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The humoral immune response of turbot, Scophthalmus maximus L., to spore-surface antigens of microsporidian parasites
Veterinary
immunology and
ELSEVIER
Veterinary Immunology and Immunopatbology 55 (1996) 235-242
immunopathokqy
The humoral immune response of turbot, Scophthalmus maximus L., to spore-surface antigens of microsporidian parasites J. Leiro bq*, M. Ortega b, J. Estevez ‘, M.T. Santamarina b, M.L. Sanmartin a, F.M. Ubeira b alnstituto de Irwestigacih y Aruilisis Alimentarios, Santiugo de Compostela, Spain b Departamento de Microbiologia y Parasitologia, Laboratorio de Parasitologia, Facultad de Furmacio, Universidad de Santiago de Compostela, Santiago de Compostela 15706, Spain ’ Laboratorio de Parasitologia. Facultud de Ciencius de1 Mar. Uniuersidad de Vigo, As Lagoas-Marcosende, 874, Vigo, Spain Accepted 26 March 1996
Abstract
Experiments based on enzyme-linked immunosorhent assay (ELBA) revealed considerable antigenic homology in turbot between two species of microsporidian, Tetrumicru breuifilum (a parasite of the turbot, Scophthalmus muximus) and Gfugea caulleryi (a parasite of the lesser sand-eel, Ammodytes tobianus). We next investigated whether G. caulleryi is able to suppress the turbot immune response. Intraperitoneal inoculation of turbot with G. caufleryi spores (whether heat-killed or viable) did not suppress the humoral immune response to injection of G. caulleryi spores plus adjuvant 15 days later; in fact, specific serum antibody levels (as revealed by ELBA) reached maximum levels by about Day 30 post re-exposure. Similar results were obtained with cellular enzyme-linked immunosorbent assay: 15 days after injection with G. cuulleryi spores plus adjuvant, specific antibody secretion rate was higher in turbot which had been pre-exposed to G. cauller-yi spores than in turbot which had not been pre-exposed. Keywords: Antibody; Turbot; Microsporidians; Spore antigens; ELISA; CELISA
’ Corresponding author. Laboratorio de Parasitologia, Facultad de Fannacia, Universidad de Santiago de Compostela, 15706, Santiago de Compostela, Spain. Fax: 81-5933 16; e-mail: [email protected] 01652427/%/$15.00 Published by Elsevier Science B.V. PII SO165-2427(96)05624-3
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1. Introduction Tetrumicra breuifilum is a common microsporidian parasite of farmed turbot (Matthews and Matthews, 1980; Este’vez et al., 1992). In Galicia in northwest Spain, T. breuifilum infection is generally low-intensity and benign, but epizootics causing high mortality and significant economic losses sometimes occur, most commonly among juvenile stock (Figueras et al., 1992; Este’vez et al., 1992). It is well known that the immune system of fish plays a key role in defence against many parasites, including microsporidians (Dykova and Lom, 1980; Pulsford and Matthews, 1991). However, there is little information on the humoral immune responses of fish to microsporidians (Woo, 1992). Research into fish responses to T. brevifilum is particularly problematic, because live material is difficult to obtain in quantity and because no effective procedures for experimental infection are known. A possible solution to this problem would be to use other readily available and antigenically similar microsporidian species as experimental models. One promising candidate species is Glugea cuulleryi (Van den Berghe, 1940), which is easy to obtain in quantity from the lesser sand-eel ( Ammodytes tobianus). Furthermore, lesser sand-eels are widely used as feed in turbot farms, and G. caulleryi is thus a potential pathogen of turbot. In the present study we evaluated the degree of antigenic relationship between Tetramicra brevifilum and Glugea caulleryi, and characterized the humoral immune response of turbot to surface antigens of G. caulleryi spores.
