A

sesaieiwC©

K

1

atadlTcsrt

0d

Vaccine 25 (2007) 2095–2102

Immunological characterization of a bacterial protein isolated fromsalmonid fish naturally infected with Piscirickettsia salmonis

Sergio H. Marshall a,∗, Pablo Conejeros a, Marcela Zahr b, Jorge Olivares a,Fernando Gomez a, Patricio Cataldo a, Vitalia Henrıquez a

a Laboratorio de Genetica e Inmunologıa Molecular, Instituto de Biologıa, Facultad de Ciencias Basicas y Matematicas,Pontificia Universidad Catolica de Valparaıso, Avenida Brasil 2950, Valparaıso, Chile

b Laboratorio de Microbiologıa Molecular, Universidad de Valparaıso, Avenida Gran Bretana 1111, Valparaıso, Chile

Received 8 July 2006; received in revised form 9 November 2006; accepted 13 November 2006Available online 4 December 2006

bstract

The Salmon Rickettsia syndrome (SRS) remains a major infectious disease in the Chilean aquaculture. A limited number of Piscirickettsiaalmonis proteins have been characterized so far for their use as potential candidates for vaccines studies. In this study, we identified andxpressed a highly immunogenic protein of P. salmonis extracted by selective hydrophobicity from crude-cell macerates of naturally infectedalmonid fish. One and two-D PAGE gels followed by Western blot analysis with a battery of polyclonal anti-P. salmonis antibodies havellowed the isolation of the target protein. Basic local alignment search (BLAST) done after partial sequencing of the pure protein identifiedt as a member of the heat-shock protein (HSP) family of prokaryotes. The protein, named ChaPs, was cloned as a single open reading framencoding 545 amino acid residues with a predicted molecular mass of 57.3 kDa. The amplicon representing the entire novel gene was expressed

n vitro in different heterologous systems: the PurePro Caulobacter crescentus expression system from where most of the characterizationas attained, and also in the Escherichia coli BL-21 CodonPlus model for commercially potential purposes. The immunologic potential ofhaPs was determined with serum from naturally infected fish.2006 Elsevier Ltd. All rights reserved.

ogenici

(bm[s

oitw

eywords: Intracellular bacterial pathogen; In vivo isolated protein; Immun

. Introduction

Rickettsial diseases are common and significant in humansnd in the veterinary world [1,2] and fish are not an excep-ion [3,4]. Salmon Rickettsia Syndrome (SRS), also knowns piscirickettsiosis, corresponds to an aggressive infectiousisease affecting the world of salmon aquaculture since theate 80s with a notorious impact in southern Chile [5–7].he disease is caused by a novel non-motile obligate intra-ellular Gram-negative bacteria, Piscirickettsia salmonis (P.

almonis) [8] and has been unequivocally identified as theesponsible agent for dramatic economic losses suffered byhe Chilean salmon industry in the last decade. P. salmonis

∗ Corresponding author. Tel.: +56 32 2273119; fax: +56 32 2596703.E-mail address: smarshal@ucv.cl (S.H. Marshall).

lwidsb

264-410X/$ – see front matter © 2006 Elsevier Ltd. All rights reserved.oi:10.1016/j.vaccine.2006.11.035

ty; Potential vaccine

type strain LF 89, ATCC VR 1361) survives in salt wateretter than in fresh water [9], and can be horizontally trans-issible by contact and cohabitation in both environments

10,11]. Suspicious of intermediate vectors in the transmis-ion have also been raised [12,13].

After the first description of piscirickettsiosis in Chile,ther salmonid species have begun experiencing mortalitiesn saltwater milieu [4]. In time, P. salmonis was identified ashe causative agent of diseased salmonids in other latitudes,ith special impact in western Canada, Norway and Ire-

and [14,15]. Additionally, a piscirickettsiosis-like syndromeas also reported in epizooties affecting farmed salmon

n Scotland [16], and similar piscirickettsia-like organismsisplaying comparable clinical signs have been reported ineveral number of different fish species, which in spite ofeing non-reactive to antibodies against standard P. salmonis,

2 accine

msiistr

cistbaattorsovrdn

ipP5(hopoib

2

2

2

hobpp

2

tiM

taefili12ttdwp(

2

2

(gup(a

2

E(afl

2

a8alc

2

2

at

2

at

096 S.H. Marshall et al. / V

ight very well represent antigenic variants to the prototypetrain [17]. Notwithstanding, recently a true P. salmonis wassolated in the coast of southern California from a naturallynfected white sea bass (Atractoscion nobilis), which wasuccessfully transmitted both in vivo and in vitro, suggestinghat the expected spread of the agent to other commerciallyelevant fish species has already started [18].

