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The uptake of bovine serum albumin by the skin of bath-immunised rainbow troutOncorhynchus mykiss
Fish & Shellfish Immunology (1996) 6, 321–333
The uptake of bovine serum albumin by the skin of bath-immunised rainbow trout Oncorhynchus mykiss MITSURU OTOTAKE*, GEORGE K. IWAMA†
AND
TERUYUKI NAKANISHI
National Research Institute of Aquaculture, Tamaki, Mie 519-04, Japan, †Department of Animal Science and Canadian Bacterial Diseases Network, University of British Columbia, Vancouver, B.C. Canada V6T-1Z4 (Received 22 September 1995, accepted in revised form 29 November 1995) In order to clarify the main site responsible for soluble antigen uptake in bath immunisation, the localisation of labelled bovine serum albumin (BSA) was examined in rainbow trout after bath administrations by hyperosmotic infiltration (HI) and direct immersion (DI). Quantitative analyses indicated that the main site of BSA uptake is the skin with a lesser role for the gills in both HI and DI treatments. These data were further confirmed by histological analysis. BSA was observed primarily in the intercellular spaces of the skin and gills after HI, while it was most common in epithelia of the skin and in macrophage-like cells in the gills after DI. Following either treatment, BSA was trapped by the secondary circulatory system in the skin and gills, as well as the kidney and spleen. ? 1996 Academic Press Limited Key words:
gill, rainbow trout, secondary circulation, skin, vaccination.
I. Introduction The bath immunisation method, first reported by Amend & Fender (1976), is currently used in aquaculture because of its convenience and e#ectiveness for mass vaccination. In bath immunisation, however, the exact mechanisms of antigen uptake and protection still remain unknown. Amend & Fender (1976), observing the time course of BSA concentration in various organs after bath treatment, stated that the main site responsible for uptake of antigen is the lateral line. In contrast, later studies reported that the gill is the main site of antigen uptake (Bower & Alexander, 1981; Alexander et al., 1981), and this is generally accepted at present (Smith, 1982; Tatner et al., 1984; Zapata et al., 1987; Kawahara & Kusuda, 1988). The intestine has also been proposed as a site of antigen uptake (Robohm, 1986; Rombout et al., 1985; 1986; Tatner, 1987; Robohm & Koch, 1995). Thus, the primary site responsible for antigen uptake has been a controversial topic with several organs having been implicated. This controversy seems to be attributable to the following three factors: (1) only one study has measured the antigen concentration in the gills, lateral line, and intestine after bath immunisation (Amend & Fender, 1976); (2) the two methods, direct immersion (DI) and hyperosmotic infiltration (HI), may have di#erent mechanisms of antigen uptake; (3) soluble and particulate *To whom all correspondence should be addressed. 321 1050–4648/96/050321+13 $18.00/0
? 1996 Academic Press Limited
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antigens may be taken up by di#erent mechanisms. In the present study, two experiments were carried out in order to clarify the main site of soluble antigen uptake in bath immunisation using the HI and DI methods. First, 125 I labelled BSA (125I-BSA) was used to measure BSA uptake quantitatively in each organ after bath administration, and then FITC labelled BSA (FITC-BSA) was used to observe the BSA localisation histologically in each organ. II. Materials and Methods EXPERIMENTAL ANIMALS
Juvenile rainbow trout Oncorhynchus mykiss, weighing 15&2 g (mean&standard deviation), were used in this study. They were kept in tanks supplied with running fresh water at 17·5)C and fed commercial dry pellets. QUANTITATIVE ANALYSIS
Fish were divided into the following two groups: HI (hyperosmotic infiltration) group with 60 fish and DI (direct immersion) group with 60 fish. Fish of the HI group were administered BSA (fraction V) conjugated with 125I (125I-BSA, No. 68031, ICN Co.) by the two-step HI treatment reported by Amend and Fender (1976) with slight modifications. Distilled water was used for the preparation of the two bath solutions: 5·3% NaCl solution and 2% BSA solution (50 k Bq ml "1). Fish of the HI group were immersed in a 5·3% NaCl solution for 3 min, and then placed in a 2% 125I-BSA solution for 3 min. Fish of the DI group were placed directly in a 2% BSA solution for 3 min. Following bath treatment, fish were rinsed in fresh water for 5 min. Thereafter, each fish was individually transferred to a tank with 5 l of static fresh water. Temperatures of the water and room were maintained at 18)C throughout the experiment. At 10, 30, 60, and 120 min after bath treatment, six fish of each group were anaesthetized with MS-222 (tricaine), and blood samples were withdrawn from the caudal blood vessels. Each fish was sacrificed by decapitation, and the whole skin, gills, stomach, intestine, liver, pronephros, mesonephros and spleen were taken. The skin was divided into two parts: lateral-line skin and trunk skin; the former consisting of the skin along the lateral line approximately 3 mm in width and the latter consisting of the remaining skin of the body. Each organ was weighed and then placed in 10 ml polyethylene tubes. The quantity of 125I-BSA in each tube was measured by a ã-counter (ARC-360, Aroka Co.). The measuring time was 20 min for each sample, and 120 min for the background. BSA concentration (ìg BSA g "1 tissue) and the total quantity of BSA in each tissue (mg BSA kg "1 fish) were calculated from the count for each tissue minus the background. HISTOLOGICAL ANALYSIS
Fish were divided into the following four groups: HI group (60 fish), DI group (60 fish), intravenous injection (IV) group (21 fish), and control
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unimmunised group (3 fish). Fish of the HI and DI groups were administered BSA conjugated with FITC (FITC-BSA, A9771, Sigma Co.) using the same treatments mentioned above for 125I-BSA. Fish of the IV group were administered 5 mg ml "1 of FITC-BSA in 0·85% NaCl by intravenous injection at 2·7 mg kg "1 fish, because the gross BSA release into the blood after HI treatment was estimated to be 2·7 mg kg "1 fish (Ototake & Nakanishi, 1992). The control group was left untreated. Three fish of each group were anaesthetised with MS-222, and blood samples were withdrawn from the caudal blood vessels 1, 5, 15, 30, 60, and 120 min after the bath. Then the same organs as mentioned above were dissected. Each tissue was fixed with a bu#ered 10% formalin (pH 7·2). After dehydration, tissues were embedded in resin (Technovit 7100, Kulzer Co.), and 2 ìm sections were a$xed to microscope slides. A cover glass was mounted with 50% glycerin and sections were examined under IB excitation with a fluorescence microscope (BH2-F, Olympus Co.). STATISTICS
Data were first subjected to the F test in order to detect significant di#erences between variances. When the di#erence of variances was significant, the data were subjected to the Cochran-Cox test in order to detect significant di#erences between means. The rest of the data were subjected to the Student’s unpaired t-test in order to detect significant di#erences between means. Significance level for all statistical analyses was P<0·05.
