- Email: [email protected]
Passive immunization of Nile tilapia (Oreochromis niloticus) provides significant protection against Streptococcus agalactiae
Fish & Shellfish Immunology 21 (2006) 365e371 www.elsevier.com/locate/fsi
Passive immunization of Nile tilapia (Oreochromis niloticus) provides significant protection against Streptococcus agalactiae David J. Pasnik a,*, Joyce J. Evans a, Phillip H. Klesius b a Aquatic Animal Health Research Laboratory (AAHRL), United States Department of Agriculture e Agricultural Research Service (USDA-ARS), 118B Lynchburg Street, Chestertown, MD 21620, USA b AAHRL, USDA-ARS, P.O. Box 952, Auburn, AL 36832, USA
Received 16 November 2005; revised 22 December 2005; accepted 10 January 2006 Available online 10 March 2006
Abstract A study was conducted to determine the role of specific antibodies in immunity to Streptococcus agalactiae. Adult Nile tilapia (Oreochromis niloticus) were injected i.p. with tryptic soy broth as control or with S. agalactiae vaccine. Ninety days later, fish were challenged with 1.5 104 CFU S. agalactiae fish1. Blood was drawn from all fish 90 d after vaccination and 25 d after challenge, and the acquired serum was injected i.p. in fingerling Nile tilapia. These passively immunized fish were subsequently challenged 72 h later with 1.5 104 CFU S. agalactiae fish1, and significantly less (P < 0.0001) mortalities were noted among fish administered serum containing specific anti-S. agalactiae antibodies (0.0e10.0% mortalities) than in control groups (63.3e72.7% mortalities). Heat-inactivation of serum produced no significant differences in mortalities than non-heat-treated serum in groups administered serum containing specific antibodies from vaccinated fish (P < 0.9455) or vaccinatedechallenged fish (P < 0.0781). Pre-challenge serum samples indicate that the passively immunized fish had significantly increased (P < 0.0001) specific antibody levels over control fish. A highly significant (r2 ¼ 0.5892; P < 0.0001) correlation between increased pre-challenge specific serum antibody OD levels and survival after challenge was demonstrated when analyzing the control and passive immunization groups. The results of this study indicate that specific anti-S. agalactiae antibodies play a primary role in immunity to S. agalactiae in fish. Ó 2006 Elsevier Ltd. All rights reserved. Keywords: Antibody; Oreochromis niloticus; Passive immunization; Streptococcus agalactiae; Tilapia
1. Introduction The emerging fish pathogen, group B Streptococcus agalactiae, has been shown to cause significant morbidity and mortality among a variety of freshwater and saltwater fish species throughout the world [1e4]. Because of its potential economic and ecological impacts to aquaculture and fisheries, a vaccine composed of concentrated extracellular products (ECP) and formalin-killed S. agalactiae whole cells was constructed and determined to be significantly
* Corresponding author. Tel.: þ1 410 778 4136; fax: þ1 410 778 4399. E-mail address: [email protected] (D.J. Pasnik). 1050-4648/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.fsi.2006.01.001
366
D.J. Pasnik et al. / Fish & Shellfish Immunology 21 (2006) 365e371
efficacious [5,6]. Further studies by Pasnik et al. [7,8] established the putative protective antigens of S. agalactiae and suggested that specific antibodies were involved in protective immunity. In a long-term study of vaccine efficacy, increased protection against the pathogen was significantly correlated with elevated specific anti-S. agalactiae antibody OD levels [8]. Despite providing indications that specific antibodies were important in immunity to S. agalactiae, those studies were not designed to provide a definitive answer regarding the nature of protection and did not assess other non-specific responses potentially involved. Several passive immunization studies have been used to determine the role of specific antibodies in protective immunity against fish bacterial pathogens, such as Aeromonas salmonicida [9,10], Vibrio anguillarum [11,12], Yersinia ruckeri [13], Edwardsiella tarda [14], Edwardsiella ictaluri [15], and Flavobacterium psychrophilum [16]. These researchers found variable levels of protection according to the degree to which specific antibodies play a role in protective immunity. For studies analyzing passive immunization against Streptococcus spp., variable results have also been reported. Akhlaghi et al. [17] passively immunized rainbow trout (Oncorhynchus mykiss) with antiStreptococcus spp. serum from sheep, rabbits, and rainbow trout. Protective effects were observed in fish injected with mammalian serum but not with trout serum. Eldar et al. [18] determined that rainbow trout passively immunized against Streptococcus iniae experienced significantly less mortalities than control fish. However, because mortalities among immunized fish were still considerable, the authors concluded that protective immunity was not conferred by specific antibody alone. Shelby et al. [19] injected Nile tilapia (Oreochromis niloticus) with anti-S. iniae serum and observed significant protection following challenge. This finding indicated that specific antibodies played a highly significant role in protection against S. iniae, and the conclusion was further substantiated because heat-inactivation of complement in anti-S. iniae serum also allowed for significant protection. Nonetheless, given the various outcomes of these Streptococcus spp. passive immunization studies, the role of specific antibodies in protection of fish against other Streptococcus spp. remains relatively unknown. The study presented here was designed to assess the role of specific antibodies in protection against S. agalactiae, determine whether complement is involved in protection, and further resolve whether specific antibodies play an important role in immunity against Streptococcus spp.
2. Materials and methods 2.1. Fish Nile tilapia (O. niloticus) were housed at the USDA/ARS Aquatic Animal Health Laboratory in Chestertown, Maryland. The fish were kept in 57 L (large fish) or 9 L (small fish) aquaria supplied with flow-through dechlorinated tap water and were maintained on a 12-h:12-h light:dark period. The fish were fed daily to satiation with Aquamax Grower 400 (large fish) or Aquamax Grower (small fish) (Brentwood, Missouri, USA1). Daily water temperature averaged 30.83 1.32 C; mean daily dissolved oxygen was 3.16 0.83 mg L1; mean pH was 7.03 0.13; and mean total ammonia concentration was 0.79 0.68 mg L1. 2.2. Active immunization and antiserum production Triplicate groups of fish (mean weight 104.6 6.5 g) were used to produce the anti-S. agalactiae serum. Fish were injected with an S. agalactiae vaccine prepared as previously described [5,7]. The polysaccharide-encapsulated S. agalactiae was grown in tryptic soy broth (Difco Laboratories, Sparks, Maryland, USA) at 27 C for 72e125 h. The resulting cultures were treated with 3% neutral buffered formalin for 24 h and then centrifuged to separate the cell pellet and culture fluid. The ECP fraction of the vaccine was prepared by concentrating the cell-free culture fluid containing ECP on a 3-kDa Amicon column (S3Y3) using a Millipore Proflux M12 (Millipore, Billerica, Massachusetts, USA), and sterilized using a 0.22-mm 1 L microbiological filter (Corning, Corning Inc., New York, USA). 1 Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.
