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Analysis of the vaccine potential of a laboratory Escherichia coli strain in a Japanese flounder model
Fish & Shellfish Immunology 28 (2010) 275e280
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Fish & Shellfish Immunology journal homepage: www.elsevier.com/locate/fsi
Analysis of the vaccine potential of a laboratory Escherichia coli strain in a Japanese flounder model Shuang Cheng a, b, Xu-dong Jiao a, b, Min Zhang a, b, Li Sun a, * a b
Institute of Oceanology, Chinese Academy of Sciences, 7 Nanhai Road, Qingdao 266071, PR China Graduate University of the Chinese Academy of Sciences, Beijing, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 26 April 2009 Received in revised form 24 October 2009 Accepted 2 November 2009 Available online 10 November 2009
Escherichia coli DH5a is a genetically tailored laboratory strain that is commonly used for general cloning. In this study, the vaccine potential of DH5a was investigated. It was found that when used as a live vaccine, DH5a could afford effective protection upon Japanese flounder against Aeromonas hydrophila infection. Vaccination with purified outer membrane proteins and lipopolysaccharides of DH5a failed to induce protective immunity against A. hydrophila. Specific antibody production was observed in fish immunized with DH5a, which lasted at least 8 weeks and was enhanced by a booster injection during the vaccination process. Analysis of the transcription profiles of immune-related genes showed that vaccination with DH5a heightened the expression of the genes encoding factors that are likely involved in both specific and nonspecific immunities. Furthermore, compared to the control fish, fish vaccinated with DH5a/pAQ1, which is DH5a harboring the plasmid pAQ1 that expresses the coding element of a Vibrio harveyi antigen, exhibited significantly improved survival rates following V. harveyi and A. hydrophila challenges. These results demonstrate that DH5a possesses intrinsic immunoprotective potential against A. hydrophila. This property, together with the feature of cloning friendliness, should render DH5a useful in the construction of cross-protective vaccines. Ó 2009 Elsevier Ltd. All rights reserved.
Keywords: Aeromonas hydrophila Cross-protection DH5a Escherichia coli Vibrio harveyi
1. Introduction Aeromonas hydrophila is a Gram-negative bacterium of the family Aeromonadaceae that is found widely in freshwater and marine environments. It can be pathogenic to humans and a broad range of animals, bird, and fish. For humans, A. hydrophila is considered as an opportunistic pathogen that is more likely to infect individuals with weakened immune functions, open wounds, or pre-infections by other bacterial or viral pathogens. Human diseases associated with A. hydrophila infection include gastroenteritis, cellulitis, myonecrosis, eczema, and septicemia [1,2]. A. hydrophila has long been recognized as an important aquaculture pathogen due to its ability to infect a variety of reared fish species cultured worldwide, which include Atlantic salmon [3], channel catfish [4], Indian major carp [5,6], common carp [7], rainbow trout [8,9], and Japanese flounder [10]. Fish infected by A. hydrophila often develop hemorrhagic septicemia, ulcers, skin rot, and exophthalmia. Disease outbreaks, which usually occur under stress conditions such as those caused by poor water quality, temperature changes, and handling [11,12], can cause severe economic losses to the farming industries. * Corresponding author. Tel./fax: þ86 532 82898834. E-mail address: [email protected] (L. Sun). 1050-4648/$ e see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.fsi.2009.11.003
Control of A. hydrophila infection relies chiefly on the use of chemotherapeutic agents and vaccination. Currently, vaccines that have been tried against A. hydrophila are mostly inactivated whole bacterial cells and extracted extracellular products [12,13]. Laboratory studies have identified a number of outer membrane proteins and surface components as potential subunit vaccines [14e19]. In addition, several live attenuated A. hydrophila vaccine candidates have also been reported [20e23]. Escherichia coli DH5a is a common laboratory strain derived from the Hoffman-Berling strain 1100 [24]. It has been genetically tailored to possess a number of useful features, such as the lack of the EcoKI restrictionemodification system and deficiency in homologous recombination, which renders DH5a as a suitable host strain for general cloning [25e27]. In this study, we evaluated the immunoprotective potential of this strain and found that, when used as a live vaccine, DH5a can induce high levels of protection in Japanese flounder against A. hydrophila infection. 2. Materials and methods 2.1. Bacterial strains and growth conditions E. coli DH5a was purchased from Takara, Dalian, China. DH5a/ pAQ1 is DH5a harboring the plasmid pAQ1 that expresses the Vibrio
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harveyi protein DegQ, which is a protective immunogen and can induce immunoprotection in Japanese flounder against V. harveyi infection [28]. DH5a/pBTA1 is DH5a harboring the plasmid pBTA1 that is the control vector for pAQ1 [28]. A. hydrophila AH1 (LD50 w105.5) [29], V. harveyi T4D1 (LD50 w106.1) [30], and Yersinia ruckeri YR1 (LD50 w105.6) were pathogenic strains isolated from diseased Japanese flounder in local fish farms. All strains were cultured in LuriaeBertani broth (LB) medium [31] at 37 C (for E. coli) or 28 C (for all other strains). 2.2. Bacterial cell number determination Plate count was used to determine the number of viable bacterial cells corresponding to an OD600 of 1. In subsequent vaccination experiments, the numbers of bacterial cells were all estimated based on OD600 measurements. 2.3. Fish Healthy Japanese flounder (Paralichthys olivaceus, w11 g) were purchased from a commercial fish farm (Haiyang, Shandong, China) and maintained at 20e22 C in aerated seawater that was changed twice daily. Fish were fed daily with commercial dry pellets (purchased from Shandong Sheng-suo Fish Feed Research Center, Shandong, China). Fish were anaesthetized with tricaine methanesulfonate (Sigma, USA) prior to experiments involving injection, blood collection, or sacrifice. Before each of the infection and vaccination experiments, fish were randomly sampled for the examination of bacterial recovery from blood, liver, spleen, and kidney, and no bacteria could be detected in any of the fish examined. 2.4. Preparation of lipopolysaccharides (LPS) and outer membrane proteins (OMPs) Lipopolysaccharides were prepared according to the method of Apicella et al. [32]. Outer membrane proteins were prepared as described by Chen et al. [14]. The prepared LPS and OMPs were suspended in phosphate-buffered saline (PBS) to 500 mg ml1 and 300 mg ml1, respectively.
