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The effect of growth medium salinity of Photobacterium damselae subsp. piscicida on the immune response of hybrid bass (Morone saxatilis×M. chrysops)
Fish & Shellfish Immunology Fish & Shellfish Immunology 16 (2004) 107–116 www.elsevier.com/locate/fsi
The effect of growth medium salinity of Photobacterium damselae subsp. piscicida on the immune response of hybrid bass (Morone saxatilisM. chrysops) S. Nitzan 1*, B. Shwartsburd 2, E.D. Heller 2 2
1 Central Fish Health Laboratory, Department of Fisheries and Agriculture and Rural Development, Nir-David 19150, Israel Department of Animal Sciences, Faculty of Agricultural, Food and Environmental Quality Sciences, The Hebrew University of Jerusalem, Rehovot 76100, Israel
Received 27 August 2002; received in revised form 10 March 2003; accepted 7 April 2003
Abstract Photobacterium damselae subsp. piscicida (P. damselae) was grown on various media and the effect of media salinity on certain immune responses of hybrid bass was studied. In Israel, pasteurellosis outbreaks have not been reported at water salinities below 1.38‰. During vaccination experiments the salinity of the medium on which P. damselae is grown, was shown to affect stimulation of the immune system. No correlation was found between antibody response and protection. Bacterial envelopes separated by electrophoresis and subjected to western blot analysis revealed an antibody response against some protein bands. Band sequencing was performed to identify the protein stimulating the immune response. Sequence identity of 80% was seen in 10-amino-acid overlap of the 36-kDa band with a specific gene of alkalophilic Bacillus firmus. A preparation of P. damselae grown in a 2.5% NaCl medium at 25 (C is the most effective vaccine against pasteurellosis, providing hybrid bass with quite good protection. 2003 Elsevier Ltd. All rights reserved. Keywords: Hybrid bass; Pasteurellosis; Bacterial envelope preparation; ELISA; Salinity; Vaccination
1. Introduction Hybrid bass was introduced into Israel in 1993. In the spring of 1994, when the water temperature in the fish ponds rose to 22–24 (C, an outbreak of pasteurellosis occurred. The causative agent was identified as Photobacterium damselae subsp. piscicida (P. damselae) [1], formerly Pasteurella piscicida [2]. Since the first outbreak of pasteurellosis was reported in the US [3], it has appeared in different countries and in a variety of fish species. Pasteurellosis outbreaks have occurred in fish species that are economically important for mariculture and brackish-water fish farms [4–11]. Many factors influence the expression/production of * Corresponding author E-mail address: [email protected] (S. Nitzan). 1050-4648/04/$ - see front matter 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S1050-4648(03)00045-7
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various virulence factors, as with temperature [1]. Pasteurellosis outbreaks occur at 24 (C and no mortality occurs at 15–16 (C or 30 (C. Additional factors that influence virulence of P. damselae are preinoculation with hemin and hemoglobin which increase virulence, the effect being stronger in avirulent strains [12]. Another factor that influences virulence is the capsular layer [13]. Barnes et al. [14] characterised additional important virulence factors, enzymes which neutralise reactive oxygen species produced in fish macrophages (SOD) and a cytoplasmic catalase in P. damselae. Since hybrid bass can be raised in either fresh, brackish or salt water, and pasteurellosis has never been seen in freshwater farms, the aim of this study was to determine the effect of salinity of P. damselae growth medium on its ability to stimulate the immune system and protect hybrid bass from the disease. 2. Materials and methods 2.1. Bacteria P. damselae was isolated from the spleen of moribund hybrid bass grown on a farm in the coastal Mediterranean region of Israel in 1995. This isolate, 2207, was cultured on brain heart infusion (BHI) agar (Oxoid, Hampshire, UK) supplemented with 0.1% (w/v) yeast extract (Difco, Detroit, MI, USA) and 2% (w/v) NaCl (1.5% NaCl was added to the original content of 0.5%), or the different salinities detailed below, and incubated at 25 (C, unless otherwise indicated. Some colonies of P. damselae were transferred and incubated for 6 to 8 h in BHI medium. Bacteria were grown for 18 to 20 h during the exponential phase, and the required amount adjusted to an optical density (OD) of approximately 1.00 at 540 nm (Spectronic 1201, Milton Roy, Rochester, NY, USA); at this OD, 2.5109 bacteria ml1 were found. The required bacterial concentration was diluted accordingly in phosphate-buffered saline (PBS) pH 7.4. Formalin-killed bacterial antigens (bacterins) were prepared according to Stolen et al. [15], and adjusted to an OD of 1.00. When adjuvant (incomplete Freund’s adjuvant; IFA; Sigma, St. Louis, MO, USA) was used, the bacterial antigen preparation or envelope preparation was mixed at equal volumes with the IFA. Microcapsules were produced from the polysaccharide alginate (2% w/v; Keltone, LV, San Diego, CA, USA) and polarisation was performed with 0.5% (w/v) CaCl2 (Sigma) in water. Bacterial number for capsules was the same as for the bacterin, grown in BHI medium (0.5% NaCl). 