D.J. Pasnik et al. / Fish & Shellfish Immunology 21 (2006) 365e371
367
Triplicate groups of five fish each were injected intraperitoneally (i.p.) with 0.1 mL of the S. agalactiae vaccine, and additional triplicate groups of five fish each were injected i.p. with 0.1 mL sterile tryptic soy broth (TSB) to serve as control groups. After injection, all fish were sequestered in groups of vaccinated or control fish in separate aquaria and maintained as previously described. Blood was drawn for passive immunization and ELISA from vaccinated and control fish 90 d after vaccination or injection with TSB, respectively. The time period of 90 d after vaccination was chosen because it corresponded to the highest measured antibody OD levels post-S. agalactiae vaccination as determined in a previous study [8]. On the same day, all fish were challenged by i.p. injection with 1.5 104 colony-forming units (CFU) S. agalactiae fish1. After challenge, fish were observed for 25 d and blood was drawn again for passive immunization and ELISA. During all bleeding procedures, fish were bled from the caudal vein and the sampled blood was held at 25 C for 1 h. Control serum or serum containing specific anti-S. agalactiae (ASA) antibodies was separated with centrifugation at 400 g for 6 min and then stored at 70 C until use. 2.3. Passive immunization and bacterial challenge In the passive immunization study, triplicate tanks of 10 fish (3.3 0.4 g) each were used. Fish in each group were passively immunized by i.p. injection with 100 mL of serum from control fish (CON), tryptic soy broth (TSB group), serum from 90 d vaccinated fish (VAX), serum from 90 d vaccinatede25 d challenged fish (VCH), heat-inactivated serum from 90 d vaccinated fish (HIVAX), or heat-inactivated serum from 90 d vaccinatede25 d challenged fish (HIVCH). Serum was heat-inactivated by heating at 56 C for 30 min in a water bath. After 72 h following the passive immunization, all fish were challenged by i.p. injection with 1.5 104 CFU S. agalactiae fish1. During the subsequent 14-d challenge period, fish were monitored daily for clinical signs of disease and mortality. Moribund and dead fish were removed twice daily, and bacterial samples were aseptically obtained from the nares, brain, head kidney, and intestine of 10% of morbid and dead fish to confirm the presence of S. agalactiae. Samples were cultured at 35 C for 24 h on 5% de-fibrinated sheep blood agar (SBA; Remel, Lenexa, Kansas, USA), and isolate identity was confirmed as S. agalactiae using the BIOLOG MicroLog Microbial Identification System (BIOLOG, Hayward, California, USA) according to the manufacturer’s instructions. Positive cultures were beta-haemolytic, oxidase-negative, catalase-negative, and Gram-positive cocci [3]. 2.4. Enzyme-linked immunosorbent assay (ELISA) All acquired tilapia serum were tested for antibodies against the vaccine ECP fraction by indirect ELISA based on the methods of Shelby et al. [19]. The total protein content of this fraction was determined by the bicinchoninic acid method and adjusted to 500 mg protein mL1. One hundred microlitres of antigen was added to each well of a 96-well microtitre plate, which was incubated overnight at 4 C. The plates were washed with phosphate-buffered saline plus 0.05% tween-20 (PBSeT). Nile tilapia serum samples were diluted 1:100 in PBSeT, and 100 mL of the resulting solution was added to three replicate wells of the microtitre plate. The plate was incubated at 25 C for 2 h and washed with PBSeT. Mouse anti-tilapia IgM heavy chain specific monoclonal antibody 1H1 [19] was diluted 1:5000 in PBSe T and 100 mL of this solution added to each well. The plate was incubated at 25 C for 1 h and washed with PBSe T. Peroxidase-conjugated goat anti-mouse IgG (Pierce Biotechnology, Rockford, Illinois, USA) was diluted 1:5000 in PBSeT and added to each well. After incubation at 25 C for 1 h, the plate was washed again and 100 mL of OneStep Ultra TMB-ELISA (Pierce) was added to each well. The ELISA reaction was stopped after 20 min with 50 mL 3 M H2SO4, and the ELISA optical density (OD) of the reactions was read at 450 nm with a Bio-Tek Automated Microplate Reader (Bio-Tek Instruments, Winooski, Vermont, USA). The relative amount of specific antibody was measured as the OD value. 2.5. Statistical analysis All statistical analyses were performed using the SAS program (SAS Institute, Cary, North Carolina, USA). Survival data were compared by the proc lifetest with Wilcoxon comparison. ELISA results (mean S.E.) were compared by one-way analysis of variance (ANOVA) and Duncan’s multiple range test. Significant differences between groups and between tanks within groups were accepted at P < 0.05.