2. Materials and methods 2.1. Fish Juvenile turbot (40-50 g) were obtained from a local farm (Insuiila, El Grove, Pontevedra, Spain) and maintained in the laboratory at 18 + 1°C in 10 1 tanks with constant aeration. 2.2. Microsporidian
spores
Spores of Tetrumicru breuifilum were isolated from the muscle of naturally infected turbot, and spores of Glugea caulleryi from nodules in the liver of naturally infected lesser sand-eels, as described previously (Estevez et al., 1992). The spores were then freeze-dried and stored at room temperature until use. 2.3. immunization
and sera
Ten turbot were immunized by intraperitoneal (i.p.) injection of 0.2 ml of a 1: 1 mixture of Freund’s complete adjuvant (FCA; Sigma, USA) and phosphate-buffered saline (PBS) containing lo8 T. brev.@lum spores (five fish; for cx-Tetramicra brevifilum serum) or lo8 G. caulleryi spores (five fish; for cx-Glugea caulleryi serum). These fish
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were bled via the caudal vein (following anaesthesia with 0.1 g 1-l of MS 222, Sigma, USA) 45 days post-immunization. In addition, three groups of five turbot were injected i.p. with 0.2 ml of PBS containing lo* G. cuulleryi spores (Group A), or with 0.2 ml of PBS containing lo* G. caulleryi spores previously autoclaved for 10 min at 121°C (Group B), or with PBS alone (Group C). Fifteen days later, the fish in all three groups were injected i.p. with 0.2 ml of a 1:l mixture of FCA and PBS containing lo8 G. caulleryi spores. Appropriate controls were also run (see figure legends). These fish were bled 15, 30, 60 or 90 days after the second injection. In all cases, serum was separated by centrifugation (2000 g for 10 min), mixed 1:1 with glycerol and stored at - 30°C until use. 2.4. Enzyme-linked immunorbent assay (ELBA) To assay levels of anti-microsporidian antibodies in serum, we used ELISA as described previously (Leiro et al., 1993; Leiro et al., 1994). Spores were coupled to 96well PVC plates previously coated with poly-L-lysine (Sigma, USA). Antibodies were detected with UR3, an IgG, monoclonal antibody specific for the turbot Ig heavy chain (Este’vez et al., 1994a), and a peroxidase-conjugated rabbit anti-mouse Ig polyclonal antibody (Dakopatts A/S, Denmark). 2.5. Cellular enzyme-linked immunosorbent assay (CELISA) To assay the rate of secretion of anti-microsporidian serum antibodies, we used a slightly modified version of the cellular ELISA (CELISA) procedure described by 0.5 -
EZ
Preimmune serum
0.4 5 z t
0.3-
s P Jj
oz-
k 9 0.1 -
0.0 -
G. caukryi
1. brevifilum Antigen
Fig. 1. Reactivity in ELBA of anti-T. breu$lwn sera (cross-hatched bars) and anti-G. caulleryi sera (white bars) with surface antigens of 7’. brevifilum or G. caulleryi spores. Preimmune sera, and sera from fish which had been injected with Freund’s complete adjuvant alone, were assayed as controls. Values shown are mean absorbance (*SE) for individual assays of sera from five turbot.
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Effros et al. (1985) and Anmachalam et al. (1990). The turbot antibody-secreting cells used in this assay were from the spleen and anterior kidney. First, these organs were disaggregated by pushing through a steel mesh into Earles’ Balanced Salts Solution (Gibco, UK) containing 10% (v/v) 0.15 M phosphate buffer (pH 7.3). After mixing by drawing into and out of a pipette, the cell suspension was filtered (80 pm pore size) to remove nondisaggregated tissue fragments. The CELISA procedure was as follows. First, 50 ~1 of a solution of poly-L-lysine (1 mg in 100 ml of PBS) and 50 ~1 of a suspension of spores in PBS (10’ spores per ml) were mixed and poured onto PVC plates (Costar, USA). Plates were then centrifuged (500 g for 10 min) and washed three times with Tris-buffered saline (TBS). Nonspecific binding sites were blocked with TBS containing 0.2% Tween 20, 5% nonfat skimmed milk and 1% bovine serum albumin. Plates were then washed again with TBS. Turbot antibody-secreting cells (1.0 X lo7 cells ml- ‘> were added and plates were incubated for 2 h at 22.5”C under 5% CO,. Plates were then incubated with the appropriate dilution of UR3 (as used in ELISA; see above), then washed several times with TBS, then incubated with peroxidase-conjugated rabbit anti-mouse Ig polyclonal antibody (Dakopatts). After five washes in TBS, o-phenylenediamine at a concentration of 0.04% in phosphate-citrate buffer (pH 5.0) containing 0.0001% H,O, was added to 0.90
0.75
0 Group A Group _U Group
A B C
0.00
I
I
I
I
15
30
60
90
Days post-lmmunlzatlon Fig. 2. Kinetics of production of specific antibodies to G. cauUeryi by turbot which had been preexposed to the parasite. Pre-exposure was by intraperitoneal injection with 0.2 ml of PBS containing 10’ G. caulferyi spores (Group A), or with 0.2 ml of PBS containing lo* G. caulleryi spores previously autoclaved for 10 min at 121°C (Group B); Group C received 0.2 ml of PBS alone. Fifteen days later, the fish in all three groups were injected i.p. with 0.2 ml of a 1: 1 mixture of FCA and PBS containing 10s G. caulleryi spores. The fish were bled 15, 30, 60 or 90 days after the second injection, and serum antibody levels (to G. caulleryi spore-surface antigens) were determined by ELISA. Values shown arc mean absorbances ( 4~SE) for individual assays of sera from five turbot.