2.2. Bacterial sonicate and enzyme-linked immunosorbent assay (ELISA) Sonicate was prepared from 1.5 l of pelleted bacterial suspension and the pellet was washed and diluted 1:10 (v/v) with sonication buffer (50 mMol Tris buffer; pH 7.4). The suspension was sonicated for 15 min (550 W, 20 kHz) until a clear suspension was obtained, using an ultrasonic sonicator (Misonix XL 2020, Farmingdale, NY, USA). The obtained suspension was stored at 73 (C. For verification of complete disruption of cells, one drop of sonicate suspension was smeared on the BHI agar. Sonicate protein concentration was determined by means of the Lowry assay (Bio-Rad DC protein assay, Hercules, CA, USA). The immune responses were evaluated by ELISA [16] adapted to our requirements. Ninety-six-well immunoplates (Kartell, Milano, Italy) were coated with 2 µg sonicate protein per well, diluted in coating buffer. Bovine serum albumin (200 µl/well; BSA diluent/blocking solution, KPL, Gaithersburg, MD, USA) was added as a blocking solution. Fish sera were diluted in BSA diluent solution. Two New Zealand albino rabbits were immunised with bass immunoglobulin [15] purified from sera by means of ammonium sulphate precipitation. A mixture of equal volume bass serum and PBS was prepared, and an equal volume of saturated ammonium sulphate (pH 7.0) was added to the mixture. Centrifugation was performed twice by discarding the supernatant and suspending the deposit in half-saturated ammonium sulphate, and the pellet was dialysed against a saline solution three times. The rabbits were intramuscularly (IM) injected with 1 mg of fish immunoglobulins five times. In the first injection the immunoglobulins were emulsified in complete
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Freund’s adjuvant (CFA) and subsequently in IFA. Satisfactory antibody titre was confirmed by means of the Ouchterlony serological test. The substrate reagent was azinobis (ethylbenzthiazoline-6-sulphonic acid) (ABTS; KPL, Gaithersburg, MD, USA). The plates were read in a microplate autoreader (Tecan Spectra, Austria) at 405 nm. 2.3. Envelope preparation for gel electrophoresis P. damselae was grown in BHI with 0.5, 2.5 or 3.5% NaCl, and harvested by centrifugation (4000 g for 10 min at 4 (C). The sonicate pellet was resuspended in PBS, washed three times and resuspended 1:10 in sonication buffer. After centrifugation of the sonicate at 4000 g for 15 min, the supernatant was centrifuged by ultra-centrifugation (Centricon T-1170, Kontron Instruments, Zu¨rich, Switzerland) at 100,000 g for 1 h at 4 (C. The pellet was resuspended in 10 ml of sonication buffer and centrifuged again under the same conditions. The pellet was resuspended and incubated at 37 (C for 2 h with shaking, in a minimal amount of solution made up of sonication buffer, 2% (w/v) SDS, 0.5 M NaCl and 5 mM EDTA. Protein concentration was determined by means of the Lowry assay. The envelope preparation of P. damselae for vaccination was obtained as for gel electrophoresis. Bacterial growth conditions were at the different salinities and temperatures detailed below. Electrophoresis and western blot analysis were performed using 10% SDS–PAGE according to Laemmei [17] and Towbin et al. [18]. 2.4. Fish Hybrid bass from a farm with a water salinity of 0.66‰ (weight range 60–100 g), diagnosed as free of parasites and bacteria, were transferred to the laboratory. Immunisation experiments were performed in 100-l containers with water at 25 (C and a salinity of 3.5‰ in a recirculation system. 2.5. Vaccination Fish vaccination was performed by injecting 0.5 ml of the antigenic solution (bacterin or envelope preparation) into the upper peritoneal cavity with a 22G needle. The antigen was mixed at equal volumes with the adjuvant (IFA). A booster vaccination was performed 21 days after the first vaccination (except for the first experiment), using the same method. Sera for antibody titre determinations were obtained aseptically 10 days post-boost (or 21 days post-vaccination as in the first experiment) by dorsal aorta puncture, allowed to clot at 4 (C, separated by centrifugation at 4000 g for 5 min and stored at 4 (C with 0.02% (w/v) sodium azide for a few days until antibody evaluation. 2.6. First experiment Antibody titre comparison between fish vaccinated with bacterin and microcapsules. One group of fish was vaccinated with bacterin and a second one with microcapsules, as detailed below. The bacterial vaccine dose was 5105 bacteria per g fish. Fish were divided into four groups: group I (four fish), the control, was vaccinated with adjuvant only; group II (five fish) was vaccinated with microcapsules (bacteria grown in BHI—0.5% NaCl); group III (twelve fish) was vaccinated with bacterin (bacteria grown in BHI—0.5% NaCl) emulsified in IFA, and group IV (nine fish) with bacterin (bacteria grown in BHI—2.5% NaCl) emulsified in IFA. Sera for antibody titre determination were collected 21 days post-vaccination. 2.7. Second experiment Antibody titre and protection of fish vaccinated with envelope prepared from P. damselae grown under different condition were determined. To choose the bacterial growth conditions that provide the best
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Table 1 Second experiment design Group no.
I II III IV V VI a
Growth conditions (C
% salinity
– – 25 25 30 30
– – 0.5 2.5 0.5 2.5
Vaccination dose: µg g1a
No. of fish
Non-treated Adjuvant 1.5 1.5 1.5 1.5
9 9 10 14 6 13
µg protein per g fish.