D.J. Pasnik et al. / Fish & Shellfish Immunology 21 (2006) 365e371
368
3. Results 3.1. Active immunization and antiserum production Fish were injected i.p. with tryptic soy broth or S. agalactiae vaccine, and blood was sampled 90 d later for control or specific anti-S. agalactiae (ASA) antibodies. After challenge with 1.5 104 CFU S. agalactiae fish1, fish blood was sampled again 25 d later. The mean percent mortalities among control fish after challenge was 60%, while that of the vaccinated fish was 40%. Mortalities began within 48 h, and some of the fish exhibited lethargy, anorexia, discoloration, and changes in swimming patterns before death. Some mortalities among the vaccinated fish were attributed to observed severe aggression among tankmates, a common behavioral phenomenon among Nile tilapia [20]. Overall, sufficient ASA serum was obtained from the control, vaccinated, and vaccinatedechallenged fish for the passive immunization study. ELISA results (optical density; OD) indicated that no pre-existing specific S. agalactiae antibodies were present in the test fish. However, 90 d after vaccination the vaccinated fish antibody OD levels (0.116 0.008) were significantly increased (P < 0.0001) over pre-vaccination OD levels and over unvaccinated control fish antibody OD levels (0.042 0.008). The specific antibody levels among the vaccinated fish increased significantly (P < 0.0034) again to 0.221 0.029 OD at 25 d post-challenge with S. agalactiae. A significant (r2 ¼ 0.7678; P < 0.0157) correlation between increased specific serum antibody OD levels among the vaccinated fish (90 d post-vaccination) and survival after challenge was noted. 3.2. Passive immunization and bacterial challenge Within 24 h of challenge with 1.5 104 CFU S. agalactiae fish1, some fish in each control and passive immunization group exhibited clinical signs such as lethargy and anorexia. Most mortalities occurred during this time period, and clinical signs among the clinically affected fish in the antiserum groups generally resolved after the 24-h period. The mean percent cumulative mortalities (Table 1) among the controls were 72.7 6.4 (CON) and 63.3 6.7 (TSB) percent and among the ASA groups were 10.0 5.8 (VCH), 6.7 3.3 (VAX and HIVAX), and 0.0 0.0 (HIVCH). No significant differences in cumulative mortalities were noted between the two control groups (P < 0.7178), and no significant differences were noted between the groups injected with anti-S. agalactiae serum (P < 0.4302). Heatinactivation of serum also produced no significant differences in mortalities than non-heat-treated serum in groups administered serum containing specific antibodies from vaccinated fish (P < 0.9455) or vaccinatedechallenged fish (P < 0.0781). However, mortalities in the control groups were significantly higher than that in the groups injected with anti-S. agalactiae serum (P < 0.0001). All sampled organs from dead fish were culture-positive for S. agalactiae. Serum obtained from fish 72 h after passive immunization exhibited increased ASA OD levels, ranging from 0.094 0.003 OD (HIVAX) to 0.114 0.009 OD (VAX), and were not significantly different. However, the levels Table 1 Specific anti-Streptococcus agalactiae antibody levels (ELISA optical density; OD) in tilapia 72 h after passive immunization (pre-challenge)a and 14 d after challenge with S. agalactiae (post-challenge), and cumulative percent survival 14 d post-challenge Group CON TSB VAX VCH HIVAX HIVCH
Pre-challenge OD (mean S.E.) x
0.047 0.001 0.055 0.004x 0.114 0.009y 0.111 0.011y 0.094 0.003y 0.103 0.003y
Post-challenge OD (mean S.E.) x*
0.099 0.003 0.103 0.005x* 0.141 0.012x 0.251 0.055y* 0.111 0.008x* 0.131 0.017x
Cumulative percent survival 27.3 6.4 36.7 6.7 93.3 3.3 90.0 5.8 93.3 3.3 100.0 0.0
Significant differences between groups within pre-challenge or post-challenge samples are noted by different letters, and significant differences between post-challenge and corresponding pre-challenge samples are noted by an asterisk. Significant differences were accepted at P < 0.05. a Tilapia were passively immunized by i.p. injection with serum from control fish (CON), tryptic soy broth (TSB group), serum from 90 d vaccinated fish (VAX), serum from 90 d vaccinatede25 d challenged fish (VCH), heat-inactivated serum from 90 d vaccinated fish (HIVAX), or heat-inactivated serum from 90 d vaccinatede25 d challenged fish (HIVCH). Pre-challenge serum samples were obtained 72 h later, and all fish were then challenged i.p. with 1.5 104 CFU S. agalactiae fish1. Fish were monitored for mortalities for 14 d after challenge, and post-challenge serum samples were obtained 14 d after challenge.
D.J. Pasnik et al. / Fish & Shellfish Immunology 21 (2006) 365e371
369
were significantly higher (P < 0.0001) than those of the controls (CON: 0.047 0.001 OD; TSB: 0.055 0.004 OD). Fourteen days after the challenge, the control ASA levels (CON: 0.099 0.003 OD; TSB: 0.103 0.005 OD) had increased significantly (P < 0.0001) roughly to the pre-challenge levels of the passively immunized groups. The VCH (0.251 0.055 OD) and HIVAX (0.111 0.008 OD) groups also significantly increased (P < 0.0100 and P < 0.0434, respectively) above their corresponding pre-challenge levels. The ASA antibody OD levels among the VAX (0.141 0.012 OD) and HIVCH (0.131 0.017 OD) groups increased, but not significantly (P < 0.0848 and P < 0.1383, respectively). While the post-challenge specific antibody OD levels of the controls and passively immunized fish were not significantly different, the VCH group had a significantly higher level. A highly significant (r2 ¼ 0.5892; P < 0.0001) correlation between increased pre-challenge specific serum antibody levels and survival after challenge was demonstrated when analyzing the control and passive immunization groups. 4. Discussion Significantly higher protective effects were observed in the actively vaccinated fish, as previously demonstrated [5e8]. However, a higher percentage of mortalities was observed in these fish than expected based on this prior research, but this can be attributed to the observed severe aggression among tankmates. Even so, fish actively vaccinated with the S. agalactiae vaccine [5] exhibited a significant increase in specific antibody levels over 90 d, and these levels were roughly similar to those found in a previous S. agalactiae vaccine study [8]. As in this previous vaccine study paper, increased pre-challenge OD levels among the actively vaccinated fish were significantly correlated with postchallenge survival. The fish injected with TSB did not exhibit such an increase in antibody levels. The post-challenge specific antibody OD levels among the vaccinated and control groups did increase significantly over pre-challenge levels, but there were still significantly less mortalities among the vaccinated fish than among control fish after challenge. The ASA serum samples from these time points were used to passively immunize tilapia against S. agalactiae. In the passive immunization part of this study, most mortalities occurred in the control groups within 24 h after challenge with 1.5 104 CFU S. agalactiae fish1. This mortality pattern corresponded with previous S. agalactiae challenges [5e8]. Overall, the groups injected i.p. with ASA serum experienced 0.0e10.0% mortalities and control groups experienced 63.3e72.7% mortalities. These significant differences in mortalities between immunized and control fish indicate that specific antibodies play a significant role in immunity against S. agalactiae. The immunity appears to be relatively immediate, as passively immunized fish were protected and exhibited significantly increased serum ASA antibody levels 72 h after ASA serum injection. This is consistent with Akhlaghi et al. [17] and Shelby et al. [19], who observed increased antibody levels 2e48 h after injection with anti-streptococcal serum. However, when serum was sampled here from the challenged survivors 14 d later, control ASA antibody levels were