In vitrodifferentiation of eosinophilic granular cells inRenibacterium salmoninarum-infected gill cultures from rainbow trout

Fish & Shellfish Immunology (1996) 6, 173–184

In vitro differentiation of eosinophilic granular cells in Renibacterium salmoninarum-infected gill cultures from rainbow trout E. FLAÑO, P. LÓPEZ-FIERRO, B. E. RAZQUIN

AND

A. VILLENA*

Departamento de Biología Celular y Anatomı´a, Facultad de Biología, Universidad de León, 24071-León, Spain (Received 30 June 1995, accepted 16 October 1995) Gill explants were maintained for one week in culture, and infected with R. salmoninarum. The explants were collected at two weeks post-infection and processed for ultrastructural examination. A marked tissue reaction occurred in the infected explants in contrast with the control (non-infected) cultures, partially resembling an inflammatory response, and involving a marked increase in the number of eosinophilic granular cells. Several stages were identified from non-granulated precursors to mature eosinophilic granular cells, the earlier ones being closely apposed to fibroblasts. The ultrastructural images suggest that mature eosinophilic granular cells showed a chronic, non-anaphylactic, degranulation. Although the major extracellular products of R. salmoninarum may elicit the di#erentiation and degranulation of the EGCs, the possibility of these phenomena being cytokine-driven is discussed. These results demonstrate that eosinophilic granular cells are able to di#erentiate in situ, thus reinforcing the analogies between this cell type and mast cells. ? 1996 Academic Press Limited Key words:

eosinophilic granular cells, gill, tissue culture, BKD, Renibacterium salmoninarum, rainbow trout, Oncorhynchus mykiss.

I. Introduction Eosinophilic granular cells (EGCs) are leukocyte-like cells of fish, which characteristically contain numerous large, eosinophilic granules (Ellis, 1977). They have been described in several tissues and organs of salmonids, including the lamina propria of the gut (Ezeasor & Stokoe, 1980; Bergeron & Woodward, 1982, 1983; Powell et al., 1991), the skin (Blackstock & Pickering, 1980), and the gills (Powell et al., 1990). Some cytochemical characteristics and their involvement in histaminergical and pathological reactions have led to the suggestion that they are analogous to mammalian mast cells (Ellis, 1985; Powell et al., 1991, Powell et al., 1993). However, the eosinophilic staining of the EGC granules, and some morphological, cytochemical, and functional characteristics of the EGCs di#er from those of typical mast cells (Ezeasor & Stokoe, 1980; Bielek, 1981; Ishizeki et al., 1984; Vallejo & Ellis, 1989). Although several putative roles in physiological (Powell et al., 1991) and pathological *Author to whom correspondence should be addressed 173 1050–4648/96/030173+12 $18.00/0

? 1996 Academic Press Limited

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reactions (Ellis, 1985; Vallejo & Ellis, 1989; Lamas et al., 1991) have been attributed to the EGCs, their origin and functions remain unclear. In mammals, organ cultures are useful experimental models for the study of pathogenicity mechanisms, which may help to assess the respective contributions of a pathogen and the host to tissue damage during infection (McGee & Woods, 1987). In the present study this approach has been used to examine the pathogenic mechanisms of Renibacterium salmoninarum, which causes Bacterial Kidney Disease in salmonids (Fryer & Sanders, 1981). A methodology to obtain cultures from rainbow trout organs and to infect them in vitro with R. salmonimarum has been developed in order to analyse the histo- and physio-pathological responses to the pathogen without external interferences.

II. Materials and Methods ANIMALS

Yearling rainbow trout, Oncorhynchus mykiss, from a commercial fish farm, were kept in 600 l tanks supplied with running well water at a temperature of 14&1) C and a constant photoperiod of 12 h light/darkness. After anaesthesia with 0·2 mg l "1 of MS-222 (Sandoz), the trout were bled from the caudal sinus, and the gills dissected in sterile conditions and kept in cold RPMI-1640 (BioWhittaker).

CULTURE CONDITIONS

Cultures were initiated from gill explants. A portion of the gill was washed several times in cold sterile phosphate-bu#ered saline (PBS) firstly under gentle agitation and later flushed with a syringe, in order to eliminate as much mucous as possible. Individual gill filaments were then separated, washed again with PBS, and transferred to 24-well culture plates (Costar) with 1 ml of culture medium per well. The plates were incubated at 15&1) C in air, the medium being renewed every week.