III. Results QUANTITATIVE ANALYSIS
HI group In the trunk skin, the BSA concentration was a maximum of 110&12 ìg BSA g "1 tissue (mean&standard error) 10 min after the bath, then significantly decreased to 49&7 ìg g "1 60 min after the bath (P<0·01), and subsequently showed little change [Fig. 1(a)]. There was little di#erence in BSA concentration between the lateral-line skin and trunk skin throughout the experiment. In the gills, a maximum BSA concentration of 67&7 ìg g "1 was observed at the initial sampling time, and the level significantly decreased by 30 min after the bath (P<0·01). In contrast, the BSA level in the blood was a minimum of 23&2 ìg g "1 at the initial sampling time, and gradually increased to 60&7 ìg g "1 120 min after the bath. Also in the mesonephros, pronephros, spleen, liver and intestine, the BSA levels were minimal at the initial sampling time, and gradually increased [Fig. 1(b)]. In the stomach, BSA concentrations varied greatly between individuals and no significant change was detected after the bath [Fig. 1(a)]. The total amount of BSA in each organ is shown in Fig. 2. At the initial sampling time, the trunk skin had the highest concentration (11·2&1·2 mg BSA kg "1 fish), followed by the gills (2·5&0·3 mg kg "1), lateral-line skin (1·0&0·1 mg kg "1), stomach (0·9&0·3 mg kg "1), and intestine (0·1&0·0 mg kg "1). Values for the trunk skin remained significantly
M. OTOTAKE ET AL.
324 140
(a)
120 100 Lateral-line skin Gills Blood Trunk skin Stomach
80
BSA (µ g g–1)
60 40 20 30
0
60
90
120
30 (b) Mesonephros Liver Intestine Pronephros Spleen
20 10
0
30
60 Minutes after HI
90
120
Fig. 1. Changes in bovine serum albumin (BSA) concentrations in organs of rainbow trout Oncorhynchus mykiss after hyperosmotic infiltration (HI) treatment. Data are presented as mean&standard error. 12
BSA (mg kg–1 fish)
10 8
Lateral-line skin Gills Intestine Trunk skin Stomach
6 4 2
0
30
60 Minutes after HI
90
120
Fig. 2. Changes in total bovine serum albumin (BSA) present in organs of rainbow trout Oncorhynchus mykiss after hyperosmotic infiltration (HI) treatment. Data are presented as mean&standard error.
higher than those for the gills, stomach, lateral-line skin and intestine throughout the experiment (P<0·01), and decreased by 6·8&1·0 mg kg "1 during the experiment.
BOVINE SERUM ALBUMIN UPTAKE
40
325
(a)
35 30 Lateral-line skin Gills Blood Trunk skin Stomach
25
BSA (µ g g–1)
20 15 10 5 0
30
60
90
120
10 8
(b) Mesonephros Liver Intestine Pronephros Spleen
6 4 2 0
30
60 Minutes after HI
90
120
Fig. 3. Changes in bovine serum albumin (BSA) concentrations in organs of rainbow trout Oncorhynchus mykiss after direct immersion (DI) treatment. Data are presented as mean&standard error.
DI group The BSA concentrations in this group were significantly lower than those of the HI group for all the tissues except the stomach (P<0·05, Figs 1 & 3). There was little di#erence in BSA concentration between the trunk skin and lateral-line skin throughout the experiment. The total amount of BSA in each organ is shown in Fig. 4. At the initial sampling time, the trunk skin was the highest (1·5&0·0 mg kg "1), followed by the stomach (0·3&0·2 mg kg "1), gills (0·2&0·0 mg kg "1) and lateral-line skin (0·1&0·0 mg kg "1). Values for the trunk skin remained significantly higher than those for the gills, stomach, lateral-line skin and intestine throughout the experiment (P<0·01), and decreased by 0·8&0·1 mg kg "1 during the experiment. HISTOLOGICAL ANALYSIS
Unimmunised control group Green auto-fluorescence was observed in some scales, and yellow autofluorescent granules were observed in some renal tubules and the splenic cord. HI group Table 1 shows the temporal changes in FITC fluorescence observed in each tissue. Strong fluorescence was observed 1 min after HI treatment in the
M. OTOTAKE ET AL.
326 1.6
BSA (mg kg–1 fish)
1.4 1.2 Lateral-line skin Gills Intestine Trunk skin Stomach
1.0 0.8 0.6 0.4 0.2 0
30
60 Minutes after HI
90
120
Fig. 4. Changes in total bovine serum albumin (BSA) present in organs of rainbow trout Oncorhynchus mykiss after direct immersion (DI) treatment. Data are presented as mean&standard error.
following locations in the skin: portions of the surface of the epidermis; the intercellular space in the epithelium; the collagenous connective tissue around scale pockets immediately beneath the epithelium; and a very limited number of epithelial cells [Fig. 5(a)]. In the gills, FITC signals were observed in the basement membrane under the epithelium of gill filaments, the connecting basement membrane of gill lamellae, and collagenous fibres in pillar cells [Fig. 5(c)]. Five minutes after HI treatment, the positive area extended along the basement membrane in gill filaments and lamellae and along the collagenous connective tissue around scale pockets in the skin. Fluorescence became visible in the endothelial cells of the central venous sinus (CVS) in the gill and the secondary circulation vessel (SCV) under the lateral line (the lateral lymph trunk) 5 and 15 min after HI treatment, respectively [Fig. 5(e)], and it remained visible until 120 min after the bath. In contrast, no fluorescence was observed in the endothelial cells of veins. Strongly-fluorescent granules were observed in the endothelial cells of the peritubular capillary network in the mesonephros and in the endothelial cells of the capillaries in the pronephros 15–120 min after treatment. The granules were also observed in the endothelial cells of the sinus of the spleen. DI group The relative strength of FITC fluorescence was remarkably lower in the skin, gills, pronephros, mesonephros and spleen of the DI group fish compared with those of the HI group (Table 1). In contrast to HI group fish, no fluorescence was visible in the intercellular spaces and collagenous connective tissue of the gills or skin, and strong fluorescence was seen in macrophage-like cells in the gills and skin 1–30 min after the treatment [Figs 5(b) & (d)].
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Table 1. The localisation of FITC-BSA administered by intravenous injection (IV), hyperosmotic infiltration (HI), or direct immersion (DI) Organ
Tissue
Administration Skin IV HI
DI Gills IV
S.C.V. I.S.E. C.C. E. S.C.V. Mö S.C.V.
P.C. C.V.S. HI Mö B.M. P.C. C.V.S. DI Mö C.V.S. Mesonephros IV P.C.N. HI P.C.N. DI P.C.N. Pronephros IV C. HI C. DI C. Spleen IV S. HI S. DI S.
Minutes after administration 1
5
15
30
60
120
" ++ ++ + " " "
+ + +++ + " + "
++ " ++++ + + + +
++ " ++++ + +++ + ++
+ " ++ " +++ " ++
+ " ++ " +++ " +
" " ++ ++++ + " ++ "
" + ++ ++++ ++ + ++ "
+ ++ ++ +++ + ++ ++ +
" + + ++ + ++ + ++
" " " ++ + ++ " ++
" " " " " + " +
" " "
++ + "
++++ ++++ +
++++ ++++ +
+++ ++++ +
+++ ++++ +
" " "
+ " "
++++ ++++ +
++++ ++++ +
+++ ++++ +
+++ ++++ +
" " "
+ " "
++ " "
++ + +
+ ++ +
+ ++ +
Note: S.C.V., endothelial cells of secondary circulatory vessel; I.S.E., intercellular space of epithelium; C.C., collagenous connective tissue; Mö, macrophage; B.M., basement membrane; E., epithelial cell; P.C., pillar cell; C.V.S., endothelial cells of central venous sinus; P.C.N., endothelial cells of peritubular capillary network; C., endothelial cells of capillary; S., endothelial cells of sinus.
