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Immune response of gilt-head seabream (Sparus aurata) to antigens from Photobacterium damselae subsp. piscicida
Fish & Shellfish Immunology Fish & Shellfish Immunology 16 (2004) 65–70 www.elsevier.com/locate/fsi
Short communication
Immune response of gilt-head seabream (Sparus aurata) to antigens from Photobacterium damselae subsp. piscicida Salvador Arijo, Carmen Balebona, Eduardo Martinez-Manzanares, Miguel Angel Morin˜igo* Department of Microbiology, Faculty of Sciences, University of Malaga, Malaga 29071, Spain Received 19 December 2002; received in revised form 3 March 2003; accepted 17 March 2003 Keywords: Gilthead seabream; Photobacterium damselae subsp. piscicida; Immunogenic preparations; Antibodies; Leucocyte proliferation
Photobacterium damselae subsp. piscicida (formerly Pasteurella piscicida) is the causative agent of pseudotuberculosis (also referred to as pasteurellosis), which is one of the most important diseases affecting the culture of gilt-head seabream (Sparus aurata) in the Mediterranean countries [1]. Several studies have reported the protection against Ph. damselae subsp. piscicida by different designs of vaccines in cultured fish [2–5]. The ability of fish vaccines to induce a protective immunity is for the most part based on experimental challenge studies and/or field experiments, but the laboratory research focused on determining which antigens are the most immunogenic in fish is very scarce [6], despite this objective it is critical to identify the individual components involved in inducing protection. This kind of data could be very useful in order to develop subunit vaccines. In this way, different studies showed that vaccines consisting of immunogenic fractions can induce higher protection than inactivated whole cell bacteria in fish [7]. Measures of the antibody response have been used to help describe the impact of physiological changes, environmental conditions and vaccination [8,9]. Antibodies in the circulation constitute an important part of the resistance against pathogenic microorganisms [10]. The objective of this study is to identify the immunodominant antigens of Ph. damselae subsp. piscicida by evaluating the antigenic specificity of the humoral response in gilt-head seabream vaccinated with a bacterin in oil adjuvant, and the potential mitogenic effect of different bacterial antigens on the leucocytes of this fish. Gilt-head seabream of 100 g body weight were maintained in 100 l tanks of seawater at 20 (C with aeration. Twenty of these fish were immunised by intraperitoneal injection (i.p.) with 106 formalin-killed cells of Ph. damselae subsp. piscicida Pp8 suspended in 0.1 ml of phosphate-buffered saline (PBS) and emulsified in Freund’s incomplete adjuvant (FIA). This strain, virulent for this fish (LD50 for gilt-head seabream was 1.8105 cfu g1), was isolated in our laboratory from diseased gilt-head seabream and was grown on Brain Heart Infusion Agar (OXOID) supplemented with 2% (w/v) NaCl (BHIAS) at 22 (C. * Corresponding author. E-mail address: [email protected] (M.A. Morin˜igo). 1050-4648/04/$ - see front matter 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S1050-4648(03)00032-9
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Control fish were inoculated with PBS in FIA. Four weeks after the immunisation a group of ten vaccinated fish received a booster injection (i.p.) with 106 formalin-killed cells of Ph. damselae subsp. piscicida Pp8 suspended in 0.1 ml of PBS and emulsified in FIA. Four once vaccinated, four twice vaccinated and six unvaccinated fish were anaesthetised with MS-222 and peripheral blood was sampled by caudal puncture four weeks after the first inoculation and after the booster injection. Sera from the specimens were obtained by allowing the blood to clot for 2 h at room temperature and centrifuged at 500g for 30 min at 4 (C. Sera were collected and stored as single samples at 20 (C until assayed (within one month). Erythrocyte-free leucocyte suspensions from blood of the unvaccinated fish were prepared by centrifugation at 500g for 30 min at 4 (C using Percoll (Pharmacia) density gradient 40%. The leucocytes were collected at the interface layer, washed three times and suspended in RPMI 1640 medium (Gibco) supplemented with 100 IU penicillin ml1, 100 µg streptomycin ml1, 10 IU heparin ml1 and 2% foetal calf serum (FCS). Leucocyte viability was determined by trypan blue exclusion test. The extracellular products (ECPs) of Ph. damselae subsp. piscicida were obtained using the cellophane technique described by Liu [11]. Briefly, a sterilised cellophane sheet was placed on the surface of a BHIAS plate and inoculated by spreading 0.5 ml of a 36–48 h old broth culture of the strain with a sterile swab. The plate were incubated at 22 (C for 48 h. After incubation bacterial cells were harvested off the cellophane with a minimum volume of PBS, and the cell suspension was centrifuged at 13,000g for 20 min at 4 (C. The supernatant were filtered through a 0.45 µm pore size membrane filter (Millipores) and used as crude ECPs. The total protein concentration was determined following the Bradford method [12], using bovine serum albumin (Sigma) as the standard. Outer (OM) and cytoplasmic membrane (CM) preparations were obtained following the techniques described by Al-Harbi and Austin [13]. Briefly, the cells were washed three times in PBS, and suspended in 10 mM Tris–HCl buffer (pH 8.0) containing 0.3% NaCl. The cells were disrupted by sonication (ten pulses of 30 s at 50 W). Cell debris was removed by centrifugation at 100,000g for 1 h at 4 (C. CM was solubilised selectively with sodium lauryl sarcosinate (Sigma) by the method of Filip et al. [14]. OM was sedimented