CULTURE MEDIUM

This was based on the formulation of Diago et al. (1993). It consisted of RPMI-1640 (BioWhittaker) supplemented with 10% FCS (Boehringer Mannheim), 10% allogeneic trout serum, and 5 mM L-glutamine, and was supplemented with 2·5 mM sodium pyruvate (Merck); nonessential aminoacids (Seromed); 25 mM nucleosides—guanosine, cytidine, adenosine, uridine— (Sigma); 5 mM 2-mercaptoethanol (Merck), 100 ìg ml "1 gentamycin (Flow); and 2 ìg ml "1 amphotericin B (Seromed).

IN VITRO INFECTIONS

The FT10 strain of Renibacterium salmoninarum, kindly provided by Dr. B. Austin, was used for infection. After an in vitro passage by intra-peritoneal

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(i.p.) inoculation of yearling rainbow trout, bacteria were re-isolated in SKDM (Austin et al., 1983) at 15) C. For use, they were resuspended in 0·02 M sterile saline at a density of 108 bacteria ml "1. Week-old gill cultures from three di#erent fish were used for the in vitro infections. For each animal, three infected cultures and three control (noninfected) cultures were examined. Cultures were infected by adding 107 bacteria in 100 ìl to each well, and control cultures were set up by adding 100 ìl per well of sterile saline solution. The plates were centrifuged at 200 g for 10 min at 4) C to facilitate contact between the bacteria and cells. After 1 h of incubation at 15) C, free bacteria were removed by washing the cultures twice with RPMI-1640 under gentle agitation, and 1 ml of fresh culture medium was added to each well. The cultures were then incubated as indicated above.

TRANSMISSION ELECTRON MICROSCOPY

At two weeks post-infection (21 days in culture), the explants were fixed with 2% glutaraldehyde in cold cacodylate bu#er, pH 7·2, for 2 h, and postfixed with 1% osmium tetroxide solution in the same bu#er for 1 h. The explants were given consistency using 2% agar, dehydrated in acetone series, contrasted with 1% uranyl acetate in 70% acetone, and embedded in Araldite (Durcupan). Semithin sections, 1 ìm thick, were stained with an aqueous solution of toluidine blue in borax. Ultrathin sections were obtained with a Reichert-Jung UM-3 ultratome, counterstained with lead citrate, and observed in a JEOL EM1010 electron microscope at 60 kV.

III. Results CONTROL CULTURES

After 21 days in culture the major components of the gill were still discernible, although the structure of the secondary lamellae became disorganised. The primary lamella consisted of a cartilaginous rod surrounded by a thin perichondrium, a layer of loose connective tissue, and the epithelium [Fig. 1(a)]. The connective tissue contained a network of fusiform fibroblasts, and numerous free cells, mainly undi#erentiated fibroblasts, and a few EGCs [Fig. 1(a)]. There were still identifiable blood vessels (venules and capillaries) formed by a lumen surrounded by fibroblasts and endothelial cells [Fig. 1(a)] and occasional macrophages. The epithelium of the primary lamella retained its continuity, although with signs of spongiosis, and contained few mucous cells and chloride cells [Fig. 1(a)]. It was resting on a thickened basal membrane [Fig. 1(a)]. In some cases, wandering macrophages were observed migrating out of the lamella [Fig. 1(a)]. Most of the secondary lamellae were distorted, with enlargement of the capillaries located at the tip, some lamellae being fused in this zone. They were generally devoid of the respiratory epithelium, and their major cell component was pillar cells, which were covered by a thick basal membrane [Fig. 1(a)].