protection against the bacteria, fish were divided into six groups of acclimatised fish. Group I was made up of non-treated controls, II (nine fish each) formed the control group injected with adjuvant only. Four groups, III, IV, V and VI, were vaccinated with bacterial envelope preparations emulsified in adjuvant grown under the following conditions: group III grown at 25 (C and in 0.5% NaCl; group IV grown at 25 (C and in 2.5% NaCl; group V grown at 30 (C and in 0.5% NaCl; and group VI grown at 30 (C and in 2.5% NaCl. A booster vaccination was performed 21 days after the first vaccination, using the same method. All fish were challenged 15 days post-booster vaccination (36 days post-first vaccination) by injecting with 8.3105 live bacteria per g fish (Table 1). Mortality was followed for 10 days after the challenge. Kidney from moribund fish was streaked on to BHI agar to confirm the reisolation of P. damselae. The potency of the vaccines was calculated as relative percent survival (RPS) [19] using the formula: RPS=[1(%vaccinated group mortality/% non-vaccinated group mortality)]100. 2.8. Third experiment Bacterial envelopes from bacteria grown at 25 (C with 2.5% NaCl were used in a larger-scale vaccination experiment. Fish were acclimatised for a few days in four 1000-l containers, three groups for vaccination (groups IVa, IVb, and IVc) and with the same vaccination dose as in the second experiment, and one group (I) as a control injected with adjuvant only. Booster vaccination was performed 21 days post-first vaccination, and challenge was performed 7 days post-booster. The potency of the vaccines was calculated as RPS. 2.9. Sequencing protein fractions Envelope preparations from P. damselae 2207 grown at 25 (C on BHI (2.5% or 0.5% NaCl) were used. The 22-kDa band from lane 2 (0.5% NaCl) and the 36-kDa band from lane 3 (2.5% NaCl) (Fig. 3) were eluted from the gel, blotted on to polyvinylidene difluoride (PVDF) membranes (Immobilon Transfer, Millipore, Bedford, MA), stained with Coomassie Blue R-250 (Bio-Rad) and sequenced directly as described by [20]. The membranes were not chemically activated or pretreated with Polybrene before usage, and PVDF membranes proved superior supports for sequence analysis of picomole protein quantities. 3. Results The amount of antibody produced in fish sera by exposure to the bacterin or microcapsules was determined by ELISA. Results of the first experiment are summarised in Fig. 1, which shows that
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Fig. 1. First vaccination experiment: antibody titre of fish vaccinated with bacterin or microencapsulated vaccine. Group I, the control, was vaccinated with adjuvant only; group II was vaccinated with microcapsules (bacteria grown in BHI—0.5% NaCl); group III was vaccinated with bacterin (bacteria grown in BHI—0.5% NaCl) emulsified in IFA, and group IV with bacterin (bacteria grown in BHI—2.5% NaCl) emulsified in IFA. Data with different letters are significantly different (P<0.05).
vaccination with microcapsules prepared from bacteria grown in 0.5% NaCl (group II) resulted in a slight increase of antibody titre (AT) compared to the control group: 6.63% of maximum AT (group I). Bacterin prepared from bacteria grown in a 0.5% NaCl medium (group III) showed a slight increase of AT: 28.71% compared to group II, whereas vaccination with bacterin prepared from bacteria grown in a 2.5% NaCl medium (group IV), gave the highest AT: 54.11% of maximum AT. In the second vaccination experiment (Fig. 2), immunogenic components from the envelope preparate gave different ATs. The two control groups did give a certain background AT: the non-treated control (group I) gave a mean value of 12.25% compared to the second control, which was vaccinated with adjuvant (group II). Its value increased to 22.19% of maximum AT. Groups IV, V and VI gave slight, non-significant increases of mean AT: 33.75%, 37.26% and 46.38% of maximum AT, respectively. Envelopes prepared from bacteria grown in 0.5% NaCl at 25 (C (group III) gave the highest AT: 56.8% of maximum AT. Challenge results, summarised in Table 2, show that 100% mortality occurred in both control groups, the non-treated one (group I) and group II which was vaccinated only with adjuvant. The protection level of group VI (RPS 54) was relatively low, increasing slightly among groups III and V (RPS 70 and 67, respectively). The highest protection level (RPS 79) occurred in group IV, which was vaccinated with envelopes produced from P. damselae grown in 2.5% NaCl at 25 (C. In the third vaccination experiment, summarised in Table 3, the three groups (making up a larger number of fish than in the former experiments) were vaccinated with only one envelope product prepared from P. damselae grown in 2.5% NaCl at 25 (C. Challenge results showed higher RPS values than in the former experiment: 86, 81 and 91 (groups IVa, IVb and IVc respectively) while mortality in the control injected with adjuvant (group I) was 74.2%. Fig. 3 shows the results of the electrophoresis of bacterial envelope preparate. Electrophoresis lanes represent (from right to left): molecular weight standards, envelope preparate grown in 0.5 and in 2.5% NaCl. The western blot of the envelope preparate used for sequencing the protein fraction is shown on the right side. Lane 1 is the molecular weight standards, while lanes 2, 3 and 4 are envelope preparates grown in 0.5, 2.5 and 3.5% NaCl, respectively. The 36-kDa band appeared significantly in all three lanes (2, 3 and 4), whereas the 22-kDa band was found only in lane 2 and was absent from the other lanes.
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Fig. 2. Second vaccination experiment: antibody titre of fish vaccinated with envelopes prepared from P. damselae grown under different conditions. Group I was made up of non-treated controls, II formed the control group injected with adjuvant only. Four groups, III, IV, V and VI, were vaccinated with bacterial envelope preparations emulsified in adjuvant grown under the following conditions: group III grown at 25 (C and in 0.5% NaCl; group IV grown at 25 (C and in 2.5% NaCl; group V grown at 30 (C and in 0.5% NaCl; and group VI grown at 30 (C and in 2.5% NaCl. Data with different letters are significantly different (P<0.05).
Table 2 Second vaccination experiment: protection of fish vaccinated with envelopes prepared from P. damselae grown under different conditions Group no.
No. of fish
Specific loss
% Fish mortality
RPSa
I II III IV V VI
9 9 10 14 6 13
9 9 3 3 2 6
100 100 30 21.4 33.3 46.2
– – 70 79 67 54
a
RPS=relative percent survival.
3.1. Sequencing protein fractions (from the N terminal) The 22-kDa band from lane 2 (0.5% NaCl) was extracted and transferred for sequencing. The 15-amino-acid sequence of this band is LAGPYIGVNIGSGGM. The same process was performed for the 36-kDa band from lane 3 (2.5% NaCl) and the 15-amino-acid sequence of this band is ADATATTDNNVVAAK. The second sequence, according to the Genbank sequence from the genome server at: vega.igh.cnrs.fr; locus BFU89914, 7169 bp DNA, showed 80.00% identity in a 10-amino-acid overlap with a Bacillus firmus hypothetical 34.0-kDa protein and Na+/ H+ antiporter homolog gene.