IV group FITC-positive granules were observed in the endothelial cells of the CVS in the gills and the endothelial cells of the SCV under the lateral line [Fig. 5(f)]. In the pronephros, mesonephros and spleen, positive granules were observed in the same tissues as those of the HI group [Figs 5(g) & (h)]. Weakly stained granules and more di#use fluorescence were observed 60–120 min after injection. No FITC fluorescence was observed in the liver, pseudobranch, pyloric portion of the stomach, muscle, or intestine in all groups throughout the experiment.
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M. OTOTAKE ET AL.
IV. Discussion It has been suggested that the gills are the main site of antigen uptake in bath immunisation (Bower & Alexander, 1981; Alexander et al., 1981; Smith, 1982; Tatner et al., 1984; Zapata et al., 1987; Kawahara & Kusuda, 1988). However, our quantitative analyses indicate that the skin is the main site of antigen uptake both in hyperosmotic infiltration (HI) treatment and in direct immersion (DI) treatment, although some uptake also occurs in the gills. We have reached this conclusion for the following reasons: (1) The skin contained a significantly larger amount of BSA than the lateral line, gills, stomach or
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intestine after both DI and HI treatments (P<0·01, Figs 2 & 4); (2) in the skin and gills, BSA concentration decreased quickly during the first two hours after the bath [Figs 1(a) & 3(a)]; (3) the decrease of BSA in the skin, which was 6·8 mg BSA kg "1 fish after HI treatment and 0·8 mg kg "1 after DI treatment, is su$cient to account for the increase of BSA level in the plasma after HI and DI treatments, because the estimated gross BSA that was released into the plasma from sites of antigen uptake was 2·7–3·8 mg kg "1 after the HI treatment (Ototake & Nakanishi, 1992) and 0·6–0·8 mg kg "1 after the DI treatment (unpublished data). The present results are the first quantitative data for antigen uptake by the skin, although there have been other histological investigations that reported the presence of antigens in the skin after bath immunisation (Smith, 1982; Tatner et al., 1984; Hockney, 1985; Kawahara & Kusuda, 1988). Furthermore, our histological analyses using FITC-BSA confirmed that BSA is taken up by the skin as well as the gills (Table 1). Specifically, BSA was observed in the intercellular spaces of the skin epithelium, the collagenous fibrous connective tissue in the skin, and the basement membrane of the gill filaments and lamellae after HI treatment [Figs 5(a) & (c)]. BSA was also observed in macrophage-like cells in the skin and gills after both HI and DI treatments (Figs 5(b) & (d)]. The possibility that BSA was transported to these tissues from other organs, such as the intestine, by the blood was unlikely, because these tissues were BSA negative after IV administration. Therefore, BSA appears to be initially incorporated into the skin and to a lesser extent into the gills during 3 min of bath treatment in a 2% BSA solution, and released to the blood thereafter. Amend & Fender (1976) examined BSA concentration in the lateral line, which probably included some portion of the skin, in addition to the gills, skin mucus, stomach and intestine after HI treatment, and concluded that the lateral line was the main site of antigen uptake in HI treatment. In the present study, however, the BSA concentration in the skin was very similar to that of
Fig. 5. Fluorescence micrographs of tissue sections of rainbow trout administered FITC labelled BSA by intravenous injection (IV), hyperosmotic infiltration (HI), and direct immersion (DI). Scale bar=50 ìm. (a) Skin tissue one min after HI treatment. Strong fluorescence is observed on the surface (c) of the epidermis (E), intercellular spaces in the epithelium, and the collagenous connective tissue (C) around scale pocket (S). (b) Skin tissue one min after DI treatment. The fluorescence is visible in the macrophage-like cell (]) and on the surface of the skin (c). E, epidermis. (c) Gill tissue one min after HI treatment. FITC signals are observed in the basement membrane (c) under the epithelium of gill filaments (F) and the connecting basement membrane (c) of the gill lamella (L). (d) Gill tissue 15 min after DI treatment. Strong fluorescence is seen in macrophage-like cells (]) in the gill filament (F). L, gill lamella. (e) Cross section of the secondary circulatory vessel (SCV) under the lateral line (left hand side) 60 min after HI treatment. Fluorescence is visible in the endothelial cells (c) of SCV. M, muscle. (f) Gill tissue 5 min after IV administration. Fluorescence is visible in the endothelial cells of the central venous sinus (c). L, gill lamella; F, gill filament. (g) The mesonephros 15 min after IV administration. Strongly-fluorescent granules are observed in the endothelial cells of the peritubular capillary network. R, renal tubule. (h) The pronephros 15 min after IV administration. Strongly-fluorescent granules are observed in the endothelial cells of the capillaries.
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M. OTOTAKE ET AL.
the lateral line, which also contained some portion of the skin, after either HI or DI treatments. Furthermore, BSA was histologically detected in the skin, but not in the lateral line after either bath treatment. These results indicate that the skin itself is responsible for the uptake of antigen and not the lateral line. As for the stomach, the BSA concentration was very di#erent for each fish at each sampling time, and had no relationship to the blood BSA concentration in either HI or DI treatments (Figs 1 & 3). Furthermore, no BSA was histologically observed in the stomach. Thus the BSA was apparently present only in the contents of the stomach and not absorbed. Regarding the intestine, there are many studies on the absorption of intact protein (Rombout et al., 1985; 1986; Georgopoulou et al., 1986; 1988; McLean & Ash, 1986; 1987; Suzuki et al., 1988a; 1988b; Fujino & Nagai, 1988; Jenkins et al., 1992). Accordingly, intact BSA might be absorbed into the blood through the mucosal epithelium of the posterior intestine after treatment. In the present study, however, the intestine contained an insu$cient amount of BSA to maintain the blood BSA level after both HI and DI treatments (Figs 2 & 4). Moreover, no BSA was histologically observed in the intestine within 2 h after treatment. Therefore, we believe that the intestine is not the main site of antigen uptake in bath immunisation. Recently, Robohm & Koch (1995) suggested that the intestine was the principal site of antigen uptake, because plugging fish oesophagi with a dental-impression compound reduced the uptake of botulinum toxin nearly sixfold in bath treatment. However, their suggestion seems to be inapplicable to ordinary bath immunisation for the following two reasons. First, they used a lethal dose of toxin that may a#ect the function of antigen uptake in the skin, gills and intestine. Second, their method seems inappropriate to investigate the site of antigen uptake in typical bath treatment, because they immersed fish in the toxin solution for 23–269 h, periods which are much longer than ordinary HI and DI methods. In addition, it is the initial several hours after bath treatment that is considered the most important period to examine antigen uptake, since 90% of the gross BSA release into the blood occurred within 4 h after treatment in four species (Ototake & Nakanishi, 1992). The BSA localisation in the skin and gills di#ered dramatically between HI and DI treatments. Namely, BSA was observed in the macrophage-like cells, but not in intercellular spaces of the skin or gills after DI treatment. Thus, BSA uptake and transport are probably carried out by macrophage-like cells in DI. In contrast, with HI treatment most BSA was observed in the intercellular spaces of the skin and gills immediately after the bath. The antigen then spread into the basement membrane and the collagenous fibre connective tissue under the epithelium. This result shows that the majority of BSA infiltrated the intercellular spaces of the skin and gill epithelium, and then was transported by the tissue fluid through the intercellular space of the connective tissue after HI treatment. Therefore, it seems that the mechanism of antigen uptake by the skin and the gill di#ered between HI and DI. Amend & Fender (1976) hypothesised that the hyperosmotic treatment step of HI (2 min immersion in 5·3% NaCl solution) had an e#ect of dehydration on
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the epithelium, which facilitated BSA entrance. This hypothesis is supported by the results of the present study. Further experiments with the electron microscope will make it possible to observe structural changes in the skin and gill epithelia resulting from hyperosmotic treatment, and to verify this hypothesis. It has been reported that foreign antigens are trapped by the pronephros, the mesonephros and the spleen (Ellis, 1980; Smith, 1982; McLean et al., 1989). In the present study, we found that BSA was trapped not only by those organs but also by the secondary circulatory systems in the skin and gills. There seems to be little possibility of BSA release into the blood from these systems after HI and DI treatments, because BSA appeared in these systems sooner after IV treatment than after HI and DI treatments (Table 1). Thus BSA probably entered the secondary circulatory systems via blood circulation, and was then trapped by the endothelial cells of those tissues. Although there are some reports on the structure of the secondary circulatory system in fish (Vogel, 1981; Ste#ensen & Lomholt, 1992), there is only one report on its function. Ishimatsu et al. (1992) demonstrated that the secondary circulatory system plays an important role in the acid-base balance of rainbow trout exposed to environmental hypercapnia. Therefore, antigen trapping appears to be a newly described function of the secondary circulatory system. Clearly, more study is warranted on the immunological functions of this system in fish. We would like to thank Dr Yasuo Inui and Dr James D. Moore for critical reviewing of the manuscript. We also acknowledge the sta# of Nagano Prefectural Institute of Fisheries for supplying us with the rainbow trout used in this study. This study was supported in part by a grant-in-aid (Bio Media Program) from the Ministry of Agriculture, Forestry and Fisheries (BMP 96-V-2-2-3) and an operating grant to G.K.I. from the Canadian Bacterial Diseases Network.