generally similar to those of passively immunized fish. This indicates that the immediate immunity following ASA serum injection was crucial for the significant increase in protection among the immunized fish. The ASA levels among the control fish would nonetheless increase to similar levels of those among the immunized fish over 14 d, though control fish would initially experience higher mortalities in the beginning of the challenge. In addition, heat-inactivated serum provided significantly increased protection over controls, but still provided statistically similar protection to the non-heat-inactivated serum. Shelby et al. [19] observed the same phenomenon and suggested that complement or cytokines were thus not responsible for protection against S. iniae. The findings here would likewise suggest that other immune factors play inconsequential or no roles in protection against S. agalactiae, though the activity of already-existing endogenous immune factors in the small fish cannot be completely discounted. While this study corresponds with the Shelby et al. [19] S. iniae passive immunization study, other passive immunization studies have suggested that cell-mediated and/or non-specific responses may also be important against piscine Streptococcus sp. [17,18]. Passive immunization has been shown to provide limited or no protection against some piscine pathogens [15,16,18] and complete protection against others [10,19]. Some authors have associated the levels of protection with specific minimum serum antibody levels. Akhlaghi et al. [17] found that trout anti-Streptococcus sp. serum did not protect fish against challenge, though antiserum from mammalian sources was protective. The authors suggested that this occurred because the antibody levels of the fish serum (ELISA OD of approximately 0.2 at 405 nm with a 1:100 serum dilution) was lower than that of the mammalian serum (ELISA OD of approximately 0.5e0.9). Shelby et al. [19] determined that anti-S. iniae antibody levels above an ELISA OD of 0.1 at 490 nm after a 1:1000 serum dilution likely signified a protective level. LaFrentz et al. [16] suggested that significant protection
370
D.J. Pasnik et al. / Fish & Shellfish Immunology 21 (2006) 365e371
against F. psychrophilum required a specific antibody titre of at least 102,400. The results in the present study indicate that an ELISA OD of 0.094 after a 1:100 serum dilution signifies sufficient ASA antibody levels for protection against S. agalactiae. A highly significant correlation between increased pre-challenge ASA levels above this OD and protection was exhibited in this study. Thus this minimum ASA level could be used to determine the potential S. agalactiae-related immunity of a fish. Overall, specific ASA antibody responses appear primarily responsible for protection against S. agalactiae challenge, with limited impact from non-specific immune factors. Protective antibody responses are generated against protective antigens; Western blot analysis of the S. agalactiae vaccine ECP components determined that the 54 and 55 kDa antigens are important in a protective immune response against S. agalactiae [7]. This indicates that the serum administered to the passively immunized fish contained sufficient antibody generated against such immunodominant antigens, resulting in protection against challenge. The findings in this study further confirm the protective mechanism of the S. agalactiae vaccine developed by Evans et al. [5] that contains an ECP fraction and formalin-killed whole cells of S. agalactiae. Passive immunization may also be regarded as a prophylactic or therapeutic tool against S. agalactiae infection in fish. Acknowledgements The authors would like to thank Daniel Brougher and Patrick Horley for their technical assistance with the study and Lisa Biggar for her editorial work on the manuscript. References [1] Robinson JA, Meyer FP. Streptococcal fish pathogen. Journal of Bacteriology 1966;92:512. [2] Plumb JA, Schachte JH, Gaines JL, Peltier W, Carroll B. Streptococcus sp. from marine fishes along the Alabama and Northwest Florida coast of the Gulf of Mexico. Transactions of the American Fisheries Society 1974;103:358e61. [3] Evans JJ, Klesius PH, Glibert PM, Shoemaker CA, Al Sarawi MA, Landsberg J, et al. Characterization of beta-haemolytic Group B Streptococcus agalactiae in cultured seabream, Sparus auratus (L.) and wild mullet, Liza klunzingeri (Day), in Kuwait. Journal of Fish Diseases 2002;25:505e13. [4] Glibert PM, Landsberg J, Evans JJ, Al Sarawi MA, Faraj M, Al Jarallah MA, et al. A fish kill of massive proportion in Kuwait Bay, Arabian Gulf, 2001: the roles of disease, harmful algae, and eutrophication. Harmful Algae 2002;12:1e17. [5] Evans JJ, Shoemaker CA, Klesius PH. Efficacy of Streptococcus agalactiae (Group B) vaccine in tilapia (Oreochromis niloticus) by intraperitoneal and bath immersion administration. Vaccine 2004;22:3769e73. [6] Evans JJ, Klesius PH, Shoemaker CA, Fitzpatrick BT. Streptococcus agalactiae vaccination and infection stress in Nile tilapia, Oreochromis niloticus. Journal of Applied Aquaculture 2004;16:105e15. [7] Pasnik DJ, Evans JJ, Panangala VS, Klesius PH, Shelby RA, Shoemaker CA. Antigenicity of Streptococcus agalactiae extracellular products and vaccine efficacy. Journal of Fish Diseases 2005;28:205e12. [8] Pasnik DJ, Evans JJ, Klesius PH. Duration of protective antibodies and correlation with survival in Nile tilapia (Oreochromis niloticus) following Streptococcus agalactiae vaccination. Diseases of Aquatic Organisms 2005;66:129e34. [9] Spence KD, Fryer JL, Pilcher KS. Active and passive immunization of certain salmonid fishes against Aeromonas salmonicida. Canadian Journal of Microbiology 1965;11:397e405. [10] Marquis H, Lallier R. Efficacy studies of passive immunization against Aeromonas salmonicida in brook trout, Salvelinus fontinalis (Mitchell). Journal of Fish Diseases 1989;12:233e40. [11] Harrell LW, Etlinger HM, Hodgins HO. Humoral factors important in resistance of salmonid fish to bacterial disease. I. Serum antibody protection of rainbow trout (Salmo gairdneri) against vibriosis. Aquaculture 1975;6:211e9. [12] Akhlaghi M. Passive immunization of fish against vibriosis, comparison of intraperitoneal, oral and immersion routes. Aquaculture 1999;180:191e205. [13] Olesen NJ. Detection of the antibody response in rainbow trout following immersion vaccination with Yersinia ruckeri bacterins by ELISA and passive immunization. Journal of Applied Ichthyology 1991;7:36e43. [14] Gutierrez MA, Miyazaki T, Harta H, Kim M. Protective properties of egg yolk IgY containing anti-Edwardsiella tarda antibody against paracolo disease in the Japanese eel, Anguilla japonica Temminck & Schlegel. Journal of Fish Diseases 1993;16:113e22. [15] Klesius PH, Sealey WM. Characteristics of serum antibody in enteric septicemia of catfish. Journal of Aquatic Animal Health 1995;7:205e10. [16] LaFrentz BR, LaPatra SE, Jones GR, Cain KD. Passive immunization of rainbow trout, Oncorhynchus mykiss (Walbaum), against Flavobacterium psychrophilum, the causative agent of bacterial coldwater disease and rainbow trout fry syndrome. Journal of Fish Diseases 2003;26:377e84. [17] Akhlaghi M, Munday B, Whittington R. Comparison of passive and active immunization of fish against streptococcosis (enterococcosis). Journal of Fish Diseases 1996;19:251e8.
D.J. Pasnik et al. / Fish & Shellfish Immunology 21 (2006) 365e371
371
[18] Eldar A, Horovitcz A, Bercovier H. Development and efficacy of a vaccine against Streptococcus iniae infection in farmed rainbow trout. Veterinary Immunology and Immunopathology 1997;56:175e83. [19] Shelby RA, Klesius PH, Shoemaker CA, Evans JJ. Passive immunization of tilapia, Oreochromis niloticus (L.), with anti-Streptococcus iniae whole sera. Journal of Fish Diseases 2002;25:1e6. [20] Barki A, Volpato GL. Early social environment and the fighting behaviour of young Oreochromis niloticus (Pisces, Cichlidae). Behaviour 1998;135:913e29.