by centrifugation at 100,000g for 1 h at 4 (C and the supernatant (containing CM) dialysed against distilled water for 24 h at 4 (C with 6–8 kDa molecular weight cut-off membrane (Spectrum) and stored at 20 (C. OM was washed three times in PBS and stored at 20 (C. Lipopolysaccharide (LPS) preparations were obtained following the methodology described by Chandan et al. [15]. Ten ml of the cell suspension (109 cfu ml1) in PBS were mixed with 0.1 ml of 0.5 M ethylediaminetetraacetate (EDTA) in PBS (pH 7.2). The mixture was autoclaved at 121 (C for 10 min, cooled to room temperature and centrifuged for 10 min at 10,000g. The supernatant was dialysed against distilled water using a Spectra/Por membrane (molecular weight cut-off 6–8 kDa) (Spectrum) and freeze-dried. The extract was suspended in 1 ml of H2O for chemical analyses. The concentrations of LPS were determined following the technique described by Keller and Nowotny [16] using Escherichia coli LPS as the standard. O-antigens (Ag-O) were obtained following the technique described by Edwards and Ewing [17]. Microbial cells were pelleted by centrifugation at 1600g for 25 min at 4 (C, suspended at 10% (v/v) in PBS, heat-killed by placing in a boiling water bath for 60 min and then twice centrifuged at 8000g for 10 min at 4 (C. The pellet was resuspended in ethanol at room temperature for 24 h. Then, the suspension was centrifuged at 3000g for 10 min and washed twice in acetone. The pellet was dried overnight at room temperature, and crushed. The antigen-O was stored at 4 (C until required for use. In the experiment, the antigen-O was resuspended in PBS at 1 mg ml1, and was maintained in a boiling water bath for 2 h with shaking. Extracellular material (EM) preparations were obtained following the technique described by Bonet et al. [18]. Cells of the microorganism were centrifuged at 6000g for 20 min, and the supernatants discarded. The cell pellets were suspended in 1 N KOH and extraction was carried out by stirring for 4 h at room
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Table 1 Serum antibody titres (medians of groups of vaccinated and unvaccinated fish) to antigenic preparations of Photobacterium damselae subsp. piscicida in gilt-head sea bream vaccinated with formalin-killed cells of Photobacterium damselae subsp. piscicida Antigenic preparations
4 weeks after vaccination 4 weeks after booster Controls
Bacterin
ECPs
OM
CM
EM
Ag-O
LPS
1:800 1:2400 <1:50
1:1200 1:2400 <1:50
1:2400 1:3200 1:300
1:1600 1:2400 1:50
1:300 1:100 <1:50
1:1600 1:2400 1:100
1:200 1:400 <1:50
ECPs: extracellular products; OM: outer membrane; CM: cytoplasmic membrane; EM: extracellular material; Ag-O: O-antigen; LPS: lipopolysaccharide
temperature. The cell residue was separated by centrifugation at 8000g for 30 min at 10 (C, and the clear KOH extract was precipitated by addition of three volumes ethanol. Then, the precipitate was diluted in distilled water and lyophilised and stored at 4 (C until use. Antibody titres in gilt-head seabream serum against whole cells, subcellular fractions and ECPs of Ph. damselae subsp. piscicida Pp8 were determined in gilt-head seabream by using an indirect enzyme-linked immunoadsorbent assay (ELISA). Wells of the microtitre plates (Costar E.I.A. plates) were coated with 50 µl of a suspension of 108 cfu ml1 of the bacterial strain or with a solution of antigenic preparations (20 µg of protein ml1), and incubated overnight at 4 (C. Then, the wells were washed three times with PBS supplemented with 0.05% of Tween 20 (PBS-T), blocked for 2 h at room temperature with 300 µl of PBS-T supplemented with 0.25% (w/v) of bovine serum albumin (PBS-T-BSA), and washed three times with PBS-T. Antigen-coated wells were incubated at room temperature for 2 h with 50 µl of serial dilutions of fish serum in PBS-T-BSA, washed three times with PBS-T, and incubated for 2 h at room temperature with 50 µl of rabbit anti gilt-head seabream immunoglobulin serum diluted in PBS-T-BSA (1:500). Finally, the wells were incubated for 1 h at room temperature with peroxidase-conjugated anti-rabbit immunoglobulin (Sigma) diluted in PBS-T-BSA (1:5000), washed three times with PBS-T, and then 50 µl of the developing solution (sodium acetate 0.1 M, pH 6 with 1% tetramethylbenzydine solution (Sigma) and hydrogen peroxide) were added and incubated in the dark at room temperature for 15 min. The reaction was stopped by adding 50 µl 1 M H2SO4. Absorbance values were determined using a microplate reader at 450 nm. Serum from non-immunised gilt-head seabream treated in the same way as the serum from vaccinated fish was used for negative controls. The reaction was considered positive in those wells with an optical density higher than that obtained for negative controls. Leucocytes were cultured in flat-bottomed microtitre plates with 5105 cells per well. Each well was added with 100 µl RPMI 1640 medium with antibiotics, heparin and 10% FCS. Antigens were used at the following concentrations: formalin-killed Ph. damselae subsp. piscicida (106 whole cells ml1), 10 µg ml1 of ECPs, EM, OM, 50 µg ml1 of Ag-O and CM, 100 µg ml1 of LPS. 2.5 µg ml1 of phytohaemagglutinin (PHA) and 12.5 µg ml1 of LPS of Escherichia coli (Sigma) were used as positive controls. All tests were carried out in triplicate. Cultures were incubated for 3 days at 20 (C. At 24 h prior to harvest, 0.5 µCi of [3H] thymidine (Amersham) was added to each well, the liquid was removed and the cells were washed four times with HBSS. The cells were harvested on to fibreglass paper and washed four times, and the incorporated radioactivity was measured in a liquid scintillation counter. The proliferative responses were expressed as counts per minute (cpm). Results were analysed by an ANOVA and Student’s t-test using a Statistical Software Statview for the Macintosh (SAS Institute Inc.). Differences were considered statistically significant at P<0.01. The serum antibody titres to antigenic preparations of Ph. damselae subsp. piscicida are summarised in Table 1. In this study, it has been demonstrated that the vaccination of gilt-head seabream with a single injection of formalin-killed Ph. damselae subsp. piscicida produced a humoral immune response after four