(the control) was injected with 100 ml of PBS. The fish were boosted and challenged with AH1 as described above. For vaccination with DH5a/pAQ1 and DH5a/pBTA1, the cells were cultured in LB medium and resuspended in PBS as described above for vaccination with DH5a. Japanese flounder were divided randomly into four groups named AeD; groups A and B were administered via i.p. injection with 100 ml of DH5a/pAQ1, while groups C and D were administered with 100 ml of DH5a/pBTA1 (vector control). Fish were boosted as above. Five weeks after the initial immunization, groups A and C were challenged with T4D1, while groups B and D were challenged with AH1 as described above. For all vaccinations, fish were monitored for mortality for 18 days post-challenge, and relative percent of survival (RPS) was calculated according to the following formula: RPS ¼ {1 (% mortality in vaccinated fish/% mortality in control fish)} 100 [34]. All vaccinations, except those with LPS and OMPs, were replicated, and the mean accumulated mortality and RPS are given in the results. 2.7. Analysis of bacterial dissemination in fish blood and tissues DH5a was cultured in LB medium and resuspended in PBS as described above. Japanese flounder were i.p. injected with 100 ml of DH5a suspension or PBS. The peritoneal fluids, blood, liver, kidney, and spleen of the fish (4 at each time point) were taken aseptically at 2- to 4-day intervals for a period of 16 days post-infection as described previously [28]. The tissues were homogenized in PBS. The homogenates, blood, and peritoneal fluids were diluted serially in PBS and plated in triplicate on LB agar plates. After incubation at 37 C for 36 h, the colonies that appeared on the plates were enumerated. The genetic nature of the colonies was verified by PCR analysis using primers specific to E. coli. The PCR products were randomly selected for DNA sequencing. 2.8. Western immunoblotting analysis
Histopathology was performed exactly as described previously [33].
Western immunoblotting was performed as described previously [28]. Briefly, proteins and LPS were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE); after electrophoresis, the proteins and LPS were transferred to nitrocellulose membranes. Immunoblotting was performed as described previously [28] using serum from fish vaccinated with DH5a.
2.6. Vaccination
2.9. Whole cell enzyme-linked immunosorbent assay (ELISA)
For all vaccination experiments, the group size is 20. For vaccination with live DH5a without boost, DH5a was cultured to an OD600 of 0.8 in LB medium, washed, and resuspended in PBS to w109 CFU ml1. Japanese flounder were divided randomly into six groups named AeF; groups AeC were administered via intraperitoneal (i.p.) injection with 100 ml of DH5a suspension, while groups DeF (the controls) were injected with 100 ml of PBS. Five weeks post-immunization, groups A and D, B and E, and C and F were challenged via i.p. injection with A. hydrophila AH1 (8 106 CFU), Y. ruckeri YR1 (1 107 CFU), and V. harveyi T4D1 (2.5 107 CFU). Vaccination with live DH5a with boost was performed in the same fashion, except that a booster injection was carried out at the 20th day after the initial immunization. For vaccination with LPS and OMPs, Japanese flounder were divided randomly into three groups named AeC. Groups A and B were administered via i.p. injection with 100 ml of LPS and OMP suspension (prepared as described above), respectively. Group C
Sera were collected from vaccinated fish (five at each time point) at various time points post-vaccination. As a control, sera were also collected from pre-immune fish. Sera were diluted serially in two-fold in PBS. Whole cell ELISA was performed as described previously [30]. Briefly, AH1 was cultured in LB medium to midlogarithmic phase and resuspended in PBS. The AH1 suspension was added to an ELISA plate. After blocking with bovine serum albumin (BSA), the cells in the plate were treated with PBS (the control) or diluted serum from vaccinated or unvaccinated fish. The cells were then treated with mouse anti-Japanese flounder IgM monoclonal antibody (Aquatic Diagnostic Ltd., UK). After incubation at 37 C, horse-radish peroxidase-conjugated goat anti-mouse IgG (Bios, Beijing, China) was added to the plate. Color development was performed using the TMB Kit (Bios, Beijing, China). The plate was read at 450 nm, and positive readings were defined as at least twice that of the control. Antibody titer was presented as the highest dilution that gave rise to positive reading.
2.5. Histopathological analysis
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2.10. Quantitative real-time reverse transcriptase PCR (qRT-PCR) Japanese flounder were vaccinated with DH5a or PBS as described above. Spleen was taken from five fish at 24 h postchallenge. Total RNA was extracted from the spleen by using the RNAprep Tissue/Bacteria Kit (Tiangen, Beijing, China). One microgram of total RNA was used for cDNA synthesis with the Superscript II reverse transcriptase (Invitrogen, USA). qRT-PCR was carried out in an ABI 7300 Real-time Detection System (Applied Biosystems) by using the SYBR ExScript qRT-PCR Kit (Takara, Dalian, China) as described previously [30]. Each assay was performed in triplicate with b-actin RNA as the control. The primers used for qRT-PCR of bactin and the immune-related genes were as described previously [30]. All data are given in terms of relative mRNA, expressed as means plus or minus standard errors of the means (SE). 2.11. Statistical analysis All statistical analyses were performed by using SPSS 15.0 software (SPSS Inc., USA). Differences in antibody titers and transcription levels of the immune-related genes were analyzed with Student's t-test; differences in mortality were determined with Chi-square test. In all cases, the significance level was defined as P < 0.05.