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Table 3 Third vaccination experiment: protection of fish vaccinated with envelopes prepared from P. damsela grown at 25 (C and 2.5% NaCl Group no.
No. of fish
Specific loss
% Fish mortality
RPSa
I IVa IVb IVc
31 29 28 29
23 3 4 2
74.2 10.3 14.3 6.9
– 86 81 91
a
RPS=relative percent survival.
Fig. 3. Electrophoresis (left) and western blot (right) of bacterial envelope preparate used for sequencing protein fractions. Electrophoresis lanes represent (from right to left): molecular weight standards, envelope preparate grown in 0.5 and in 2.5% NaCl. The western blot of the envelope preparate used for sequencing the protein fraction is shown on the right side. Blot lanes: 1. Molecular weight standards. 2. Envelope preparate grown in 0.5% NaCl. The 36 and 22-kDa bands appeared. 3. Envelope preparate grown in 2.5% NaCl. The 36-kDa band appeared. 4. Envelope preparate grown in 3.5% NaCl. The 36-kDa band appeared.
4. Discussion Since 1994, Israeli aquaculture farms growing hybrid bass in brackish water have suffered substantial economic losses due to pasteurellosis outbreaks. Antibiotics have been used widely to cure the disease, but preventing it remains most important to the fish grower. Since pasteurellosis has never caused disease in freshwater fish farms, our assumption was that P. damselae growing at high salinity (over 1.38‰ water salinity) gain pathogenic mechanisms which enable the
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bacteria to penetrate the organism, cause disease and avoid the immune system. The major contribution to P. damselae virulence is the capsule’s ability to reduce phagocytosis by macrophages [21] and to confer resistance to serum killing [13]; alternatively it could be protected against changes in cell turgor. The strain DI21 was able to survive and multiply in two fish species’ non-immune sera [13]. Hawke [22] reported that P. damselae resists the bactericidal effects of normal hybrid striped bass serum. Disease prevention by vaccination could eliminate the severe losses. In a preliminary study (first experiment) three bacterial preparations were used: P. damselae grown in BHI supplemented with two final concentrations of NaCl, 0.5% and 2.5% (bacterins) and 0.5% (microcapsules). Different AT resulted from vaccinating the fish with these preparations (Fig. 1). The conclusion was that hybrid bass respond with high AT to vaccination with P. damselae bacterins from bacteria grown on BHI with either 0.5% or 2.5% NaCl. The microcapsule preparation (containing bacterin) stimulated a low antibody response. In the second experiment, AT and survival after challenge with four membrane preparations from P. damselae bacteria grown under four different conditions were compared (see Table 1). Vaccination with any of these preparations resulted in an antibody response (Fig. 2). Challenge with live bacteria after vaccination resulted in 100% mortality of the control groups, whereas the four vaccinated groups showed partial protection (Table 2). The envelope preparation from P. damselae gave 79% survival. Taking into consideration that the challenge dose resulted in 100% mortality, it was concluded that this preparation demonstrates high potential as a vaccine. This experiment’s results showed no direct correlation between average AT of a group and the protection against challenge conferred by the vaccination. It can be assumed that in protecting hybrid bass against pasteurellosis, activation of the cell-mediated immune response is more dominant than the humoral response. Similar results have been reported for other fish, such as gilthead seabream [10], or for other fish challenged with Vibrio anguillarum [23–25] and Aeromonas salmonicida [26]. The third experiment was aimed at verifying the results of the previous experiment. Vaccinating three groups of fish with envelope preparations from the bacteria grown on BHI with 2.5% NaCl at 25 (C, it was demonstrated that this vaccination gives a high protection to challenge with pathogenic bacteria (74.2% mortality in the control, non-vaccinated group compared to 6.9–14.3% mortality of the vaccinated groups). Solubilising the envelopes and separating the proteins by electrophoresis revealed many proteins. When these proteins were subjected to western blot analysis using serum from convalescent hybrid bass, it was obvious that the fish’s immune response to the vaccine resulted in antibody against a limited number of proteins. While the antibody responded to proteins of about 36 kDa from the bacterial envelopes grown in 0.5% and 2.5% NaCl, the antibody responded strongly to an additional protein of 22 kDa in the 0.5% NaCl envelope preparation. In order to identify the protein stimulating the immune response these proteins were then characterised further. Since western blotting (Fig. 3) showed the same pattern for 2.5 and 3.5% NaCl, which was different from that of 0.5% NaCl, the 3.5% NaCl lane was not used for sequencing proteins. The sequence of the first 15 amino acids from the N terminal of the 22 kDa band could not be identified as any known protein. A 10-amino-acid overlap of the 36-kDa band (2.5% NaCl) showed 80% sequence identity to the specific gene of the alkaliphilic Bacillus firmus Na+/H+ antiporter homolog gene. It may provide an additional explanation for P. damselae virulence, i.e. its ability to maintain homeostasis in extreme environments, e.g., P. damselae which was capable of intracellular growth in the phagocytic cell of a yellowtail [7]. The existence of a Na+/H+ antiporter gene may be responsible for the physiological challenge confronting the P. damselae undergoing stress-dependent activities, e.g. at the upper edge of their pH range for growth [27,28]. This study showed that the envelope preparation of P. damselae grown at 25 (C in 2.5% NaCl can be used as an effective vaccine to protect hybrid bass from the disease. It is postulated that the growth of P. damselae at different salinities results in changes in the outer envelope which cause the bacteria to become more invasive and pathogenic. A vaccine which includes these envelopal changes is expected to provide protection for the vaccinated fish.