Passive immunization of Nile tilapia (Oreochromis niloticus) provides significant protection against Streptococcus agalactiae David J. Pasnik a,*, Joyce J. Evans a, Phillip H. Klesius b a Aquatic Animal Health Research Laboratory (AAHRL), United States Department of Agriculture e Agricultural Research Service (USDA-ARS), 118B Lynchburg Street, Chestertown, MD 21620, USA b AAHRL, USDA-ARS, P.O. Box 952, Auburn, AL 36832, USA
Received 16 November 2005; revised 22 December 2005; accepted 10 January 2006 Available online 10 March 2006
Abstract A study was conducted to determine the role of specific antibodies in immunity to Streptococcus agalactiae. Adult Nile tilapia (Oreochromis niloticus) were injected i.p. with tryptic soy broth as control or with S. agalactiae vaccine. Ninety days later, fish were challenged with 1.5 104 CFU S. agalactiae fish1. Blood was drawn from all fish 90 d after vaccination and 25 d after challenge, and the acquired serum was injected i.p. in fingerling Nile tilapia. These passively immunized fish were subsequently challenged 72 h later with 1.5 104 CFU S. agalactiae fish1, and significantly less (P < 0.0001) mortalities were noted among fish administered serum containing specific anti-S. agalactiae antibodies (0.0e10.0% mortalities) than in control groups (63.3e72.7% mortalities). Heat-inactivation of serum produced no significant differences in mortalities than non-heat-treated serum in groups administered serum containing specific antibodies from vaccinated fish (P < 0.9455) or vaccinatedechallenged fish (P < 0.0781). Pre-challenge serum samples indicate that the passively immunized fish had significantly increased (P < 0.0001) specific antibody levels over control fish. A highly significant (r2 ¼ 0.5892; P < 0.0001) correlation between increased pre-challenge specific serum antibody OD levels and survival after challenge was demonstrated when analyzing the control and passive immunization groups. The results of this study indicate that specific anti-S. agalactiae antibodies play a primary role in immunity to S. agalactiae in fish. Ó 2006 Elsevier Ltd. All rights reserved. Keywords: Antibody; Oreochromis niloticus; Passive immunization; Streptococcus agalactiae; Tilapia
1. Introduction The emerging fish pathogen, group B Streptococcus agalactiae, has been shown to cause significant morbidity and mortality among a variety of freshwater and saltwater fish species throughout the world [1e4]. Because of its potential economic and ecological impacts to aquaculture and fisheries, a vaccine composed of concentrated extracellular products (ECP) and formalin-killed S. agalactiae whole cells was constructed and determined to be significantly
* Corresponding author. Tel.: þ1 410 778 4136; fax: þ1 410 778 4399. E-mail address: [email protected] (D.J. Pasnik). 1050-4648/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.fsi.2006.01.001
366
D.J. Pasnik et al. / Fish & Shellfish Immunology 21 (2006) 365e371
efficacious [5,6]. Further studies by Pasnik et al. [7,8] established the putative protective antigens of S. agalactiae and suggested that specific antibodies were involved in protective immunity. In a long-term study of vaccine efficacy, increased protection against the pathogen was significantly correlated with elevated specific anti-S. agalactiae antibody OD levels [8]. Despite providing indications that specific antibodies were important in immunity to S. agalactiae, those studies were not designed to provide a definitive answer regarding the nature of protection and did not assess other non-specific responses potentially involved. Several passive immunization studies have been used to determine the role of specific antibodies in protective immunity against fish bacterial pathogens, such as Aeromonas salmonicida [9,10], Vibrio anguillarum [11,12], Yersinia ruckeri [13], Edwardsiella tarda [14], Edwardsiella ictaluri [15], and Flavobacterium psychrophilum [16]. These researchers found variable levels of protection according to the degree to which specific antibodies play a role in protective immunity. For studies analyzing passive immunization against Streptococcus spp., variable results have also been reported. Akhlaghi et al. [17] passively immunized rainbow trout (Oncorhynchus mykiss) with antiStreptococcus spp. serum from sheep, rabbits, and rainbow trout. Protective effects were observed in fish injected with mammalian serum but not with trout serum. Eldar et al. [18] determined that rainbow trout passively immunized against Streptococcus iniae experienced significantly less mortalities than control fish. However, because mortalities among immunized fish were still considerable, the authors concluded that protective immunity was not conferred by specific antibody alone. Shelby et al. [19] injected Nile tilapia (Oreochromis niloticus) with anti-S. iniae serum and observed significant protection following challenge. This finding indicated that specific antibodies played a highly significant role in protection against S. iniae, and the conclusion was further substantiated because heat-inactivation of complement in anti-S. iniae serum also allowed for significant protection. Nonetheless, given the various outcomes of these Streptococcus spp. passive immunization studies, the role of specific antibodies in protection of fish against other Streptococcus spp. remains relatively unknown. The study presented here was designed to assess the role of specific antibodies in protection against S. agalactiae, determine whether complement is involved in protection, and further resolve whether specific antibodies play an important role in immunity against Streptococcus spp.
2. Materials and methods 2.1. Fish Nile tilapia (O. niloticus) were housed at the USDA/ARS Aquatic Animal Health Laboratory in Chestertown, Maryland. The fish were kept in 57 L (large fish) or 9 L (small fish) aquaria supplied with flow-through dechlorinated tap water and were maintained on a 12-h:12-h light:dark period. The fish were fed daily to satiation with Aquamax Grower 400 (large fish) or Aquamax Grower (small fish) (Brentwood, Missouri, USA1). Daily water temperature averaged 30.83 1.32 C; mean daily dissolved oxygen was 3.16 0.83 mg L1; mean pH was 7.03 0.13; and mean total ammonia concentration was 0.79 0.68 mg L1. 2.2. Active immunization and antiserum production Triplicate groups of fish (mean weight 104.6 6.5 g) were used to produce the anti-S. agalactiae serum. Fish were injected with an S. agalactiae vaccine prepared as previously described [5,7]. The polysaccharide-encapsulated S. agalactiae was grown in tryptic soy broth (Difco Laboratories, Sparks, Maryland, USA) at 27 C for 72e125 h. The resulting cultures were treated with 3% neutral buffered formalin for 24 h and then centrifuged to separate the cell pellet and culture fluid. The ECP fraction of the vaccine was prepared by concentrating the cell-free culture fluid containing ECP on a 3-kDa Amicon column (S3Y3) using a Millipore Proflux M12 (Millipore, Billerica, Massachusetts, USA), and sterilized using a 0.22-mm 1 L microbiological filter (Corning, Corning Inc., New York, USA). 1 Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.