References Amend, D. F. & Fender, D. C. (1976). Uptake of bovine serum albumin by rainbow trout from hyperosmotic infiltration: a model for vaccinating fish. Science 192, 793–794. Alexander, J. B., Bowers, A. & Shamshoon, S. M. (1981). Hyperosmotic infiltration of bacteria into trout: Route of entry and the fate of infiltrated bacteria. Developments in Biological Standardization 49, 441–445. Bower, A. & Alexander, J. B. (1981). Hyperosmotic infiltration: Immunological demonstration of infiltrating bacteria in brown trout, Salmo trutta L. Journal of Fish Biology 18, 9–13. Ellis, A. E. (1980). Antigen-trapping in the spleen and kidney of the plaice Pleuronectes platessa L. Journal of Fish Diseases 3, 413–426. Fujino, Y. & Nagai, A. (1988). The ingestion of bovine serum albumin into the serum through the intestine of chum salmon, Oncorhynchus keta Walbaum. Journal of Faculty of Marine Science Technology, Tokai University 26, 155–166. Georgopoulou, U., Sire, M. F. & Vernier, J. M. (1986). Immunological determination of intestinal absorption and digestion of protein macromolecules in the trout, Salmo gairdneri. Cell and Tissue Research 245, 387–395. Georgopoulou, U., Dabrowski, K, Sire, M. F. & Vernier, J. M. (1988). Absorption of intact protein by the intestinal epithelium of trout Salmo gairdneri: a luminescence enzyme immunoassay and cytochemical study. Cell and Tissue Research 251, 145–152.
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Hockney, M. J. (1985). An investigation of the skin of rainbow trout, Salmo gairdneri Richardson, for antigen uptake mechanisms following spray vaccination. In: Fish Immunology (M. J. Manning & M. F. Tatner, eds) pp. 195–205. New York: Academic Press. Ishimatsu, A., Iwama, G. K., Bentley, T. M. & Heisler, N. (1992). Contribution of the secondary circulatory system to acid-base regulation during hypercapnia in rainbow trout (Oncorhynchus mykiss). Journal of Experimental Biology 170, 43–56. Jenkins, P. G., Harris, J. E. & Pulsford, A. L. (1992). Quantitative serological aspects of the enhanced enteric uptake of human gamma globulin by Quil-A saponin in Oreochromis mossambicus. Fish & Shellfish Immunology 2, 193–210. Kawahara, E. & Kusuda, R. (1988). Location of Pasteurella piscicida antigen in tissue of yellowtail Seriola quinqueradiata vaccinated by immersion. Nippon Suissan Gakkaishi 54, 1101–1105. McLean, E. & Ash, R. (1986). The time-course of appearance and net accumulation of horseradish peroxidase (HRP) presented orally to juvenile carp Cyprinus carpio. Comparative Biochemistry and Physiology 84A, 687–690. McLean, E. & Ash, R. (1987). The time-course of appearance and net accumulation of horseradish peroxidase (HRP) presented orally to rainbow trout Salmo gairdneri (Richardson). Comparative Biochemistry and Physiology 88A, 507–517. McLean, E., Ash, R. & Westcott, P. A. B. (1989). The appearance of soluble antigen, horseradish peroxidase (HRP), in tissue of the rainbow trout (Salmo gairdneri Richardson), subsequent to variable dip-immersion procedures. Aquaculture 79, 411–415. Ototake, M. & Nakanishi, T. (1992). Kinetics of bovine serum albumin in fish plasma after hyperosmotic infiltration treatment: comparison between marine and freshwater fish. Aquaculture 103, 229–240. Robohm, R. A. (1986). Evidence that intestine is the principal route of antigen uptake in bath immunized fish. Development and Comparative Immunology 10, 145. (abstract). Robohm, R. A. & Koch, R. A. (1995). Evidence for oral ingestion as the principal route of antigen entry in bath-immunized fish. Fish & Shellfish Immunology 5, 137–150. Rombout, J. W. H. M., Blok, L. J., Lamers, C. H. K., Helfrich, M. H., Dekker, A. & Taverne-Thiele, J. J. (1985). Uptake and transport of intact macromolecules in the intestinal epithelium of carp (Cyprinus carpio L.) and the possible immunological implications. Cell and Tissue Research 239, 519–530. Rombout, J. W. H. M., Blok, L. J., Lamers, C. H. K. & Egberts, E. (1986). Immunization of carp (Cyprinus carpio) with a Vibrio anguillarum bacterin: indications for a common mucosal immune system. Development and Comparative Immunology 10, 341–352. Smith, P. D. (1982). Analysis of the hyperosmotic and bath methods for fish vaccination comparison of uptake of particulate and non-particulate antigens. Development and Comparative Immunology 2, 181–186. Ste#ensen, J. F. & Lomholt, J. P. (1992). The secondary vascular system. In: Fish Physiology, vol. 12 Cardiovascular System (W. S. Hoar, D. J. Randall & A. P. Farrell, eds.) pp. 185–218. New York: Academic Press. Suzuki, Y., Kobayashi, M., Aida, K. & Hanyu, I. (1988). Transport of physically active salmon gonadotropin into the circulation in goldfish, following oral administration of salmon pituitary extract. Journal of Comparative Physiology, B 157, 753–758. Suzuki, Y., Kobayashi, M., Nakamura, O., Aida, K. & Hanyu, I. (1988). Induced ovulation of the goldfish by oral administration of salmon pituitary extract. Aquaculture 74, 379–384. Tatner, M. F., Johnson, C. M. & Horne, M. T. (1984). The tissue localization of Aeromonas salmonicida in rainbow trout, Salmo gairdneri Richardson, following three methods of administration. Journal of Fish Biology 25, 95–108.
