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Different concentrations of Edwardsiella tarda ghost vaccine induces immune responses in vivo and protects Sparus macrocephalus against a homologous challenge
Fish and Shellfish Immunology 80 (2018) 467–472
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Different concentrations of Edwardsiella tarda ghost vaccine induces immune responses in vivo and protects Sparus macrocephalus against a homologous challenge
T
Maocang Yana,b,1, Jinyu Liua,2, Yu Lia,2, Xuepeng Wanga,c,∗, Heng Jianga, Hao Fanga, Zhiming Guoa, Yongcan Suna a
Shandong Provincial Key Laboratory of Animal Biotechnology and Disease Control and Prevention & Shandong Provincial Engineering Technology Research Center of Animal Disease Control and Prevention, Shandong Agricultural University, Taian, 271018, PR China Zhejiang Mariculture Research Institute, Zhejiang Key Laboratory of Exploitation and Preservation of Coastal Bio-Resource, Wenzhou, 325005, PR China c Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao National Laboratory for Marine Science and Technology, Qingdao, 272000, PR China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Edwardsiella tarda Bacterial ghosts Sparus microcephalus Humoral immunity Cellular immunity Non-specific immunity
Bacterial ghosts (BGs) can be generated by the controlled expression of the PhiX174 lysis gene E in gramnegative bacteria. They are intriguing vaccine candidates since ghosts retain functional antigenic cellular determinants often lost during traditional inactivation procedures. Here we prepared Edwardsiella tarda ghost (ETG) and tested different concentrations in vaccination trials. The results showed that serum IgM antibody titers were significantly higher in three different concentration immunization groups than control group (P < 0.05), However, there was no significant (P > 0.05) difference between the immunized groups. The phagocytic percentage (PP) was significantly higher (P < 0.05) in ETG immunized groups than in the control group from 3 days post-treatment. The PP continued to rise with time until day 21, when the values of three ETG immunized groups were 45.7%,51.2% and 50.7%, respectively. In addition, phagocytic index (PI) was significantly higher (P < 0.05) in ETG immunized groups than in the control group after 7 days post-treatment. However, there was no significant (P > 0.05) difference of PP or PI between immunized groups. In addition, non-specific immune immunity, such as acid phosphatase, alkaline phosphatase, superoxide dismutase and lysozyme activities displayed a similar pattern in all immunized groups, all immunized fish showed significantly higher activities than control group fish (P < 0.05). Most importantly three ETG immunized groups were all significantly more protected against the E. tarda challenge (19/25, 76% survival), (21/25, 84% survival) and (20/25, 80% survival) respectively, compared to (9/25, 36% survival) survival in the control group, but there was no significant (P > 0.05) difference of survival rate (SR) or relative percent survival (RPS) between immunized groups. All these results suggest that an ETG could stimulate cellular and humoral immunity, and could be used as a vaccine candidate in S.m. In summary, ETG can protect fish from Edwardsiellosis, and there is no significant difference in SR and RPS when three different concentrations of ETG are used, so it can easily be developed as a vaccine for mechanical and artificial operations.
1. Introduction Sparus macrocephalus (S.m) belongs to Sparus, and is mainly distributed in the western Pacific, extending from Hokkaido, Japan in the north to Taiwan and Hainan Island in the south of China. It is an economically important fish cultured in parts of Asia including Japan, Korea, China, and some other countries of Southeast Asia [1]. With the scale development of the aquaculture, the frequent occurrence of
∗
Corresponding author. Shandong Agricultural University, Taian, 271018, PR China. E-mail address: [email protected] (X. Wang). First Author: Maocang Yan. 2 Co-first author: Jinyu Liu, Yu Li. 1
https://doi.org/10.1016/j.fsi.2018.06.034 Received 29 March 2018; Received in revised form 11 June 2018; Accepted 15 June 2018 Available online 19 June 2018 1050-4648/ © 2018 Elsevier Ltd. All rights reserved.
diseases has become one of the key points which restricts the development of aquaculture industry. It has been confirmed that Edwardsiella tarda (E. tarda), is the main pathogen of S.m [2]. As we know, E. tarda is the intracellular, rod-shaped Gram-negative, non-capsulated, motile, facultative anaerobic bacteria, and is widely distributed in aquatic environments and is infectious to a variety of animals including humans, fish, amphibians, reptiles, and birds [3]. In recent years, chemotherapy has been used effectively in
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controlling fish infections [4], however, there is significant concern regarding food safety following chemotherapeutic interventions in addition to the danger of selecting for antibiotic-resistant E. tarda isolates which have been reported worldwide [5]. These concerns have prompted the development of novel vaccination strategies for the control of E. tarda infections. In order to better prevent and control the infection of E. tarda, a biological method, such as vaccines, must be developed to control it. Over the past ten years, vaccines have become one of the most important methods to prevent the infection of fish and other animals [3]. As it is reported, vaccines for prevention of E. tarda are various and show different protective efficiency. These vaccines, in common use, are formalin treated or heat-inactivated, effecting the physical and chemical structure of bacterial surface antigen, changing the original anti immunogenicity and affecting the immune effects of vaccine [6,7]. In recent years, the bacterial ghost vaccine, a new type of vaccine, has attracted the considerable attention of research workers. Bacterial ghosts may be generated by the controlled expression of the PhiX174 lysis gene E in Gram-negative bacteria to form the free cytoplasm and reproduction of bacterial shell. Thus it preserves the original bacterial cell morphology, bacterial surface antigen, and adhesion properties. At the same time, bacterial ghosts contain LPS, adipose, peptidoglycans, and other natural immunostimulating complexes. It can induce stronger humoral immunity, cellular immunity, and mucosal immune response without the use of the adjuvant [3]. Although studies have reported the superiority of E. tarda bacterial ghost vaccine at home and abroad, their non-specific immune parameters have not been reported [3,8]. In this study, we analyze changes in specific and non-specific immune parameters in fish vaccinated with different concentrations of bacterial ghost vaccine, using S.m. These immunological techniques and concentration selection would be useful in the development of E. tarda ghost vaccine. The protective ability of E. tarda ghost vaccine was investigated on S.m to provide the theoretical basis for the application of E. tarda ghost vaccine and to provide new ideas for the immunization of marine fish bacterial diseases.
Staphylococcus aureus was inoculated into common broth agar slants for 24 h, inactivated by 0.5% formaldehyde for 24 h, washed with sterile saline 3 times, adjusted to 1.0 × 108 CFU/fish and stored in 4 °C [10]. The phagocytosis was used as the phagocytic activity of leukocytes, which was determined using the same method following our earlier research [9]. Then 100 μL of anticoagulant was added to 100 μL of S. aureus. Then shacked it and put it into the water at 25 °C for 60min with shacking once every 10 min. After this, the mixture was drawn with a pipette on the slides, dried and fixed with methanol for 10 min, and then Giemsa stained for 1 h. Finally, slides were washed and dried to observe with an oil microscope. The phagocytic percentage (PP) and phagocytic index (PI) were calculated according to the following equations.