D.J. Pasnik et al. / Fish & Shellfish Immunology 21 (2006) 365e371
367
Triplicate groups of five fish each were injected intraperitoneally (i.p.) with 0.1 mL of the S. agalactiae vaccine, and additional triplicate groups of five fish each were injected i.p. with 0.1 mL sterile tryptic soy broth (TSB) to serve as control groups. After injection, all fish were sequestered in groups of vaccinated or control fish in separate aquaria and maintained as previously described. Blood was drawn for passive immunization and ELISA from vaccinated and control fish 90 d after vaccination or injection with TSB, respectively. The time period of 90 d after vaccination was chosen because it corresponded to the highest measured antibody OD levels post-S. agalactiae vaccination as determined in a previous study [8]. On the same day, all fish were challenged by i.p. injection with 1.5 104 colony-forming units (CFU) S. agalactiae fish1. After challenge, fish were observed for 25 d and blood was drawn again for passive immunization and ELISA. During all bleeding procedures, fish were bled from the caudal vein and the sampled blood was held at 25 C for 1 h. Control serum or serum containing specific anti-S. agalactiae (ASA) antibodies was separated with centrifugation at 400 g for 6 min and then stored at 70 C until use. 2.3. Passive immunization and bacterial challenge In the passive immunization study, triplicate tanks of 10 fish (3.3 0.4 g) each were used. Fish in each group were passively immunized by i.p. injection with 100 mL of serum from control fish (CON), tryptic soy broth (TSB group), serum from 90 d vaccinated fish (VAX), serum from 90 d vaccinatede25 d challenged fish (VCH), heat-inactivated serum from 90 d vaccinated fish (HIVAX), or heat-inactivated serum from 90 d vaccinatede25 d challenged fish (HIVCH). Serum was heat-inactivated by heating at 56 C for 30 min in a water bath. After 72 h following the passive immunization, all fish were challenged by i.p. injection with 1.5 104 CFU S. agalactiae fish1. During the subsequent 14-d challenge period, fish were monitored daily for clinical signs of disease and mortality. Moribund and dead fish were removed twice daily, and bacterial samples were aseptically obtained from the nares, brain, head kidney, and intestine of 10% of morbid and dead fish to confirm the presence of S. agalactiae. Samples were cultured at 35 C for 24 h on 5% de-fibrinated sheep blood agar (SBA; Remel, Lenexa, Kansas, USA), and isolate identity was confirmed as S. agalactiae using the BIOLOG MicroLog Microbial Identification System (BIOLOG, Hayward, California, USA) according to the manufacturer’s instructions. Positive cultures were beta-haemolytic, oxidase-negative, catalase-negative, and Gram-positive cocci [3]. 2.4. Enzyme-linked immunosorbent assay (ELISA) All acquired tilapia serum were tested for antibodies against the vaccine ECP fraction by indirect ELISA based on the methods of Shelby et al. [19]. The total protein content of this fraction was determined by the bicinchoninic acid method and adjusted to 500 mg protein mL1. One hundred microlitres of antigen was added to each well of a 96-well microtitre plate, which was incubated overnight at 4 C. The plates were washed with phosphate-buffered saline plus 0.05% tween-20 (PBSeT). Nile tilapia serum samples were diluted 1:100 in PBSeT, and 100 mL of the resulting solution was added to three replicate wells of the microtitre plate. The plate was incubated at 25 C for 2 h and washed with PBSeT. Mouse anti-tilapia IgM heavy chain specific monoclonal antibody 1H1 [19] was diluted 1:5000 in PBSe T and 100 mL of this solution added to each well. The plate was incubated at 25 C for 1 h and washed with PBSe T. Peroxidase-conjugated goat anti-mouse IgG (Pierce Biotechnology, Rockford, Illinois, USA) was diluted 1:5000 in PBSeT and added to each well. After incubation at 25 C for 1 h, the plate was washed again and 100 mL of OneStep Ultra TMB-ELISA (Pierce) was added to each well. The ELISA reaction was stopped after 20 min with 50 mL 3 M H2SO4, and the ELISA optical density (OD) of the reactions was read at 450 nm with a Bio-Tek Automated Microplate Reader (Bio-Tek Instruments, Winooski, Vermont, USA). The relative amount of specific antibody was measured as the OD value. 2.5. Statistical analysis All statistical analyses were performed using the SAS program (SAS Institute, Cary, North Carolina, USA). Survival data were compared by the proc lifetest with Wilcoxon comparison. ELISA results (mean S.E.) were compared by one-way analysis of variance (ANOVA) and Duncan’s multiple range test. Significant differences between groups and between tanks within groups were accepted at P < 0.05.
D.J. Pasnik et al. / Fish & Shellfish Immunology 21 (2006) 365e371
368
3. Results 3.1. Active immunization and antiserum production Fish were injected i.p. with tryptic soy broth or S. agalactiae vaccine, and blood was sampled 90 d later for control or specific anti-S. agalactiae (ASA) antibodies. After challenge with 1.5 104 CFU S. agalactiae fish1, fish blood was sampled again 25 d later. The mean percent mortalities among control fish after challenge was 60%, while that of the vaccinated fish was 40%. Mortalities began within 48 h, and some of the fish exhibited lethargy, anorexia, discoloration, and changes in swimming patterns before death. Some mortalities among the vaccinated fish were attributed to observed severe aggression among tankmates, a common behavioral phenomenon among Nile tilapia [20]. Overall, sufficient ASA serum was obtained from the control, vaccinated, and vaccinatedechallenged fish for the passive immunization study. ELISA results (optical density; OD) indicated that no pre-existing specific S. agalactiae antibodies were present in the test fish. However, 90 d after vaccination the vaccinated fish antibody OD levels (0.116 0.008) were significantly increased (P < 0.0001) over pre-vaccination OD levels and over unvaccinated control fish antibody OD levels (0.042 0.008). The specific antibody levels among the vaccinated fish increased significantly (P < 0.0034) again to 0.221 0.029 OD at 25 d post-challenge with S. agalactiae. A significant (r2 ¼ 0.7678; P < 0.0157) correlation between increased specific serum antibody OD levels among the vaccinated fish (90 d post-vaccination) and survival after challenge was noted. 