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weeks against different antigenic preparations of this microorganism. Four weeks after vaccination the fish assayed showed significant increases (P<0.01) of the serum antibody titres for all antigenic preparations assayed with respect to those obtained for unvaccinated fish, the highest titres of antibodies being those obtained against OM, CM, Ag-O and ECPs. This result is in concordance with the data recorded by Santarem and Figueras [19], who reported a peak antibody titre in turbot (Scophthalmus maximus) 28 days after immunisation with O-antigen from Ph. damselae subsp. piscicida. In this study, the advantage of a booster vaccination has been demonstrated to achieve high levels of antibodies against the different antigenic preparations. These results are in agreement with those data reported by Magarin˜os et al. [4], who recommended an initial gilt-head seabream vaccination for Ph. damselae subsp. piscicida and then a booster vaccination. In the group of fish that received a booster vaccination four weeks after the first immunisation, significant increases (P<0.01) of the serum antibody titres to bacterin, ECPs, OM and LPS were observed with respect to the titres detected with only one vaccination. These significant increases were detected three and four weeks after the booster. In contrast, the serum antibody titres for Ag-O, EM and CM were not higher than those obtained in the first immunisation. The booster vaccination improved significantly (P<0.05) the titres of antibodies against LPS, but not those detected against EM. This increase of the titre of antibodies against LPS could be a factor to consider in a future vaccine because there are data that suggest the addition of LPS to a chloroform-killed vaccine against Ph. damselae subsp. piscicida stimulated the production of serum antibodies and improved the effectiveness of the vaccine [5]. Previous studies have indicated that the extracellular products (ECPs) and the capsule of Ph. damselae subsp. piscicida play an important role in the virulence of this microorganism [20,21]. It is interesting to remark that it has been possible to detect high levels of circulating antibody against ECPs four weeks after the immunisation, when fish were inoculated only with washed formalin-killed cells of the pathogenic microorganism. This result is in concordance with the results observed by Boesen et al. [6], who also reported high levels of circulating serum antibody against ECPs of Vibrio anguillarum after inoculation of rainbow trout specimens with formalin-killed cells of this bacterial species. These antibodies could be synthesised against some of the ECPs that were associated with the bacterial surface, in this way, Magarin˜os et al. [4] demonstrated a similar pattern of enzymatic and hydrolytic activities detected in the ECPs and the whole cells of Ph. damselae subsp. piscicida. However, Magarin˜os et al. [3] carried out vaccination trials on gilt-head seabream against Ph. damselae subsp. piscicida using two vaccine formulations, a whole-cell bacterin and a toxoid enriched whole-cell vaccine, and they reported that only the toxoid-enriched vaccine conferred protection against Ph. damselae subsp. piscicida, perhaps because the most virulent products are in higher concentration in the extracellular products and they are not associated with the bacterial cell. There is no agreement on the correlation between the level of serum antibodies and protection against pasteurellosis. Several studies have reported that serum antibodies play an important role in this protection and low levels of agglutinating antibodies have been associated with prolonged epizootics of pasteurellosis in yellowtail [22]. On the contrary, Magarin˜os et al. [3] found no correlation between the level of agglutinating antibodies and protection in vaccinated seabream. However, high levels of antibodies against OM have been correlated with protection against several fish pathogens such as Cytophaga-like [13] and Flavobacterium psychrophilum [23]. The titres obtained with soluble and particulated (bacterin) antigens after the booster are similar, and these results are not in agreement with other data that reported that the immune response is more pronounced against soluble than particulated antigens [13]. Leucocytes from unvaccinated fish were stimulated in vitro with the antigenic preparations, and the proliferative responses were compared with that of leucocytes not stimulated with the same preparations (Fig. 1). A mitogenic effect of the Ph. damselae subsp. piscicida antigens has been demonstrated for LPS, OM and Ag-O, whereas the other antigenic preparations induced low stimulation, although these differences were not-significant statistically (P>0.01) due to variations among individual fish. These results are in agreement with those obtained by Boesen et al. [6], who reported in the case of the immune response of rainbow trout to Vibrio anguillarum serogroup O1, proliferative responses for preparations of the
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Fig. 1. Proliferative responses of leucocytes of unvaccinated gilt-head seabream specimens elicited by different antigenic preparations obtained from Photobacterium damselae subsp. piscicida.