.I. L&o et al./ Veterinary Immunology and Immunopathology
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each well. After 20 min, the reaction was stopped with 3 N H,SO,, and absorbance at 492 nm was measured with a Titertek Multiscan ELISA reader (Labsystem, Finland). 2.6. Statistical analysis All statistical comparisons were by means of unpaired Student’s t-tests (a = 0.05).
3. Results 3.1. Antigenic relationships between T. brevijlum and G. caulleryi The results of ELISA (Fig. 1) indicate that serum antibodies produced by turbot immunized with T. brevi$lum spores cross-reacted extensively with surface antigens of G. caulleryi spores; likewise, the anti-G. caulleryi serum cross-reacted extensively with T. breviflum antigens.
3.2. Kinetics of the humoral immune response to G. caulleryi spores Some microsporidians are able to suppress the host immune response (see Discussion). To determine whether G. caulleryi is able to modulate the turbot humoral
E c
0.4
-
0.3
-
0.2
-
0.1
-
0.0
-
5 s z s e c: s
A
B
C
Control
Group Fig. 3. Rate of secretion of specific antibodies to G. cuulleryi
by spleen/kidney
exposed to the parasite 30 days previously and re-exposed
15 days previously. The treatments received by
cells of turbot which had been
Groups A, B and C are as detailed for Fig. 2. Amount of antibody secreted over the 2 h assay period was The control value is for spleen/kidney cells from turbot which had not been exposed to the parasite. Values shown are mean absorbance ( + SE) for individual assays of spleen/kidney cells from
determined by CELISA. five turbot.
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response, we used ELISA to investigate the kinetics of production of serum antibodies to G. caulleryi spores following pre-exposure to the parasite. Turbot were pre-exposed to viable or heat-killed spores, and re-exposed 15 days later to viable spores plus FCA; blood samples were taken 15, 30, 60 or 90 days after the second injection. The second injection was with spores plus FCA in order to maximize the antibody response (Leiro et al., 1993). The results (Fig. 2) show that the response after pre-exposure was greater than the response without pre-exposure, and that the response was very similar regardless of whether the fish had been pre-exposed to viable or heat-killed spores. Similar results were obtained with CELISA. Fifteen days after the second injection, spleen/kidney cells from turbot that had been pre-exposed to viable or heat-killed spores secreted specific antibodies at a much higher rate than spleen/kidney cells from turbot which had not been pre-exposed to the parasite (Fig. 3).
4. Discussion Tetramicra brevi$lum is a common parasite of farmed turbot, and may cause heavy mortalities (Figueras et al., 1992; Estevez et al., 1992). Unfortunately, research into the turbot immune response to T. brevifilum has been hindered by the difficulty of obtaining live material in quantity, and by the lack of effective procedures for experimental infection. It is thus of interest to look for experimental models of T. brevi$lum infection. Many microsporidians show considerable antigenic similarity. For example, close antigenic homologies have been reported among Encephalitozoon cuniculi, Glugea hertwigi, Glugea stephani and Nosema plodiae (Niederkom et al., 1980), between two different isolates of Nosema necatrix (Knell and Zam, 19781, and between Glugea atherinae and Enterocytozoon bieneusi (Ombrouck et al., 1995). The results of the present study indicate close similarities between the surface antigens of T. breviflum and G. caulleryi. As intracellular parasites, microsporidians can be expected to have developed mechanisms for avoidance or suppression of the host immune response (Moulder, 19851. For example, some microsporidians invade phagocytes, implying that they are able to manipulate the normal phagocytotic response (Schmidt and Shadduck, 1984). Laudan et al. (1986a); Laudan et al. (1986b) reported that Glugea-infected winter flounder (i.e. flounders naturally infected with Glugea stephani or injected with G. stephani spores) had reduced serum immunoglobulin levels and showed a reduced humoral response to other antigens (horse erythrocytes