3.2. Passive immunization and bacterial challenge Within 24 h of challenge with 1.5 104 CFU S. agalactiae fish1, some fish in each control and passive immunization group exhibited clinical signs such as lethargy and anorexia. Most mortalities occurred during this time period, and clinical signs among the clinically affected fish in the antiserum groups generally resolved after the 24-h period. The mean percent cumulative mortalities (Table 1) among the controls were 72.7 6.4 (CON) and 63.3 6.7 (TSB) percent and among the ASA groups were 10.0 5.8 (VCH), 6.7 3.3 (VAX and HIVAX), and 0.0 0.0 (HIVCH). No significant differences in cumulative mortalities were noted between the two control groups (P < 0.7178), and no significant differences were noted between the groups injected with anti-S. agalactiae serum (P < 0.4302). Heatinactivation of serum also produced no significant differences in mortalities than non-heat-treated serum in groups administered serum containing specific antibodies from vaccinated fish (P < 0.9455) or vaccinatedechallenged fish (P < 0.0781). However, mortalities in the control groups were significantly higher than that in the groups injected with anti-S. agalactiae serum (P < 0.0001). All sampled organs from dead fish were culture-positive for S. agalactiae. Serum obtained from fish 72 h after passive immunization exhibited increased ASA OD levels, ranging from 0.094 0.003 OD (HIVAX) to 0.114 0.009 OD (VAX), and were not significantly different. However, the levels Table 1 Specific anti-Streptococcus agalactiae antibody levels (ELISA optical density; OD) in tilapia 72 h after passive immunization (pre-challenge)a and 14 d after challenge with S. agalactiae (post-challenge), and cumulative percent survival 14 d post-challenge Group CON TSB VAX VCH HIVAX HIVCH
Pre-challenge OD (mean S.E.) x
0.047 0.001 0.055 0.004x 0.114 0.009y 0.111 0.011y 0.094 0.003y 0.103 0.003y
Post-challenge OD (mean S.E.) x*
0.099 0.003 0.103 0.005x* 0.141 0.012x 0.251 0.055y* 0.111 0.008x* 0.131 0.017x
Cumulative percent survival 27.3 6.4 36.7 6.7 93.3 3.3 90.0 5.8 93.3 3.3 100.0 0.0
Significant differences between groups within pre-challenge or post-challenge samples are noted by different letters, and significant differences between post-challenge and corresponding pre-challenge samples are noted by an asterisk. Significant differences were accepted at P < 0.05. a Tilapia were passively immunized by i.p. injection with serum from control fish (CON), tryptic soy broth (TSB group), serum from 90 d vaccinated fish (VAX), serum from 90 d vaccinatede25 d challenged fish (VCH), heat-inactivated serum from 90 d vaccinated fish (HIVAX), or heat-inactivated serum from 90 d vaccinatede25 d challenged fish (HIVCH). Pre-challenge serum samples were obtained 72 h later, and all fish were then challenged i.p. with 1.5 104 CFU S. agalactiae fish1. Fish were monitored for mortalities for 14 d after challenge, and post-challenge serum samples were obtained 14 d after challenge.
D.J. Pasnik et al. / Fish & Shellfish Immunology 21 (2006) 365e371
369
were significantly higher (P < 0.0001) than those of the controls (CON: 0.047 0.001 OD; TSB: 0.055 0.004 OD). Fourteen days after the challenge, the control ASA levels (CON: 0.099 0.003 OD; TSB: 0.103 0.005 OD) had increased significantly (P < 0.0001) roughly to the pre-challenge levels of the passively immunized groups. The VCH (0.251 0.055 OD) and HIVAX (0.111 0.008 OD) groups also significantly increased (P < 0.0100 and P < 0.0434, respectively) above their corresponding pre-challenge levels. The ASA antibody OD levels among the VAX (0.141 0.012 OD) and HIVCH (0.131 0.017 OD) groups increased, but not significantly (P < 0.0848 and P < 0.1383, respectively). While the post-challenge specific antibody OD levels of the controls and passively immunized fish were not significantly different, the VCH group had a significantly higher level. A highly significant (r2 ¼ 0.5892; P < 0.0001) correlation between increased pre-challenge specific serum antibody levels and survival after challenge was demonstrated when analyzing the control and passive immunization groups. 4. Discussion Significantly higher protective effects were observed in the actively vaccinated fish, as previously demonstrated [5e8]. However, a higher percentage of mortalities was observed in these fish than expected based on this prior research, but this can be attributed to the observed severe aggression among tankmates. Even so, fish actively vaccinated with the S. agalactiae vaccine [5] exhibited a significant increase in specific antibody levels over 90 d, and these levels were roughly similar to those found in a previous S. agalactiae vaccine study [8]. As in this previous vaccine study paper, increased pre-challenge OD levels among the actively vaccinated fish were significantly correlated with postchallenge survival. The fish injected with TSB did not exhibit such an increase in antibody levels. The post-challenge specific antibody OD levels among the vaccinated and control groups did increase significantly over pre-challenge levels, but there were still significantly less mortalities among the vaccinated fish than among control fish after challenge. The ASA serum samples from these time points were used to passively immunize tilapia against S. agalactiae. In the passive immunization part of this study, most mortalities occurred in the control groups within 24 h after challenge with 1.5 104 CFU S. agalactiae fish1. This mortality pattern corresponded with previous S. agalactiae challenges [5e8]. Overall, the groups injected i.p. with ASA serum experienced 0.0e10.0% mortalities and control groups experienced 63.3e72.7% mortalities. These significant differences in mortalities between immunized and control fish indicate that specific antibodies play a significant role in immunity against S. agalactiae. The immunity appears to be relatively immediate, as passively immunized fish were protected and exhibited significantly increased serum ASA antibody levels 72 h after ASA serum injection. This is consistent with Akhlaghi et al. [17] and Shelby et al. [19], who observed increased antibody levels 2e48 h after injection with anti-streptococcal serum. However, when serum was sampled here from the challenged survivors 14 d later, control ASA antibody levels were generally similar to those of passively immunized fish. This indicates that the immediate immunity following ASA serum injection was crucial for the significant increase in protection among the immunized fish. The ASA levels among the control fish would nonetheless increase to similar levels of those among the immunized fish over 14 d, though control fish would initially experience higher mortalities in the beginning of the challenge. In addition, heat-inactivated serum provided significantly increased protection over controls, but still provided statistically similar protection to the non-heat-inactivated serum. Shelby et al. [19] observed the same phenomenon and suggested that complement or cytokines were thus not responsible for protection against S. iniae. The findings here would likewise suggest that other immune factors play inconsequential or no roles in protection against S. agalactiae, though the activity of already-existing endogenous immune factors in the small fish cannot be completely discounted. While this study corresponds with the Shelby et al. [19] S. iniae passive