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Table 1 Summary of the cumulative mortality and relative percent survival (RPS) of Japanese flounder immunized with different vaccines. Vaccine
Challenging organism
Challenging timea
Cumulative RPS (%) mortality (%)
PBS PBS PBS DH5a DH5a DH5a PBS with boost DH5a with boost DH5a with boost DH5a with boost DH5a with boost LPS OMPs DH5a/pAQ1 DH5a/pAQ1 DH5a/pBTA1 DH5a/pBTA1
Aeromonas hydrophila Yersinia ruckeri Vibrio harveyi A. hydrophila Y. ruckeri V. harveyi A. hydrophila A. hydrophila A. hydrophila A. hydrophila Y. ruckeri A. hydrophila A. hydrophila A. hydrophila V. harveyi A. hydrophila V. harveyi
5 week p.v. 5 week p.v. 5 week p.v. 5 week p.v. 5 week p.v. 5 week p.v. 5 week p.v. 5 week p.v. 8 week p.v. 12 week p.v. 5 week p.v. 5 week p.v. 5 week p.v. 5 week p.v. 5 week p.v. 5 week p.v. 5 week p.v.
85 93 78 23 60 73 88 10 13 23 55 85 80 13 5 83 75
a
73 35 6 89 83 74 41 11 16 84 93
p.v., post-vaccination.
recovered from the blood and tissues of DH5a-vaccinated fish at any of the examined time points.
3. Results
3.3. Effect of boost on the immunoprotectivity of DH5a
3.1. Immunoprotective analysis of DH5a as a live vaccine
Since DH5a appeared to be retained transiently in vivo, we examined whether a booster during the vaccination process would enhance the immunoprotective effect of DH5a. The results showed that, with a booster injection, DH5a-immunized fish exhibited an accumulated mortality of 10% upon challenging with A. hydrophila at 5 weeks post-vaccination, whereas the control fish exhibited an accumulated mortality of 88%. Hence, vaccination with DH5a followed by a boost produces an RPS of 89% (Table 1). To examine the duration of protection, fish were also challenged at 8 and 12 weeks post-vaccination, and the resulting cumulative mortalities were 13% and 23%, respectively, which correspond to an RPS of 83% and 74%, respectively (Table 1). The effect of boost on the immunoprotectivity of DH5a against Y. ruckeri was also examined. The results showed that, with
Preliminary studies in our laboratory showed that Japanese flounder pre-exposed to DH5a challenge exhibited enhanced survival against subsequent infection by A. hydrophila (Cheng S and Sun L, unpublished data). This observation led us to investigate the possibility of using DH5a as a vaccine. For this purpose, we first analyzed the virulence potential of DH5a using Japanese flounder as an animal model. The results showed that i.p. injection of 109 CFU of DH5a into Japanese flounder caused no mortality over a period of 20 days post-challenge. Morphological and histopathological examinations indicated that fish survived the challenge displayed no apparent damages in the spleen, liver, and kidney. We next examined the immunoprotective potential of DH5a. To this end, Japanese flounder were vaccinated via i.p. injection with live DH5a, followed by challenging with A. hydrophila and two other aquaculture pathogens, Y. ruckeri and V. harveyi, respectively. The results showed that the cumulative mortalities of the DH5avaccinated groups exposed to A. hydrophila, Y. ruckeri, and V. harveyi challenges were 23%, 60%, and 73%, respectively, while the cumulative mortalities of the control groups, which had been mockvaccinated with PBS, were 85%, 93%, and 78%, respectively (Table 1). Hence, the immunoprotective efficacies, in terms of RPS, of DH5a against A. hydrophila, Y. ruckeri, and V. harveyi were 73%, 35%, and 6%, respectively. 3.2. Survival of DH5a in vivo the vaccinated fish To determine the in vivo dissemination and survival ability of DH5a after i.p. injection, DH5a- and PBS-vaccinated fish were examined for bacterial recovery from the peritoneal fluids, blood, liver, spleen, and kidney at various times post-vaccination. The results showed that for PBS-vaccinated fish, no bacteria could be recovered from the blood or tissues at any of the examined time points. For DH5a-vaccinated fish, DH5a was recovered from peritoneal fluids in the first 12 days post-vaccination, during which time the numbers of recovered cells decreased rapidly with time, especially during the first 6 days (Fig. 1). No bacterial cells could be
Fig. 1. Recovery of DH5a from the peritoneal cavity of vaccinated fish. Japanese flounder were vaccinated via intraperitoneal injection with DH5a, and bacterial recovery from the peritoneal fluids was determined at various time points postvaccination. Data are means for four fish and presented as the means SE.
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a booster injection, the accumulated mortality of the vaccinated group was reduced to 55%, which corresponds to an RPS of 41%. 3.4. Immunoprotective analysis of DH5a lipopolysaccharides (LPS) and outer membrane proteins (OMPs) Since vaccination with live DH5a afforded significant protection upon flounder against A. hydrophila infection, we examined whether the LPS and OMPs of DH5a were also immunoprotective. The results showed that the accumulated mortalities of the fish immunized with LPS, OMPs, and PBS (the control) were 85, 80, and 95% respectively. Hence, the levels of protection induced by vaccination with DH5a LPS and OMPs were, in terms of RPS, 11 and 16% respectively. Western immunoblotting analysis showed that some OMPs and cytoplasmic proteins, but not periplasmic proteins or LPS, could react with the serum from fish vaccinated with DH5a (Fig. S1 in the supplemental material). 3.5. Immune response elicited by vaccination with DH5a 3.5.1. Serum antibody production Whole cell ELISA indicated that production of serum antibodies against A. hydrophila occurred in Japanese flounder vaccinated with DH5a with or without boost (Fig. 2). In both cases, specific antibodies were detected at five to eight weeks post-vaccination. The antibody titers of the boosted fish were the same as those of unboosted fish at seven weeks post-vaccination but were 2- to 4fold higher than those of the unboosted fish at 5, 6, and 8 weeks post-vaccination. For the boosted fish, specific antibodies could still be detected at 12 weeks post-vaccination but at a lower level than that observed at 8 weeks post-vaccination. 3.5.2. Expression of immune-related genes To investigate the effect of DH5a vaccination on the expression of immune-related genes, qRT-PCR was carried out to analyze the transcription levels of the genes encoding CD8a, type I interferon (IFN), interleukin 1b (IL-1b), major histocompatibility complex (MHC) class Ia and class IIa, immunoglobulin M (IgM), IFN-g, and IFN-induced Mx gene (Mx) in the spleen of DH5a- and PBS-vaccinated fish. The results showed that expressions of all the examined genes, especially that of IgM, were significantly enhanced in DH5avaccinated fish compared to those in PBS-vaccinated fish (Fig. 3). 3.6. DH5a as a cross-protective vaccine in the form of a carrier of a heterologous antigen In a previous study, we have constructed a plasmid pAQ1 that expresses the V. harveyi protein DegQ, which is a protective
Fig. 2. Serum antibody titers in DH5a-vaccinated fish. Japanese flounder were vaccinated with DH5a with or without a booster injection, and sera were collected at 5e8 weeks post-vaccination. As a control, sera were also collected from unvaccinated fish. Serum antibodies against AH1 were determined by whole cell ELISA. Antibody titers represent the highest dilutions that gave rise to positive readings.