2. Materials and methods
PP =
The number of cells involved in phagocytosis in one hundred phagocytes × 100% 100
PI =
The total number of bacteria in phagocytes × 100% The number of cells that had phagocyted bacteria
Fish were divided into four groups; three immunization groups (A to C groups were respectively immunized with ETG at 1 × 105 CFU/fish, 1 × 106 CFU/fish, 1 × 107 CFU/fish, n = 60 for each group), and control fish (group D), treated with phosphate-buffered saline (PBS), respectively. Before the experiment, fish were not fed for 24 h. Fish in immunization groups were intraperitoneally injected immunization with 0.2 ml bacterial ghost vaccine while fish in the control group were injected with 0.2 ml saline. Immune responses were measured after 0, 3, 7, 14, 21, 28 days of treating. From each group, six fish were taken to draw blood from the caudal vein (on day 0, two fish were taken randomly from each group before treatment). The blood of each fish was divided into two parts. One part of the blood was allowed to stand at room temperature for 2 h and was centrifuged at 4000 r/min for 10 min at 4 °C. After that, the upper serum was collected for the determination of serum antibody level. The other part of blood was made to the anticoagulant with heparin for the determination of phagocytic activity of blood leucocytes. During the experiment, the water was controlled at about 24 °C, salinity at 25–26, pH 8.2, and the air was continuously aerated. 2.4. Phagocytic activity
2.1. Bacterial strains and culture conditions E. tarda strain SM (GenBank accession number MH390702) was isolated from S.m which were cultured in the net cage in the Yueqing Gulf [2]. E. tarda was grown in tryptic soy broth (TSB) (Oxoid Ltd., Basingstoke, Hampshire, England) at 28 °C. Transformed E. tarda cells were grown in TSB containing 100 μg/ml ampicillin (Sigma, MO, USA) at 28 °C. Incubation temperatures for repression and expression of the lysis gene during transformation were 28 °C and 42 °C, respectively.
2.5. Antibody response assessment The antibody level was determined by an enzyme-linked immunosorbent assay (ELISA) following our earlier research [3]. Briefly, 100 μL carbonate-bicarbonate buffer (pH 9.6) containing 1 × 105 formalin-killed E. tarda were added to respective microtiter plate wells and incubated for 20 h at 37 °C. The plates were washed three times with PBS containing 0.05% Tween 20 (PBS-T) and blocked for 24 h at 4 °C with blocking buffer (0.5% BSA in PBS-T). After the plate was washed three times, sera from immunized and control fish was added to the plates, then incubated at 37 °C for 1 h and washed 3 times with PBS-T, then probed with 100 μL self-made rabbit anti-sparus-IgM antibody incubated at 37 °C for 1 h, and washed 3 times with PBS-T, added the horseradish peroxidase-conjugated goat anti-rabbit IgG for 1 h at 37 °C. Plates were washed four times with PBS-T and binding visualized by adding TMB (Tiangen, Beijing, China) according to the manufacturer's instructions (100 μL/well). The plates were incubated at room temperature for 20 min and the reaction stopped with 100 μL of 2 M H2SO4 and the absorbance read at 450 nm.
2.2. Production of bacterial ghost vaccine Bacterial ghost vaccines were generated following our earlier research paper using the same method [3]. Morphological features of E. tarda and ETG were examined by scanning electron microscopy (Hitachi S-2400) and transmission electron microscopy (7650; Hitachi) as previously described [3]. There were 1 × 1010 cells per milliliter of ETG vaccine were prepared. 2.3. Immunization protocol and challenge infection S.m were fed in Zhejiang Marine Fisheries Research Institute. They were the same size and had no diseases, their weight was 150 g ± 10 g. These fish were fed daily using the compound feed bought from Fuzhou Haima Feed Co. Ltd. The water was changed every morning and the water was kept running for 1 h. The temperature of the water was at about 24 °C. The final experiment was carried out after two-weeks of feeding observation.
2.6. Non-specific immune parameters assay Acid phosphatase (ACP), alkaline phosphatase (AKP), superoxide dismutase (SOD) and lysozyme (LZM) activity were determined at 0, 3, 468
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7, 14, 21, 28 days using the detection kits (Nanjing Jiancheng Bioengineering Institute, China) according to the manufacturer's protocols. One ACP and AKP activity unit was expressed as the production of 1 mg phenol by reaction between every 100 ml of serum and substance in 30 min.
The morphology of the cell, including all cell surface structures, were unaffected by lysis. Pores ranging from 200 to 500 nm in diameter were observed in E. tarda ghost by scanning electron microscopy (Fig. 1A). The loss of cytoplasmic material and structural integrity were observed in E. tarda ghost by transmission electron microscopy (Fig. 1A, C).
2.7. Challenge
3.2. Antibody response analysis
The vaccine groups were injected with 100 μL containing E. tarda SM 2.0 × 107 CFU/fish (about 5-fold LD50). The cumulative mortality was recorded for 7 days post-challenge, and the relative percent survival (RPS) was calculated as follows: make an equation RPS = 1–(% mortality of vaccinated fish) × (% mortality of control fish) – 1 × 100% [9].
S.m fish were immunized with different concentrations of ETG (groups A, B, and C) on day 0, then the sera were collected and IgMspecific E. tarda antibody levels were examined by ELISA. Specific antiE. tarda IgM antibodies were detected in the serum of fish immunized with ETG, and no antibody reactivity could be detected in the serum collected from control fish (Fig. 2). The results showed a statistical IgM antibody (P < 0.05) increase in all fish immunized with ETG, and that they developed significantly (P < 0.05) higher IgM antibody titers than fish treated with PBS throughout the whole period. However, there was no significant (P > 0.05) difference of antibodies between immunized groups A, B, and C. From the 7 t h day of the experiment, the antibody level of each experimental group was significantly higher than that of the control group until the 28 t h day, and the antibody level reached the highest level at the 21st day.
2.8. Statistical analyses Student's t-test was used, with significance set at the 95% level, and the different alphabet marked on groups indicated P < 0.05. Error bars on graphs indicate the S.D. 3. Results 3.1. Identification of bacterial ghosts
3.3. Effects on the phagocytic activity of leukocytes
E. tarda ghost were successfully generated and identified by an electron microscopic analysis (EMA, Fig. 1). EMA of protein E-lysed E. tarda cells revealed no gross alterations in cellular morphology compared to unlysed cells (Fig. 1B, D) except for the lysis pore (Fig. 1A).
The phagocytic activity of leukocytes in the blood of fish improved significantly after immunization with ETG vaccine (Fig. 3). The PP in the control group was 13.6%–15.8% and the PI was 1.95–2.19. There was no significant (P > 0.05) change. After 3 days of immunization,
Fig. 1. Evaluation of E. tarda and E. tarda ghosts by SEM (A and B) and TEM (C and D). 469
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control group at the 7 t h day after immunization. At the 14 t h day after immunization, the PI reached the highest level in the three experimental groups and maintained a high level until the 28 t h day after immunization. After 7 days, PP and PI in group B and group C were significantly higher than those in group A and there was no significant (P > 0.05) difference between group B and group C. To summarize, the result showed a statistical PP and PI (P < 0.05) increase in all fish immunized with ETG, and developed significantly higher (P < 0.05) PP and PI than fish treated with PBS during the whole period. 3.4. Change of non-specific immune parameters assay The ACP, AKP, LMZ and SOD activities were recorded in different groups, as shown in Fig. 4. ACP activity (ACPa) of control group D was not significantly different from 4.04 to 4.44 U/100 ml during the whole period. However, ACPa of group A was significantly higher than group B (P < 0.05, Fig. 4A), which was significantly higher than group C (P < 0.05, Fig. 4A), and group C was significantly higher than group D (P < 0.05, Fig. 4A). AKP activity (AKPa, Fig. 4B) of serum in vaccinated groups initially remained unchanged until after 7 days and then it increased as time extended to 28 days, whereas the AKPa of the control group (D) was not significantly different from 1.51 to 1.92 U/100 ml during the whole experiment. However, the AKPa of immunized groups (A and B) were increased 7 days post vaccine, and all vaccinated groups were significantly higher than group D after 14 days (P < 0.05). AKPa reached the highest level on 28 days post-treatment. Meanwhile, the AKPa of group A was significantly more increased than groups B and C in the whole experiments. Lysozyme activity (LZMa) of the control group (D) was not changed significantly during the whole period from 6.22 to 6.99 U/fish (Fig. 4C). However, the LZMa of vaccinated groups (A, B, and C) was increased after being vaccinated with ETG, and all vaccine groups were significantly higher than D after 3 days (P < 0.05), LZMa reached the highest level on the 21st day and maintained high levels until day 28. Whereas, the LZMa of group A, B and C were not different (P > 0.05). The SOD activity (SODa, Fig. 4D) of the control group (D) was not significantly different from 162.10 to 166.78 U/100 ml during the whole experiment. However, SODa of all vaccine groups was significantly higher than D after 7 days (P < 0.05), which reached the highest level on day 21. In addition, SODa of vaccinated groups (A and B) showed significantly higher than group C (P < 0.05), which was significantly higher than group D (P < 0.05) throughout the whole experiment period.