bacterial cell envelope, but low stimulation for ECPs. In contrast, these authors observed an elevated proliferative response for formalin-killed bacteria, whereas in this study a low stimulation has been reported, although in the Boesen’s study the differences observed in the proliferative responses elicited by different antigenic preparations were also statistically non-significant. In conclusion, the results of this study suggest that the OM preparation was one of the most immunodominant antigens assayed which produced an immunological response, and this information could be useful to design prophylactic strategies. The ability of the outer membrane fraction (OM) to produce a high immunological response is consistent with other reports in rainbow trout, goldfish and ayu vaccinated against Aeromonas salmonicida and F. psychrophilum [23,24].
Acknowledgements This work was funded by grants of the Comisio´n Interministerial de Ciencia y Tecnologı´a (C.I.C.Y.T.) of the Spanish Government (P.E.T.R.I. 95-0181-OP). The authors thank Dr M.D. Castro Lo´pez of the Department of Microbiology of the University of Malaga for providing the rabbit gilt-head seabream anti-immunoglobulin serum.
References [1] Magarin˜os B, Toranzo AE, Romalde JL. Phenotypic and pathobiological characteristics of Pasteurella piscicida. Annu Rev Fish Dis 1996;6:41–64. [2] Kusuda R, Hamaguchi M. The efficacy of attenuated live bacterin of Pasteurella piscicida against pseudotuberculosis in yellowtail. Bull Eur Ass Fish Pathol 1989;8:51–3.
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[3] Magarin˜os B, Romalde JL, Santos Y, Casal JF, Barja JL, Toranzo AE. Vaccination trials on gilt-head sea bream (Sparus aurata) against Pasteurella piscicida. Aquaculture 1994;120:201–8. [4] Magarin˜os B, Romalde JL, Barja JL, Nu´n˜ez S, Toranzo AE. Protection of gilthead bream against pasteurellosis at the larval stages. Bull Eur Ass Fish Pathol 1999;19:159–61. [5] Kawakami H, Shinohara N, Fukuda Y, Yamashita H, Kihara H, Sakai M. The efficacy of lipopolysaccharide mixed chloroform-killed cell (LPS-ckc) bacterin of Pasteurella piscicida of yellowtail, Seriola quinqueradiata. Aquaculture 1997; 154:95–105. [6] Boesen HT, Pedersen K, Koch C, Larsen JL. Immune response of rainbow trout (Oncorhynchus mykiss) to antigenic preparations from Vibrio anguillarum serogroup O1. Fish Shellfish Immunol 1997;7:543–53. [7] Gudmundsdottir BK, Magnadottir B. Protection of atlantic salmon (Salmo salar L.) against an experimental infection of Aeromonas salmonicida subsp. achromogenes. Fish Shellfish Immunol 1997;7:55–69. [8] Traxler GS, Roome JR, Landa KA, LaPatra S. Appearance of infections hematopoietic necrosis virus (IHNV) and neutralizing antibodies in sockeye salmon Oncorhynchus nerka during their migration and maturation period. Dis Aquat Organ 1997;28:31–8. [9] Palm RC Jr, Landolt ML, Busch RA. Route of vaccine administration: effects on the specific humoral response in rainbow trout Oncorhynchus mykiss. Dis Aquat Organ 1998;33:157–66. [10] Aaltonen TM, Jokinen EI, Valtonen ET. Antibody synthesis in roach (Rutilus rutilus); analysis of antibody secreting cells in lymphoid organs with ELISPOT-assay. Fish Shellfish Immunol 1994;4:129–40. [11] Liu PV. Survey of hemolysin production among species of Pseudomonas. J Bacteriol 1957;74:718–27. [12] Bradford NM. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Annu Rev Biochem 1976;72:248–54. [13] Al-Harbi AH, Austin B. The immune response of turbot (Scopthalmus maximus L.) to formalized and heat inactivated cells, and subcellular components of a fish pathogenic Cytophaga-like bacterium (CLB). Bull Eur Ass Fish Pathol 1993;13:21–4. [14] Filip C, Fletcher G, Wulff JL, Earhart CF. Solubilization of the cytoplasmic membrane of Escherichia coli by the ionic detergent sodium lauryl sarcosinate. J Bacteriol 1973;115:717–22. [15] Chandan V, Fraser ADE, Brooks BW, Yamazaki H. Simple extraction of Campylobacter lipopolysacharide and protein antigens and production of their antibodies in egg yolk. Int J Food Microbiol 1994;22:189–200. [16] Keller TK, Nowotny A. Metachromatic assay for the quantitative determination of bacterial endotoxins. Anal Biochem 1985; 156:189–93. [17] Edwards PR, Ewing WH. Identification of Enterobacteriaceae. . Minneapolis: Burgess Publishing Company, 1972. [18] Bonet R, Magarin˜os B, Romalde JL, Simon-Pujol MD, Toranzo AE, Congregado F. Capsular polysaccharide expressed by Pasteurella piscicida grown in vitro. FEMS Microbiol Lett 1994;124:285–90. [19] Santarem MM, Figueras A. Kinetics of phagocytic activity, plaque-forming cells and specific agglutinins of turbot (Scophthalmus maximus L.) immunised with O antigen of Vibrio damsela and Pasteurella piscicida. Fish Shellfish Immunol 1994;4:527–37. [20] Magarin˜os B, Santos Y, Romalde JL, Rivas C, Barja