Fig. 3. Expression of immune-related genes in DH5a-vaccinated fish. Japanese flounder were vaccinated with DH5a and challenged with AH1. Total RNA was extracted from the spleens of the fish at 24 h post-challenge and used for qRT-PCR. The mRNA level of each gene was normalized to that of b-actin. For each gene, the mRNA level of the control fish was set as 1. Data are means for five assays and presented as the means SE. **, P < 0.001; *, P < 0.05.
immunogen [28]. DH5a harboring pAQ1 (DH5a/pAQ1) can secrete recombinant DegQ into the extracellular milieu and is highly effective against V. harveyi infection when used as a live vaccine [28]. In this study, we examined the cross-protective potential of DH5a/pAQ1 against infections by V. harveyi and A. hydrophila. The results showed that the cumulative mortalities of DH5a/pAQ1- and DH5a/pBTA1 (the vector control)-vaccinated fish were 5% and 75% respectively, upon V. harveyi challenge and 13% and 83% respectively, upon A. hydrophila challenge. Hence, fish vaccinated with DH5a/pAQ1 exhibited an RPS of 93% and 84%, respectively, upon challenging with V. harveyi and A. hydrophila. 4. Discussion In this study, we demonstrated that vaccination with live E. coli DH5a could confer significant protection in Japanese flounder against A. hydrophila infection. Several live A. hydrophila vaccines, in the form of attenuated A. hydrophila cells, have been reported previously [20e22]. DH5a was comparable to these vaccines in terms of immunoprotection efficacy; however, DH5a has one advantage over these attenuated vaccines in that it is much less risky, because, as demonstrated in our study, DH5a lacks infectivity and could not disseminate into the blood and tissues of Japanese flounder even following i.p. injection. The rapid decrease in the number of bacterial cells recovered from the peritoneal cavity following i.p. injection and the eventual clearance of the inoculated bacterial population from the fish suggest that DH5a cannot replicate inside the vaccinated fish. The transient in vivo retention of DH5a may in part account for the relatively weaker protection elicited by a single immunization. A booster injection enhances the immunoprotection most likely by renewing the presence of DH5a and thus prolonging and heightening the immune response. In line with the enhanced immunity induced by vaccination with a booster injection, the boosted fish exhibited higher levels of antibody titers compared to unboosted fish, which suggests that the humoral immune response may play an important role in DH5a-mediated protection. Consistent with the production of specific antibodies in fish vaccinated with DH5a, the expression of the IgM gene was significantly enhanced in these fish. In addition to the IgM gene, genes encoding MHC Ia, MHC IIa, CD8a, IFN, IL-1b, IFN-g, and Mx were also upregulated, though to lower levels, by
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DH5a. These results suggest the possibility that immunization with DH5a provokes humoral, cellular, and innate immune responses. Cross-species immunoprotection involving A. hydrophila has been reported previously by Fang et al. [15] and Vivas et al. [23], the former showed that vaccination with an A. hydrophila major adhesin conferred protection upon blue gourami against A. hydrophila and Vibrio anguillarum challenges, while the latter showed that an aroA mutant of A. hydrophila elicited protective immunity in rainbow trout against infection by Aeromonas salmonicida. In our study, the observed strong immunoprotection of DH5a against A. hydrophila suggests that DH5a may possess certain antigenic elements that are shared by A. hydrophila. It is known that surfaceexposed cellular components, especially outer membrane proteins and LPS, can be immunogenic and in some cases are protective. A study by Hirst et al. has demonstrated that the iron-regulated outer membrane proteins of A. salmonicida are good vaccine candidates and could afford protection against furunculosis [16]. In our case, we found that some OMPs and cytoplasmic proteins were antigenic, yet the extracted OMPs, as well as LPS, failed to induce immunoprotectivity. These results suggest that the OMPs and LPS of DH5a, at least when used alone, cannot elicit effective immune responses that are required to block A. hydrophila infection. However, it is possible that LPS and OMPs may contribute to the overall immunoprotectivity of live DH5a. E. coli serving as a producer and delivery vehicle of heterologous antigens has been documented previously [35,36]. Onate et al. [37] and Andrews et al. [38] have reported that vaccination of mice with live E. coli producing a Brucella abortus antigen elicited strong protective immunity. In our study, we found that DH5a/pAQ1, a carrier of a V. harveyi antigen, is protective not only against V. harveyi but also against A. hydrophila challenges. The fact that the level of protection against A. hydrophila induced by DH5a/pAQ1 is comparable to that induced by DH5a indicates that production of the V. harveyi antigen does not affect the natural vaccine property of DH5a. With these results, it is reasonable to speculate that, since DH5a is a strain suitable for plasmid maintenance and thus a producer of potentially any antigenic proteins, cross-protective vaccines against A. hydrophila and other pathogens might be constructed in the form of recombinant DH5a engineered to produce the antigen of interest. In conclusion, the results of this study demonstrate that the E. coli strain DH5a is an effective vaccine candidate against A. hydrophila infection in Japanese flounder, and that the unique genetic features and the immunoprotective potential of DH5a can be exploited for the construction of vaccines with cross-protective qualities.
Acknowledgements This work was supported by the National Basic Research Program of China grant 2006CB101807.
Appendix. Supplementary material Supplementary data associated with this article can be found in the online version at doi:10.1016/j.fsi.2009.11.003.