Fig. 2. Anti-E. tarda titers in fish following immunization with either ETG or PBS. Data are means for at least five independent assays and presented as the means ± SD. Different alphabet a, b and c indicate significant difference of antibody levels at P < 0.05.
the PP in the immunized group was significantly (P < 0.05) higher than that in the control group. With the prolongation of time, the PP in each experimental group continued to increase. At the 7 t h day after immunization, the PP in each group was significantly (P < 0.01) higher than in the control group and maintained a higher level. At the 21st day after immunization, the highest PP values were 45.7%, 51.2%, and 50.7% respectively in the three experimental groups A, B and C. At the 28 t h day after immunization, the PP in the three experimental groups decreased, but there was still significant (P < 0.05) difference from the control group. PI was slightly different from the control group at the 3rd day after immunization and significantly higher than that in
3.5. Bacterial challenge Survival differences (P < 0.01 to 0.05) were observed between the vaccination groups (Table 1). ETG-immunized fish of group B showed the highest survival rates (21/25, 84% survival rate, RS) and group C (20/25, 80% RS) and group A (19/25, 76% RS) were better protected than PBS-treated controls (9/25, 36% survival). The relative percent survival (RPS) of group A throughout C was 61.7%, 71.7%, and 68.3%, respectively, were significantly higher than PBS-treated controls. However there was no significant (P > 0.05) difference of SR or RPS between immunized groups. During challenge trials, dead fish showed typical clinical symptoms of E. tarda infection, E. tarda infection was confirmed in all dead fish by finding black pigments on SSA. 4. Discussions
Fig. 3. The phagocytic activity of leukocytes in Sparus microcephalus post-immunization with different concentrate ETG or PBS. (A): phagocytic percentage (PP) and (B): phagocytic index (PI). Values are representative of at least five independent experiments and are expressed as Mean ± Standard Error. Different alphabet a, b and c indicate significant difference values of phagocytic activity at P < 0.05.
Bacterial ghost was a complete cell shell of bacteria without contents, which can be used as an important means of prevention and control of fish bacterial and viral diseases [8–11]. Because of its bacterial membrane containing intact membrane proteins, fimbriae and 470
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Fig. 4. Change of non-specific immune parameters of Sparus microcephalus post-immunization with different concentrate ETG or PBS and intraperitoneally challenge with E. tarda. (A) acid phosphatase (ACP) activity, (B) alkaline phosphatase (AKP) activity, (C) lysozyme (LZM) activity and (D) superoxide dismutase (SOD) activity. Data are means for at least five assays and presented as the means ± SD. Different alphabet a, b, c and d indicate significant difference values of non-specific immune parameters at P < 0.05.
the different concentrations of E. tarda ghost vaccine, this suggests that the concentrations of ETG affect the immune response slightly. In addition, the results showed that the immunization of ETG vaccine can effectively improve the level of antibody in S.m, and reached the highest level on the 21st day post-treatment. It is reported that ETG protected tilapia from Edwardsiellosis with reaching the highest antibody level on the 21st day post-treatment too [8]. In addition, the RS and RPS of groups A throughout C were significantly higher than control group fish. All these results suggested that the ETG vaccine could protect fish from Edwardsiellosis. Phagocytosis is commonly considered as one of the most important cellular immune reactions to pathogen invasion in invertebrates [13]. The phagocytosis of tilapia is the most important non-specific cellular immunity [14]. In addition to phagocytic cells with specific immune function, the key ingredient is composed of nonspecific defense system [15]. Our earlier research found that the phagocytic activity of leucocytes of zebrafish immunized with ETG vaccine was significantly higher than the other formalin-inactivated E. tarda, the results showed that ETG vaccine can effectively activate the phagocytic ability of the organism [9]. In the present study, PP and PI increased in all fish immunized with ETG and developed significantly more PP and PI than fish treated with PBS throughout the whole period. Although there was no significant difference of PP and PI in immunized groups A, B and C during the whole observation, the PP of group B showed significantly higher than group A on days 7, 14, 21 and 28, and the PI of group B and C showed significantly higher than group A on days 14, 21 and 28. Besides the specific immune function, phagocytes play an important role in constituting non-specific defense systems. It can capture and digest pathogens that invade the body at all stages of microbial infection [16,17]. The leucocytes digesting pathogens can retain the relevant antigen information and transmit the information to the relevant lymphocytes, thereby inducing the humoral and cellular immunity in the host [18]. Bacterial ghost vaccine can effectively stimulate antibody in fish,
Table 1 Survival rates following three concentrations of ETG treated and E. tarda challenge.a Groups
A B C D
Subgroups (n=5)b 1
2
3
4
5
1 1 1 4
2 0 1 3
0 2 0 3
2 0 2 4
1 1 1 2
Survival rate (%)c
Relative percent survival (%)c
76.0a 84.0a 80.0a 36.0b
61.7a 71.7a 68.3a /b
a
Fish were infected intraperitoneally. Number of dead fish/subgroup post-challenge. c Alphabet a and b indicate significant difference of the survival rate and the relative percent survival (P < 0.05). b
other immunostimulatory components, such as lipopolysaccharide, peptidoglycan and so on can be discriminated by the related membrane receptor of dendritic cells and macrophages and be swallowed to stimulate the immune effects effectively [3]. It has been reported that fish immunity trials primarily included oral treatments, intraperitoneal injections and bath immersion [12]. E. tarda ghost vaccine usages were reported in immunized intraperitoneally in tilapia and orally immunized in mice [3,8]. Many studies have confirmed that bacterial ghost vaccine can effectively stimulate more antibody than formalininactivated E. tarda [3,8,11]. Although these vaccines were able to stimulate high antibody titer, which produced in the ETG vaccine groups had better bactericidal activity and more protective immunity to fish or mice than that produced in formalin-inactivated E. tarda group. Here we focus on the different concentrations of ETG vaccine to S.m. In this study, we found that the antibody level of S.m in immunized groups with different concentrations of ETG vaccine was significantly (P < 0.05) higher than that in the control group. However, there was no significant difference in the antibody level in fish vaccinated with 471
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rats, and other animals. Bacterial ghosts itself is also a good adjuvant, it also can induce immune enzyme and other humeral factors at the same time [9]. Although the specific immune mechanism of fish is the deficiency, a large number of studies have proved that the non-specific immune system plays a more key role than the specific immune system in the fish's anti-infection. Therefore, to defend against pathogen invasion in fish, the non-specific immunity is considered as the primary defense [19]. ACP and AKP have been used as a symbol of macrophage activation for the ability of intracellular digestion of phagocytized antigens in the immune system of invertebrates [20]. Superoxide dismutase (SOD) is a vital antioxidant enzyme, which could eliminate superoxide radicals to reduce intracellular oxidant stress levels [21]. Lysozyme is an important defense molecule of the fish innate immune system, it can activate lyse acetyl peptidoglycan in the cell wall hydrolysis of gram-positive bacteria in polysaccharide which is released, causing the formation of a hydrolase system, destruction and elimination of invading foreign objects inside the body to assume the host defense function [22]. It was found that innate immune parameters, e.g., ACP, AKP, SOD, and LMZ activities were significantly increased in all ETG immunized groups (Fig. 4). It's possible that the increased numbers of cells involved in the process or enhanced pathogen resistance led to the increase of innate immune parameter activities [23]. Besides the fact that bacterial ghosts are viable vaccine candidates, it also can be used as a carrier of drugs, nucleic acids, viral and/or bacterial antigens comprising multivalent vaccines designed to protect against animal and human diseases [24]. Ghost formulations represent an improved nonliving bacterial vaccine delivery strategy that is safe and highly immunogenic [3,11]. The data presented in this report suggests that intraperitoneal immunization with ETG provided effective immune protection against infection, suggesting that it is possible to develop an E. tarda-based bacterial-ghost vaccine for use in aquaculture.