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Tatner, M. F. (1987). The quantitative relationship between vaccine dilution, length of immersion time and antigen uptake, using a radiolabelled Aeromonas salmonicida bath in direct immersion experiments with rainbow trout, Salmo gairdneri. Aquaculture 62, 173–185. Vogel, W. O. P. (1981). Struktur und organisationsprinzip im gefäßsystem der knchenfische. Gegenbaurs Morph. Jb. 127, 772–784. Zapata, A. G., Torroba, M., Alvarez, F., Anderson, D. P., Dixon, O. W. & Wisiniewski, M. (1987). Electron microscopic examination of antigen uptake by salmonid gill cells after bath immunization with a bacterin. Journal Fish Biology 31, 209–217.
The uptake of bovine serum albumin by the skin of bath-immunised rainbow trout Oncorhynchus mykiss MITSURU OTOTAKE*, GEORGE K. IWAMA†
AND
TERUYUKI NAKANISHI
National Research Institute of Aquaculture, Tamaki, Mie 519-04, Japan, †Department of Animal Science and Canadian Bacterial Diseases Network, University of British Columbia, Vancouver, B.C. Canada V6T-1Z4 (Received 22 September 1995, accepted in revised form 29 November 1995) In order to clarify the main site responsible for soluble antigen uptake in bath immunisation, the localisation of labelled bovine serum albumin (BSA) was examined in rainbow trout after bath administrations by hyperosmotic infiltration (HI) and direct immersion (DI). Quantitative analyses indicated that the main site of BSA uptake is the skin with a lesser role for the gills in both HI and DI treatments. These data were further confirmed by histological analysis. BSA was observed primarily in the intercellular spaces of the skin and gills after HI, while it was most common in epithelia of the skin and in macrophage-like cells in the gills after DI. Following either treatment, BSA was trapped by the secondary circulatory system in the skin and gills, as well as the kidney and spleen. ? 1996 Academic Press Limited Key words:
gill, rainbow trout, secondary circulation, skin, vaccination.
I. Introduction The bath immunisation method, first reported by Amend & Fender (1976), is currently used in aquaculture because of its convenience and e#ectiveness for mass vaccination. In bath immunisation, however, the exact mechanisms of antigen uptake and protection still remain unknown. Amend & Fender (1976), observing the time course of BSA concentration in various organs after bath treatment, stated that the main site responsible for uptake of antigen is the lateral line. In contrast, later studies reported that the gill is the main site of antigen uptake (Bower & Alexander, 1981; Alexander et al., 1981), and this is generally accepted at present (Smith, 1982; Tatner et al., 1984; Zapata et al., 1987; Kawahara & Kusuda, 1988). The intestine has also been proposed as a site of antigen uptake (Robohm, 1986; Rombout et al., 1985; 1986; Tatner, 1987; Robohm & Koch, 1995). Thus, the primary site responsible for antigen uptake has been a controversial topic with several organs having been implicated. This controversy seems to be attributable to the following three factors: (1) only one study has measured the antigen concentration in the gills, lateral line, and intestine after bath immunisation (Amend & Fender, 1976); (2) the two methods, direct immersion (DI) and hyperosmotic infiltration (HI), may have di#erent mechanisms of antigen uptake; (3) soluble and particulate *To whom all correspondence should be addressed. 321 1050–4648/96/050321+13 $18.00/0
? 1996 Academic Press Limited
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antigens may be taken up by di#erent mechanisms. In the present study, two experiments were carried out in order to clarify the main site of soluble antigen uptake in bath immunisation using the HI and DI methods. First, 125 I labelled BSA (125I-BSA) was used to measure BSA uptake quantitatively in each organ after bath administration, and then FITC labelled BSA (FITC-BSA) was used to observe the BSA localisation histologically in each organ. II. Materials and Methods EXPERIMENTAL ANIMALS
Juvenile rainbow trout Oncorhynchus mykiss, weighing 15&2 g (mean&standard deviation), were used in this study. They were kept in tanks supplied with running fresh water at 17·5)C and fed commercial dry pellets. QUANTITATIVE ANALYSIS
Fish were divided into the following two groups: HI (hyperosmotic infiltration) group with 60 fish and DI (direct immersion) group with 60 fish. Fish of the HI group were administered BSA (fraction V) conjugated with 125I (125I-BSA, No. 68031, ICN Co.) by the two-step HI treatment reported by Amend and Fender (1976) with slight modifications. Distilled water was used for the preparation of the two bath solutions: 5·3% NaCl solution and 2% BSA solution (50 k Bq ml "1). Fish of the HI group were immersed in a 5·3% NaCl solution for 3 min, and then placed in a 2% 125I-BSA solution for 3 min. Fish of the DI group were placed directly in a 2% BSA solution for 3 min. Following bath treatment, fish were rinsed in fresh water for 5 min. Thereafter, each fish was individually transferred to a tank with 5 l of static fresh water. Temperatures of the water and room were maintained at 18)C throughout the experiment. At 10, 30, 60, and 120 min after bath treatment, six fish of each group were anaesthetized with MS-222 (tricaine), and blood samples were withdrawn from the caudal blood vessels. Each fish was sacrificed by decapitation, and the whole skin, gills, stomach, intestine, liver, pronephros, mesonephros and spleen were taken. The skin was divided into two parts: lateral-line skin and trunk skin; the former consisting of the skin along the lateral line approximately 3 mm in width and the latter consisting of the remaining skin of the body. Each organ was weighed and then placed in 10 ml polyethylene tubes. The quantity of 125I-BSA in each tube was measured by a ã-counter (ARC-360, Aroka Co.). The measuring time was 20 min for each sample, and 120 min for the background. BSA concentration (ìg BSA g "1 tissue) and the total quantity of BSA in each tissue (mg BSA kg "1 fish) were calculated from the count for each tissue minus the background. HISTOLOGICAL ANALYSIS
Fish were divided into the following four groups: HI group (60 fish), DI group (60 fish), intravenous injection (IV) group (21 fish), and control
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unimmunised group (3 fish). Fish of the HI and DI groups were administered BSA conjugated with FITC (FITC-BSA, A9771, Sigma Co.) using the same treatments mentioned above for 125I-BSA. Fish of the IV group were administered 5 mg ml "1 of FITC-BSA in 0·85% NaCl by intravenous injection at 2·7 mg kg "1 fish, because the gross BSA release into the blood after HI treatment was estimated to be 2·7 mg kg "1 fish (Ototake & Nakanishi, 1992). The control group was left untreated. Three fish of each group were anaesthetised with MS-222, and blood samples were withdrawn from the caudal blood vessels 1, 5, 15, 30, 60, and 120 min after the bath. Then the same organs as mentioned above were dissected. Each tissue was fixed with a bu#ered 10% formalin (pH 7·2). After dehydration, tissues were embedded in resin (Technovit 7100, Kulzer Co.), and 2 ìm sections were a$xed to microscope slides. A cover glass was mounted with 50% glycerin and sections were examined under IB excitation with a fluorescence microscope (BH2-F, Olympus Co.). STATISTICS
Data were first subjected to the F test in order to detect significant di#erences between variances. When the di#erence of variances was significant, the data were subjected to the Cochran-Cox test in order to detect significant di#erences between means. The rest of the data were subjected to the Student’s unpaired t-test in order to detect significant di#erences between means. Significance level for all statistical analyses was P<0·05.