and formalin-killed Klebsiella pneumoniae). The immunosuppressive factor was transferable to healthy fish by injection of serum (Laudan et al., 1987). Suppression of the response to other antigens was most evident during the initial stages of infection (Laudan et al., 1989). In the present study we did not detect any evidence of immunomodulation by Glugea caulleryi; on the contrary, pre-exposure to spores (whether viable or heat-killed) significantly stimulated the subsequent response to injection of spores plus FCA (see Fig. 2). A similar result was obtained with CELISA. This assay allows quantification of the rate of antibody secretion at time t, as opposed to serum antibody level reached by time 1. The rate of antibody secretion reflects the proportion of the antibody-secreting-cell population producing that
J. Leiro et al./ Veterinary Immunology and Immunopathology 55 (1996) 235-242
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antibody. Fifteen days after the second injection, spleen/kidney cells from turbot which had been pre-exposed to viable or heat-killed G. cuulleryi spores secreted specific antibodies at a faster rate than cells from turbot which had not been previously exposed to the parasite. In both ELISA and CELISA, the response of fish which had been pre-exposed to viable spores was of greater magnitude than that of fish which had been pre-exposed to heat-killed spores (though in neither case was the difference statistically significant). This suggests that heat treatment may cause slight modifications to surface antigens, making them less immunogenic. The results of ELISA (Fig. 2) indicate that specific serum antibody levels in primed fish increased more or less exponentially until about Day 30 after the second injection, after which they remained more or less constant or declined. We have observed similar kinetics in previous studies of the turbot responses to T. breuijlum (Leiro et al., 1993) and to inactivated Vibrio anguiflarum (Estevez et al., 1994b). In the latter study, the maximum response was reached about 30-45 days after re-exposure, and antibody levels remained more or less constant until about 90 days after re-exposure. In conclusion, the results of this study indicate (a) that there is considerable homology between the surface antigens of Tetrczmicra brevifilum and Glugea caulleryi and (b) that inoculation of turbot with viable Glugea caulleryi spores does not cause any suppression of (and indeed stimulates) the subsequent humoral response to surface antigens of this parasite.
Acknowledgements
This study was supported by grants XUGA20303B95 and XUGA20308B95 (Xunta de Galicia, Spain). We would like to thank Insuitia (El Grove, Pontevedra) for the generous supply of turbot.
References Arunachalam, B., Talwar, G.P. and Raghupathy, R., 1990. A simplified cellular ELISA (CELISA) for the detection of antibodies reacting with cell-surface antigens. J. Immunol. Meth., 135: 181-189. Dykova, I. and Lom, J., 1980. Tissue reactions to microsporidian infections in fish. J. Fish Dis., 3: 265-283. Effros, R.B., ZelIer, E., DiIIard, L. and Walford, R.L., 1985. Detection of antibodies to cell-surface antigens by a simplified cellular ELISA (CELISA). Tissue Antigens, 25: 204. Estevez, J., lglesias, R., Leiro, J., Ubeira, F.M. and Sanmartin, M.L., 1992. An unusual site of infection by a microsporean in the turbot Scophthalmus maximus. Dis. Aquat. Org., 13: 139-142. Estevez, J., Leiro, J., Santamarina, M.T., Dominguez, J. and Ubeira, F.M., 1994a. Monoclonal antibodies to turbot (Scophthulmus maximus) immunoglobulins: characterization and applicability in immunoassays. Vet. Immunol. Immunopathol., 41: 353-366. EstCvez, J., Leiro, J., Toranzo, A.E., Barja, J.L. and Ubeira, F.M., 1994b. Kinetics of antibody production against Vibrio anguilkurum antigens in turbot. Aquaculture, 123: 191- 196. Figueras, A., Novoa, B., Santarem, M., Martinez, E., Alvarez. J.M., Toranzo, A.E. and Dykovi, I., 1992. Terramicra breuifilum, a potential threat to farmed turbot Scophthalmus maximus. Dis. Aquat. Org., 14: 127-135. Knell, J.D. and Zam, S.G., 1978. A serological comparison of some species of microsporidia. J. Invertebr. Pathol., 31: 280-288.
