ac n s SA Article history: Received 19 September 2013 Received in revised form 19 November 2013 Accepted 22 November 2013 Available online 8 December 2013 a b s t r a c t 1. Introduction Indianmajor carp [6e8], and rainbow trout [9]. Live and attenuated A. hydrophila cells generated via gene inactivation, transposon insertion or antibiotic resistance were assessed for protection efficacy in rainbow trout [10], swordtail fish [11], and channel onal or structural ere evaluated for ourami [13], outer European eel [15], gellar protein for components from nated fish, such as for common carp, rmalin-inactivated of immune protection against A. hydrophila were achieved among these studies though variations of performance remained to be addressed; search for critical immunogens to improve vaccination efficiency is still needed. Strains of A. hydrophila have been shown to be heterogeneous biochemically and serologically [21] and virulence determinants may also vary among clinical and environmental strains [22]. Additionally, ECP secreted by the bacterium was considered to be * Corresponding author. Tel.: þ1 (334) 887 3741; fax: þ1 (334) 887 2983. E-mail addresses:
[email protected],
[email protected] Contents lists availab Fish & Shellfish w. Fish & Shellfish Immunology 36 (2014) 270e275 (D. Zhang). killed whole cells of A. hydrophila (baterins) for Nile tilapia [5], extracellular products [7] for Indian major carp. Various degrees Aeromonas hydrophila, a gram-negative bacterium, had long been implicated as the etiological agent in a wide range of fresh water fish diseases worldwide [1] and was lately culpable for some disease outbreaks in aquaculture, causing severe economic losses [2e4]. To control this emerging disease, research has been carried out to develop vaccines against the pathogen as a sustainable preven- tion method. Attempts have been made to use formalin- or heat- catfish [12], respectively. Alternatively, functi proteins of A. hydrophila in recombinant form w vaccine candidates, such as adhesin for blue g membrane protein for Indianmajor carp [14] and S-layer protein for common carp [16], and fla channel catfish [17]. Additionally, complex A. hydrophila cells or cultures were used to vacci crude lipopolysaccharide [18] or biofilm [19] whole cell lysate [20] for rainbow trout and fo Keywords: Aeromonas hydrophila Extracellular products Vaccination Agglutination Passive immunization 1050-4648/$ e see front matter Published by Elsevie http://dx.doi.org/10.1016/j.fsi.2013.11.015 Aeromonas hydrophila, a Gram-negative bacterium, is one of the economically-important pathogens in modern aquaculture. Among various traits, extracellular products (ECP) secreted by the bacterium are considered to be essential factors for virulence. Whether vaccination with the ECP could produce im- mune protection in catfish against the pathogen was determined in this study. The results showed that fish vaccinated with ECP had 100% of relative percent survival (RPS) when challenged with the pathogen two weeks post vaccination. The anti-ECP serum from vaccinated fish could aggregate cells of homo- geneous bacteria as well as other virulent strains (isolates) of A. hydrophila but not an A. veronii isolate and a low virulent field isolate. The agglutination titers increased from two weeks to four weeks post immunization and sustained a high level at week seven when the RPS remained at 100%. The anti-ECP serum could also provide naïve fish with immediate protection against A. hydrophila as evidenced by passive immunization. Immunoblotting analysis showed that the anti-ECP serum contained antibodies that bound to specific targets, including protein and lipopolysaccharide-like molecules, in the ECP. Mass spectrometric analysis identified following putative proteins that may serve as important immunogens: chitinase, chitodextrinase, outer membrane protein85, putative metalloprotease, extracellular lipase, hemolysin and elastase. Findings revealed in this study suggest that, while ECP prepared in a conven- tional and convenient way could be a vaccine candidate, further characterization of antibody-mediated targets in the ECP would uncover quintessential antigens for the future development of highly efficacious vaccines. Published by Elsevier Ltd. a r t i c l e i n f o Full length article Vaccination of channel catfish with extr Aeromonas hydrophila provides protectio the pathogen Dunhua Zhang*, Julia W. Pridgeon, Phillip H. Klesiu Aquatic Animal Health Research