immunization study, other passive immunization studies have suggested that cell-mediated and/or non-specific responses may also be important against piscine Streptococcus sp. [17,18]. Passive immunization has been shown to provide limited or no protection against some piscine pathogens [15,16,18] and complete protection against others [10,19]. Some authors have associated the levels of protection with specific minimum serum antibody levels. Akhlaghi et al. [17] found that trout anti-Streptococcus sp. serum did not protect fish against challenge, though antiserum from mammalian sources was protective. The authors suggested that this occurred because the antibody levels of the fish serum (ELISA OD of approximately 0.2 at 405 nm with a 1:100 serum dilution) was lower than that of the mammalian serum (ELISA OD of approximately 0.5e0.9). Shelby et al. [19] determined that anti-S. iniae antibody levels above an ELISA OD of 0.1 at 490 nm after a 1:1000 serum dilution likely signified a protective level. LaFrentz et al. [16] suggested that significant protection
370
D.J. Pasnik et al. / Fish & Shellfish Immunology 21 (2006) 365e371
against F. psychrophilum required a specific antibody titre of at least 102,400. The results in the present study indicate that an ELISA OD of 0.094 after a 1:100 serum dilution signifies sufficient ASA antibody levels for protection against S. agalactiae. A highly significant correlation between increased pre-challenge ASA levels above this OD and protection was exhibited in this study. Thus this minimum ASA level could be used to determine the potential S. agalactiae-related immunity of a fish. Overall, specific ASA antibody responses appear primarily responsible for protection against S. agalactiae challenge, with limited impact from non-specific immune factors. Protective antibody responses are generated against protective antigens; Western blot analysis of the S. agalactiae vaccine ECP components determined that the 54 and 55 kDa antigens are important in a protective immune response against S. agalactiae [7]. This indicates that the serum administered to the passively immunized fish contained sufficient antibody generated against such immunodominant antigens, resulting in protection against challenge. The findings in this study further confirm the protective mechanism of the S. agalactiae vaccine developed by Evans et al. [5] that contains an ECP fraction and formalin-killed whole cells of S. agalactiae. Passive immunization may also be regarded as a prophylactic or therapeutic tool against S. agalactiae infection in fish. Acknowledgements The authors would like to thank Daniel Brougher and Patrick Horley for their technical assistance with the study and Lisa Biggar for her editorial work on the manuscript. References [1] Robinson JA, Meyer FP. Streptococcal fish pathogen. Journal of Bacteriology 1966;92:512. [2] Plumb JA, Schachte JH, Gaines JL, Peltier W, Carroll B. Streptococcus sp. from marine fishes along the Alabama and Northwest Florida coast of the Gulf of Mexico. Transactions of the American Fisheries Society 1974;103:358e61. [3] Evans JJ, Klesius PH, Glibert PM, Shoemaker CA, Al Sarawi MA, Landsberg J, et al. Characterization of beta-haemolytic Group B Streptococcus agalactiae in cultured seabream, Sparus auratus (L.) and wild mullet, Liza klunzingeri (Day), in Kuwait. Journal of Fish Diseases 2002;25:505e13. [4] Glibert PM, Landsberg J, Evans JJ, Al Sarawi MA, Faraj M, Al Jarallah MA, et al. A fish kill of massive proportion in Kuwait Bay, Arabian Gulf, 2001: the roles of disease, harmful algae, and eutrophication. Harmful Algae 2002;12:1e17. [5] Evans JJ, Shoemaker CA, Klesius PH. Efficacy of Streptococcus agalactiae (Group B) vaccine in tilapia (Oreochromis niloticus) by intraperitoneal and bath immersion administration. Vaccine 2004;22:3769e73. [6] Evans JJ, Klesius PH, Shoemaker CA, Fitzpatrick BT. Streptococcus agalactiae vaccination and infection stress in Nile tilapia, Oreochromis niloticus. Journal of Applied Aquaculture 2004;16:105e15. [7] Pasnik DJ, Evans JJ, Panangala VS, Klesius PH, Shelby RA, Shoemaker CA. Antigenicity of Streptococcus agalactiae extracellular products and vaccine efficacy. Journal of Fish Diseases 2005;28:205e12. [8] Pasnik DJ, Evans JJ, Klesius PH. Duration of protective antibodies and correlation with survival in Nile tilapia (Oreochromis niloticus) following Streptococcus agalactiae vaccination. Diseases of Aquatic Organisms 2005;66:129e34. [9] Spence KD, Fryer JL, Pilcher KS. Active and passive immunization of certain salmonid fishes against Aeromonas salmonicida. Canadian Journal of Microbiology 1965;11:397e405. [10] Marquis H, Lallier R. Efficacy studies of passive immunization against Aeromonas salmonicida in brook trout, Salvelinus fontinalis (Mitchell). Journal of Fish Diseases 1989;12:233e40. [11] Harrell LW, Etlinger HM, Hodgins HO. Humoral factors important in resistance of salmonid fish to bacterial disease. I. Serum antibody protection of rainbow trout (Salmo gairdneri) against vibriosis. Aquaculture 1975;6:211e9. [12] Akhlaghi M. Passive immunization of fish against vibriosis, comparison of intraperitoneal, oral and immersion routes. Aquaculture 1999;180:191e205. [13] Olesen NJ. Detection of the antibody response in rainbow trout following immersion vaccination with Yersinia ruckeri bacterins by ELISA and passive immunization. Journal of Applied Ichthyology 1991;7:36e43. [14] Gutierrez MA, Miyazaki T, Harta H, Kim M. Protective properties of egg yolk IgY containing anti-Edwardsiella tarda antibody against paracolo disease in the Japanese eel, Anguilla japonica Temminck & Schlegel. Journal of Fish Diseases 1993;16:113e22. [15] Klesius PH, Sealey WM. Characteristics of serum antibody in enteric septicemia of catfish. Journal of Aquatic Animal Health 1995;7:205e10. [16] LaFrentz BR, LaPatra SE, Jones GR, Cain KD. Passive immunization of rainbow trout, Oncorhynchus mykiss (Walbaum), against Flavobacterium psychrophilum, the causative agent of bacterial coldwater disease and rainbow trout fry syndrome. Journal of Fish Diseases 2003;26:377e84. [17] Akhlaghi M, Munday B, Whittington R. Comparison of passive and active immunization of fish against streptococcosis (enterococcosis). Journal of Fish Diseases 1996;19:251e8.
D.J. Pasnik et al. / Fish & Shellfish Immunology 21 (2006) 365e371
371
[18] Eldar A, Horovitcz A, Bercovier H. Development and efficacy of a vaccine against Streptococcus iniae infection in farmed rainbow trout. Veterinary Immunology and Immunopathology 1997;56:175e83. [19] Shelby RA, Klesius PH, Shoemaker CA, Evans JJ. Passive immunization of tilapia, Oreochromis niloticus (L.), with anti-Streptococcus iniae whole sera. Journal of Fish Diseases 2002;25:1e6. [20] Barki A, Volpato GL. Early social environment and the fighting behaviour of young Oreochromis niloticus (Pisces, Cichlidae). Behaviour 1998;135:913e29.