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Laudan, R., Stolen, J.S. and Cali, A., 1986a. The immune response of a marine teleost, Pseudopleuronectes americcrnus (winter flounder) to the protozoan parasite, Glugea stephani. Vet. Immunol. Immunopathol., 12: 403-412. Laudan, R., Stolen, J.S. and Cali, A., 1986b. Immunoglobulin levels of the winter flounder (Pseudopleuronectes americanus) and the summer flounder (Pnralichthys dentatus) injected with the microsporidart parasite (Glugea stephand. Dev. Comp. Immunol., 10: 33 I-340. Laudan, R., Stolen, J.S. and Cali, A., 1987. The immunomodulating effect of the microsporidan Glugea stephani on the humoral response and immunoglobulin levels in winter flounder, Pseudopleuronectes americanus. J. Fish Biol., 31 (Supplement A): 155- 160. Laudan, R., Stolen, J.S. and Cali, A., 1989. The effect of the microsporida Glugea stephani on the immunoglobulin levels of juvenile and adult winter flounder (Pseudopleuronectes americanus). Dev. Comp. Immunol., 13: 35-41. Leiro, J., Esdvez, J., Santamarina, M.T., Sanmartin, M.L. and Ubeira, F.M., 1993. Humoral immune response of turbot, Scophthulmus muximus CL.), to antigens from Tetramicra breuifilum Matthews and Matthews. 1980 (Microsporal. I. Fish Dis., 16: 577-584. Leiro, J., Estevez, J., Ubeira, F.M., Santamarina, M.T., and Sanmartin, M.L., 1994. Serological relationships between two microsporidian parasites of fish. Aquaculture, 125: l-9. Matthews, R.A. and Matthews, B.F., 1980. Cell and tissue reactions of turbot, Scophthalmus maximus (L-1, to Tetramicra breoijlum gen. n. sp. n. (Microspora). J. Fish Dis., 3: 495-515. Moulder, J.W., 1985. Comparative biology of intracellular parasitism. Microbial. Rev., 49: 298-337. Niederkom, J.Y., Shadduck, J.A. and Weidner, E., 1980. Antigenic cross-reactivity among different microsporidian spores as determined by immunofluorescence. J. Parasitol., 66: 677-679. Ombrouck, C., Romestand, B., Dacosta, J.M.C., Desporteslivage, 1.. Datry. A., Coste, F., Bouix, G. and Gentilini, M., 1995. Use of cross-reactive antigens of the microsporidian Glugea atherinue for the possible detection of Enterocytozoon bieneusi by Western blot. Am. J. Trop. Med. Hyg., 52: 89-93. Pulsford, A. and Matthews, R.A., 1991. Macrophages and giant cells associated with a microsporidian parasite causing liquefaction of the slceletal muscle of the Norway pout, Trisoprerus esmarkii (Nilsson). J. Fish Dis., 14: 67-78. Schmidt, E.C. and Shadduck, J.A., 1984. Mechanism of resistance to the intracellular protozoan Encephalitoloon cuniculi in mice. J. Immunol., 133: 2712-2719. Van den Berghe, L., 1940. Glugea caulleryi nom. n. for Glugea microsporu. J. Parasitol., 26: 238. Woo, P.T.K., 1992. Immunological responses of fish to parasitic organisms. Ann. Rev. Fish Dis., 2: 339-366.
immunology and
ELSEVIER
Veterinary Immunology and Immunopatbology 55 (1996) 235-242
immunopathokqy
The humoral immune response of turbot, Scophthalmus maximus L., to spore-surface antigens of microsporidian parasites J. Leiro bq*, M. Ortega b, J. Estevez ‘, M.T. Santamarina b, M.L. Sanmartin a, F.M. Ubeira b alnstituto de Irwestigacih y Aruilisis Alimentarios, Santiugo de Compostela, Spain b Departamento de Microbiologia y Parasitologia, Laboratorio de Parasitologia, Facultad de Furmacio, Universidad de Santiago de Compostela, Santiago de Compostela 15706, Spain ’ Laboratorio de Parasitologia. Facultud de Ciencius de1 Mar. Uniuersidad de Vigo, As Lagoas-Marcosende, 874, Vigo, Spain Accepted 26 March 1996
Abstract
Experiments based on enzyme-linked immunosorhent assay (ELBA) revealed considerable antigenic homology in turbot between two species of microsporidian, Tetrumicru breuifilum (a parasite of the turbot, Scophthalmus muximus) and Gfugea caulleryi (a parasite of the lesser sand-eel, Ammodytes tobianus). We next investigated whether G. caulleryi is able to suppress the turbot immune response. Intraperitoneal inoculation of turbot with G. caufleryi spores (whether heat-killed or viable) did not suppress the humoral immune response to injection of G. caulleryi spores plus adjuvant 15 days later; in fact, specific serum antibody levels (as revealed by ELBA) reached maximum levels by about Day 30 post re-exposure. Similar results were obtained with cellular enzyme-linked immunosorbent assay: 15 days after injection with G. cuulleryi spores plus adjuvant, specific antibody secretion rate was higher in turbot which had been pre-exposed to G. cauller-yi spores than in turbot which had not been pre-exposed. Keywords: Antibody; Turbot; Microsporidians; Spore antigens; ELISA; CELISA