Unit, USDA-ARS, 990 Wire Road, Auburn, AL 36832, U journal homepage: ww r Ltd. ellular products of against infection by le at ScienceDirect Immunology elsevier .com/locate/ fs i h Im associated with not only pathogenicity but also environmental adaptability [23]. Toxicity of A. hydrophila ECP has been reported to be lethal in tilapia [24], rohu [25], and catfish [26]. Therefore, to generate a wide spectrum of protective immune response against various heterogeneous isolates, use of extracellular products (ECP) of A. hydrophila would be a better choice among different immu- nogens studied. The aim of this study was to assess the protective efficacy of A. hydrophila ECP-mediated vaccination in channel catfish and evaluate activities of anti-ECP serum in agglutination, passive im- munization and immunoblotting. 2. Materials and methods 2.1. Bacterial culture and ECP preparation Avirulent strain of A. hydrophila, ML-10-51K [26,27], was used in this study. The bacteriumwas regularly cultured from glycerol stock at �80 �C and routinely maintained in tryptic soy agar (TSA) at 24e 26 �C. To prepare ECP, the bacterium was inoculated in liquid me- dium, tryptic soy broth (TSB), and shake-incubated at 26 �C for 16e 18 h when optical density at 600 nm (OD600) reached to about 5e6. The bacterial suspension was centrifuged at 5000 rpm for 30 min and the supernatant recovered was filtered through a 0.22 mm PES membrane (Millipore, Billerica, MA, USA). The filtered supernatant was, then, concentrated to approximately 1000 times using 20K- MWCO centrifugal concentrator (Pierce/Thermo Scientific, Rock- ford, IL, USA). The concentrated supernatant was referred to ECP hereafter and kept at 4 �C (for less one week before use) or �80 �C (for more than one week storage). For control, uncultured TSB was processed in the sameway and had the similar concentration factor. Protein quantities in the concentrated ECP and TSB were estimated with Bradford Reagent (SigmaeAldrich, St. Louis, MO, USA) using bovine serum albumin as the standard protein. 2.2. Immunization, challenge of pathogen and serum preparation Catfish fingerlings, weighing 10.8 � 1.5 g, were maintained in aquaria tanks (57 L water) with 20 fish per tank. Protocols estab- lished for fish rearing and treatment were followed as described previously [28]. Immunogens were prepared by emulsification of equal volumes of Freund’s complete adjuvant (Sigma) and ECP solution (16 mg ml�1 of total protein) in sterile phosphate-buffer saline (PBS; Sigma). For control, the concentrated TSB with the same volume of the ECP applied was diluted in sterile PBS and emulsified in the same way. One hundred twenty fish were vacci- nated by intraperitoneal (IP) injectionwith 100 ml of the emulsified ECP and other 120 were sham-immunized with the same amount of emulsified TSB. Two weeks after immunization, 30 fish were randomly chosen from the ECP-treated pool, IP-injectedwith 100 ml of fresh-cultured A. hydrophila in TSB [with 2 � 108 cfu (colony forming units) ml�1; the same bacterial concentration was used when mentioned challenge of pathogen below], and equally divided into 3 tanks. The same challenge of pathogen was carried out at weeks 4 and 7 with 3 � 10 fish from the ECP-treated pool each time. For sham-immunized fish, the exact same challenging procedure was followed. Mortality of fish was recorded daily for 1 week for each individual challenge. The protective efficacy of ECP vaccination was expressed as relative percent survival (RPS) of fish, comparing with the sham-immunized, after challenge of pathogen {RPS¼ [1� (% mortality in immunized group/% mortality in control group)] � 100}. Additionally, 5 fish were randomly chosen and subjected to blood collection from both ECP- and sham-immunized fish (all unchallenged) weekly (from 2 weeks to 7 weeks post im- D. Zhang et al. / Fish & Shellfis munization). Blood samples from each set of 5 fish were pooled and allowed to clot at 4 �C for 2 h. Sera were collected after centrifu- gation at 4 �C (7000 rpm) for 20 min and stored at�80 �C until use. All of above experiments were repeated once and partial of them were twice. To assess the effect of repeated challenges of pathogen on fish survival and serum response, a group of the same size fish (30 fish � 2 replicates), which had RPS equal to 100% after challenge of pathogen twoweeks post immunizationwith ECP, was subjected to 2 more challenges at two-week intervals. In the meanwhile, five fish from each replicate were sampled for serum collection before challenge and one week after each challenge. 