Although the causative agent of this disease has been suc-essfully grown in tissue culture cells [19,4], almost nothings known about its biology and pathogenesis. Nonetheless,ince infected fish are immunoreactive to crude extracts con-aining the bacteria, the possibility of either purifying theacteria and/or searching for single antigens appears to bereasonable way to evaluate potential protective immunity

gainst the disease. Several reports indicate that the use of par-ially purified-inactivated bacteria (bacterins) does not seemo be an effective vaccination alternative [19–21]. On thether hand, several potential bacterial antigens have beeneported [22–26]. Though only until recently, the use of aingle antigen (OspA) as putative vaccine plus the additionf T cell epitopes has had some stimulating results when theaccine was administered to coho salmon. A post-challengeelative percent of survival (RPS) as high as 59% was attained,emonstrating the potential efficacy of a putative recombi-ant vaccine based upon a single antigen [27,28].

In this study, we report the cloning and partial character-zation of the ChaPs gene, which encodes an immunogenicrotein of P. salmonis. DNA sequence analysis reveals that the. salmonis ChaPs gene has an open reading frame encoding45 amino acid residues. Basic local alignment search toolBLAST) analysis shows that the putative protein is highlyomologous to members of the HSP chaperone-like familyf other bacteria (cpn). Finally, we are considering that thisrotein may very well represent a new target for the devel-pment of an efficacious vaccine to control piscirickettsiosisn favor of a sustainable development of the salmon industryoth in Chile and worldwide.

. Materials and methods

.1. In vivo purification

.1.1. AnimalsLiver and kidney from 30 adult coho salmon (Oncor-

ynchus kisutch) specimens were recovered after an epizootyf P. salmonis in southern Chile. Organs were received in HNuffer (0.5 M NaCl, 50 mM Tris, 1 mM EDTA pH 7.8) in theresence of 1 mM phenylmethylsulfonyl fluoride (PMSF) asrotease inhibitor.

.1.2. Protein extraction procedures

Samples homogenized in 10 mM Tris–Cl (pH 8.0) con-

aining 1 mM PMSF and 1 ml/20 g sample of a proteasenhibitor cocktail (Sigma Chemical Company, St. Louis,

O) were exposed to hydroxiapatite to a final concentra-

C

2i

25 (2007) 2095–2102

ion of 10%, incubated for 5 h at 4 ◦C, followed by 60 mint 30 ◦C. After centrifugation at 12000 × g, 12 min at 4 ◦C,ach resulting fraction was exposed to Triton X-114 to anal concentration of 2% and thoroughly homogenized fol-

owed by 2 h incubation at 4 ◦C. The mixture was loadednto a 10× volume sucrose cushion (0.3 M + 0.06% Triton X-14), incubated for 2 h at 30 ◦C and centrifuged at 12000 × g,0 min at 30 ◦C. The upper phase was recovered, dialyzedhree times against 10 mM Tris pH 8.0 + 0.2% Amberlite andwo times against 1 mM Tris pH 8.0 + 0.2% Amberlite. Theialyzed sample was concentrated in the same dialysis tubingith dried Polyethylene Glycol 8000 (Sigma Chemical Com-any) and was the starting material for SDS-PAGE analysisSanchez-Ferrer procedure modified by S. Marshall) [29].

.2. In vitro purification

.2.1. Cell lineCHSE-214 (ATCC CRL 1681), a chinook salmon

Oncorhynchus tschawytscha) embryo derived cell line wasrown at 17 ◦C in monolayers in 75 cm2 tissue culture flaskssing antibiotic-free minimal essential medium (MEM) sup-lemented with 7.5% heat-inactivated fetal bovine serumFBS) and buffered to pH 7.2 with 10 mM sodium bicarbonatend 15 mM Hepes.

.2.2. Bacterial strains and growth conditionsP. salmonis type strain LF-89 ATCC VR 1361 as well as

M-90, CI-95, NOR-92 were grown in the chinook salmonOncorhynchus tshawytscha) embryo cell line CHSE-214s described previously [12]. P. salmonis obtained from 20asks (75 cm2 each) 15 days post-infection were used.

.2.3. P. salmonis for protein extractionCHSE-214 cells were grown in 75 cm2 tissue culture flasks

s described earlier. At day 6, when each culture flask reached0% confluence (day 0), P. salmonis infection occurred bydding growing medium from infected cells to 10 of the flaskseaving the other 10 as control and processed at day 15, whenytophatic effect (CPE) was close to 90% in infected cells.