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[37] Onate AA, Vemulapalli R, Andrews E, Schurig GG, Boyle S, Folch H. Vaccination with live Escherichia coli expressing Brucella abortus Cu/Zn superoxide dismutase protects mice against virulent B. abortus. Infect Immun 1999;67:986e8. [38] Andrews E, Salgado P, Folch H, Onate A. Vaccination with live Escherichia coli expressing Brucella abortus Cu/Zn superoxide-dismutase: II. Induction of specific CD8þ cytotoxic lymphocytes and sensitized CD4þIFN-g-producing cell. Microbiol Immunol 2006;50:389e93.
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Analysis of the vaccine potential of a laboratory Escherichia coli strain in a Japanese flounder model Shuang Cheng a, b, Xu-dong Jiao a, b, Min Zhang a, b, Li Sun a, * a b
Institute of Oceanology, Chinese Academy of Sciences, 7 Nanhai Road, Qingdao 266071, PR China Graduate University of the Chinese Academy of Sciences, Beijing, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 26 April 2009 Received in revised form 24 October 2009 Accepted 2 November 2009 Available online 10 November 2009
Escherichia coli DH5a is a genetically tailored laboratory strain that is commonly used for general cloning. In this study, the vaccine potential of DH5a was investigated. It was found that when used as a live vaccine, DH5a could afford effective protection upon Japanese flounder against Aeromonas hydrophila infection. Vaccination with purified outer membrane proteins and lipopolysaccharides of DH5a failed to induce protective immunity against A. hydrophila. Specific antibody production was observed in fish immunized with DH5a, which lasted at least 8 weeks and was enhanced by a booster injection during the vaccination process. Analysis of the transcription profiles of immune-related genes showed that vaccination with DH5a heightened the expression of the genes encoding factors that are likely involved in both specific and nonspecific immunities. Furthermore, compared to the control fish, fish vaccinated with DH5a/pAQ1, which is DH5a harboring the plasmid pAQ1 that expresses the coding element of a Vibrio harveyi antigen, exhibited significantly improved survival rates following V. harveyi and A. hydrophila challenges. These results demonstrate that DH5a possesses intrinsic immunoprotective potential against A. hydrophila. This property, together with the feature of cloning friendliness, should render DH5a useful in the construction of cross-protective vaccines. Ó 2009 Elsevier Ltd. All rights reserved.
Keywords: Aeromonas hydrophila Cross-protection DH5a Escherichia coli Vibrio harveyi
1. Introduction Aeromonas hydrophila is a Gram-negative bacterium of the family Aeromonadaceae that is found widely in freshwater and marine environments. It can be pathogenic to humans and a broad range of animals, bird, and fish. For humans, A. hydrophila is considered as an opportunistic pathogen that is more likely to infect individuals with weakened immune functions, open wounds, or pre-infections by other bacterial or viral pathogens. Human diseases associated with A. hydrophila infection include gastroenteritis, cellulitis, myonecrosis, eczema, and septicemia [1,2]. A. hydrophila has long been recognized as an important aquaculture pathogen due to its ability to infect a variety of reared fish species cultured worldwide, which include Atlantic salmon [3], channel catfish [4], Indian major carp [5,6], common carp [7], rainbow trout [8,9], and Japanese flounder [10]. Fish infected by A. hydrophila often develop hemorrhagic septicemia, ulcers, skin rot, and exophthalmia. Disease outbreaks, which usually occur under stress conditions such as those caused by poor water quality, temperature changes, and handling [11,12], can cause severe economic losses to the farming industries. * Corresponding author. Tel./fax: þ86 532 82898834. E-mail address: [email protected] (L. Sun). 1050-4648/$ e see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.fsi.2009.11.003
Control of A. hydrophila infection relies chiefly on the use of chemotherapeutic agents and vaccination. Currently, vaccines that have been tried against A. hydrophila are mostly inactivated whole bacterial cells and extracted extracellular products [12,13]. Laboratory studies have identified a number of outer membrane proteins and surface components as potential subunit vaccines [14e19]. In addition, several live attenuated A. hydrophila vaccine candidates have also been reported [20e23]. Escherichia coli DH5a is a common laboratory strain derived from the Hoffman-Berling strain 1100 [24]. It has been genetically tailored to possess a number of useful features, such as the lack of the EcoKI restrictionemodification system and deficiency in homologous recombination, which renders DH5a as a suitable host strain for general cloning [25e27]. In this study, we evaluated the immunoprotective potential of this strain and found that, when used as a live vaccine, DH5a can induce high levels of protection in Japanese flounder against A. hydrophila infection. 2. Materials and methods 2.1. Bacterial strains and growth conditions E. coli DH5a was purchased from Takara, Dalian, China. DH5a/ pAQ1 is DH5a harboring the plasmid pAQ1 that expresses the Vibrio
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harveyi protein DegQ, which is a protective immunogen and can induce immunoprotection in Japanese flounder against V. harveyi infection [28]. DH5a/pBTA1 is DH5a harboring the plasmid pBTA1 that is the control vector for pAQ1 [28]. A. hydrophila AH1 (LD50 w105.5) [29], V. harveyi T4D1 (LD50 w106.1) [30], and Yersinia ruckeri YR1 (LD50 w105.6) were pathogenic strains isolated from diseased Japanese flounder in local fish farms. All strains were cultured in LuriaeBertani broth (LB) medium [31] at 37 C (for E. coli) or 28 C (for all other strains). 2.2. Bacterial cell number determination Plate count was used to determine the number of viable bacterial cells corresponding to an OD600 of 1. In subsequent vaccination experiments, the numbers of bacterial cells were all estimated based on OD600 measurements. 2.3. Fish Healthy Japanese flounder (Paralichthys olivaceus, w11 g) were purchased from a commercial fish farm (Haiyang, Shandong, China) and maintained at 20e22 C in aerated seawater that was changed twice daily. Fish were fed daily with commercial dry pellets (purchased from Shandong Sheng-suo Fish Feed Research Center, Shandong, China). Fish were anaesthetized with tricaine methanesulfonate (Sigma, USA) prior to experiments involving injection, blood collection, or sacrifice. Before each of the infection and vaccination experiments, fish were randomly sampled for the examination of bacterial recovery from blood, liver, spleen, and kidney, and no bacteria could be detected in any of the fish examined. 2.4. Preparation of lipopolysaccharides (LPS) and outer membrane proteins (OMPs) Lipopolysaccharides were prepared according to the method of Apicella et al. [32]. Outer membrane proteins were prepared as described by Chen et al. [14]. The prepared LPS and OMPs were suspended in phosphate-buffered saline (PBS) to 500 mg ml1 and 300 mg ml1, respectively.