[2]
[3] [4]
[5]
[6] [7] [8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
5. Conclusions
[16]
ETG vaccine can significantly stimulate the specific immune function and the non-specific immune system in S.m, it can protect fish from Edwardsiellosis. In addition, the concentration of ETG affects the immune response slightly, so it can easily be developed as a vaccine for mechanical and artificial operations.
[17]
[18]
Acknowledgments [19]
This work was supported by the National Natural Science Foundation of China (31402325), Funds of Shandong “Double Tops” Program, the earmarked fund for the Modern Agro-industry Technology Research System in Shandong Province (SDAIT-14-07 and SDAIT-1302), fund of the China Scholarship Council (201708370021), and special funds from the central finance to support the development of local universities. We thank Chengming Wang and Taylor Novak at Auburn University for performing manuscript editing.
[20]
[21]
[22] [23]
Appendix A. Supplementary data [24]
Supplementary data related to this article can be found at http://dx. doi.org/10.1016/j.fsi.2018.06.034. References [1] J. Zhang, F. Zhou, L.i. Wang, Q. Shao, Z. Xu, Dietary protein requirement of juvenile
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black sea bream, Sparusmacroce phallus, J. World Aquacult. Soc. 41 (2) (2010) 151–164. X. Wang, M. Yan, W. Hu, S. Chen, S. Zhang, Q. Xie, Visualization of Sparus macrocephalus infection by GFP-labeled Edwardsiella tarda, Isr. J. Aquacult. Bamidgeh 64 (2012) doi: IJA_64.2012.693. X. Wang, C. Lu, Mice orally vaccinated with Edwardsiella tarda ghosts are significantly protected against infection, Vaccine 27 (2009) 1571–1578. N. Pirarat, T. Kobayashi, T. Katagiri, M. Maita, M. Endo, Protective effects and mechanisms of a probiotic bacterium lactobacillus rhamnosus against experimental Edwardsiella tarda infection in Tilapia (Oreochromis niloticus), Vet. Immunol. Immunopathol. 113 (3–4) (2006) 339–347. T. Aoki, T. Kitao, M. Fukudome, Chemotherapy against infection with multiple drug resistance strains of Edwardsiella tarda in cultured eels, Fish Pathol. 24 (3) (1989) 161–168. X. Huang, C. Lu, Characterization of antigenicity of the outer membrane protein from Edwardsiella tarda, Chinese Journal of Immunology 18 (6) (2002) 385–387. Q. Xiong, C. Lu, Immune effect of Edwardsiella tarda on mice and Xiphophorus belleri, Chin. J. Vet. Sci. 22 (3) (2002) 251–253. R.K. Se, K.N. Yoon, Protection of Tilapia (Oreochromis mosambicus) from Edwardsiellosis by vaccination with Edwardsiella Tarda ghosts, Fish Shellfish Immunol. 20 (2006) 621–626. T. Pan, M. Yan, S. Chen, X. Wang, Effects of ten traditional Chinese herbs on immune response and disease resistance of Sciaenops ocellatus, Acta Ichthyol. Piscatoria 43 (2013) 41–49. P.K. Sahoo, B.R. Pillai, J. Mohanty, J. Kumari, S. Mohanty, B.K. Mishra, In vivo humoral and cellular reactions, and fate of injected Bacteria Aeromonas Hydrophila in freshwater prawn Macrobrachium rosenbergii, Fish Shellfish Immunol. 23 (2007) 327–340. K. Hao, X. Chen, X. Qi, X. Yu, E. Du, F. Ling, B. Zhu, G. Wang, Protective immunity of grass carp induced by DNA vaccine encoding capsid protein gene (vp7) of grass carp reovirus using bacterial ghost as delivery vehicles, Fish Shellfish Immunol. 64 (2017) 414–425. J.J. Evans, P.H. Klesius, A. Craig Shoemaker, Efficacy of Streptococcus agalactiae (group B) vaccine in Tilapia (Oreochromis niloticus) by intraperitoneal and bath immersion administration, Vaccine 22 (2004) 3769–3773. Y. Hong, X. Yang, G. Yan, Y. Huang, F. Zuo, Y. Shen, Y. Ding, Y. Cheng, Effects of glyphosate on immune responses and haemocyte DNA damage of Chinese mitten crab, Eriocheir sinensis, Fish Shellfish Immunol. 71 (2017) 19–27. S. Gallage, T. Katagiri, M. Endo, M. Maita, Comprehensive evaluation of immunomodulation by moderate hypoxia in S. Agalactiae vaccinated Nile tilapia, Fish Shellfish Immunol. 66 (2017) 445–454. A. Fazio, R. Cerezuela, M.R. Panuccio, A. Cuesta, M.A. Esteban, Vitro effects of Italian lavandula Multifida L. leaf extracts on gilthead seabream (Sparus aurata) leucocytes and SAF-1 cells, Fish Shellfish Immunol. 66 (2017) 334–344. C. Xu, E. Li, Y. Suo, Y. Su, M. Lu, Q. Zhao, J.G. Qin, L. Chen, Histological and transcriptomic responses of two immune organs, the spleen and head kidney, in nile tilapia (Oreochromis niloticus) to long-term hypersaline stress, Fish Shellfish Immunol. 76 (2018) 48–57. Q. Huang, M. Yu, H. Chen, M. Zeng, Y. Sun, T.T. Saha, D. Chen, LRFN (Leucine-rich repeat and fibronectin type-III domain-containing protein) recognizes bacteria and promotes hemocytic phagocytosis in the pacific oyster Crassostrea gigas, Fish Shellfish Immunol. 72 (2018) 622–628. Y. Shi, K. Chen, W. Ma, J. Chen, A C-reactive protein/serum amyloid P agglutinates bacteria and inhibits complement-mediated opsonophagocytosis by monocytes/ macrophages, Fish Shellfish Immunol. 76 (2018) 58–67. T. Behera, P. Swain, Alginate-chitosan-PLGA composite microspheres induce both innate and adaptive immune response through parenteral immunization in fish, Fish. Shellfish Immunology 35 (2013) 785–791. F. Yin, H. Gong, Q. Ke, A. Li, Stress antioxidant defence and mucosal immune responses of the large yellow croaker Pseudosciaena crocea challenged with Cryptocaryon irritans, Fish Shellfish Immunol. 47 (2015) 344–351. J. Tian, J. Yu, Poly(Lactic-Co-Glycolic acid) nanoparticles as candidate DNA vaccine Carrier for oral immunization of Japanese flounder (Paralichthys olivaceus) against lymphocystis disease virus, Fish Shellfish Immunol. 30 (2011) 109–117. S. Saurabh, P.K. Sahoo, Lysozyme: an amportant defence molecule of fish innate immune system, Aquacult. Res. 39 (2008) 223–239. B. Zhu, G.L. Liu, Y.X. Gong, F. Ling, G.X. Wang, Protective immunity of grass carp immunized with DNA vaccine encoding the Vp7 gene of grass carp reovirus using carbon nanotubes as a carrier molecule, Fish Shellfish Immunol. 42 (2015) 325–334. C.A. Tabrizi, P. Walcher, U.B. Mayr, T. Stiedl, M. Binder, J McGrath, Bacterial ghosts–biological particles as delivery systems for antigens, Nucleic Acids and Drugs. Curr Opin Microbiol 15 (6) (2004) 530–537.
