter parasite behaviour and induce an escape reaction, whereby specific IgT (or other classes of immunoglobulin in fish surfaces) takes a central role in protection against the parasite.

K E Y W O R D S
adaptive, fish, immunity, immunoglobulin, innate activated following infection. 15,16 This suggests that host protection is based on a more differentiated and complicated immune response than previously outlined.

| Life cycle
The life cycle of I multifiliis comprises four stages 17,18 (Figure 2). The feeding stage in the epidermis is termed the trophont, and it is richly equipped with cilia ( Figure 3). When reaching a size of 0.1-1.0 mm, it can break out of its infection focus and attain a new stage, termed the tomont, which actively (still by ciliary action) moves in water for minutes to hours before it settles on firm substrates (glass, plastic, wood, plants and fish tank wall). Here, it attains a tomocyst stage as it produces an external protective jelly-like substance, whereafter it initiates a series of mitotic divisions resulting in several hundreds of tomites. These ciliated tomites move vividly inside the tomocyst and escape continuously through openings in the gelatinous coating ( Figure 4). The liberated and free-swimming ciliated cell, termed the theront, seeks and penetrates the fish host surface and attains the early trophont stage ( Figure 5).

| Protection
Protective immunity against the parasitic ciliate as previously de- Antibody reactions have been documented by ELISA and Western blotting, and histological and immunohistochemical analyses have shown the direct interaction between parasite and host factors (immunoglobulin and lymphocytes). 35,36 Gene expression analyses (quantitative QPCR) have elucidated a varied response to invasion, and establishment in specific hosts for which assays (primers and probes) have been developed to elucidate involvement of specific immune genes. 24,27 General transcriptomic analyses have been applied in order to provide an overall picture of regulation of thousands of sequences which can be compared to annotated genes associated with different physiological pathways and compartments. 16 By analysing peptides and proteins, the proteomic approach can supplement the expression studies by presenting variations in effector molecules. 15

| Immune cells and tissues
Shortly after penetration of the host surface, it is possible to measure regulation of a series of immune related molecules. 33,34 Teleost surfaces are covered by mucosal tissue with associated lymphoid cell aggregations corresponding partly to the gut surface architecture in higher vertebrates including mammals. 36 The different conglomerates of immune reactive cells in fish surfaces have been termed SALT (skin-associated lymphoid tissue), GALT (gut-associated lymphoid tissue), gill-associated lymphoid tissue (GIALT), NALT (nasal-associated lymphoid tissue) and ILT (intrabranchial lymphoid tissue). [36][37][38][39] However, as the lymphoid cells in these surfaces are not (apart from interbrancial lymphoid tissue) organized in discrete tissues (as in head kidney and spleen), it may be suggested to replace, in these abbreviations, 'T' (for tissue) with 'C' (for cells). Thereby, terms such as SALC, GALC, GIALC and NALC, respectively, may be preferred for specification of the cells. The immediate response to parasite penetration is associated with expression of genes encoding inflammatory cytokines and acute phase reactants, 40

| IgT and its role in protection
Immunoglobulin T (IgT) is a prominent antibody isotype in some teleost species which was described by Hansen et al 45 46 The antibody is present in both internal organs and surfaces of fish at even very early developmental stages of the trout. 47 The dense layer of IgT in the mucous lining of naïve trout larvae may be protective-but merely partly-as yolksac larvae exposed to a high I multifiliis pressure become infected. 48 F I G U R E 2 Schematic view of the life cycle stages of Ichthyophthirius multifiliis F I G U R E 3 Trophont of Ichthyophthirius multifiliis escaped from trout skin epidermis attaining the tomont stage. Scale bar 100 µm. From 58 F I G U R E 4 Opening of the tomocyst wall with appearing theront. Scale bar 10 µm. From 58 Juvenile rainbow trout on the other hand (with a relatively less dense layer of IgT in the surface lining) are highly susceptible until they develop specific immunity, but the early trophont get into close contact with IgT and IgT producing cells even in naïve trout. The IgT-I multifiliis interaction was documented by several studies 35,36,49 and indicated to be involved in host protection as judged from the stronger binding of IgT to the parasite surface in immunized fish compared to naïve fish. IgT is also produced by Atlantic salmon, 50 turbot, 51 Nile tilapia, 52 zebrafish (IgZ), 43 stickleback and carp. 46 However, channel catfish Ictalurus punctatus, which develop a strong immunity against the parasite, does not possess IgT genes and seems to rely on various forms of IgM and possibly other immune mechanisms during the combat against Ich. 53 The local responses in this ictalurid fish host are highly developed and seems to be determined by B-cell clones communicating between different mucosal tissues. 54

| D ISCUSS I ON AND CON CLUS I ON
Development of protective immunity in fish against infections with the ciliated protozoan I multifiliis has been well known and recognized for more than a century. The main scientific challenge has been to describe the protective immunological mechanisms in the host and develop techniques to study the reactions. The history during the latest four decades, therefore, reflects the available methodologies which have been taken in action for the purpose.
With the advent of new immunological techniques for various fish species, it has been possible in a stepwise manner to build layer on layer on our understanding of immune mechanisms. Invading I multifiliis theronts induce a series of physiological changes 15 including inflammatory reactions in the affected fish surfaces, and in naïve fish, they increase with growth of the trophont (Figure 6). There is basis to suggest that messengers, antigen sampling and probably antigen presenting cells, 55  view and the European Union cannot be held responsible for any use that may be made of the information contained herein.

D I SCLOS U R E S
None.