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INFECTED CULTURES

The primary and the secondary lamellae of the infected gill cultures contained the same cell types as the control cultures, but they showed a patent tissue reaction, comprising an increase in the thickness and cellularity of the connective tissue [Figs 1(b) & (c)], in which many cells, especially fibroblasts, appeared hypertrophic [Figs 1(b) & (c)]. The intercellular spaces of the connective tissue appeared swollen and filled with connective tissue matrix materials [Figs 1(b) & (c)]. A remarkable change observed in most infected gill cultures consisted of a notable increase in the number of EGCs, as well as of monocyte/macrophages, which appeared scattered throughout the connective tissue [Figs 1(b) & (c)]. Most EGCs were mature cells but by electron microscopy it was possible to observe several stages of EGC di#erentiation [Figs 2(a) & 3(a)]. The earlier EGC precursors were identified as rounded or ovoid cells which showed scant cytoplasm and very few, if any, small cytoplasmic granules [Fig. 2(a)]. These cells had a polygonal, sometimes bilobulated, nucleus, with sparse masses of heterochromatin and prominent nucleoli [Figs 2(a) & (b)]. The cytoplasm contained numerous ribosomes, short profiles of rough endoplasmic reticulum, and some characteristic electron-lucent multivesicular bodies, with small ovoid dense vesicles apposed to the inner surface of the membrane [Fig. 2(b)]. Later stages of EGC di#erentiation were characterised by the presence of a low number of typical EGC-cytoplasmic granules, while retaining some of the features of the EGC precursors [Figs 3(a) & 4(a)]. At this stage, the developing EGCs showed an abundant cytoplasm [Fig. 3(a)], the Golgi apparatus was more developed, and there were numerous small electron dense granules associated to the dictiosomes [Figs 3(b) & 4(a)]. They also had short profiles of smooth and rough endoplasmic reticulum, and large ovoid mitochondria with an electron-dense matrix and irregular laminar crests [Figs 3(b) & 4(a)]. The granules of the immature EGCs were heterogeneous in electron-density and size, some of them having an electron-lucent halo, and others with fuzzy edges [Figs 3(a), 3(b) & 4(a)]. The di#erentiating EGCs appeared closely apposed to fibroblastic cells [Figs 2(a) & 3(a)], sometimes with structures resembling gap junctions. Mature EGCs had similar structural features, but with more and larger granules (0·5 ìm average size), most of them having a core with homogeneous material of high electron-density, and an electron-lucent halo [Fig. 4(b)].

Fig. 1. Semithin sections of gill explants. (a) Control explant cultured for 21 days. The connective tissue, epithelium of the primary lamella (E), and three secondary lamellae (L) are shown. Note the presence of a blood vessel (➧), EGCs (➝) and fibroblasts (*) in the connective tissue. Arrowheads (c) indicate the basal membrane. Mucus cell (➝). Macrophage (9). Pillar cells (G).#828. (b) Primary lamella of a R. salmoninarum-infected gill explant (at two weeks postinfection). The connective tissue is colonized by numerous EGCs (➝), and monocyte/ macrophages (9). Note the hypertrophy of the fibroblasts (*) and the matrix materials (c). Epithelium (E).#832. (c) R. salmoninarum-infected gill explant showing part of the primary lamella and two secondary lamellae. The connective tissue has similar features to those shown in (b). (Signs as above.)#823.

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Fig. 2. Ultrastructure of developing EGCs in the connective tissue of an infected gill. (a) Two stages of di#erentiation of the EGCs are shown. The earlier, non-granulated EGC precursor shows a bilobulated nucleus, and poorly developed rough endoplasmic reticulum (➧). Note that the two EGCs are closely apposed to the surrounding fibroblasts (F).#10 300. (b) High power magnification of the cytoplasm of the non-granulated EGC precursor shown in the previous figure. Note the typical vesicles (V), and the numerous free ribosomes.#25 700.

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Fig. 3. Ultrastructure of developing EGCs in the connective tissue of an infected gill. (a) An EGC showing the ultrastructure features of a later stage of di#erentiation. Note the heterogeneity of the granules, and the well developed Golgi apparatus (9). The EGC shows close cell–cell interactions (c) with the surrounding fibroblasts (F).#14 600. (b) High power magnification of the cytoplasm of the EGC shown in the previous figure. Note the presence of developing granules (➧) in the Golgi apparatus, and the tubule of the smooth endoplasmic reticulum (➝).#33 500.

Fig. 4. Ultrastructure of developing and mature EGCs in the connective tissue of an infected gill. (a) High power magnification of the cytoplasm of a late stage of EGC di#erentiation. Note the development of the Golgi apparatus (9), to which the developing granules (➧) are connected, the tubules of the smooth endoplasmic reticulum (➝) and the large, laminar mitochondria (*), which are typical features of immature EGCs.#32 700. (b) Two mature EGCs showing characteristic granules with electron-lucent haloes, and evidence of degranulation. Note fusion of adjacent granules (c), and disintegration of their electron-dense materials.#9 900.