2.3. Passive immunization and challenge of pathogen The ECP-immunized sera with high bacterial cell agglutination titers (see below) and those of the sham-immunized were sepa- rately pooled and used for passive immunization as described previously [29]. Briefly, 108 fish (12.5 � 1.7 g) were equally divided into 9 aquaria tanks (57 L water) and subdivided into 3 groups (36 fish in 3 replicate tanks per group). Fish in each of the groups were IP-injected 100 ml of either ECP-immunized serum, heat-treated ECP-immunized serum (incubated at 56 �C for 1 h) or sham- immunized serum. After taking 2 fish from each tank for serum collection 2 days post the passive immunization, all fish were challenged by IP-injection of 100 ml of A. hydrophila cell suspension in TSB. Mortality was recorded daily for 2 weeks. This experiment was repeated once. 2.4. Bacterial cell agglutination and titration Serum collected from each set samplewas serially diluted in PBS in wells of a 96-well microtiter plate. Cells of A. hydrophila cultured onTSA for 24e48 hwere suspended in PBS. The cell suspensionwas centrifuged at RT (5000 rpm) for 5 min. The cell pellet was re- suspended in PBS and precipitated by centrifugation for two times. After re-suspended in PBS, bacterial cells were adjusted to 7.5 � 0.2 � 108 cells ml�1 in PBS by reading at OD600. Aliquots of 50 ml of this bacterial cell suspension were, then, mixed into indi- vidual wells containing 50 ml of undiluted or diluted serum. After sealed with a 3 M Scotch Pad sealer, the microtiter plate was kept still at RT for at least 4 h. The titer of a serumwas determined by the reciprocal of the highest dilution factor that resulted in visible clumping of bacterial cells. For each serum sample, the agglutina- tion test was repeated twice. With ML-10-51K, used in this study, as a reference strain of A. hydrophila, a serum sample with known agglutination titer was also used to test relative titration for other five strains (isolates) of Aeromonas species which were characterized or partially charac- terized (Table 3). 2.5. SDS-PAGE, immunoblotting and mass spectrometric analysis Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was conducted to separate samples of ECP and concentrated TSB for protein profiling and western analysis, using NuPAGE 4e12% Bis-Tris precast gel, MES/SDS running buffer and associated protocols (Invitrogen, Carlsbad, CA, USA). The SeeBlue Plus2 Prestained Standard (Invitrogen) was used as reference mo- lecular weight marker. After electrophoresis, the gel was either stained using SimpleBlue Safestain (Invitrogen) or subjected to western blot using Novex Mini-cell apparatus and associated pro- tocols (Invitrogen). For western analysis, the blot was first probed using either the ECP-immunized serum or the sham (TSB)-immu- nized serum and, then, incubated with peroxidase-conjugated goat munology 36 (2014) 270e275 271 anti-catfish-IgM IgG (produced by Rockland, Gilbertsville, PA, USA), followed by colorimetrical development using 4-chloro-1- naphthol/H2O2 (Bio-RAD, Hercules, CA, USA). Additionally, protein bands in gel after SDS-PAGE, approximately corresponding to pro- teins revealed by immunoblotting, were excised and subjected to LC-MS/MS (liquid chromatography with tandem mass spectrom- etry) analysis and identification (Scafford program, Proteome Software, Portland, OR, USA). 2.6. Statistic analysis Differences of mortality between different vaccination treat- ments were analyzed using one-way analysis of variance with Turkey’s method. 3. Results 3.1. Relative percent survival of fish immunized with ECP Two weeks post immunization with ECP, all experimental fish survived the challenge of pathogen at dosage of 2�107 cfu fish�1, at which all sham-immunized fish died within 4e5 h (Table 1). No mortality occurred for those survived the challenge in following (mean � SD), 37.8 � 3.9 and 100, respectively, after challenge of pathogen (Table 2). In terms of RPS, the untreated anti-ECP serum couldprovide about85%protectionagainst infectionwhile theheated one did about 62%. And, notably, all fish received control serum died within 4 h after challenge of pathogen while mortality of those received anti-ECP serumwas delayed for 1e3 days. 3.5. Cross agglutination of different strains (isolates) of A. hydrophila and related species Serum samples generated from fish immunized with ECP of strain ML-10-51K (used