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Full length article
Different concentrations of Edwardsiella tarda ghost vaccine induces immune responses in vivo and protects Sparus macrocephalus against a homologous challenge
T
Maocang Yana,b,1, Jinyu Liua,2, Yu Lia,2, Xuepeng Wanga,c,∗, Heng Jianga, Hao Fanga, Zhiming Guoa, Yongcan Suna a
Shandong Provincial Key Laboratory of Animal Biotechnology and Disease Control and Prevention & Shandong Provincial Engineering Technology Research Center of Animal Disease Control and Prevention, Shandong Agricultural University, Taian, 271018, PR China Zhejiang Mariculture Research Institute, Zhejiang Key Laboratory of Exploitation and Preservation of Coastal Bio-Resource, Wenzhou, 325005, PR China c Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao National Laboratory for Marine Science and Technology, Qingdao, 272000, PR China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Edwardsiella tarda Bacterial ghosts Sparus microcephalus Humoral immunity Cellular immunity Non-specific immunity
Bacterial ghosts (BGs) can be generated by the controlled expression of the PhiX174 lysis gene E in gramnegative bacteria. They are intriguing vaccine candidates since ghosts retain functional antigenic cellular determinants often lost during traditional inactivation procedures. Here we prepared Edwardsiella tarda ghost (ETG) and tested different concentrations in vaccination trials. The results showed that serum IgM antibody titers were significantly higher in three different concentration immunization groups than control group (P < 0.05), However, there was no significant (P > 0.05) difference between the immunized groups. The phagocytic percentage (PP) was significantly higher (P < 0.05) in ETG immunized groups than in the control group from 3 days post-treatment. The PP continued to rise with time until day 21, when the values of three ETG immunized groups were 45.7%,51.2% and 50.7%, respectively. In addition, phagocytic index (PI) was significantly higher (P < 0.05) in ETG immunized groups than in the control group after 7 days post-treatment. However, there was no significant (P > 0.05) difference of PP or PI between immunized groups. In addition, non-specific immune immunity, such as acid phosphatase, alkaline phosphatase, superoxide dismutase and lysozyme activities displayed a similar pattern in all immunized groups, all immunized fish showed significantly higher activities than control group fish (P < 0.05). Most importantly three ETG immunized groups were all significantly more protected against the E. tarda challenge (19/25, 76% survival), (21/25, 84% survival) and (20/25, 80% survival) respectively, compared to (9/25, 36% survival) survival in the control group, but there was no significant (P > 0.05) difference of survival rate (SR) or relative percent survival (RPS) between immunized groups. All these results suggest that an ETG could stimulate cellular and humoral immunity, and could be used as a vaccine candidate in S.m. In summary, ETG can protect fish from Edwardsiellosis, and there is no significant difference in SR and RPS when three different concentrations of ETG are used, so it can easily be developed as a vaccine for mechanical and artificial operations.
1. Introduction Sparus macrocephalus (S.m) belongs to Sparus, and is mainly distributed in the western Pacific, extending from Hokkaido, Japan in the north to Taiwan and Hainan Island in the south of China. It is an economically important fish cultured in parts of Asia including Japan, Korea, China, and some other countries of Southeast Asia [1]. With the scale development of the aquaculture, the frequent occurrence of
∗
Corresponding author. Shandong Agricultural University, Taian, 271018, PR China. E-mail address: [email protected] (X. Wang). First Author: Maocang Yan. 2 Co-first author: Jinyu Liu, Yu Li. 1
https://doi.org/10.1016/j.fsi.2018.06.034 Received 29 March 2018; Received in revised form 11 June 2018; Accepted 15 June 2018 Available online 19 June 2018 1050-4648/ © 2018 Elsevier Ltd. All rights reserved.
diseases has become one of the key points which restricts the development of aquaculture industry. It has been confirmed that Edwardsiella tarda (E. tarda), is the main pathogen of S.m [2]. As we know, E. tarda is the intracellular, rod-shaped Gram-negative, non-capsulated, motile, facultative anaerobic bacteria, and is widely distributed in aquatic environments and is infectious to a variety of animals including humans, fish, amphibians, reptiles, and birds [3]. In recent years, chemotherapy has been used effectively in
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controlling fish infections [4], however, there is significant concern regarding food safety following chemotherapeutic interventions in addition to the danger of selecting for antibiotic-resistant E. tarda isolates which have been reported worldwide [5]. These concerns have prompted the development of novel vaccination strategies for the control of E. tarda infections. In order to better prevent and control the infection of E. tarda, a biological method, such as vaccines, must be developed to control it. Over the past ten years, vaccines have become one of the most important methods to prevent the infection of fish and other animals [3]. As it is reported, vaccines for prevention of E. tarda are various and show different protective efficiency. These vaccines, in common use, are formalin treated or heat-inactivated, effecting the physical and chemical structure of bacterial surface antigen, changing the original anti immunogenicity and affecting the immune effects of vaccine [6,7]. In recent years, the bacterial ghost vaccine, a new type of vaccine, has attracted the considerable attention of research workers. Bacterial ghosts may be generated by the controlled expression of the PhiX174 lysis gene E in Gram-negative bacteria to form the free cytoplasm and reproduction of bacterial shell. Thus it preserves the original bacterial cell morphology, bacterial surface antigen, and adhesion properties. At the same time, bacterial ghosts contain LPS, adipose, peptidoglycans, and other natural immunostimulating complexes. It can induce stronger humoral immunity, cellular immunity, and mucosal immune response without the use of the adjuvant [3]. Although studies have reported the superiority of E. tarda bacterial ghost vaccine at home and abroad, their non-specific immune parameters have not been reported [3,8]. In this study, we analyze changes in specific and non-specific immune parameters in fish vaccinated with different concentrations of bacterial ghost vaccine, using S.m. These immunological techniques and concentration selection would be useful in the development of E. tarda ghost vaccine. The protective ability of E. tarda ghost vaccine was investigated on S.m to provide the theoretical basis for the application of E. tarda ghost vaccine and to provide new ideas for the immunization of marine fish bacterial diseases.
Staphylococcus aureus was inoculated into common broth agar slants for 24 h, inactivated by 0.5% formaldehyde for 24 h, washed with sterile saline 3 times, adjusted to 1.0 × 108 CFU/fish and stored in 4 °C [10]. The phagocytosis was used as the phagocytic activity of leukocytes, which was determined using the same method following our earlier research [9]. Then 100 μL of anticoagulant was added to 100 μL of S. aureus. Then shacked it and put it into the water at 25 °C for 60min with shacking once every 10 min. After this, the mixture was drawn with a pipette on the slides, dried and fixed with methanol for 10 min, and then Giemsa stained for 1 h. Finally, slides were washed and dried to observe with an oil microscope. The phagocytic percentage (PP) and phagocytic index (PI) were calculated according to the following equations.