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Many of the EGCs showed ultrastructural features of degranulation, some of the granules being fused together, swollen and/or partially disintegrated [Fig. 4(b)]. IV. Discussion Several authors have developed methods of establishing short-term gill cultures (Stadländer & Kirchho#, 1989). Our culture system, however, is capable of maintaining long-term cultures of gill explants. This is necessary to allow the development of the in vitro infection with R. salmoninarum, which proceeds very slowly, as it does in vivo (Evenden et al., 1993). In this system, the primary lamella of the control gill explants retained its basic structure, as well as its major cell populations, for a long time (at least 3 weeks). However, the secondary lamellae became disorganised, and the respiratory epithelia and the capillaries were severely distorted. In the infected explants there occurred a marked tissue reaction, which partially resembled an inflammatory response, with a marked fibroblastic activation, synthesis of matrix components, and a remarkable increase in the number of EGCs. An inflammatory response also occurs in fish with BKD (Wood & Yasutake, 1956), and it may partially explain the changes observed in culture. The main aim of the present study was to report on EGC di#erentiation and degranulation observed in the infected gill cultures. In this sense, two major results deserve particular discussion regarding EGC biology. Firstly, the study demonstrates that EGCs can di#erentiate in situ, as their numbers increased in the infected gill explants. The origin of the EGCs in vivo is unclear, although it has been suggested that they di#erentiate in some unidentified organs and reach the di#erent target organs by way of the blood circulation (Bergeron & Woodward, 1983; Powell et al., 1990; Lamas et al., 1991). However, EGCs have not usually been found in trout blood (Blaxhall & Daisley, 1973), although Lamas et al. (1991) observed some circulating EGCs after i.p. injection with Vibrio anguillarum. In culture, gill explants cannot be populated by migrating EGCs and, therefore, the new EGCs must be formed in the gill connective tissue from resident precursors. However, mitosis was not observed in presumptive EGC precursors, which would suggest that the connective tissues house immature EGCs, which are non-proliferative. Mitosis in EGCs has never been described in vivo in normal tissues, but they have been reported to be capable of regeneration after degranulation (Vallejo & Ellis, 1989). These data therefore indicate that the EGC precursors may be formed elsewhere (probably in the haemopoietic organs), and reach the organs via the blood circulation as immature, non-granulated cells, as has been demonstrated for mammalian mast cells (Kirshenbaum et al., 1991). The early stage of the EGC di#erentiation identified here shows the structural features of immature, but not of blast, cells. There are few clear structural markers of the early stages of the EGCs, but they could be di#erentiated from fibroblasts by the myelopoietic-like aspect of the nucleus, the poorly developed rough endoplasmic reticulum, and the occasional

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presence of multivesicular bodies. These bodies resemble those found in mature EGCs (Vallejo & Ellis, 1989; Powell et al., 1993), and in in vitro developing human connective tissue mast cells co-cultured with 3T3 fibroblasts (Ishizaka et al., 1993). More advanced EGCs were characterised by the presence of heterogeneous granules, whose structural features resembled those of the mature EGCs. At this stage, the immature EGCs had a prominent Golgi apparatus with apposed small electron-dense granules, and tubules of smooth endoplasmic reticulum. An extensive development of these two organelles has also been described in regenerating EGCs after degranulation (Vallejo & Ellis, 1989; Powell et al., 1993). The structure of the mature EGCs in the gill cultures was similar to that previously described in vivo (Roberts et al., 1971; Ezeasor & Stokoe, 1980; Vallejo & Ellis, 1989; Powell et al., 1993). Also, the degranulation of the EGCs appeared to occur as described in vivo (Vallejo & Ellis, 1989; Powell et al., 1993), except that the release of intact granules into the connective tissue was not observed. The reason for this di#erence might be explained by the di#erent experimental models used. In the present in vitro system, degranulation appeared to be a chronic process, eliciting a slow degranulation of the EGCs (see below). The second point concerns the possible role of EGCs in BKD pathology. A possible homology of the EGCs with mammalian mast cells has been suggested (Ezeasor & Stokoe, 1980; Ellis, 1985, 1977; Vallejo & Ellis, 1989; Powell et al., 1990; Powell et al., 1993), and this cell type is probably involved in the release of histamine during inflammatory responses in rainbow trout (Ellis, 1985; Vallejo & Ellis, 1989). Ezeasor & Stokoe (1980) hypothesized that EGCs in the gut of rainbow trout were involved in immune defense mechanisms. The present in vitro results on the behaviour of the EGCs in response to infection clearly do not resemble those reported in vivo (Vallejo & Ellis, 1989; Lamas et al., 1991; Powell et al., 1993). Although the in vitro infection with R. salmoninarum was able to promote EGC di#erentiation from resident immature precursors in gill tissue, there is no anaphylactic-type degranulation, as reported after the i.p. injection of extracellular products of Aeromonas salmonicida or of histamine liberators (Vallejo & Ellis, 1989; Powell et al., 1993). It is unclear what mechanism(s) elicit EGC recruitment and degranulation. Although the major extracellular product of R. salmoninarum, the p57 protein (Evenden et al., 1993), may elicit their di#erentiation, it is more likely that this phenomenon is cytokine-driven, rather than a direct response to the bacteria. There are two major reasons that support this hypothesis. Firstly, at 2 weeks’ post-infection, few R. salmoninarum was present in the infected cultures, as demonstrated by immunostaining using a monoclonal antibody anti-p57 (BW3.6, kindly provided by Dr. B. Austin) (data not shown). Moreover, owing to the weekly changes of the culture medium, the levels of p57 would obviously remain very low in the cultures. Secondly, in contrast with the present results, most of the in vivo studies reported a reduction in the number of EGCs after injection with bacteria, with their extracellular products or with histamine liberators (Ellis, 1985; Powell et al., 1993). For this reason EGC di#erentiation