in this study) were able to cross- agglutinate cells of other 3 strains (isolates) of A. hydrophila but not those of 2 related species (Table 3). The agglutination titers for ML-09-73, ML-10-208K and ML-10-20S were the same as that of ML-10-51K. TheML-09-73 and theML-10-208K had been positively identified as virulent strains of A. hydrophila while the ML-10-205 was also virulent but partially characterized to be an isolate of A. hydrophila (unpublished data). For those that could not be agglutinated, the ALG-10-89 was identified as A. veronii, a species closely related to A. hydrophila, based on partial genomic DNA se- quences (unpublished data) while the AL-98-C1B was a very low D. Zhang et al. / Fish & Shellfish Im272 two weeks and thereafter (at least seven weeks later as observed). Likewise, those challenged 4 and 7 weeks post immunization had RPS equal to 100% (Table 1). 3.2. Agglutination of A. hydrophila cells and the serum titration All serum samples collected from ECP-immunized fishwere able to cause aggregates of A. hydrophila cells, resulting in visible clumping (i. e. agglutination), while no agglutination ( Table 2 Passive immunization of catfish with anti-ECP serum.a Fish treatment Cumulative percentage of mortality (mean � SD) 4 h 24 h 2 day 3 Anti-ECP serum 0 0 8.9 � 3.9 15 Heated anti-ECP serumb 4.5 � 3.9 17.8 � 3.9 37.8 � 3.9 37 Shamed serum 100 100 100 10 a gerli wit use. ts. D. Zhang et al. / Fish & Shellfish Im 3.6. SDS-PAGE, immunoblotting and mass spectrometry Profiles of samples of ECP and concentrated TSB used for im- munization of fish were shown in Fig. 2A after SDS-PAGE and staining. The ECP had complex stainable bands rangingmostly from 3 to 98 kDa (Lane 1 of Fig. 2A) while no distinct band was seen in concentrated TSB (Lane 2 of Fig. 2A). The same samples, upon blotted to PVDF membrane, showed different profiles recognized by fish serum. There was no visible band shown for both ECP and TSB samples which were probed by the sham-immunized fish serum (1� antibody source; Fig. 2B). A cluster of prominent bands mostly ranging from 38 to 98 kDa was seen in ECP but not TSB sample when the blot was probed by ECP-immunized fish serum (Fig. 2C). Among these visible bands, some were intensely stained while others were in light shade. While the lightly stained bands were apparently arranged in an orderly, ladder-like pattern with resemblance to lipopolysaccharide profile (Supplemental Fig. 2), the intensely-stained bands (indicated by arrows in Fig. 2C) were most likely protein molecules. Mass spectrometric analyses of the corresponding bands excised from the protein gel (Fig. 2A) revealed that they were chitinase (accession #A0KGX3) & chitodextrinase (#A0KNS8); outer membrane protein85 (#A0KHH1) & putative metalloprotease (#A0KGX4); extracellular lipase (#A5A359); and hemolysin (#D2XPP9) & elastase (#A0KGK2), respectively (arrow-pointed bands from top to bottom; protein identification probability ¼ 100%). 4. Discussion A. hydrophila secreted an array of extracellular products [23,30], including not only well-known virulence factors (such as metal- loproteases, glycerophospholipid-cholesterol acyltransferase, he- molysin, aerolysin, and lipases) but also proteins for digestion (such as amylases and chitinases) and host cell attachment (such as S- layer, and flagella), which are considered to contribute to their wide distribution, great adaptability and pathogenicity. The ECP pre- pared in this study showed a complex profile (Fig. 2A) and may Mixed anti-ECP serum with agglutination titer of 64 was IP-injected to catfish fin the sham-immunized fish. At 48 h post passive immunization, fish were challenged b The serum was heated at 56 �C for 1 h and cooled to room temperature before c The overall mortality was significantly different (p < 0.0001) between treatmen represent majority of products the bacterium secreted in milieu. Vaccination of fish with this ECP preparation apparently provided immune protection against fatal challenge of A. hydrophila. The success of 100% of RPS of vaccinated fish may be attributed to the representation of antigens in the ECP preparation and, Table 3 Cross agglutination of Aeromonas species by anti-ECP serum. Strain (isolate) Agglutination titer Identification Reference ML-10-51K 64 A. hydrophila This study ML-09-73 64 A. hydrophila [3] ML-10-208K 64 A. hydrophila [26] ML-10-205 64 A. hydrophila Unpublished ALG-10-089 h Im A. 188 98 62 49 38 28 188 98 62 49 38 28 B. D. Zhang et