’ Corresponding author. Laboratorio de Parasitologia, Facultad de Fannacia, Universidad de Santiago de Compostela, 15706, Santiago de Compostela, Spain. Fax: 81-5933 16; e-mail: [email protected] 01652427/%/$15.00 Published by Elsevier Science B.V. PII SO165-2427(96)05624-3
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1. Introduction Tetrumicra breuifilum is a common microsporidian parasite of farmed turbot (Matthews and Matthews, 1980; Este’vez et al., 1992). In Galicia in northwest Spain, T. breuifilum infection is generally low-intensity and benign, but epizootics causing high mortality and significant economic losses sometimes occur, most commonly among juvenile stock (Figueras et al., 1992; Este’vez et al., 1992). It is well known that the immune system of fish plays a key role in defence against many parasites, including microsporidians (Dykova and Lom, 1980; Pulsford and Matthews, 1991). However, there is little information on the humoral immune responses of fish to microsporidians (Woo, 1992). Research into fish responses to T. brevifilum is particularly problematic, because live material is difficult to obtain in quantity and because no effective procedures for experimental infection are known. A possible solution to this problem would be to use other readily available and antigenically similar microsporidian species as experimental models. One promising candidate species is Glugea cuulleryi (Van den Berghe, 1940), which is easy to obtain in quantity from the lesser sand-eel ( Ammodytes tobianus). Furthermore, lesser sand-eels are widely used as feed in turbot farms, and G. caulleryi is thus a potential pathogen of turbot. In the present study we evaluated the degree of antigenic relationship between Tetramicra brevifilum and Glugea caulleryi, and characterized the humoral immune response of turbot to surface antigens of G. caulleryi spores.
2. Materials and methods 2.1. Fish Juvenile turbot (40-50 g) were obtained from a local farm (Insuiila, El Grove, Pontevedra, Spain) and maintained in the laboratory at 18 + 1°C in 10 1 tanks with constant aeration. 2.2. Microsporidian
spores
Spores of Tetrumicru breuifilum were isolated from the muscle of naturally infected turbot, and spores of Glugea caulleryi from nodules in the liver of naturally infected lesser sand-eels, as described previously (Estevez et al., 1992). The spores were then freeze-dried and stored at room temperature until use. 2.3. immunization
and sera
Ten turbot were immunized by intraperitoneal (i.p.) injection of 0.2 ml of a 1: 1 mixture of Freund’s complete adjuvant (FCA; Sigma, USA) and phosphate-buffered saline (PBS) containing lo8 T. brev.@lum spores (five fish; for cx-Tetramicra brevifilum serum) or lo8 G. caulleryi spores (five fish; for cx-Glugea caulleryi serum). These fish
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were bled via the caudal vein (following anaesthesia with 0.1 g 1-l of MS 222, Sigma, USA) 45 days post-immunization. In addition, three groups of five turbot were injected i.p. with 0.2 ml of PBS containing lo* G. cuulleryi spores (Group A), or with 0.2 ml of PBS containing lo* G. caulleryi spores previously autoclaved for 10 min at 121°C (Group B), or with PBS alone (Group C). Fifteen days later, the fish in all three groups were injected i.p. with 0.2 ml of a 1:l mixture of FCA and PBS containing lo8 G. caulleryi spores. Appropriate controls were also run (see figure legends). These fish were bled 15, 30, 60 or 90 days after the second injection. In all cases, serum was separated by centrifugation (2000 g for 10 min), mixed 1:1 with glycerol and stored at - 30°C until use. 2.4. Enzyme-linked immunorbent assay (ELBA) To assay levels of anti-microsporidian antibodies in serum, we used ELISA as described previously (Leiro et al., 1993; Leiro et al., 1994). Spores were coupled to 96well PVC plates previously coated with poly-L-lysine (Sigma, USA). Antibodies were detected with UR3, an IgG, monoclonal antibody specific for the turbot Ig heavy chain (Este’vez et al., 1994a), and a peroxidase-conjugated rabbit anti-mouse Ig polyclonal antibody (Dakopatts A/S, Denmark). 2.5. Cellular enzyme-linked immunosorbent assay (CELISA) To assay the rate of secretion of anti-microsporidian serum antibodies, we used a slightly modified version of the cellular ELISA (CELISA) procedure described by 0.5 -
EZ
Preimmune serum
0.4 5 z t
0.3-
s P Jj
oz-
k 9 0.1 -
0.0 -
G. caukryi
1. brevifilum Antigen
Fig. 1. Reactivity in ELBA of anti-T. breu$lwn sera (cross-hatched bars) and anti-G. caulleryi sera (white bars) with surface antigens of 7’. brevifilum or G. caulleryi spores. Preimmune sera, and sera from fish which had been injected with Freund’s complete adjuvant alone, were assayed as controls. Values shown are mean absorbance (*SE) for individual assays of sera from five turbot.