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in the infected gill cultures might be under the control of cytokine/growth factors, as described for mammalian mast cells (Ishizaka et al., 1993). A role for such factors is also suggested by the increase in number of monocyte/ macrophages in the infected explants. Infection with R. salmoninarum may result in the activation of the monocyte/macrophages, which are involved in the release of several cytokines, including IL-1 (Secombes, 1991). This interleukin may in turn elicit the observed inflammatory-like response, and fibroblast activation, as well as the activation of other leukocytes, all of which in mammals release growth factors necessary for mast cell maturation (Ishizaka et al., 1993). On the other hand, it is worth noting that in our study the immature EGCs are closely apposed to fibroblasts. This kind of relationship between these two cell types has also been described in vivo (Ezeasor & Stokoe, 1980; Powell et al., 1993). Although it has been claimed that such cell–cell interactions can be incidental, owing to the dense connective tissue environment in which EGCs are located (Vallejo & Ellis, 1989), this is not the case in the gill cultures, where intercellular spaces were enlarged. Moreover, such EGC-fibroblast association was observed in a lesion in which an unusual EGC proliferation was found in Oncorhynchus mykiss (Kent et al., 1993). Remarkably, such a relationship closely resembles the specific supporting role of 3T3 fibroblasts for human mast cell di#erentiation in vitro (Ishizaka et al., 1993). Therefore, the di#erentiation of the EGCs in the gill cultures, as well as their chronic degranulation, appear to be indirect results of the infection. Albeit speculatively, it is suggested that such phenomena are probably mediated throughout the cytokine network, which may be activated in response to the pathogen. This work has been supported by EU Project Contract No AIR-1-CT920036, and CICYT project MAR91-0851. FT10 strain of Renibacterium salmoninarum and BW3.6 monoclonal antibody were kindly provided by Dr. Brian Austin of Heriot-Watt University, Edinburgh. The authors thank the owners of Piscifactoria Los Leoneses (Castrillo del Porma, León) for providing the trout.

References Austin, B., Embley, T. M. & Goodfellow, M. (1983). Selective isolation of Renibacterium salmoninarum. FEMS Microbiology Letters 17, 111–114. Bergeron, T. C. M. & Woodward, W. (1982). Development of the stratum granulosum of the small intestine of the rainbow trout (Salmo gairdneri). Canadian Journal of Zoology 60, 1513–1516. Bergeron, T. C. M. & Woodward, W. (1983). Ultrastructure of the granule cells in the small intestine of the rainbow trout (Salmo gairdneri) before and after stratum granulosum formation. Canadian Journal of Zoology 61, 133–138. Bielek, E. (1981). Development stages and localization of peroxidatic activity in the leucocytes of three teleost species (Cyprinus carpio L; Tinca tinca L; Salmo gairdneri Richardson). Cell and Tissue Research 220, 163–180. Blackstock, N. & Pickering, A. D. (1980). Acidophilic granule cells in the epidermis of brown trout Salmo trutta L. Cell and Tissue Research 210, 359–369. Blaxhall, P. C. & Daisley, K. W. (1973). Routine haematological methods for use with fish blood. Journal of Fish Biology 5, 771–781. Diago, M. L., López-Fierro, M. P., Razquin, B. & Villena, A. (1993). Long-term myelopoietic cultures from the renal hematopoietic tissue of the rainbow trout,