JL, Toranzo AE. Pathogenic activities of live cells and extracellular products of the fish pathogen Pasteurella piscicida. J Gen Microbiol 1992;138:2491–8. [21] Arijo S, Borrego JJ, Zorrilla I, Balebona MC, Morin˜igo MA. Role of the capsule of Photobacterium damsela subsp. piscicida in protection against phagocytosis and killing by gilt-head seabream (Sparus aurata, L.) macrophages. Fish Shellfish Immunol 1998; 8:63–72. [22] Satoh K, Fukuda Y, Nakaro S. Changes in agglutinin titer against Pasteurella pisicicida in cultured yellowtail during the epizootics of pseudotuberculosis in 1993 and 1994. Fish Pathol 1995;30:291–2. [23] Rahman MH, Kuroda A, Dijistra JM, Kiryu I, Nakanishi T, Ototake M. The outer membrane fraction of Flavobacterium psychrophilum induces protective immunity in rainbow trout and ayu. Fish Shellfish Immunol 2002;12:169–79. [24] Rahman MH, Kawai K. Outer membrane protein of Aeromonas hydrophila induce protective immunity in goldfish. Fish Shellfish Immunol 2000;10:379–82.
Short communication
Immune response of gilt-head seabream (Sparus aurata) to antigens from Photobacterium damselae subsp. piscicida Salvador Arijo, Carmen Balebona, Eduardo Martinez-Manzanares, Miguel Angel Morin˜igo* Department of Microbiology, Faculty of Sciences, University of Malaga, Malaga 29071, Spain Received 19 December 2002; received in revised form 3 March 2003; accepted 17 March 2003 Keywords: Gilthead seabream; Photobacterium damselae subsp. piscicida; Immunogenic preparations; Antibodies; Leucocyte proliferation
Photobacterium damselae subsp. piscicida (formerly Pasteurella piscicida) is the causative agent of pseudotuberculosis (also referred to as pasteurellosis), which is one of the most important diseases affecting the culture of gilt-head seabream (Sparus aurata) in the Mediterranean countries [1]. Several studies have reported the protection against Ph. damselae subsp. piscicida by different designs of vaccines in cultured fish [2–5]. The ability of fish vaccines to induce a protective immunity is for the most part based on experimental challenge studies and/or field experiments, but the laboratory research focused on determining which antigens are the most immunogenic in fish is very scarce [6], despite this objective it is critical to identify the individual components involved in inducing protection. This kind of data could be very useful in order to develop subunit vaccines. In this way, different studies showed that vaccines consisting of immunogenic fractions can induce higher protection than inactivated whole cell bacteria in fish [7]. Measures of the antibody response have been used to help describe the impact of physiological changes, environmental conditions and vaccination [8,9]. Antibodies in the circulation constitute an important part of the resistance against pathogenic microorganisms [10]. The objective of this study is to identify the immunodominant antigens of Ph. damselae subsp. piscicida by evaluating the antigenic specificity of the humoral response in gilt-head seabream vaccinated with a bacterin in oil adjuvant, and the potential mitogenic effect of different bacterial antigens on the leucocytes of this fish. Gilt-head seabream of 100 g body weight were maintained in 100 l tanks of seawater at 20 (C with aeration. Twenty of these fish were immunised by intraperitoneal injection (i.p.) with 106 formalin-killed cells of Ph. damselae subsp. piscicida Pp8 suspended in 0.1 ml of phosphate-buffered saline (PBS) and emulsified in Freund’s incomplete adjuvant (FIA). This strain, virulent for this fish (LD50 for gilt-head seabream was 1.8105 cfu g1), was isolated in our laboratory from diseased gilt-head seabream and was grown on Brain Heart Infusion Agar (OXOID) supplemented with 2% (w/v) NaCl (BHIAS) at 22 (C. * Corresponding author. E-mail address: [email protected] (M.A. Morin˜igo). 1050-4648/04/$ - see front matter 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S1050-4648(03)00032-9
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Control fish were inoculated with PBS in FIA. Four weeks after the immunisation a group of ten vaccinated fish received a booster injection (i.p.) with 106 formalin-killed cells of Ph. damselae subsp. piscicida Pp8 suspended in 0.1 ml of PBS and emulsified in FIA. Four once vaccinated, four twice vaccinated and six unvaccinated fish were anaesthetised with MS-222 and peripheral blood was sampled by caudal puncture four weeks after the first inoculation and after the booster injection. Sera from the specimens were obtained by allowing the blood to clot for 2 h at room temperature and centrifuged at 500g for 30 min at 4 (C. Sera were collected and stored as single samples at 20 (C until assayed (within one month). Erythrocyte-free leucocyte suspensions from blood of the unvaccinated fish were