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Acknowledgements The authors thank Mrs Rima Vaiman for her professional technical help. This work was supported by the Israel Ministry of Agriculture and Rural Development.
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The effect of growth medium salinity of Photobacterium damselae subsp. piscicida on the immune response of hybrid bass (Morone saxatilisM. chrysops) S. Nitzan 1*, B. Shwartsburd 2, E.D. Heller 2 2
1 Central Fish Health Laboratory, Department of Fisheries and Agriculture and Rural Development, Nir-David 19150, Israel Department of Animal Sciences, Faculty of Agricultural, Food and Environmental Quality Sciences, The Hebrew University of Jerusalem, Rehovot 76100, Israel
Received 27 August 2002; received in revised form 10 March 2003; accepted 7 April 2003
Abstract Photobacterium damselae subsp. piscicida (P. damselae) was grown on various media and the effect of media salinity on certain immune responses of hybrid bass was studied. In Israel, pasteurellosis outbreaks have not been reported at water salinities below 1.38‰. During vaccination experiments the salinity of the medium on which P. damselae is grown, was shown to affect stimulation of the immune system. No correlation was found between antibody response and protection. Bacterial envelopes separated by electrophoresis and subjected to western blot analysis revealed an antibody response against some protein bands. Band sequencing was performed to identify the protein stimulating the immune response. Sequence identity of 80% was seen in 10-amino-acid overlap of the 36-kDa band with a specific gene of alkalophilic Bacillus firmus. A preparation of P. damselae grown in a 2.5% NaCl medium at 25 (C is the most effective vaccine against pasteurellosis, providing hybrid bass with quite good protection. 2003 Elsevier Ltd. All rights reserved. Keywords: Hybrid bass; Pasteurellosis; Bacterial envelope preparation; ELISA; Salinity; Vaccination
1. Introduction Hybrid bass was introduced into Israel in 1993. In the spring of 1994, when the water temperature in the fish ponds rose to 22–24 (C, an outbreak of pasteurellosis occurred. The causative agent was identified as Photobacterium damselae subsp. piscicida (P. damselae) [1], formerly Pasteurella piscicida [2]. Since the first outbreak of pasteurellosis was reported in the US [3], it has appeared in different countries and in a variety of fish species. Pasteurellosis outbreaks have occurred in fish species that are economically important for mariculture and brackish-water fish farms [4–11]. Many factors influence the expression/production of * Corresponding author E-mail address: [email protected] (S. Nitzan). 1050-4648/04/$ - see front matter 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S1050-4648(03)00045-7
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various virulence factors, as with temperature [1]. Pasteurellosis outbreaks occur at 24 (C and no mortality occurs at 15–16 (C or 30 (C. Additional factors that influence virulence of P. damselae are preinoculation with hemin and hemoglobin which increase virulence, the effect being stronger in avirulent strains [12]. Another factor that influences virulence is the capsular layer [13]. Barnes et al. [14] characterised additional important virulence factors, enzymes which neutralise reactive oxygen species produced in fish macrophages (SOD) and a cytoplasmic catalase in P. damselae. Since hybrid bass can be raised in either fresh, brackish or salt water, and pasteurellosis has never been seen in freshwater farms, the aim of this study was to determine the effect of salinity of P. damselae growth medium on its ability to stimulate the immune system and protect hybrid bass from the disease. 2. Materials and methods 2.1. Bacteria P. damselae was isolated from the spleen of moribund hybrid bass grown on a farm in the coastal Mediterranean region of Israel in 1995. This isolate, 2207, was cultured on brain heart infusion (BHI) agar (Oxoid, Hampshire, UK) supplemented with 0.1% (w/v) yeast extract (Difco, Detroit, MI, USA) and 2% (w/v) NaCl (1.5% NaCl was added to the original content of 0.5%), or the different salinities detailed below, and incubated at 25 (C, unless otherwise indicated. Some colonies of P. damselae were transferred and incubated for 6 to 8 h in BHI medium. Bacteria were grown for 18 to 20 h during the exponential phase, and the required amount adjusted to an optical density (OD) of approximately 1.00 at 540 nm (Spectronic 1201, Milton Roy, Rochester, NY, USA); at this OD, 2.5109 bacteria ml1 were found. The required bacterial concentration was diluted accordingly in phosphate-buffered saline (PBS) pH 7.4. Formalin-killed bacterial antigens (bacterins) were prepared according to Stolen et al. [15], and adjusted to an OD of 1.00. When adjuvant (incomplete Freund’s adjuvant; IFA; Sigma, St. Louis, MO, USA) was used, the bacterial antigen preparation or envelope preparation was mixed at equal volumes with the IFA. Microcapsules were produced from the polysaccharide alginate (2% w/v; Keltone, LV, San Diego, CA, USA) and polarisation was performed with 0.5% (w/v) CaCl2 (Sigma) in water. Bacterial number for capsules was the same as for the bacterin, grown in BHI medium (0.5% NaCl). 