al. / Fish & Shellfis274 observation that the similar ladder-like banding pattern was revealed by immunoblotting of purified A. hydrophila LPS using anti-ECP serum as primary antibody (Supplemental Fig. 2). LPS was known as one of the major structural molecules of the outer membrane and as an endotoxin associated with pathogenesis [34]; extracellular LPS was, however, rarely examined except for the report by Horisberger and Dentan [35], in which cellular LPS and extracellular LPS of Moraxella glucidolytica were found to have minor structural differences but were similar in animal toxicity. The extracellular LPS in A. hydrophila was not reported before. Earlier study showed that vaccination of common carp with the LPS, extracted from A. hydrophila cells, could provide some protection against A. hydrophila but no antibody in vaccinated fish serum was found in agglutination assay [18]. The presence of anti-LPS antibody in the anti-ECP serum, observed in this study, would probably result from combinationwith other ECP components [36] and have conferred the fish with better immunity against A. hydrophila. Protein identities revealed by mass spectrometry (Fig. 2A and C) were likely the antibody’s targets and may serve as important immunogens although individual roles has yet to be determined using recombinant proteins. In addition, the possible synergic ef- fects between protein antigens and LPS merit further investigation. In conclusion, fish could develop immunity against fatal chal- lenge of A. hydrophila when vaccinated with ECP secreted by the 17 14 6 kDa 17 14 6 kDa Fig. 2. SDS-PAGE and western analysis of A. hydrophila ECP and catfish anti-ECP sera. Approx individual lanes in 4e12% NuPAGE Bis-Tris gel (Invitrogen). After electrophoresis, the gel wa to PVDF membrane (Panels B & C). Panel B: the blot was probed using sham-immunized s labeled 1 were ECP and those in lanes labeled 2 were concentrated TSB medium while lan analyzed by mass spectrometry (detailed in Section 3.6). 28 38 49 62 188 98 C. munology 36 (2014) 270e275 pathogen and the immunity could be transferred via serum to provide naïve fish with immediate protection. The anti-ECP serum could have differential agglutination activities toward different virulent isolates within A. hydrophila and different species in genus Aeromonas. Fish immunized with the ECP of ML-10-51K could have immunity against various virulent isolates of A. hydrophila in the fields. Antibodies in the anti-ECP serum were shown to target specific components in the complex ECP. Further characterization of these antibody’s targets would help uncover quintessential an- tigens for the development of higher efficient vaccines. Acknowledgements We thank Drs. Perng-Kuang Chang and Victor Panangala for critical review of this manuscript, and Mrs. Ning Qin and Beth Peterman for their technical support. This study was funded by USDA-ARS CRIS project #6420-32000-024-00D. Appendix A. Supplementary data Supplementary data related to this article can be found online at http://dx.doi.org/10.1016/j.fsi.2013.11.015. kDa 6 14 17 imately 9 mg of ECP or equivalent volumes of concentrated TSB mediumwere loaded to s stained (Panel A) using SimpleBlue SafeStain (Invitrogen) or subjected to western blot erum. Panel C: the blot was probed using ECP-immunized serum. All samples in lanes es in M were molecular weight markers (Invitrogen). Bands indicated by arrows were References [1] Austin B, Adams C. Fish pathogens. In: Austin, B, Altwegg, M, Gosling, P.J, Joseph, S, editors. 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Vaccination of channel catfish with extracellular products of Aeromonas hydrophila provides protection against infection by ... 1 Introduction 2 Materials and methods 2.1 Bacterial culture and ECP preparation 2.2 Immunization, challenge of pathogen and serum preparation 2.3 Passive immunization and challenge of pathogen 2.4 Bacterial cell agglutination and titration 2.5 SDS-PAGE, immunoblotting and mass spectrometric analysis 2.6 Statistic analysis 3 Results 3.1 Relative percent survival of fish immunized with ECP 3.2 Agglutination of A. hydrophila cells and the serum titration 3.3 Agglutination titer changes following challenge of A. hydrophila 3.4 Passive immunization and challenge of pathogen 3.5 Cross agglutination of different strains (isolates) of A. hydrophila and related species 3.6 SDS-PAGE, immunoblotting and mass spectrometry 4 Discussion Acknowledgements Appendix A Supplementary data References