2. Materials and methods
PP =
The number of cells involved in phagocytosis in one hundred phagocytes × 100% 100
PI =
The total number of bacteria in phagocytes × 100% The number of cells that had phagocyted bacteria
Fish were divided into four groups; three immunization groups (A to C groups were respectively immunized with ETG at 1 × 105 CFU/fish, 1 × 106 CFU/fish, 1 × 107 CFU/fish, n = 60 for each group), and control fish (group D), treated with phosphate-buffered saline (PBS), respectively. Before the experiment, fish were not fed for 24 h. Fish in immunization groups were intraperitoneally injected immunization with 0.2 ml bacterial ghost vaccine while fish in the control group were injected with 0.2 ml saline. Immune responses were measured after 0, 3, 7, 14, 21, 28 days of treating. From each group, six fish were taken to draw blood from the caudal vein (on day 0, two fish were taken randomly from each group before treatment). The blood of each fish was divided into two parts. One part of the blood was allowed to stand at room temperature for 2 h and was centrifuged at 4000 r/min for 10 min at 4 °C. After that, the upper serum was collected for the determination of serum antibody level. The other part of blood was made to the anticoagulant with heparin for the determination of phagocytic activity of blood leucocytes. During the experiment, the water was controlled at about 24 °C, salinity at 25–26, pH 8.2, and the air was continuously aerated. 2.4. Phagocytic activity
2.1. Bacterial strains and culture conditions E. tarda strain SM (GenBank accession number MH390702) was isolated from S.m which were cultured in the net cage in the Yueqing Gulf [2]. E. tarda was grown in tryptic soy broth (TSB) (Oxoid Ltd., Basingstoke, Hampshire, England) at 28 °C. Transformed E. tarda cells were grown in TSB containing 100 μg/ml ampicillin (Sigma, MO, USA) at 28 °C. Incubation temperatures for repression and expression of the lysis gene during transformation were 28 °C and 42 °C, respectively.
2.5. Antibody response assessment The antibody level was determined by an enzyme-linked immunosorbent assay (ELISA) following our earlier research [3]. Briefly, 100 μL carbonate-bicarbonate buffer (pH 9.6) containing 1 × 105 formalin-killed E. tarda were added to respective microtiter plate wells and incubated for 20 h at 37 °C. The plates were washed three times with PBS containing 0.05% Tween 20 (PBS-T) and blocked for 24 h at 4 °C with blocking buffer (0.5% BSA in PBS-T). After the plate was washed three times, sera from immunized and control fish was added to the plates, then incubated at 37 °C for 1 h and washed 3 times with PBS-T, then probed with 100 μL self-made rabbit anti-sparus-IgM antibody incubated at 37 °C for 1 h, and washed 3 times with PBS-T, added the horseradish peroxidase-conjugated goat anti-rabbit IgG for 1 h at 37 °C. Plates were washed four times with PBS-T and binding visualized by adding TMB (Tiangen, Beijing, China) according to the manufacturer's instructions (100 μL/well). The plates were incubated at room temperature for 20 min and the reaction stopped with 100 μL of 2 M H2SO4 and the absorbance read at 450 nm.
2.2. Production of bacterial ghost vaccine Bacterial ghost vaccines were generated following our earlier research paper using the same method [3]. Morphological features of E. tarda and ETG were examined by scanning electron microscopy (Hitachi S-2400) and transmission electron microscopy (7650; Hitachi) as previously described [3]. There were 1 × 1010 cells per milliliter of ETG vaccine were prepared. 2.3. Immunization protocol and challenge infection S.m were fed in Zhejiang Marine Fisheries Research Institute. They were the same size and had no diseases, their weight was 150 g ± 10 g. These fish were fed daily using the compound feed bought from Fuzhou Haima Feed Co. Ltd. The water was changed every morning and the water was kept running for 1 h. The temperature of the water was at about 24 °C. The final experiment was carried out after two-weeks of feeding observation.
2.6. Non-specific immune parameters assay Acid phosphatase (ACP), alkaline phosphatase (AKP), superoxide dismutase (SOD) and lysozyme (LZM) activity were determined at 0, 3, 468
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7, 14, 21, 28 days using the detection kits (Nanjing Jiancheng Bioengineering Institute, China) according to the manufacturer's protocols. One ACP and AKP activity unit was expressed as the production of 1 mg phenol by reaction between every 100 ml of serum and substance in 30 min.
The morphology of the cell, including all cell surface structures, were unaffected by lysis. Pores ranging from 200 to 500 nm in diameter were observed in E. tarda ghost by scanning electron microscopy (Fig. 1A). The loss of cytoplasmic material and structural integrity were observed in E. tarda ghost by transmission electron microscopy (Fig. 1A, C).
2.7. Challenge
3.2. Antibody response analysis
The vaccine groups were injected with 100 μL containing E. tarda SM 2.0 × 107 CFU/fish (about 5-fold LD50). The cumulative mortality was recorded for 7 days post-challenge, and the relative percent survival (RPS) was calculated as follows: make an equation RPS = 1–(% mortality of vaccinated fish) × (% mortality of control fish) – 1 × 100% [9].
S.m fish were immunized with different concentrations of ETG (groups A, B, and C) on day 0, then the sera were collected and IgMspecific E. tarda antibody levels were examined by ELISA. Specific antiE. tarda IgM antibodies were detected in the serum of fish immunized with ETG, and no antibody reactivity could be detected in the serum collected from control fish (Fig. 2). The results showed a statistical IgM antibody (P < 0.05) increase in all fish immunized with ETG, and that they developed significantly (P < 0.05) higher IgM antibody titers than fish treated with PBS throughout the whole period. However, there was no significant (P > 0.05) difference of antibodies between immunized groups A, B, and C. From the 7 t h day of the experiment, the antibody level of each experimental group was significantly higher than that of the control group until the 28 t h day, and the antibody level reached the highest level at the 21st day.
2.8. Statistical analyses Student's t-test was used, with significance set at the 95% level, and the different alphabet marked on groups indicated P < 0.05. Error bars on graphs indicate the S.D. 3. Results 3.1. Identification of bacterial ghosts
3.3. Effects on the phagocytic activity of leukocytes
E. tarda ghost were successfully generated and identified by an electron microscopic analysis (EMA, Fig. 1). EMA of protein E-lysed E. tarda cells revealed no gross alterations in cellular morphology compared to unlysed cells (Fig. 1B, D) except for the lysis pore (Fig. 1A).
The phagocytic activity of leukocytes in the blood of fish improved significantly after immunization with ETG vaccine (Fig. 3). The PP in the control group was 13.6%–15.8% and the PI was 1.95–2.19. There was no significant (P > 0.05) change. After 3 days of immunization,
Fig. 1. Evaluation of E. tarda and E. tarda ghosts by SEM (A and B) and TEM (C and D). 469
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control group at the 7 t h day after immunization. At the 14 t h day after immunization, the PI reached the highest level in the three experimental groups and maintained a high level until the 28 t h day after immunization. After 7 days, PP and PI in group B and group C were significantly higher than those in group A and there was no significant (P > 0.05) difference between group B and group C. To summarize, the result showed a statistical PP and PI (P < 0.05) increase in all fish immunized with ETG, and developed significantly higher (P < 0.05) PP and PI than fish treated with PBS during the whole period. 3.4. Change of non-specific immune parameters assay The ACP, AKP, LMZ and SOD activities were recorded in different groups, as shown in Fig. 4. ACP activity (ACPa) of control group D was not significantly different from 4.04 to 4.44 U/100 ml during the whole period. However, ACPa of group A was significantly higher than group B (P < 0.05, Fig. 4A), which was significantly higher than group C (P < 0.05, Fig. 4A), and group C was significantly higher than group D (P < 0.05, Fig. 4A). AKP activity (AKPa, Fig. 4B) of serum in vaccinated groups initially remained unchanged until after 7 days and then it increased as time extended to 28 days, whereas the AKPa of the control group (D) was not significantly different from 1.51 to 1.92 U/100 ml during the whole experiment. However, the AKPa of immunized groups (A and B) were increased 7 days post vaccine, and all vaccinated groups were significantly higher than group D after 14 days (P < 0.05). AKPa reached the highest level on 28 days post-treatment. Meanwhile, the AKPa of group A was significantly more increased than groups B and C in the whole experiments. Lysozyme activity (LZMa) of the control group (D) was not changed significantly during the whole period from 6.22 to 6.99 U/fish (Fig. 4C). However, the LZMa of vaccinated groups (A, B, and C) was increased after being vaccinated with ETG, and all vaccine groups were significantly higher than D after 3 days (P < 0.05), LZMa reached the highest level on the 21st day and maintained high levels until day 28. Whereas, the LZMa of group A, B and C were not different (P > 0.05). The SOD activity (SODa, Fig. 4D) of the control group (D) was not significantly different from 162.10 to 166.78 U/100 ml during the whole experiment. However, SODa of all vaccine groups was significantly higher than D after 7 days (P < 0.05), which reached the highest level on day 21. In addition, SODa of vaccinated groups (A and B) showed significantly higher than group C (P < 0.05), which was significantly higher than group D (P < 0.05) throughout the whole experiment period.