2.2. Bacterial sonicate and enzyme-linked immunosorbent assay (ELISA) Sonicate was prepared from 1.5 l of pelleted bacterial suspension and the pellet was washed and diluted 1:10 (v/v) with sonication buffer (50 mMol Tris buffer; pH 7.4). The suspension was sonicated for 15 min (550 W, 20 kHz) until a clear suspension was obtained, using an ultrasonic sonicator (Misonix XL 2020, Farmingdale, NY, USA). The obtained suspension was stored at 73 (C. For verification of complete disruption of cells, one drop of sonicate suspension was smeared on the BHI agar. Sonicate protein concentration was determined by means of the Lowry assay (Bio-Rad DC protein assay, Hercules, CA, USA). The immune responses were evaluated by ELISA [16] adapted to our requirements. Ninety-six-well immunoplates (Kartell, Milano, Italy) were coated with 2 µg sonicate protein per well, diluted in coating buffer. Bovine serum albumin (200 µl/well; BSA diluent/blocking solution, KPL, Gaithersburg, MD, USA) was added as a blocking solution. Fish sera were diluted in BSA diluent solution. Two New Zealand albino rabbits were immunised with bass immunoglobulin [15] purified from sera by means of ammonium sulphate precipitation. A mixture of equal volume bass serum and PBS was prepared, and an equal volume of saturated ammonium sulphate (pH 7.0) was added to the mixture. Centrifugation was performed twice by discarding the supernatant and suspending the deposit in half-saturated ammonium sulphate, and the pellet was dialysed against a saline solution three times. The rabbits were intramuscularly (IM) injected with 1 mg of fish immunoglobulins five times. In the first injection the immunoglobulins were emulsified in complete
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Freund’s adjuvant (CFA) and subsequently in IFA. Satisfactory antibody titre was confirmed by means of the Ouchterlony serological test. The substrate reagent was azinobis (ethylbenzthiazoline-6-sulphonic acid) (ABTS; KPL, Gaithersburg, MD, USA). The plates were read in a microplate autoreader (Tecan Spectra, Austria) at 405 nm. 2.3. Envelope preparation for gel electrophoresis P. damselae was grown in BHI with 0.5, 2.5 or 3.5% NaCl, and harvested by centrifugation (4000 g for 10 min at 4 (C). The sonicate pellet was resuspended in PBS, washed three times and resuspended 1:10 in sonication buffer. After centrifugation of the sonicate at 4000 g for 15 min, the supernatant was centrifuged by ultra-centrifugation (Centricon T-1170, Kontron Instruments, Zu¨rich, Switzerland) at 100,000 g for 1 h at 4 (C. The pellet was resuspended in 10 ml of sonication buffer and centrifuged again under the same conditions. The pellet was resuspended and incubated at 37 (C for 2 h with shaking, in a minimal amount of solution made up of sonication buffer, 2% (w/v) SDS, 0.5 M NaCl and 5 mM EDTA. Protein concentration was determined by means of the Lowry assay. The envelope preparation of P. damselae for vaccination was obtained as for gel electrophoresis. Bacterial growth conditions were at the different salinities and temperatures detailed below. Electrophoresis and western blot analysis were performed using 10% SDS–PAGE according to Laemmei [17] and Towbin et al. [18]. 2.4. Fish Hybrid bass from a farm with a water salinity of 0.66‰ (weight range 60–100 g), diagnosed as free of parasites and bacteria, were transferred to the laboratory. Immunisation experiments were performed in 100-l containers with water at 25 (C and a salinity of 3.5‰ in a recirculation system. 2.5. Vaccination Fish vaccination was performed by injecting 0.5 ml of the antigenic solution (bacterin or envelope preparation) into the upper peritoneal cavity with a 22G needle. The antigen was mixed at equal volumes with the adjuvant (IFA). A booster vaccination was performed 21 days after the first vaccination (except for the first experiment), using the same method. Sera for antibody titre determinations were obtained aseptically 10 days post-boost (or 21 days post-vaccination as in the first experiment) by dorsal aorta puncture, allowed to clot at 4 (C, separated by centrifugation at 4000 g for 5 min and stored at 4 (C with 0.02% (w/v) sodium azide for a few days until antibody evaluation. 2.6. First experiment Antibody titre comparison between fish vaccinated with bacterin and microcapsules. One group of fish was vaccinated with bacterin and a second one with microcapsules, as detailed below. The bacterial vaccine dose was 5105 bacteria per g fish. Fish were divided into four groups: group I (four fish), the control, was vaccinated with adjuvant only; group II (five fish) was vaccinated with microcapsules (bacteria grown in BHI—0.5% NaCl); group III (twelve fish) was vaccinated with bacterin (bacteria grown in BHI—0.5% NaCl) emulsified in IFA, and group IV (nine fish) with bacterin (bacteria grown in BHI—2.5% NaCl) emulsified in IFA. Sera for antibody titre determination were collected 21 days post-vaccination. 2.7. Second experiment Antibody titre and protection of fish vaccinated with envelope prepared from P. damselae grown under different condition were determined. To choose the bacterial growth conditions that provide the best
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Table 1 Second experiment design Group no.
I II III IV V VI a
Growth conditions (C
% salinity
– – 25 25 30 30
– – 0.5 2.5 0.5 2.5
Vaccination dose: µg g1a
No. of fish
Non-treated Adjuvant 1.5 1.5 1.5 1.5
9 9 10 14 6 13
µg protein per g fish.