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and immunopathofogy
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Effros et al. (1985) and Anmachalam et al. (1990). The turbot antibody-secreting cells used in this assay were from the spleen and anterior kidney. First, these organs were disaggregated by pushing through a steel mesh into Earles’ Balanced Salts Solution (Gibco, UK) containing 10% (v/v) 0.15 M phosphate buffer (pH 7.3). After mixing by drawing into and out of a pipette, the cell suspension was filtered (80 pm pore size) to remove nondisaggregated tissue fragments. The CELISA procedure was as follows. First, 50 ~1 of a solution of poly-L-lysine (1 mg in 100 ml of PBS) and 50 ~1 of a suspension of spores in PBS (10’ spores per ml) were mixed and poured onto PVC plates (Costar, USA). Plates were then centrifuged (500 g for 10 min) and washed three times with Tris-buffered saline (TBS). Nonspecific binding sites were blocked with TBS containing 0.2% Tween 20, 5% nonfat skimmed milk and 1% bovine serum albumin. Plates were then washed again with TBS. Turbot antibody-secreting cells (1.0 X lo7 cells ml- ‘> were added and plates were incubated for 2 h at 22.5”C under 5% CO,. Plates were then incubated with the appropriate dilution of UR3 (as used in ELISA; see above), then washed several times with TBS, then incubated with peroxidase-conjugated rabbit anti-mouse Ig polyclonal antibody (Dakopatts). After five washes in TBS, o-phenylenediamine at a concentration of 0.04% in phosphate-citrate buffer (pH 5.0) containing 0.0001% H,O, was added to 0.90
0.75
0 Group A Group _U Group
A B C
0.00
I
I
I
I
15
30
60
90
Days post-lmmunlzatlon Fig. 2. Kinetics of production of specific antibodies to G. cauUeryi by turbot which had been preexposed to the parasite. Pre-exposure was by intraperitoneal injection with 0.2 ml of PBS containing 10’ G. caulferyi spores (Group A), or with 0.2 ml of PBS containing lo* G. caulleryi spores previously autoclaved for 10 min at 121°C (Group B); Group C received 0.2 ml of PBS alone. Fifteen days later, the fish in all three groups were injected i.p. with 0.2 ml of a 1: 1 mixture of FCA and PBS containing 10s G. caulleryi spores. The fish were bled 15, 30, 60 or 90 days after the second injection, and serum antibody levels (to G. caulleryi spore-surface antigens) were determined by ELISA. Values shown arc mean absorbances ( 4~SE) for individual assays of sera from five turbot.
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each well. After 20 min, the reaction was stopped with 3 N H,SO,, and absorbance at 492 nm was measured with a Titertek Multiscan ELISA reader (Labsystem, Finland). 2.6. Statistical analysis All statistical comparisons were by means of unpaired Student’s t-tests (a = 0.05).
3. Results 3.1. Antigenic relationships between T. brevijlum and G. caulleryi The results of ELISA (Fig. 1) indicate that serum antibodies produced by turbot immunized with T. brevi$lum spores cross-reacted extensively with surface antigens of G. caulleryi spores; likewise, the anti-G. caulleryi serum cross-reacted extensively with T. breviflum antigens.
3.2. Kinetics of the humoral immune response to G. caulleryi spores Some microsporidians are able to suppress the host immune response (see Discussion). To determine whether G. caulleryi is able to modulate the turbot humoral
E c
0.4
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0.3
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0.2
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5 s z s e c: s
A
B
C
Control
Group Fig. 3. Rate of secretion of specific antibodies to G. cuulleryi
by spleen/kidney
exposed to the parasite 30 days previously and re-exposed
15 days previously. The treatments received by
cells of turbot which had been
Groups A, B and C are as detailed for Fig. 2. Amount of antibody secreted over the 2 h assay period was The control value is for spleen/kidney cells from turbot which had not been exposed to the parasite. Values shown are mean absorbance ( + SE) for individual assays of spleen/kidney cells from
determined by CELISA. five turbot.