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Oncorhynchus mykiss W.: phenotypic characteristics of the stromal cells. Experimental Hematology 21, 1277–1287. Ellis, A. E. (1977). The leucocytes of fish: A review. Journal of Fish Biology 11, 453–491. Ellis, A. E. (1985). Eosinophilic granule cells (EGC) and histamine response to Aeromonas salmonicida toxins in rainbow trout. Developmental and Comparative Immunology 9, 251–260. Evenden, A. J., Grayson, T. H., Gilpin, M. L. & Munn, C.B. (1993). Renibacterium salmoninarum and bacterial kidney disease—the unfinished jigsaw. Annual Review of Fish Diseases 3, 87–104. Ezeasor, D. N. & Stokoe, W. M. (1980). A cytochemical, light and electron microscopic study of eosinophilic granule cells in the gut of rainbow trout, Salmo gairdneri Richardson. Journal of Fish Biology 17, 619–634. Fryer, J. L. & Sanders, J. E. (1981). Bacterial kidney disease of salmonid fish. Annual Review of Microbiology 35, 273–298. Ishizaka, T., Mitsui, H., Miura, T. & Dvorak, A. M. (1993). Development of human mast cells from their progenitors. Current Opinion in Immunology 5, 937–943. Ishizeki, K., Nawa, T., Tachibana, T., Sakakura, Y. & Ida, S. (1984). Hemopoietic sites and development of eosinophil granulocytes in the loach, Misgurnus anguillicaudatus. Cell and Tissue Research 235, 419–426. Kent, M. L., Powell, M. D., Kieser, D., Hoskins, G. E., Speare, D. J., Burka, J. F., Bagshaw, J. & Fournie, J. W. (1993). Unusual eosinophilic granule cell proliferation in coho salmon (Oncorhynchus kisutch). Journal of Comparative Pathology 109, 129–140. Kirshenbaum, A. S., Kessler, S. W., Go#, J. P. & Metcalfe, D. D. (1991). Demonstration of the origin of human mast cells from CD34 + bone marrow progenitor cells. Journal of Immunology 146, 1410–1415. Lamas, J., Bruno, D. W., Santos, Y., Anadón, R. & Ellis, A. E. (1991). Eosinophilic granular cell response to intraperitoneal injection with Vibrio anguillarum and its extracellular products in rainbow trout, Oncorhynchus mykiss. Fish & Shellfish Immunology 1, 187–194. McGee, Z. A. & Woods, M. L. (1987). Use of organ cultures in microbiological research. Annual Review of Microbiology 41, 291–300. Powell, M. D., Wright, G. M. & Burka, J. F. (1990). Eosinophilic granule cell in the gills of rainbow trout (Oncorhynchus mykiss): evidence of migration? Journal of Fish Biology 37, 495–497. Powell, M. D., Wright, G. M. & Burka, J. F. (1991). Degranulation of eosinophilic granule cells induced by capsaicin and substance P in the intestine of the rainbow trout (Oncorhynchus mykiss Walbaum). Cell and Tissue Research 266, 469–474. Powell, M. D., Briand, H. A., Wright, G. M. & Burka, J. F. (1993). Rainbow trout (Oncorhynchus mykiss Walbaum) intestinal eosinophilic granule cell (EGC) response to Aeromonas salmonicida and Vibrio anguillarum extracellular products. Fish & Shellfish Immunology 3, 279–289. Roberts, R. J., Young, H. & Milne, J. A. (1971). Studies on the skin of plaice (Pleuronectes platessa L.) I. The structure and ultrastructure of normal plaice skin. Journal of Fish Biology 4, 87–98. Secombes, C. J. (1991). The phylogeny of cytokines. In The Cytokine Handbook (A. Thomson, ed.) pp. 387–412. London: Academic Press Ltd. Stadtländer, C. & Kirchho#, H. (1989). Gill organ culture of rainbow trout, Salmo gairdneri Richardson: an experimental model for the study of pathogenicity of Mycoplasma mobile 163 K. Journal of Fish Diseases 12, 79–86. Vallejo, A. N. & Ellis, A. E. (1989). Ultrastructural study of the response of eosinophil granule cells to Aeromonas salmonicida extracellular products and histamine liberators in rainbow trout Salmo gairdneri Richardson. Developmental and Comparative Immunology 13, 133–148. Wood, E. M. & Yasutake, W. T. (1956). Histopathology of kidney disease in fish. American Journal of Pathology 32, 845–857.