protection against the bacteria, fish were divided into six groups of acclimatised fish. Group I was made up of non-treated controls, II (nine fish each) formed the control group injected with adjuvant only. Four groups, III, IV, V and VI, were vaccinated with bacterial envelope preparations emulsified in adjuvant grown under the following conditions: group III grown at 25 (C and in 0.5% NaCl; group IV grown at 25 (C and in 2.5% NaCl; group V grown at 30 (C and in 0.5% NaCl; and group VI grown at 30 (C and in 2.5% NaCl. A booster vaccination was performed 21 days after the first vaccination, using the same method. All fish were challenged 15 days post-booster vaccination (36 days post-first vaccination) by injecting with 8.3105 live bacteria per g fish (Table 1). Mortality was followed for 10 days after the challenge. Kidney from moribund fish was streaked on to BHI agar to confirm the reisolation of P. damselae. The potency of the vaccines was calculated as relative percent survival (RPS) [19] using the formula: RPS=[1(%vaccinated group mortality/% non-vaccinated group mortality)]100. 2.8. Third experiment Bacterial envelopes from bacteria grown at 25 (C with 2.5% NaCl were used in a larger-scale vaccination experiment. Fish were acclimatised for a few days in four 1000-l containers, three groups for vaccination (groups IVa, IVb, and IVc) and with the same vaccination dose as in the second experiment, and one group (I) as a control injected with adjuvant only. Booster vaccination was performed 21 days post-first vaccination, and challenge was performed 7 days post-booster. The potency of the vaccines was calculated as RPS. 2.9. Sequencing protein fractions Envelope preparations from P. damselae 2207 grown at 25 (C on BHI (2.5% or 0.5% NaCl) were used. The 22-kDa band from lane 2 (0.5% NaCl) and the 36-kDa band from lane 3 (2.5% NaCl) (Fig. 3) were eluted from the gel, blotted on to polyvinylidene difluoride (PVDF) membranes (Immobilon Transfer, Millipore, Bedford, MA), stained with Coomassie Blue R-250 (Bio-Rad) and sequenced directly as described by [20]. The membranes were not chemically activated or pretreated with Polybrene before usage, and PVDF membranes proved superior supports for sequence analysis of picomole protein quantities. 3. Results The amount of antibody produced in fish sera by exposure to the bacterin or microcapsules was determined by ELISA. Results of the first experiment are summarised in Fig. 1, which shows that
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Fig. 1. First vaccination experiment: antibody titre of fish vaccinated with bacterin or microencapsulated vaccine. Group I, the control, was vaccinated with adjuvant only; group II was vaccinated with microcapsules (bacteria grown in BHI—0.5% NaCl); group III was vaccinated with bacterin (bacteria grown in BHI—0.5% NaCl) emulsified in IFA, and group IV with bacterin (bacteria grown in BHI—2.5% NaCl) emulsified in IFA. Data with different letters are significantly different (P<0.05).
vaccination with microcapsules prepared from bacteria grown in 0.5% NaCl (group II) resulted in a slight increase of antibody titre (AT) compared to the control group: 6.63% of maximum AT (group I). Bacterin prepared from bacteria grown in a 0.5% NaCl medium (group III) showed a slight increase of AT: 28.71% compared to group II, whereas vaccination with bacterin prepared from bacteria grown in a 2.5% NaCl medium (group IV), gave the highest AT: 54.11% of maximum AT. In the second vaccination experiment (Fig. 2), immunogenic components from the envelope preparate gave different ATs. The two control groups did give a certain background AT: the non-treated control (group I) gave a mean value of 12.25% compared to the second control, which was vaccinated with adjuvant (group II). Its value increased to 22.19% of maximum AT. Groups IV, V and VI gave slight, non-significant increases of mean AT: 33.75%, 37.26% and 46.38% of maximum AT, respectively. Envelopes prepared from bacteria grown in 0.5% NaCl at 25 (C (group III) gave the highest AT: 56.8% of maximum AT. Challenge results, summarised in Table 2, show that 100% mortality occurred in both control groups, the non-treated one (group I) and group II which was vaccinated only with adjuvant. The protection level of group VI (RPS 54) was relatively low, increasing slightly among groups III and V (RPS 70 and 67, respectively). The highest protection level (RPS 79) occurred in group IV, which was vaccinated with envelopes produced from P. damselae grown in 2.5% NaCl at 25 (C. In the third vaccination experiment, summarised in Table 3, the three groups (making up a larger number of fish than in the former experiments) were vaccinated with only one envelope product prepared from P. damselae grown in 2.5% NaCl at 25 (C. Challenge results showed higher RPS values than in the former experiment: 86, 81 and 91 (groups IVa, IVb and IVc respectively) while mortality in the control injected with adjuvant (group I) was 74.2%. Fig. 3 shows the results of the electrophoresis of bacterial envelope preparate. Electrophoresis lanes represent (from right to left): molecular weight standards, envelope preparate grown in 0.5 and in 2.5% NaCl. The western blot of the envelope preparate used for sequencing the protein fraction is shown on the right side. Lane 1 is the molecular weight standards, while lanes 2, 3 and 4 are envelope preparates grown in 0.5, 2.5 and 3.5% NaCl, respectively. The 36-kDa band appeared significantly in all three lanes (2, 3 and 4), whereas the 22-kDa band was found only in lane 2 and was absent from the other lanes.
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Fig. 2. Second vaccination experiment: antibody titre of fish vaccinated with envelopes prepared from P. damselae grown under different conditions. Group I was made up of non-treated controls, II formed the control group injected with adjuvant only. Four groups, III, IV, V and VI, were vaccinated with bacterial envelope preparations emulsified in adjuvant grown under the following conditions: group III grown at 25 (C and in 0.5% NaCl; group IV grown at 25 (C and in 2.5% NaCl; group V grown at 30 (C and in 0.5% NaCl; and group VI grown at 30 (C and in 2.5% NaCl. Data with different letters are significantly different (P<0.05).
Table 2 Second vaccination experiment: protection of fish vaccinated with envelopes prepared from P. damselae grown under different conditions Group no.
No. of fish
Specific loss
% Fish mortality
RPSa
I II III IV V VI
9 9 10 14 6 13
9 9 3 3 2 6
100 100 30 21.4 33.3 46.2
– – 70 79 67 54
a
RPS=relative percent survival.
3.1. Sequencing protein fractions (from the N terminal) The 22-kDa band from lane 2 (0.5% NaCl) was extracted and transferred for sequencing. The 15-amino-acid sequence of this band is LAGPYIGVNIGSGGM. The same process was performed for the 36-kDa band from lane 3 (2.5% NaCl) and the 15-amino-acid sequence of this band is ADATATTDNNVVAAK. The second sequence, according to the Genbank sequence from the genome server at: vega.igh.cnrs.fr; locus BFU89914, 7169 bp DNA, showed 80.00% identity in a 10-amino-acid overlap with a Bacillus firmus hypothetical 34.0-kDa protein and Na+/ H+ antiporter homolog gene.