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response, we used ELISA to investigate the kinetics of production of serum antibodies to G. caulleryi spores following pre-exposure to the parasite. Turbot were pre-exposed to viable or heat-killed spores, and re-exposed 15 days later to viable spores plus FCA; blood samples were taken 15, 30, 60 or 90 days after the second injection. The second injection was with spores plus FCA in order to maximize the antibody response (Leiro et al., 1993). The results (Fig. 2) show that the response after pre-exposure was greater than the response without pre-exposure, and that the response was very similar regardless of whether the fish had been pre-exposed to viable or heat-killed spores. Similar results were obtained with CELISA. Fifteen days after the second injection, spleen/kidney cells from turbot that had been pre-exposed to viable or heat-killed spores secreted specific antibodies at a much higher rate than spleen/kidney cells from turbot which had not been pre-exposed to the parasite (Fig. 3).
4. Discussion Tetramicra brevi$lum is a common parasite of farmed turbot, and may cause heavy mortalities (Figueras et al., 1992; Estevez et al., 1992). Unfortunately, research into the turbot immune response to T. brevifilum has been hindered by the difficulty of obtaining live material in quantity, and by the lack of effective procedures for experimental infection. It is thus of interest to look for experimental models of T. brevi$lum infection. Many microsporidians show considerable antigenic similarity. For example, close antigenic homologies have been reported among Encephalitozoon cuniculi, Glugea hertwigi, Glugea stephani and Nosema plodiae (Niederkom et al., 1980), between two different isolates of Nosema necatrix (Knell and Zam, 19781, and between Glugea atherinae and Enterocytozoon bieneusi (Ombrouck et al., 1995). The results of the present study indicate close similarities between the surface antigens of T. breviflum and G. caulleryi. As intracellular parasites, microsporidians can be expected to have developed mechanisms for avoidance or suppression of the host immune response (Moulder, 19851. For example, some microsporidians invade phagocytes, implying that they are able to manipulate the normal phagocytotic response (Schmidt and Shadduck, 1984). Laudan et al. (1986a); Laudan et al. (1986b) reported that Glugea-infected winter flounder (i.e. flounders naturally infected with Glugea stephani or injected with G. stephani spores) had reduced serum immunoglobulin levels and showed a reduced humoral response to other antigens (horse erythrocytes and formalin-killed Klebsiella pneumoniae). The immunosuppressive factor was transferable to healthy fish by injection of serum (Laudan et al., 1987). Suppression of the response to other antigens was most evident during the initial stages of infection (Laudan et al., 1989). In the present study we did not detect any evidence of immunomodulation by Glugea caulleryi; on the contrary, pre-exposure to spores (whether viable or heat-killed) significantly stimulated the subsequent response to injection of spores plus FCA (see Fig. 2). A similar result was obtained with CELISA. This assay allows quantification of the rate of antibody secretion at time t, as opposed to serum antibody level reached by time 1. The rate of antibody secretion reflects the proportion of the antibody-secreting-cell population producing that
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antibody. Fifteen days after the second injection, spleen/kidney cells from turbot which had been pre-exposed to viable or heat-killed G. cuulleryi spores secreted specific antibodies at a faster rate than cells from turbot which had not been previously exposed to the parasite. In both ELISA and CELISA, the response of fish which had been pre-exposed to viable spores was of greater magnitude than that of fish which had been pre-exposed to heat-killed spores (though in neither case was the difference statistically significant). This suggests that heat treatment may cause slight modifications to surface antigens, making them less immunogenic. The results of ELISA (Fig. 2) indicate that specific serum antibody levels in primed fish increased more or less exponentially until about Day 30 after the second injection, after which they remained more or less constant or declined. We have observed similar kinetics in previous studies of the turbot responses to T. breuijlum (Leiro et al., 1993) and to inactivated Vibrio anguiflarum (Estevez et al., 1994b). In the latter study, the maximum response was reached about 30-45 days after re-exposure, and antibody levels remained more or less constant until about 90 days after re-exposure. In conclusion, the results of this study indicate (a) that there is considerable homology between the surface antigens of Tetrczmicra brevifilum and Glugea caulleryi and (b) that inoculation of turbot with viable Glugea caulleryi spores does not cause any suppression of (and indeed stimulates) the subsequent humoral response to surface antigens of this parasite.
Acknowledgements
This study was supported by grants XUGA20303B95 and XUGA20308B95 (Xunta de Galicia, Spain). We would like to thank Insuitia (El Grove, Pontevedra) for the generous supply of turbot.
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