(the control) was injected with 100 ml of PBS. The fish were boosted and challenged with AH1 as described above. For vaccination with DH5a/pAQ1 and DH5a/pBTA1, the cells were cultured in LB medium and resuspended in PBS as described above for vaccination with DH5a. Japanese flounder were divided randomly into four groups named AeD; groups A and B were administered via i.p. injection with 100 ml of DH5a/pAQ1, while groups C and D were administered with 100 ml of DH5a/pBTA1 (vector control). Fish were boosted as above. Five weeks after the initial immunization, groups A and C were challenged with T4D1, while groups B and D were challenged with AH1 as described above. For all vaccinations, fish were monitored for mortality for 18 days post-challenge, and relative percent of survival (RPS) was calculated according to the following formula: RPS ¼ {1 (% mortality in vaccinated fish/% mortality in control fish)} 100 [34]. All vaccinations, except those with LPS and OMPs, were replicated, and the mean accumulated mortality and RPS are given in the results. 2.7. Analysis of bacterial dissemination in fish blood and tissues DH5a was cultured in LB medium and resuspended in PBS as described above. Japanese flounder were i.p. injected with 100 ml of DH5a suspension or PBS. The peritoneal fluids, blood, liver, kidney, and spleen of the fish (4 at each time point) were taken aseptically at 2- to 4-day intervals for a period of 16 days post-infection as described previously [28]. The tissues were homogenized in PBS. The homogenates, blood, and peritoneal fluids were diluted serially in PBS and plated in triplicate on LB agar plates. After incubation at 37 C for 36 h, the colonies that appeared on the plates were enumerated. The genetic nature of the colonies was verified by PCR analysis using primers specific to E. coli. The PCR products were randomly selected for DNA sequencing. 2.8. Western immunoblotting analysis
Histopathology was performed exactly as described previously [33].
Western immunoblotting was performed as described previously [28]. Briefly, proteins and LPS were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE); after electrophoresis, the proteins and LPS were transferred to nitrocellulose membranes. Immunoblotting was performed as described previously [28] using serum from fish vaccinated with DH5a.
2.6. Vaccination
2.9. Whole cell enzyme-linked immunosorbent assay (ELISA)
For all vaccination experiments, the group size is 20. For vaccination with live DH5a without boost, DH5a was cultured to an OD600 of 0.8 in LB medium, washed, and resuspended in PBS to w109 CFU ml1. Japanese flounder were divided randomly into six groups named AeF; groups AeC were administered via intraperitoneal (i.p.) injection with 100 ml of DH5a suspension, while groups DeF (the controls) were injected with 100 ml of PBS. Five weeks post-immunization, groups A and D, B and E, and C and F were challenged via i.p. injection with A. hydrophila AH1 (8 106 CFU), Y. ruckeri YR1 (1 107 CFU), and V. harveyi T4D1 (2.5 107 CFU). Vaccination with live DH5a with boost was performed in the same fashion, except that a booster injection was carried out at the 20th day after the initial immunization. For vaccination with LPS and OMPs, Japanese flounder were divided randomly into three groups named AeC. Groups A and B were administered via i.p. injection with 100 ml of LPS and OMP suspension (prepared as described above), respectively. Group C
Sera were collected from vaccinated fish (five at each time point) at various time points post-vaccination. As a control, sera were also collected from pre-immune fish. Sera were diluted serially in two-fold in PBS. Whole cell ELISA was performed as described previously [30]. Briefly, AH1 was cultured in LB medium to midlogarithmic phase and resuspended in PBS. The AH1 suspension was added to an ELISA plate. After blocking with bovine serum albumin (BSA), the cells in the plate were treated with PBS (the control) or diluted serum from vaccinated or unvaccinated fish. The cells were then treated with mouse anti-Japanese flounder IgM monoclonal antibody (Aquatic Diagnostic Ltd., UK). After incubation at 37 C, horse-radish peroxidase-conjugated goat anti-mouse IgG (Bios, Beijing, China) was added to the plate. Color development was performed using the TMB Kit (Bios, Beijing, China). The plate was read at 450 nm, and positive readings were defined as at least twice that of the control. Antibody titer was presented as the highest dilution that gave rise to positive reading.
2.5. Histopathological analysis
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2.10. Quantitative real-time reverse transcriptase PCR (qRT-PCR) Japanese flounder were vaccinated with DH5a or PBS as described above. Spleen was taken from five fish at 24 h postchallenge. Total RNA was extracted from the spleen by using the RNAprep Tissue/Bacteria Kit (Tiangen, Beijing, China). One microgram of total RNA was used for cDNA synthesis with the Superscript II reverse transcriptase (Invitrogen, USA). qRT-PCR was carried out in an ABI 7300 Real-time Detection System (Applied Biosystems) by using the SYBR ExScript qRT-PCR Kit (Takara, Dalian, China) as described previously [30]. Each assay was performed in triplicate with b-actin RNA as the control. The primers used for qRT-PCR of bactin and the immune-related genes were as described previously [30]. All data are given in terms of relative mRNA, expressed as means plus or minus standard errors of the means (SE). 2.11. Statistical analysis All statistical analyses were performed by using SPSS 15.0 software (SPSS Inc., USA). Differences in antibody titers and transcription levels of the immune-related genes were analyzed with Student's t-test; differences in mortality were determined with Chi-square test. In all cases, the significance level was defined as P < 0.05.