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Table 3 Third vaccination experiment: protection of fish vaccinated with envelopes prepared from P. damsela grown at 25 (C and 2.5% NaCl Group no.
No. of fish
Specific loss
% Fish mortality
RPSa
I IVa IVb IVc
31 29 28 29
23 3 4 2
74.2 10.3 14.3 6.9
– 86 81 91
a
RPS=relative percent survival.
Fig. 3. Electrophoresis (left) and western blot (right) of bacterial envelope preparate used for sequencing protein fractions. Electrophoresis lanes represent (from right to left): molecular weight standards, envelope preparate grown in 0.5 and in 2.5% NaCl. The western blot of the envelope preparate used for sequencing the protein fraction is shown on the right side. Blot lanes: 1. Molecular weight standards. 2. Envelope preparate grown in 0.5% NaCl. The 36 and 22-kDa bands appeared. 3. Envelope preparate grown in 2.5% NaCl. The 36-kDa band appeared. 4. Envelope preparate grown in 3.5% NaCl. The 36-kDa band appeared.
4. Discussion Since 1994, Israeli aquaculture farms growing hybrid bass in brackish water have suffered substantial economic losses due to pasteurellosis outbreaks. Antibiotics have been used widely to cure the disease, but preventing it remains most important to the fish grower. Since pasteurellosis has never caused disease in freshwater fish farms, our assumption was that P. damselae growing at high salinity (over 1.38‰ water salinity) gain pathogenic mechanisms which enable the
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bacteria to penetrate the organism, cause disease and avoid the immune system. The major contribution to P. damselae virulence is the capsule’s ability to reduce phagocytosis by macrophages [21] and to confer resistance to serum killing [13]; alternatively it could be protected against changes in cell turgor. The strain DI21 was able to survive and multiply in two fish species’ non-immune sera [13]. Hawke [22] reported that P. damselae resists the bactericidal effects of normal hybrid striped bass serum. Disease prevention by vaccination could eliminate the severe losses. In a preliminary study (first experiment) three bacterial preparations were used: P. damselae grown in BHI supplemented with two final concentrations of NaCl, 0.5% and 2.5% (bacterins) and 0.5% (microcapsules). Different AT resulted from vaccinating the fish with these preparations (Fig. 1). The conclusion was that hybrid bass respond with high AT to vaccination with P. damselae bacterins from bacteria grown on BHI with either 0.5% or 2.5% NaCl. The microcapsule preparation (containing bacterin) stimulated a low antibody response. In the second experiment, AT and survival after challenge with four membrane preparations from P. damselae bacteria grown under four different conditions were compared (see Table 1). Vaccination with any of these preparations resulted in an antibody response (Fig. 2). Challenge with live bacteria after vaccination resulted in 100% mortality of the control groups, whereas the four vaccinated groups showed partial protection (Table 2). The envelope preparation from P. damselae gave 79% survival. Taking into consideration that the challenge dose resulted in 100% mortality, it was concluded that this preparation demonstrates high potential as a vaccine. This experiment’s results showed no direct correlation between average AT of a group and the protection against challenge conferred by the vaccination. It can be assumed that in protecting hybrid bass against pasteurellosis, activation of the cell-mediated immune response is more dominant than the humoral response. Similar results have been reported for other fish, such as gilthead seabream [10], or for other fish challenged with Vibrio anguillarum [23–25] and Aeromonas salmonicida [26]. The third experiment was aimed at verifying the results of the previous experiment. Vaccinating three groups of fish with envelope preparations from the bacteria grown on BHI with 2.5% NaCl at 25 (C, it was demonstrated that this vaccination gives a high protection to challenge with pathogenic bacteria (74.2% mortality in the control, non-vaccinated group compared to 6.9–14.3% mortality of the vaccinated groups). Solubilising the envelopes and separating the proteins by electrophoresis revealed many proteins. When these proteins were subjected to western blot analysis using serum from convalescent hybrid bass, it was obvious that the fish’s immune response to the vaccine resulted in antibody against a limited number of proteins. While the antibody responded to proteins of about 36 kDa from the bacterial envelopes grown in 0.5% and 2.5% NaCl, the antibody responded strongly to an additional protein of 22 kDa in the 0.5% NaCl envelope preparation. In order to identify the protein stimulating the immune response these proteins were then characterised further. Since western blotting (Fig. 3) showed the same pattern for 2.5 and 3.5% NaCl, which was different from that of 0.5% NaCl, the 3.5% NaCl lane was not used for sequencing proteins. The sequence of the first 15 amino acids from the N terminal of the 22 kDa band could not be identified as any known protein. A 10-amino-acid overlap of the 36-kDa band (2.5% NaCl) showed 80% sequence identity to the specific gene of the alkaliphilic Bacillus firmus Na+/H+ antiporter homolog gene. It may provide an additional explanation for P. damselae virulence, i.e. its ability to maintain homeostasis in extreme environments, e.g., P. damselae which was capable of intracellular growth in the phagocytic cell of a yellowtail [7]. The existence of a Na+/H+ antiporter gene may be responsible for the physiological challenge confronting the P. damselae undergoing stress-dependent activities, e.g. at the upper edge of their pH range for growth [27,28]. This study showed that the envelope preparation of P. damselae grown at 25 (C in 2.5% NaCl can be used as an effective vaccine to protect hybrid bass from the disease. It is postulated that the growth of P. damselae at different salinities results in changes in the outer envelope which cause the bacteria to become more invasive and pathogenic. A vaccine which includes these envelopal changes is expected to provide protection for the vaccinated fish.
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Acknowledgements The authors thank Mrs Rima Vaiman for her professional technical help. This work was supported by the Israel Ministry of Agriculture and Rural Development.
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