III. Results QUANTITATIVE ANALYSIS
HI group In the trunk skin, the BSA concentration was a maximum of 110&12 ìg BSA g "1 tissue (mean&standard error) 10 min after the bath, then significantly decreased to 49&7 ìg g "1 60 min after the bath (P<0·01), and subsequently showed little change [Fig. 1(a)]. There was little di#erence in BSA concentration between the lateral-line skin and trunk skin throughout the experiment. In the gills, a maximum BSA concentration of 67&7 ìg g "1 was observed at the initial sampling time, and the level significantly decreased by 30 min after the bath (P<0·01). In contrast, the BSA level in the blood was a minimum of 23&2 ìg g "1 at the initial sampling time, and gradually increased to 60&7 ìg g "1 120 min after the bath. Also in the mesonephros, pronephros, spleen, liver and intestine, the BSA levels were minimal at the initial sampling time, and gradually increased [Fig. 1(b)]. In the stomach, BSA concentrations varied greatly between individuals and no significant change was detected after the bath [Fig. 1(a)]. The total amount of BSA in each organ is shown in Fig. 2. At the initial sampling time, the trunk skin had the highest concentration (11·2&1·2 mg BSA kg "1 fish), followed by the gills (2·5&0·3 mg kg "1), lateral-line skin (1·0&0·1 mg kg "1), stomach (0·9&0·3 mg kg "1), and intestine (0·1&0·0 mg kg "1). Values for the trunk skin remained significantly
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324 140
(a)
120 100 Lateral-line skin Gills Blood Trunk skin Stomach
80
BSA (µ g g–1)
60 40 20 30
0
60
90
120
30 (b) Mesonephros Liver Intestine Pronephros Spleen
20 10
0
30
60 Minutes after HI
90
120
Fig. 1. Changes in bovine serum albumin (BSA) concentrations in organs of rainbow trout Oncorhynchus mykiss after hyperosmotic infiltration (HI) treatment. Data are presented as mean&standard error. 12
BSA (mg kg–1 fish)
10 8
Lateral-line skin Gills Intestine Trunk skin Stomach
6 4 2
0
30
60 Minutes after HI
90
120
Fig. 2. Changes in total bovine serum albumin (BSA) present in organs of rainbow trout Oncorhynchus mykiss after hyperosmotic infiltration (HI) treatment. Data are presented as mean&standard error.
higher than those for the gills, stomach, lateral-line skin and intestine throughout the experiment (P<0·01), and decreased by 6·8&1·0 mg kg "1 during the experiment.
BOVINE SERUM ALBUMIN UPTAKE
40
325
(a)
35 30 Lateral-line skin Gills Blood Trunk skin Stomach
25
BSA (µ g g–1)
20 15 10 5 0
30
60
90
120
10 8
(b) Mesonephros Liver Intestine Pronephros Spleen
6 4 2 0
30
60 Minutes after HI
90
120
Fig. 3. Changes in bovine serum albumin (BSA) concentrations in organs of rainbow trout Oncorhynchus mykiss after direct immersion (DI) treatment. Data are presented as mean&standard error.
DI group The BSA concentrations in this group were significantly lower than those of the HI group for all the tissues except the stomach (P<0·05, Figs 1 & 3). There was little di#erence in BSA concentration between the trunk skin and lateral-line skin throughout the experiment. The total amount of BSA in each organ is shown in Fig. 4. At the initial sampling time, the trunk skin was the highest (1·5&0·0 mg kg "1), followed by the stomach (0·3&0·2 mg kg "1), gills (0·2&0·0 mg kg "1) and lateral-line skin (0·1&0·0 mg kg "1). Values for the trunk skin remained significantly higher than those for the gills, stomach, lateral-line skin and intestine throughout the experiment (P<0·01), and decreased by 0·8&0·1 mg kg "1 during the experiment. HISTOLOGICAL ANALYSIS
Unimmunised control group Green auto-fluorescence was observed in some scales, and yellow autofluorescent granules were observed in some renal tubules and the splenic cord. HI group Table 1 shows the temporal changes in FITC fluorescence observed in each tissue. Strong fluorescence was observed 1 min after HI treatment in the
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326 1.6
BSA (mg kg–1 fish)
1.4 1.2 Lateral-line skin Gills Intestine Trunk skin Stomach
1.0 0.8 0.6 0.4 0.2 0
30
60 Minutes after HI
90
120
Fig. 4. Changes in total bovine serum albumin (BSA) present in organs of rainbow trout Oncorhynchus mykiss after direct immersion (DI) treatment. Data are presented as mean&standard error.
following locations in the skin: portions of the surface of the epidermis; the intercellular space in the epithelium; the collagenous connective tissue around scale pockets immediately beneath the epithelium; and a very limited number of epithelial cells [Fig. 5(a)]. In the gills, FITC signals were observed in the basement membrane under the epithelium of gill filaments, the connecting basement membrane of gill lamellae, and collagenous fibres in pillar cells [Fig. 5(c)]. Five minutes after HI treatment, the positive area extended along the basement membrane in gill filaments and lamellae and along the collagenous connective tissue around scale pockets in the skin. Fluorescence became visible in the endothelial cells of the central venous sinus (CVS) in the gill and the secondary circulation vessel (SCV) under the lateral line (the lateral lymph trunk) 5 and 15 min after HI treatment, respectively [Fig. 5(e)], and it remained visible until 120 min after the bath. In contrast, no fluorescence was observed in the endothelial cells of veins. Strongly-fluorescent granules were observed in the endothelial cells of the peritubular capillary network in the mesonephros and in the endothelial cells of the capillaries in the pronephros 15–120 min after treatment. The granules were also observed in the endothelial cells of the sinus of the spleen. DI group The relative strength of FITC fluorescence was remarkably lower in the skin, gills, pronephros, mesonephros and spleen of the DI group fish compared with those of the HI group (Table 1). In contrast to HI group fish, no fluorescence was visible in the intercellular spaces and collagenous connective tissue of the gills or skin, and strong fluorescence was seen in macrophage-like cells in the gills and skin 1–30 min after the treatment [Figs 5(b) & (d)].
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Table 1. The localisation of FITC-BSA administered by intravenous injection (IV), hyperosmotic infiltration (HI), or direct immersion (DI) Organ
Tissue
Administration Skin IV HI
DI Gills IV
S.C.V. I.S.E. C.C. E. S.C.V. Mö S.C.V.
P.C. C.V.S. HI Mö B.M. P.C. C.V.S. DI Mö C.V.S. Mesonephros IV P.C.N. HI P.C.N. DI P.C.N. Pronephros IV C. HI C. DI C. Spleen IV S. HI S. DI S.