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Table 1 Summary of the cumulative mortality and relative percent survival (RPS) of Japanese flounder immunized with different vaccines. Vaccine
Challenging organism
Challenging timea
Cumulative RPS (%) mortality (%)
PBS PBS PBS DH5a DH5a DH5a PBS with boost DH5a with boost DH5a with boost DH5a with boost DH5a with boost LPS OMPs DH5a/pAQ1 DH5a/pAQ1 DH5a/pBTA1 DH5a/pBTA1
Aeromonas hydrophila Yersinia ruckeri Vibrio harveyi A. hydrophila Y. ruckeri V. harveyi A. hydrophila A. hydrophila A. hydrophila A. hydrophila Y. ruckeri A. hydrophila A. hydrophila A. hydrophila V. harveyi A. hydrophila V. harveyi
5 week p.v. 5 week p.v. 5 week p.v. 5 week p.v. 5 week p.v. 5 week p.v. 5 week p.v. 5 week p.v. 8 week p.v. 12 week p.v. 5 week p.v. 5 week p.v. 5 week p.v. 5 week p.v. 5 week p.v. 5 week p.v. 5 week p.v.
85 93 78 23 60 73 88 10 13 23 55 85 80 13 5 83 75
a
73 35 6 89 83 74 41 11 16 84 93
p.v., post-vaccination.
recovered from the blood and tissues of DH5a-vaccinated fish at any of the examined time points.
3. Results
3.3. Effect of boost on the immunoprotectivity of DH5a
3.1. Immunoprotective analysis of DH5a as a live vaccine
Since DH5a appeared to be retained transiently in vivo, we examined whether a booster during the vaccination process would enhance the immunoprotective effect of DH5a. The results showed that, with a booster injection, DH5a-immunized fish exhibited an accumulated mortality of 10% upon challenging with A. hydrophila at 5 weeks post-vaccination, whereas the control fish exhibited an accumulated mortality of 88%. Hence, vaccination with DH5a followed by a boost produces an RPS of 89% (Table 1). To examine the duration of protection, fish were also challenged at 8 and 12 weeks post-vaccination, and the resulting cumulative mortalities were 13% and 23%, respectively, which correspond to an RPS of 83% and 74%, respectively (Table 1). The effect of boost on the immunoprotectivity of DH5a against Y. ruckeri was also examined. The results showed that, with
Preliminary studies in our laboratory showed that Japanese flounder pre-exposed to DH5a challenge exhibited enhanced survival against subsequent infection by A. hydrophila (Cheng S and Sun L, unpublished data). This observation led us to investigate the possibility of using DH5a as a vaccine. For this purpose, we first analyzed the virulence potential of DH5a using Japanese flounder as an animal model. The results showed that i.p. injection of 109 CFU of DH5a into Japanese flounder caused no mortality over a period of 20 days post-challenge. Morphological and histopathological examinations indicated that fish survived the challenge displayed no apparent damages in the spleen, liver, and kidney. We next examined the immunoprotective potential of DH5a. To this end, Japanese flounder were vaccinated via i.p. injection with live DH5a, followed by challenging with A. hydrophila and two other aquaculture pathogens, Y. ruckeri and V. harveyi, respectively. The results showed that the cumulative mortalities of the DH5avaccinated groups exposed to A. hydrophila, Y. ruckeri, and V. harveyi challenges were 23%, 60%, and 73%, respectively, while the cumulative mortalities of the control groups, which had been mockvaccinated with PBS, were 85%, 93%, and 78%, respectively (Table 1). Hence, the immunoprotective efficacies, in terms of RPS, of DH5a against A. hydrophila, Y. ruckeri, and V. harveyi were 73%, 35%, and 6%, respectively. 3.2. Survival of DH5a in vivo the vaccinated fish To determine the in vivo dissemination and survival ability of DH5a after i.p. injection, DH5a- and PBS-vaccinated fish were examined for bacterial recovery from the peritoneal fluids, blood, liver, spleen, and kidney at various times post-vaccination. The results showed that for PBS-vaccinated fish, no bacteria could be recovered from the blood or tissues at any of the examined time points. For DH5a-vaccinated fish, DH5a was recovered from peritoneal fluids in the first 12 days post-vaccination, during which time the numbers of recovered cells decreased rapidly with time, especially during the first 6 days (Fig. 1). No bacterial cells could be
Fig. 1. Recovery of DH5a from the peritoneal cavity of vaccinated fish. Japanese flounder were vaccinated via intraperitoneal injection with DH5a, and bacterial recovery from the peritoneal fluids was determined at various time points postvaccination. Data are means for four fish and presented as the means SE.
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a booster injection, the accumulated mortality of the vaccinated group was reduced to 55%, which corresponds to an RPS of 41%. 3.4. Immunoprotective analysis of DH5a lipopolysaccharides (LPS) and outer membrane proteins (OMPs) Since vaccination with live DH5a afforded significant protection upon flounder against A. hydrophila infection, we examined whether the LPS and OMPs of DH5a were also immunoprotective. The results showed that the accumulated mortalities of the fish immunized with LPS, OMPs, and PBS (the control) were 85, 80, and 95% respectively. Hence, the levels of protection induced by vaccination with DH5a LPS and OMPs were, in terms of RPS, 11 and 16% respectively. Western immunoblotting analysis showed that some OMPs and cytoplasmic proteins, but not periplasmic proteins or LPS, could react with the serum from fish vaccinated with DH5a (Fig. S1 in the supplemental material). 3.5. Immune response elicited by vaccination with DH5a 3.5.1. Serum antibody production Whole cell ELISA indicated that production of serum antibodies against A. hydrophila occurred in Japanese flounder vaccinated with DH5a with or without boost (Fig. 2). In both cases, specific antibodies were detected at five to eight weeks post-vaccination. The antibody titers of the boosted fish were the same as those of unboosted fish at seven weeks post-vaccination but were 2- to 4fold higher than those of the unboosted fish at 5, 6, and 8 weeks post-vaccination. For the boosted fish, specific antibodies could still be detected at 12 weeks post-vaccination but at a lower level than that observed at 8 weeks post-vaccination. 3.5.2. Expression of immune-related genes To investigate the effect of DH5a vaccination on the expression of immune-related genes, qRT-PCR was carried out to analyze the transcription levels of the genes encoding CD8a, type I interferon (IFN), interleukin 1b (IL-1b), major histocompatibility complex (MHC) class Ia and class IIa, immunoglobulin M (IgM), IFN-g, and IFN-induced Mx gene (Mx) in the spleen of DH5a- and PBS-vaccinated fish. The results showed that expressions of all the examined genes, especially that of IgM, were significantly enhanced in DH5avaccinated fish compared to those in PBS-vaccinated fish (Fig. 3). 3.6. DH5a as a cross-protective vaccine in the form of a carrier of a heterologous antigen In a previous study, we have constructed a plasmid pAQ1 that expresses the V. harveyi protein DegQ, which is a protective
Fig. 2. Serum antibody titers in DH5a-vaccinated fish. Japanese flounder were vaccinated with DH5a with or without a booster injection, and sera were collected at 5e8 weeks post-vaccination. As a control, sera were also collected from unvaccinated fish. Serum antibodies against AH1 were determined by whole cell ELISA. Antibody titers represent the highest dilutions that gave rise to positive readings.