Fig. 2. Anti-E. tarda titers in fish following immunization with either ETG or PBS. Data are means for at least five independent assays and presented as the means ± SD. Different alphabet a, b and c indicate significant difference of antibody levels at P < 0.05.
the PP in the immunized group was significantly (P < 0.05) higher than that in the control group. With the prolongation of time, the PP in each experimental group continued to increase. At the 7 t h day after immunization, the PP in each group was significantly (P < 0.01) higher than in the control group and maintained a higher level. At the 21st day after immunization, the highest PP values were 45.7%, 51.2%, and 50.7% respectively in the three experimental groups A, B and C. At the 28 t h day after immunization, the PP in the three experimental groups decreased, but there was still significant (P < 0.05) difference from the control group. PI was slightly different from the control group at the 3rd day after immunization and significantly higher than that in
3.5. Bacterial challenge Survival differences (P < 0.01 to 0.05) were observed between the vaccination groups (Table 1). ETG-immunized fish of group B showed the highest survival rates (21/25, 84% survival rate, RS) and group C (20/25, 80% RS) and group A (19/25, 76% RS) were better protected than PBS-treated controls (9/25, 36% survival). The relative percent survival (RPS) of group A throughout C was 61.7%, 71.7%, and 68.3%, respectively, were significantly higher than PBS-treated controls. However there was no significant (P > 0.05) difference of SR or RPS between immunized groups. During challenge trials, dead fish showed typical clinical symptoms of E. tarda infection, E. tarda infection was confirmed in all dead fish by finding black pigments on SSA. 4. Discussions
Fig. 3. The phagocytic activity of leukocytes in Sparus microcephalus post-immunization with different concentrate ETG or PBS. (A): phagocytic percentage (PP) and (B): phagocytic index (PI). Values are representative of at least five independent experiments and are expressed as Mean ± Standard Error. Different alphabet a, b and c indicate significant difference values of phagocytic activity at P < 0.05.
Bacterial ghost was a complete cell shell of bacteria without contents, which can be used as an important means of prevention and control of fish bacterial and viral diseases [8–11]. Because of its bacterial membrane containing intact membrane proteins, fimbriae and 470
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Fig. 4. Change of non-specific immune parameters of Sparus microcephalus post-immunization with different concentrate ETG or PBS and intraperitoneally challenge with E. tarda. (A) acid phosphatase (ACP) activity, (B) alkaline phosphatase (AKP) activity, (C) lysozyme (LZM) activity and (D) superoxide dismutase (SOD) activity. Data are means for at least five assays and presented as the means ± SD. Different alphabet a, b, c and d indicate significant difference values of non-specific immune parameters at P < 0.05.
the different concentrations of E. tarda ghost vaccine, this suggests that the concentrations of ETG affect the immune response slightly. In addition, the results showed that the immunization of ETG vaccine can effectively improve the level of antibody in S.m, and reached the highest level on the 21st day post-treatment. It is reported that ETG protected tilapia from Edwardsiellosis with reaching the highest antibody level on the 21st day post-treatment too [8]. In addition, the RS and RPS of groups A throughout C were significantly higher than control group fish. All these results suggested that the ETG vaccine could protect fish from Edwardsiellosis. Phagocytosis is commonly considered as one of the most important cellular immune reactions to pathogen invasion in invertebrates [13]. The phagocytosis of tilapia is the most important non-specific cellular immunity [14]. In addition to phagocytic cells with specific immune function, the key ingredient is composed of nonspecific defense system [15]. Our earlier research found that the phagocytic activity of leucocytes of zebrafish immunized with ETG vaccine was significantly higher than the other formalin-inactivated E. tarda, the results showed that ETG vaccine can effectively activate the phagocytic ability of the organism [9]. In the present study, PP and PI increased in all fish immunized with ETG and developed significantly more PP and PI than fish treated with PBS throughout the whole period. Although there was no significant difference of PP and PI in immunized groups A, B and C during the whole observation, the PP of group B showed significantly higher than group A on days 7, 14, 21 and 28, and the PI of group B and C showed significantly higher than group A on days 14, 21 and 28. Besides the specific immune function, phagocytes play an important role in constituting non-specific defense systems. It can capture and digest pathogens that invade the body at all stages of microbial infection [16,17]. The leucocytes digesting pathogens can retain the relevant antigen information and transmit the information to the relevant lymphocytes, thereby inducing the humoral and cellular immunity in the host [18]. Bacterial ghost vaccine can effectively stimulate antibody in fish,
Table 1 Survival rates following three concentrations of ETG treated and E. tarda challenge.a Groups
A B C D
Subgroups (n=5)b 1
2
3
4
5
1 1 1 4
2 0 1 3
0 2 0 3
2 0 2 4
1 1 1 2
Survival rate (%)c
Relative percent survival (%)c
76.0a 84.0a 80.0a 36.0b
61.7a 71.7a 68.3a /b
a
Fish were infected intraperitoneally. Number of dead fish/subgroup post-challenge. c Alphabet a and b indicate significant difference of the survival rate and the relative percent survival (P < 0.05). b
other immunostimulatory components, such as lipopolysaccharide, peptidoglycan and so on can be discriminated by the related membrane receptor of dendritic cells and macrophages and be swallowed to stimulate the immune effects effectively [3]. It has been reported that fish immunity trials primarily included oral treatments, intraperitoneal injections and bath immersion [12]. E. tarda ghost vaccine usages were reported in immunized intraperitoneally in tilapia and orally immunized in mice [3,8]. Many studies have confirmed that bacterial ghost vaccine can effectively stimulate more antibody than formalininactivated E. tarda [3,8,11]. Although these vaccines were able to stimulate high antibody titer, which produced in the ETG vaccine groups had better bactericidal activity and more protective immunity to fish or mice than that produced in formalin-inactivated E. tarda group. Here we focus on the different concentrations of ETG vaccine to S.m. In this study, we found that the antibody level of S.m in immunized groups with different concentrations of ETG vaccine was significantly (P < 0.05) higher than that in the control group. However, there was no significant difference in the antibody level in fish vaccinated with 471
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rats, and other animals. Bacterial ghosts itself is also a good adjuvant, it also can induce immune enzyme and other humeral factors at the same time [9]. Although the specific immune mechanism of fish is the deficiency, a large number of studies have proved that the non-specific immune system plays a more key role than the specific immune system in the fish's anti-infection. Therefore, to defend against pathogen invasion in fish, the non-specific immunity is considered as the primary defense [19]. ACP and AKP have been used as a symbol of macrophage activation for the ability of intracellular digestion of phagocytized antigens in the immune system of invertebrates [20]. Superoxide dismutase (SOD) is a vital antioxidant enzyme, which could eliminate superoxide radicals to reduce intracellular oxidant stress levels [21]. Lysozyme is an important defense molecule of the fish innate immune system, it can activate lyse acetyl peptidoglycan in the cell wall hydrolysis of gram-positive bacteria in polysaccharide which is released, causing the formation of a hydrolase system, destruction and elimination of invading foreign objects inside the body to assume the host defense function [22]. It was found that innate immune parameters, e.g., ACP, AKP, SOD, and LMZ activities were significantly increased in all ETG immunized groups (Fig. 4). It's possible that the increased numbers of cells involved in the process or enhanced pathogen resistance led to the increase of innate immune parameter activities [23]. Besides the fact that bacterial ghosts are viable vaccine candidates, it also can be used as a carrier of drugs, nucleic acids, viral and/or bacterial antigens comprising multivalent vaccines designed to protect against animal and human diseases [24]. Ghost formulations represent an improved nonliving bacterial vaccine delivery strategy that is safe and highly immunogenic [3,11]. The data presented in this report suggests that intraperitoneal immunization with ETG provided effective immune protection against infection, suggesting that it is possible to develop an E. tarda-based bacterial-ghost vaccine for use in aquaculture.