Minutes after administration 1
5
15
30
60
120
" ++ ++ + " " "
+ + +++ + " + "
++ " ++++ + + + +
++ " ++++ + +++ + ++
+ " ++ " +++ " ++
+ " ++ " +++ " +
" " ++ ++++ + " ++ "
" + ++ ++++ ++ + ++ "
+ ++ ++ +++ + ++ ++ +
" + + ++ + ++ + ++
" " " ++ + ++ " ++
" " " " " + " +
" " "
++ + "
++++ ++++ +
++++ ++++ +
+++ ++++ +
+++ ++++ +
" " "
+ " "
++++ ++++ +
++++ ++++ +
+++ ++++ +
+++ ++++ +
" " "
+ " "
++ " "
++ + +
+ ++ +
+ ++ +
Note: S.C.V., endothelial cells of secondary circulatory vessel; I.S.E., intercellular space of epithelium; C.C., collagenous connective tissue; Mö, macrophage; B.M., basement membrane; E., epithelial cell; P.C., pillar cell; C.V.S., endothelial cells of central venous sinus; P.C.N., endothelial cells of peritubular capillary network; C., endothelial cells of capillary; S., endothelial cells of sinus.
IV group FITC-positive granules were observed in the endothelial cells of the CVS in the gills and the endothelial cells of the SCV under the lateral line [Fig. 5(f)]. In the pronephros, mesonephros and spleen, positive granules were observed in the same tissues as those of the HI group [Figs 5(g) & (h)]. Weakly stained granules and more di#use fluorescence were observed 60–120 min after injection. No FITC fluorescence was observed in the liver, pseudobranch, pyloric portion of the stomach, muscle, or intestine in all groups throughout the experiment.
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IV. Discussion It has been suggested that the gills are the main site of antigen uptake in bath immunisation (Bower & Alexander, 1981; Alexander et al., 1981; Smith, 1982; Tatner et al., 1984; Zapata et al., 1987; Kawahara & Kusuda, 1988). However, our quantitative analyses indicate that the skin is the main site of antigen uptake both in hyperosmotic infiltration (HI) treatment and in direct immersion (DI) treatment, although some uptake also occurs in the gills. We have reached this conclusion for the following reasons: (1) The skin contained a significantly larger amount of BSA than the lateral line, gills, stomach or
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intestine after both DI and HI treatments (P<0·01, Figs 2 & 4); (2) in the skin and gills, BSA concentration decreased quickly during the first two hours after the bath [Figs 1(a) & 3(a)]; (3) the decrease of BSA in the skin, which was 6·8 mg BSA kg "1 fish after HI treatment and 0·8 mg kg "1 after DI treatment, is su$cient to account for the increase of BSA level in the plasma after HI and DI treatments, because the estimated gross BSA that was released into the plasma from sites of antigen uptake was 2·7–3·8 mg kg "1 after the HI treatment (Ototake & Nakanishi, 1992) and 0·6–0·8 mg kg "1 after the DI treatment (unpublished data). The present results are the first quantitative data for antigen uptake by the skin, although there have been other histological investigations that reported the presence of antigens in the skin after bath immunisation (Smith, 1982; Tatner et al., 1984; Hockney, 1985; Kawahara & Kusuda, 1988). Furthermore, our histological analyses using FITC-BSA confirmed that BSA is taken up by the skin as well as the gills (Table 1). Specifically, BSA was observed in the intercellular spaces of the skin epithelium, the collagenous fibrous connective tissue in the skin, and the basement membrane of the gill filaments and lamellae after HI treatment [Figs 5(a) & (c)]. BSA was also observed in macrophage-like cells in the skin and gills after both HI and DI treatments (Figs 5(b) & (d)]. The possibility that BSA was transported to these tissues from other organs, such as the intestine, by the blood was unlikely, because these tissues were BSA negative after IV administration. Therefore, BSA appears to be initially incorporated into the skin and to a lesser extent into the gills during 3 min of bath treatment in a 2% BSA solution, and released to the blood thereafter. Amend & Fender (1976) examined BSA concentration in the lateral line, which probably included some portion of the skin, in addition to the gills, skin mucus, stomach and intestine after HI treatment, and concluded that the lateral line was the main site of antigen uptake in HI treatment. In the present study, however, the BSA concentration in the skin was very similar to that of
Fig. 5. Fluorescence micrographs of tissue sections of rainbow trout administered FITC labelled BSA by intravenous injection (IV), hyperosmotic infiltration (HI), and direct immersion (DI). Scale bar=50 ìm. (a) Skin tissue one min after HI treatment. Strong fluorescence is observed on the surface (c) of the epidermis (E), intercellular spaces in the epithelium, and the collagenous connective tissue (C) around scale pocket (S). (b) Skin tissue one min after DI treatment. The fluorescence is visible in the macrophage-like cell (]) and on the surface of the skin (c). E, epidermis. (c) Gill tissue one min after HI treatment. FITC signals are observed in the basement membrane (c) under the epithelium of gill filaments (F) and the connecting basement membrane (c) of the gill lamella (L). (d) Gill tissue 15 min after DI treatment. Strong fluorescence is seen in macrophage-like cells (]) in the gill filament (F). L, gill lamella. (e) Cross section of the secondary circulatory vessel (SCV) under the lateral line (left hand side) 60 min after HI treatment. Fluorescence is visible in the endothelial cells (c) of SCV. M, muscle. (f) Gill tissue 5 min after IV administration. Fluorescence is visible in the endothelial cells of the central venous sinus (c). L, gill lamella; F, gill filament. (g) The mesonephros 15 min after IV administration. Strongly-fluorescent granules are observed in the endothelial cells of the peritubular capillary network. R, renal tubule. (h) The pronephros 15 min after IV administration. Strongly-fluorescent granules are observed in the endothelial cells of the capillaries.