prepared by centrifugation at 500g for 30 min at 4 (C using Percoll (Pharmacia) density gradient 40%. The leucocytes were collected at the interface layer, washed three times and suspended in RPMI 1640 medium (Gibco) supplemented with 100 IU penicillin ml1, 100 µg streptomycin ml1, 10 IU heparin ml1 and 2% foetal calf serum (FCS). Leucocyte viability was determined by trypan blue exclusion test. The extracellular products (ECPs) of Ph. damselae subsp. piscicida were obtained using the cellophane technique described by Liu [11]. Briefly, a sterilised cellophane sheet was placed on the surface of a BHIAS plate and inoculated by spreading 0.5 ml of a 36–48 h old broth culture of the strain with a sterile swab. The plate were incubated at 22 (C for 48 h. After incubation bacterial cells were harvested off the cellophane with a minimum volume of PBS, and the cell suspension was centrifuged at 13,000g for 20 min at 4 (C. The supernatant were filtered through a 0.45 µm pore size membrane filter (Millipores) and used as crude ECPs. The total protein concentration was determined following the Bradford method [12], using bovine serum albumin (Sigma) as the standard. Outer (OM) and cytoplasmic membrane (CM) preparations were obtained following the techniques described by Al-Harbi and Austin [13]. Briefly, the cells were washed three times in PBS, and suspended in 10 mM Tris–HCl buffer (pH 8.0) containing 0.3% NaCl. The cells were disrupted by sonication (ten pulses of 30 s at 50 W). Cell debris was removed by centrifugation at 100,000g for 1 h at 4 (C. CM was solubilised selectively with sodium lauryl sarcosinate (Sigma) by the method of Filip et al. [14]. OM was sedimented by centrifugation at 100,000g for 1 h at 4 (C and the supernatant (containing CM) dialysed against distilled water for 24 h at 4 (C with 6–8 kDa molecular weight cut-off membrane (Spectrum) and stored at 20 (C. OM was washed three times in PBS and stored at 20 (C. Lipopolysaccharide (LPS) preparations were obtained following the methodology described by Chandan et al. [15]. Ten ml of the cell suspension (109 cfu ml1) in PBS were mixed with 0.1 ml of 0.5 M ethylediaminetetraacetate (EDTA) in PBS (pH 7.2). The mixture was autoclaved at 121 (C for 10 min, cooled to room temperature and centrifuged for 10 min at 10,000g. The supernatant was dialysed against distilled water using a Spectra/Por membrane (molecular weight cut-off 6–8 kDa) (Spectrum) and freeze-dried. The extract was suspended in 1 ml of H2O for chemical analyses. The concentrations of LPS were determined following the technique described by Keller and Nowotny [16] using Escherichia coli LPS as the standard. O-antigens (Ag-O) were obtained following the technique described by Edwards and Ewing [17]. Microbial cells were pelleted by centrifugation at 1600g for 25 min at 4 (C, suspended at 10% (v/v) in PBS, heat-killed by placing in a boiling water bath for 60 min and then twice centrifuged at 8000g for 10 min at 4 (C. The pellet was resuspended in ethanol at room temperature for 24 h. Then, the suspension was centrifuged at 3000g for 10 min and washed twice in acetone. The pellet was dried overnight at room temperature, and crushed. The antigen-O was stored at 4 (C until required for use. In the experiment, the antigen-O was resuspended in PBS at 1 mg ml1, and was maintained in a boiling water bath for 2 h with shaking. Extracellular material (EM) preparations were obtained following the technique described by Bonet et al. [18]. Cells of the microorganism were centrifuged at 6000g for 20 min, and the supernatants discarded. The cell pellets were suspended in 1 N KOH and extraction was carried out by stirring for 4 h at room
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Table 1 Serum antibody titres (medians of groups of vaccinated and unvaccinated fish) to antigenic preparations of Photobacterium damselae subsp. piscicida in gilt-head sea bream vaccinated with formalin-killed cells of Photobacterium damselae subsp. piscicida Antigenic preparations
4 weeks after vaccination 4 weeks after booster Controls
Bacterin
ECPs
OM
CM
EM
Ag-O
LPS
1:800 1:2400 <1:50
1:1200 1:2400 <1:50
1:2400 1:3200 1:300
1:1600 1:2400 1:50
1:300 1:100 <1:50
1:1600 1:2400 1:100
1:200 1:400 <1:50
ECPs: extracellular products; OM: outer membrane; CM: cytoplasmic membrane; EM: extracellular material; Ag-O: O-antigen; LPS: lipopolysaccharide