Fig. 3. Expression of immune-related genes in DH5a-vaccinated fish. Japanese flounder were vaccinated with DH5a and challenged with AH1. Total RNA was extracted from the spleens of the fish at 24 h post-challenge and used for qRT-PCR. The mRNA level of each gene was normalized to that of b-actin. For each gene, the mRNA level of the control fish was set as 1. Data are means for five assays and presented as the means SE. **, P < 0.001; *, P < 0.05.
immunogen [28]. DH5a harboring pAQ1 (DH5a/pAQ1) can secrete recombinant DegQ into the extracellular milieu and is highly effective against V. harveyi infection when used as a live vaccine [28]. In this study, we examined the cross-protective potential of DH5a/pAQ1 against infections by V. harveyi and A. hydrophila. The results showed that the cumulative mortalities of DH5a/pAQ1- and DH5a/pBTA1 (the vector control)-vaccinated fish were 5% and 75% respectively, upon V. harveyi challenge and 13% and 83% respectively, upon A. hydrophila challenge. Hence, fish vaccinated with DH5a/pAQ1 exhibited an RPS of 93% and 84%, respectively, upon challenging with V. harveyi and A. hydrophila. 4. Discussion In this study, we demonstrated that vaccination with live E. coli DH5a could confer significant protection in Japanese flounder against A. hydrophila infection. Several live A. hydrophila vaccines, in the form of attenuated A. hydrophila cells, have been reported previously [20e22]. DH5a was comparable to these vaccines in terms of immunoprotection efficacy; however, DH5a has one advantage over these attenuated vaccines in that it is much less risky, because, as demonstrated in our study, DH5a lacks infectivity and could not disseminate into the blood and tissues of Japanese flounder even following i.p. injection. The rapid decrease in the number of bacterial cells recovered from the peritoneal cavity following i.p. injection and the eventual clearance of the inoculated bacterial population from the fish suggest that DH5a cannot replicate inside the vaccinated fish. The transient in vivo retention of DH5a may in part account for the relatively weaker protection elicited by a single immunization. A booster injection enhances the immunoprotection most likely by renewing the presence of DH5a and thus prolonging and heightening the immune response. In line with the enhanced immunity induced by vaccination with a booster injection, the boosted fish exhibited higher levels of antibody titers compared to unboosted fish, which suggests that the humoral immune response may play an important role in DH5a-mediated protection. Consistent with the production of specific antibodies in fish vaccinated with DH5a, the expression of the IgM gene was significantly enhanced in these fish. In addition to the IgM gene, genes encoding MHC Ia, MHC IIa, CD8a, IFN, IL-1b, IFN-g, and Mx were also upregulated, though to lower levels, by
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DH5a. These results suggest the possibility that immunization with DH5a provokes humoral, cellular, and innate immune responses. Cross-species immunoprotection involving A. hydrophila has been reported previously by Fang et al. [15] and Vivas et al. [23], the former showed that vaccination with an A. hydrophila major adhesin conferred protection upon blue gourami against A. hydrophila and Vibrio anguillarum challenges, while the latter showed that an aroA mutant of A. hydrophila elicited protective immunity in rainbow trout against infection by Aeromonas salmonicida. In our study, the observed strong immunoprotection of DH5a against A. hydrophila suggests that DH5a may possess certain antigenic elements that are shared by A. hydrophila. It is known that surfaceexposed cellular components, especially outer membrane proteins and LPS, can be immunogenic and in some cases are protective. A study by Hirst et al. has demonstrated that the iron-regulated outer membrane proteins of A. salmonicida are good vaccine candidates and could afford protection against furunculosis [16]. In our case, we found that some OMPs and cytoplasmic proteins were antigenic, yet the extracted OMPs, as well as LPS, failed to induce immunoprotectivity. These results suggest that the OMPs and LPS of DH5a, at least when used alone, cannot elicit effective immune responses that are required to block A. hydrophila infection. However, it is possible that LPS and OMPs may contribute to the overall immunoprotectivity of live DH5a. E. coli serving as a producer and delivery vehicle of heterologous antigens has been documented previously [35,36]. Onate et al. [37] and Andrews et al. [38] have reported that vaccination of mice with live E. coli producing a Brucella abortus antigen elicited strong protective immunity. In our study, we found that DH5a/pAQ1, a carrier of a V. harveyi antigen, is protective not only against V. harveyi but also against A. hydrophila challenges. The fact that the level of protection against A. hydrophila induced by DH5a/pAQ1 is comparable to that induced by DH5a indicates that production of the V. harveyi antigen does not affect the natural vaccine property of DH5a. With these results, it is reasonable to speculate that, since DH5a is a strain suitable for plasmid maintenance and thus a producer of potentially any antigenic proteins, cross-protective vaccines against A. hydrophila and other pathogens might be constructed in the form of recombinant DH5a engineered to produce the antigen of interest. In conclusion, the results of this study demonstrate that the E. coli strain DH5a is an effective vaccine candidate against A. hydrophila infection in Japanese flounder, and that the unique genetic features and the immunoprotective potential of DH5a can be exploited for the construction of vaccines with cross-protective qualities.
Acknowledgements This work was supported by the National Basic Research Program of China grant 2006CB101807.
Appendix. Supplementary material Supplementary data associated with this article can be found in the online version at doi:10.1016/j.fsi.2009.11.003.
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