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the lateral line, which also contained some portion of the skin, after either HI or DI treatments. Furthermore, BSA was histologically detected in the skin, but not in the lateral line after either bath treatment. These results indicate that the skin itself is responsible for the uptake of antigen and not the lateral line. As for the stomach, the BSA concentration was very di#erent for each fish at each sampling time, and had no relationship to the blood BSA concentration in either HI or DI treatments (Figs 1 & 3). Furthermore, no BSA was histologically observed in the stomach. Thus the BSA was apparently present only in the contents of the stomach and not absorbed. Regarding the intestine, there are many studies on the absorption of intact protein (Rombout et al., 1985; 1986; Georgopoulou et al., 1986; 1988; McLean & Ash, 1986; 1987; Suzuki et al., 1988a; 1988b; Fujino & Nagai, 1988; Jenkins et al., 1992). Accordingly, intact BSA might be absorbed into the blood through the mucosal epithelium of the posterior intestine after treatment. In the present study, however, the intestine contained an insu$cient amount of BSA to maintain the blood BSA level after both HI and DI treatments (Figs 2 & 4). Moreover, no BSA was histologically observed in the intestine within 2 h after treatment. Therefore, we believe that the intestine is not the main site of antigen uptake in bath immunisation. Recently, Robohm & Koch (1995) suggested that the intestine was the principal site of antigen uptake, because plugging fish oesophagi with a dental-impression compound reduced the uptake of botulinum toxin nearly sixfold in bath treatment. However, their suggestion seems to be inapplicable to ordinary bath immunisation for the following two reasons. First, they used a lethal dose of toxin that may a#ect the function of antigen uptake in the skin, gills and intestine. Second, their method seems inappropriate to investigate the site of antigen uptake in typical bath treatment, because they immersed fish in the toxin solution for 23–269 h, periods which are much longer than ordinary HI and DI methods. In addition, it is the initial several hours after bath treatment that is considered the most important period to examine antigen uptake, since 90% of the gross BSA release into the blood occurred within 4 h after treatment in four species (Ototake & Nakanishi, 1992). The BSA localisation in the skin and gills di#ered dramatically between HI and DI treatments. Namely, BSA was observed in the macrophage-like cells, but not in intercellular spaces of the skin or gills after DI treatment. Thus, BSA uptake and transport are probably carried out by macrophage-like cells in DI. In contrast, with HI treatment most BSA was observed in the intercellular spaces of the skin and gills immediately after the bath. The antigen then spread into the basement membrane and the collagenous fibre connective tissue under the epithelium. This result shows that the majority of BSA infiltrated the intercellular spaces of the skin and gill epithelium, and then was transported by the tissue fluid through the intercellular space of the connective tissue after HI treatment. Therefore, it seems that the mechanism of antigen uptake by the skin and the gill di#ered between HI and DI. Amend & Fender (1976) hypothesised that the hyperosmotic treatment step of HI (2 min immersion in 5·3% NaCl solution) had an e#ect of dehydration on
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the epithelium, which facilitated BSA entrance. This hypothesis is supported by the results of the present study. Further experiments with the electron microscope will make it possible to observe structural changes in the skin and gill epithelia resulting from hyperosmotic treatment, and to verify this hypothesis. It has been reported that foreign antigens are trapped by the pronephros, the mesonephros and the spleen (Ellis, 1980; Smith, 1982; McLean et al., 1989). In the present study, we found that BSA was trapped not only by those organs but also by the secondary circulatory systems in the skin and gills. There seems to be little possibility of BSA release into the blood from these systems after HI and DI treatments, because BSA appeared in these systems sooner after IV treatment than after HI and DI treatments (Table 1). Thus BSA probably entered the secondary circulatory systems via blood circulation, and was then trapped by the endothelial cells of those tissues. Although there are some reports on the structure of the secondary circulatory system in fish (Vogel, 1981; Ste#ensen & Lomholt, 1992), there is only one report on its function. Ishimatsu et al. (1992) demonstrated that the secondary circulatory system plays an important role in the acid-base balance of rainbow trout exposed to environmental hypercapnia. Therefore, antigen trapping appears to be a newly described function of the secondary circulatory system. Clearly, more study is warranted on the immunological functions of this system in fish. We would like to thank Dr Yasuo Inui and Dr James D. Moore for critical reviewing of the manuscript. We also acknowledge the sta# of Nagano Prefectural Institute of Fisheries for supplying us with the rainbow trout used in this study. This study was supported in part by a grant-in-aid (Bio Media Program) from the Ministry of Agriculture, Forestry and Fisheries (BMP 96-V-2-2-3) and an operating grant to G.K.I. from the Canadian Bacterial Diseases Network.
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Hockney, M. J. (1985). An investigation of the skin of rainbow trout, Salmo gairdneri Richardson, for antigen uptake mechanisms following spray vaccination. In: Fish Immunology (M. J. Manning & M. F. Tatner, eds) pp. 195–205. New York: Academic Press. Ishimatsu, A., Iwama, G. K., Bentley, T. M. & Heisler, N. (1992). Contribution of the secondary circulatory system to acid-base regulation during hypercapnia in rainbow trout (Oncorhynchus mykiss). Journal of Experimental Biology 170, 43–56. Jenkins, P. G., Harris, J. E. & Pulsford, A. L. (1992). Quantitative serological aspects of the enhanced enteric uptake of human gamma globulin by Quil-A saponin in Oreochromis mossambicus. Fish & Shellfish Immunology 2, 193–210. Kawahara, E. & Kusuda, R. (1988). Location of Pasteurella piscicida antigen in tissue of yellowtail Seriola quinqueradiata vaccinated by immersion. Nippon Suissan Gakkaishi 54, 1101–1105. McLean, E. & Ash, R. (1986). The time-course of appearance and net accumulation of horseradish peroxidase (HRP) presented orally to juvenile carp Cyprinus carpio. Comparative Biochemistry and Physiology 84A, 687–690. McLean, E. & Ash, R. (1987). The time-course of appearance and net accumulation of horseradish peroxidase (HRP) presented orally to rainbow trout Salmo gairdneri (Richardson). Comparative Biochemistry and Physiology 88A, 507–517. McLean, E., Ash, R. & Westcott, P. A. B. (1989). The appearance of soluble antigen, horseradish peroxidase (HRP), in tissue of the rainbow trout (Salmo gairdneri Richardson), subsequent to variable dip-immersion procedures. Aquaculture 79, 411–415. Ototake, M. & Nakanishi, T. (1992). Kinetics of bovine serum albumin in fish plasma after hyperosmotic infiltration treatment: comparison between marine and freshwater fish. Aquaculture 103, 229–240. Robohm, R. A. (1986). Evidence that intestine is the principal route of antigen uptake in bath immunized fish. Development and Comparative Immunology 10, 145. (abstract). Robohm, R. A. & Koch, R. A. (1995). Evidence for oral ingestion as the principal route of antigen entry in bath-immunized fish. Fish & Shellfish Immunology 5, 137–150. Rombout, J. W. H. M., Blok, L. J., Lamers, C. H. K., Helfrich, M. H., Dekker, A. & Taverne-Thiele, J. J. (1985). Uptake and transport of intact macromolecules in the intestinal epithelium of carp (Cyprinus carpio L.) and the possible immunological implications. Cell and Tissue Research 239, 519–530. Rombout, J. W. H. M., Blok, L. J., Lamers, C. H. K. & Egberts, E. (1986). Immunization of carp (Cyprinus carpio) with a Vibrio anguillarum bacterin: indications for a common mucosal immune system. Development and Comparative Immunology 10, 341–352. Smith, P. D. (1982). Analysis of the hyperosmotic and bath methods for fish vaccination comparison of uptake of particulate and non-particulate antigens. Development and Comparative Immunology 2, 181–186. Ste#ensen, J. F. & Lomholt, J. P. (1992). The secondary vascular system. In: Fish Physiology, vol. 12 Cardiovascular System (W. S. Hoar, D. J. Randall & A. P. Farrell, eds.) pp. 185–218. New York: Academic Press. Suzuki, Y., Kobayashi, M., Aida, K. & Hanyu, I. (1988). Transport of physically active salmon gonadotropin into the circulation in goldfish, following oral administration of salmon pituitary extract. Journal of Comparative Physiology, B 157, 753–758. Suzuki, Y., Kobayashi, M., Nakamura, O., Aida, K. & Hanyu, I. (1988). Induced ovulation of the goldfish by oral administration of salmon pituitary extract. Aquaculture 74, 379–384. Tatner, M. F., Johnson, C. M. & Horne, M. T. (1984). The tissue localization of Aeromonas salmonicida in rainbow trout, Salmo gairdneri Richardson, following three methods of administration. Journal of Fish Biology 25, 95–108.
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Tatner, M. F. (1987). The quantitative relationship between vaccine dilution, length of immersion time and antigen uptake, using a radiolabelled Aeromonas salmonicida bath in direct immersion experiments with rainbow trout, Salmo gairdneri. Aquaculture 62, 173–185. Vogel, W. O. P. (1981). Struktur und organisationsprinzip im gefäßsystem der knchenfische. Gegenbaurs Morph. Jb. 127, 772–784. Zapata, A. G., Torroba, M., Alvarez, F., Anderson, D. P., Dixon, O. W. & Wisiniewski, M. (1987). Electron microscopic examination of antigen uptake by salmonid gill cells after bath immunization with a bacterin. Journal Fish Biology 31, 209–217.