temperature. The cell residue was separated by centrifugation at 8000g for 30 min at 10 (C, and the clear KOH extract was precipitated by addition of three volumes ethanol. Then, the precipitate was diluted in distilled water and lyophilised and stored at 4 (C until use. Antibody titres in gilt-head seabream serum against whole cells, subcellular fractions and ECPs of Ph. damselae subsp. piscicida Pp8 were determined in gilt-head seabream by using an indirect enzyme-linked immunoadsorbent assay (ELISA). Wells of the microtitre plates (Costar E.I.A. plates) were coated with 50 µl of a suspension of 108 cfu ml1 of the bacterial strain or with a solution of antigenic preparations (20 µg of protein ml1), and incubated overnight at 4 (C. Then, the wells were washed three times with PBS supplemented with 0.05% of Tween 20 (PBS-T), blocked for 2 h at room temperature with 300 µl of PBS-T supplemented with 0.25% (w/v) of bovine serum albumin (PBS-T-BSA), and washed three times with PBS-T. Antigen-coated wells were incubated at room temperature for 2 h with 50 µl of serial dilutions of fish serum in PBS-T-BSA, washed three times with PBS-T, and incubated for 2 h at room temperature with 50 µl of rabbit anti gilt-head seabream immunoglobulin serum diluted in PBS-T-BSA (1:500). Finally, the wells were incubated for 1 h at room temperature with peroxidase-conjugated anti-rabbit immunoglobulin (Sigma) diluted in PBS-T-BSA (1:5000), washed three times with PBS-T, and then 50 µl of the developing solution (sodium acetate 0.1 M, pH 6 with 1% tetramethylbenzydine solution (Sigma) and hydrogen peroxide) were added and incubated in the dark at room temperature for 15 min. The reaction was stopped by adding 50 µl 1 M H2SO4. Absorbance values were determined using a microplate reader at 450 nm. Serum from non-immunised gilt-head seabream treated in the same way as the serum from vaccinated fish was used for negative controls. The reaction was considered positive in those wells with an optical density higher than that obtained for negative controls. Leucocytes were cultured in flat-bottomed microtitre plates with 5105 cells per well. Each well was added with 100 µl RPMI 1640 medium with antibiotics, heparin and 10% FCS. Antigens were used at the following concentrations: formalin-killed Ph. damselae subsp. piscicida (106 whole cells ml1), 10 µg ml1 of ECPs, EM, OM, 50 µg ml1 of Ag-O and CM, 100 µg ml1 of LPS. 2.5 µg ml1 of phytohaemagglutinin (PHA) and 12.5 µg ml1 of LPS of Escherichia coli (Sigma) were used as positive controls. All tests were carried out in triplicate. Cultures were incubated for 3 days at 20 (C. At 24 h prior to harvest, 0.5 µCi of [3H] thymidine (Amersham) was added to each well, the liquid was removed and the cells were washed four times with HBSS. The cells were harvested on to fibreglass paper and washed four times, and the incorporated radioactivity was measured in a liquid scintillation counter. The proliferative responses were expressed as counts per minute (cpm). Results were analysed by an ANOVA and Student’s t-test using a Statistical Software Statview for the Macintosh (SAS Institute Inc.). Differences were considered statistically significant at P<0.01. The serum antibody titres to antigenic preparations of Ph. damselae subsp. piscicida are summarised in Table 1. In this study, it has been demonstrated that the vaccination of gilt-head seabream with a single injection of formalin-killed Ph. damselae subsp. piscicida produced a humoral immune response after four
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weeks against different antigenic preparations of this microorganism. Four weeks after vaccination the fish assayed showed significant increases (P<0.01) of the serum antibody titres for all antigenic preparations assayed with respect to those obtained for unvaccinated fish, the highest titres of antibodies being those obtained against OM, CM, Ag-O and ECPs. This result is in concordance with the data recorded by Santarem and Figueras [19], who reported a peak antibody titre in turbot (Scophthalmus maximus) 28 days after immunisation with O-antigen from Ph. damselae subsp. piscicida. In this study, the advantage of a booster vaccination has been demonstrated to achieve high levels of antibodies against the different antigenic preparations. These results are in agreement with those data reported by Magarin˜os et al. [4], who recommended an initial gilt-head seabream vaccination for Ph. damselae subsp. piscicida and then a booster vaccination. In the group of fish that received a booster vaccination four weeks after the first immunisation, significant increases (P<0.01) of the serum antibody titres to bacterin, ECPs, OM and LPS were observed with respect to the titres detected with only one vaccination. These significant increases were detected three and four weeks after the booster. In contrast, the serum antibody titres for Ag-O, EM and CM were not higher than those obtained in the first immunisation. The booster vaccination improved significantly (P<0.05) the titres of antibodies against LPS, but not those detected against EM. This increase of the titre of antibodies against LPS could be a factor to consider in a future vaccine because there are