[2]
[3] [4]
[5]
[6] [7] [8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
5. Conclusions
[16]
ETG vaccine can significantly stimulate the specific immune function and the non-specific immune system in S.m, it can protect fish from Edwardsiellosis. In addition, the concentration of ETG affects the immune response slightly, so it can easily be developed as a vaccine for mechanical and artificial operations.
[17]
[18]
Acknowledgments [19]
This work was supported by the National Natural Science Foundation of China (31402325), Funds of Shandong “Double Tops” Program, the earmarked fund for the Modern Agro-industry Technology Research System in Shandong Province (SDAIT-14-07 and SDAIT-1302), fund of the China Scholarship Council (201708370021), and special funds from the central finance to support the development of local universities. We thank Chengming Wang and Taylor Novak at Auburn University for performing manuscript editing.
[20]
[21]
[22] [23]
Appendix A. Supplementary data [24]
Supplementary data related to this article can be found at http://dx. doi.org/10.1016/j.fsi.2018.06.034. References [1] J. Zhang, F. Zhou, L.i. Wang, Q. Shao, Z. Xu, Dietary protein requirement of juvenile
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black sea bream, Sparusmacroce phallus, J. World Aquacult. Soc. 41 (2) (2010) 151–164. X. Wang, M. Yan, W. Hu, S. Chen, S. Zhang, Q. Xie, Visualization of Sparus macrocephalus infection by GFP-labeled Edwardsiella tarda, Isr. J. Aquacult. Bamidgeh 64 (2012) doi: IJA_64.2012.693. X. Wang, C. Lu, Mice orally vaccinated with Edwardsiella tarda ghosts are significantly protected against infection, Vaccine 27 (2009) 1571–1578. N. Pirarat, T. Kobayashi, T. Katagiri, M. Maita, M. Endo, Protective effects and mechanisms of a probiotic bacterium lactobacillus rhamnosus against experimental Edwardsiella tarda infection in Tilapia (Oreochromis niloticus), Vet. Immunol. Immunopathol. 113 (3–4) (2006) 339–347. T. Aoki, T. Kitao, M. Fukudome, Chemotherapy against infection with multiple drug resistance strains of Edwardsiella tarda in cultured eels, Fish Pathol. 24 (3) (1989) 161–168. X. Huang, C. Lu, Characterization of antigenicity of the outer membrane protein from Edwardsiella tarda, Chinese Journal of Immunology 18 (6) (2002) 385–387. Q. Xiong, C. Lu, Immune effect of Edwardsiella tarda on mice and Xiphophorus belleri, Chin. J. Vet. Sci. 22 (3) (2002) 251–253. R.K. Se, K.N. Yoon, Protection of Tilapia (Oreochromis mosambicus) from Edwardsiellosis by vaccination with Edwardsiella Tarda ghosts, Fish Shellfish Immunol. 20 (2006) 621–626. T. Pan, M. Yan, S. Chen, X. Wang, Effects of ten traditional Chinese herbs on immune response and disease resistance of Sciaenops ocellatus, Acta Ichthyol. Piscatoria 43 (2013) 41–49. P.K. Sahoo, B.R. Pillai, J. Mohanty, J. Kumari, S. Mohanty, B.K. Mishra, In vivo humoral and cellular reactions, and fate of injected Bacteria Aeromonas Hydrophila in freshwater prawn Macrobrachium rosenbergii, Fish Shellfish Immunol. 23 (2007) 327–340. K. Hao, X. Chen, X. Qi, X. Yu, E. Du, F. Ling, B. Zhu, G. Wang, Protective immunity of grass carp induced by DNA vaccine encoding capsid protein gene (vp7) of grass carp reovirus using bacterial ghost as delivery vehicles, Fish Shellfish Immunol. 64 (2017) 414–425. J.J. Evans, P.H. Klesius, A. Craig Shoemaker, Efficacy of Streptococcus agalactiae (group B) vaccine in Tilapia (Oreochromis niloticus) by intraperitoneal and bath immersion administration, Vaccine 22 (2004) 3769–3773. Y. Hong, X. Yang, G. Yan, Y. Huang, F. Zuo, Y. Shen, Y. Ding, Y. Cheng, Effects of glyphosate on immune responses and haemocyte DNA damage of Chinese mitten crab, Eriocheir sinensis, Fish Shellfish Immunol. 71 (2017) 19–27. S. Gallage, T. Katagiri, M. Endo, M. Maita, Comprehensive evaluation of immunomodulation by moderate hypoxia in S. Agalactiae vaccinated Nile tilapia, Fish Shellfish Immunol. 66 (2017) 445–454. A. Fazio, R. Cerezuela, M.R. Panuccio, A. Cuesta, M.A. Esteban, Vitro effects of Italian lavandula Multifida L. leaf extracts on gilthead seabream (Sparus aurata) leucocytes and SAF-1 cells, Fish Shellfish Immunol. 66 (2017) 334–344. C. Xu, E. Li, Y. Suo, Y. Su, M. Lu, Q. Zhao, J.G. Qin, L. Chen, Histological and transcriptomic responses of two immune organs, the spleen and head kidney, in nile tilapia (Oreochromis niloticus) to long-term hypersaline stress, Fish Shellfish Immunol. 76 (2018) 48–57. Q. Huang, M. Yu, H. Chen, M. Zeng, Y. Sun, T.T. Saha, D. Chen, LRFN (Leucine-rich repeat and fibronectin type-III domain-containing protein) recognizes bacteria and promotes hemocytic phagocytosis in the pacific oyster Crassostrea gigas, Fish Shellfish Immunol. 72 (2018) 622–628. Y. Shi, K. Chen, W. Ma, J. Chen, A C-reactive protein/serum amyloid P agglutinates bacteria and inhibits complement-mediated opsonophagocytosis by monocytes/ macrophages, Fish Shellfish Immunol. 76 (2018) 58–67. T. Behera, P. Swain, Alginate-chitosan-PLGA composite microspheres induce both innate and adaptive immune response through parenteral immunization in fish, Fish. Shellfish Immunology 35 (2013) 785–791. F. Yin, H. Gong, Q. Ke, A. Li, Stress antioxidant defence and mucosal immune responses of the large yellow croaker Pseudosciaena crocea challenged with Cryptocaryon irritans, Fish Shellfish Immunol. 47 (2015) 344–351. J. Tian, J. Yu, Poly(Lactic-Co-Glycolic acid) nanoparticles as candidate DNA vaccine Carrier for oral immunization of Japanese flounder (Paralichthys olivaceus) against lymphocystis disease virus, Fish Shellfish Immunol. 30 (2011) 109–117. S. Saurabh, P.K. Sahoo, Lysozyme: an amportant defence molecule of fish innate immune system, Aquacult. Res. 39 (2008) 223–239. B. Zhu, G.L. Liu, Y.X. Gong, F. Ling, G.X. Wang, Protective immunity of grass carp immunized with DNA vaccine encoding the Vp7 gene of grass carp reovirus using carbon nanotubes as a carrier molecule, Fish Shellfish Immunol. 42 (2015) 325–334. C.A. Tabrizi, P. Walcher, U.B. Mayr, T. Stiedl, M. Binder, J McGrath, Bacterial ghosts–biological particles as delivery systems for antigens, Nucleic Acids and Drugs. Curr Opin Microbiol 15 (6) (2004) 530–537.