data that suggest the addition of LPS to a chloroform-killed vaccine against Ph. damselae subsp. piscicida stimulated the production of serum antibodies and improved the effectiveness of the vaccine [5]. Previous studies have indicated that the extracellular products (ECPs) and the capsule of Ph. damselae subsp. piscicida play an important role in the virulence of this microorganism [20,21]. It is interesting to remark that it has been possible to detect high levels of circulating antibody against ECPs four weeks after the immunisation, when fish were inoculated only with washed formalin-killed cells of the pathogenic microorganism. This result is in concordance with the results observed by Boesen et al. [6], who also reported high levels of circulating serum antibody against ECPs of Vibrio anguillarum after inoculation of rainbow trout specimens with formalin-killed cells of this bacterial species. These antibodies could be synthesised against some of the ECPs that were associated with the bacterial surface, in this way, Magarin˜os et al. [4] demonstrated a similar pattern of enzymatic and hydrolytic activities detected in the ECPs and the whole cells of Ph. damselae subsp. piscicida. However, Magarin˜os et al. [3] carried out vaccination trials on gilt-head seabream against Ph. damselae subsp. piscicida using two vaccine formulations, a whole-cell bacterin and a toxoid enriched whole-cell vaccine, and they reported that only the toxoid-enriched vaccine conferred protection against Ph. damselae subsp. piscicida, perhaps because the most virulent products are in higher concentration in the extracellular products and they are not associated with the bacterial cell. There is no agreement on the correlation between the level of serum antibodies and protection against pasteurellosis. Several studies have reported that serum antibodies play an important role in this protection and low levels of agglutinating antibodies have been associated with prolonged epizootics of pasteurellosis in yellowtail [22]. On the contrary, Magarin˜os et al. [3] found no correlation between the level of agglutinating antibodies and protection in vaccinated seabream. However, high levels of antibodies against OM have been correlated with protection against several fish pathogens such as Cytophaga-like [13] and Flavobacterium psychrophilum [23]. The titres obtained with soluble and particulated (bacterin) antigens after the booster are similar, and these results are not in agreement with other data that reported that the immune response is more pronounced against soluble than particulated antigens [13]. Leucocytes from unvaccinated fish were stimulated in vitro with the antigenic preparations, and the proliferative responses were compared with that of leucocytes not stimulated with the same preparations (Fig. 1). A mitogenic effect of the Ph. damselae subsp. piscicida antigens has been demonstrated for LPS, OM and Ag-O, whereas the other antigenic preparations induced low stimulation, although these differences were not-significant statistically (P>0.01) due to variations among individual fish. These results are in agreement with those obtained by Boesen et al. [6], who reported in the case of the immune response of rainbow trout to Vibrio anguillarum serogroup O1, proliferative responses for preparations of the
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Fig. 1. Proliferative responses of leucocytes of unvaccinated gilt-head seabream specimens elicited by different antigenic preparations obtained from Photobacterium damselae subsp. piscicida.
bacterial cell envelope, but low stimulation for ECPs. In contrast, these authors observed an elevated proliferative response for formalin-killed bacteria, whereas in this study a low stimulation has been reported, although in the Boesen’s study the differences observed in the proliferative responses elicited by different antigenic preparations were also statistically non-significant. In conclusion, the results of this study suggest that the OM preparation was one of the most immunodominant antigens assayed which produced an immunological response, and this information could be useful to design prophylactic strategies. The ability of the outer membrane fraction (OM) to produce a high immunological response is consistent with other reports in rainbow trout, goldfish and ayu vaccinated against Aeromonas salmonicida and F. psychrophilum [23,24].
Acknowledgements This work was funded by grants of the Comisio´n Interministerial de Ciencia y Tecnologı´a (C.I.C.Y.T.) of the Spanish Government (P.E.T.R.I. 95-0181-OP). The authors thank Dr M.D. Castro Lo´pez of the Department of Microbiology of the University of Malaga for providing the rabbit gilt-head seabream anti-immunoglobulin serum.
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