.3. Antibodies

.3.1. Commercial fluoro test (CFT)Mixture of FITC-conjugated oligoclonal antibodies

gainst whole bacteria recovered from supernatant of infectedissue culture cells (BiosChile I.G.S.A.).

.3.2. Different forms of P. salmonisTwo highly purified in vitro grown bacteria, both intact

nd lyzed, as well as to in vitro expressed fragments ofhe reported protein as hybrid polypeptides in the PurePro

aulobacter Expression System (Invitrogen).

.3.2.1. Polyclonal anti-Ps purified-intact (Ps-I). Antibod-es elicited in rabbit against intact in vitro grown bacteria

accine

(g

2ps

2Rer

2(

E

2o

d

2

2m

2awsouscCco

2

daParwP

2

psb

0wtoC1ppc

2

wf(sPpaSA

gcIRte(sobm

2

Ceanw5iTptitaa

S.H. Marshall et al. / V

0.2 mg per animal), purified through equilibrium densityradient centrifugation [30].

.3.2.2. Polyclonal anti-Ps purified-lysed (Ps-L). As in therevious point, but intact pure bacteria were broken open byonication before injection into rabbits (0.2 mg per animal).

.3.2.3. Polyclonal monospecific anti-Ps-COOH-ChaPs-saA (Ps-C). Antibodies elicited in mice against in vitroxpressed P.s-Carboxy-RsaA fragment purified through andecovered from SDS-PAGE (0.5 mg per animal).

.3.3. To RsaA protein from Caulobacter crescentusRsaA)

Commercially available antiserum. PurePro Caulobacterxpression System.

.4. Polyacrylamide gel electrophoresis in the presencef SDS (SDS-PAGE)

One and two-D protein gels were carried out using stan-ard procedures [31].

.5. Immunodetection

.5.1. Direct and indirect immunofluorescenceicroscopyGlass coverslips containing P. salmonis-infected CHSE-

14 cells were incubated for 1 h with 1:50 dilution of eachntibody (anti-Ps-C y anti-Ps-I). Then the samples wereashed three times with wash buffer (phosphate-buffered

aline, PBS) and incubated for 45 min with 1:100 dilutionf Sigma Anti-Rabbit IgG FITC conjugate. As a control wesed commercially available rabbit FITC-conjugated-anti-P.almonis oligoclonal antibody (BiosChile I.G.S.A.). All pro-edures were carried out at room temperature in the dark.overslips were mounted onto glass slides using fluores-ent mounting medium (DAKO Corporation) and observedn fluorescent microscope (Olympus IMT2-RFL).

.5.2. ImmunoblottingPAGE-SDS gels and PAGE-SDS of a 2-D set (first

imension was electro focusing between pH gradient 3–10ccording to O’Farrell [32]) were electro transferred to C+VDF nylon membranes (Inmobilon PSQ, Millipore) andssayed with different anti-Ps sera as the first antibody. Anti-abbit—HRP was used as the second antibody. Reactivityas determined by chemiluminescence (SuperSignal® Westico Chemiluminiscent, Pierce).

.6. Immunoprecipitation of P. salmonis

Ten days post-infection CHSE-214 cells were resus-ended in 0.5 ml of phosphate-buffered saline with 4% bovineerum albumin (BSA) to reduce non-specific binding. Incu-ated with occasional gentle shaking for 2 h at 37 ◦C with

Iads

25 (2007) 2095–2102 2097

.1 ml of anti Ps-L. The complex P. salmonis/anti-Ps-Las centrifuged at 12,000 × g, 25 min at 4 ◦C and washed

wice with PBS. The pellet was then resuspended in 0.5 mlf PBS, incubated 2 h at 37 ◦C with protein A-SepharoseL-4B beads (Amersham Biosciences) and centrifuged at2,000 × g, 25 min at 4 ◦C. Supernatant and pellet were inde-endently used to infect the CHSE-214 cell line and at day 7ost-infection they were evaluated by direct immunofluores-ence microscopy with the CFT antibody.

.7. Gene expression and purification of ChaPs

The full-length gene coding for the P. salmonis ChaPs,as PCR amplified from iodixanol purified bacteria. The

ull-length gene was cloned into the plasmid pCX-TOPOInvitrogen, La Jolla, CA) generating the pPSAL12 andubcloning into pET27b+ (Novagen) generating pPSAL13.lasmids containing full-length sequences of the gene wereurified by Quantum Prep Plasmid Miniprep Kit (Bio-Rad)nd sequenced at Pontificia Universidad Catolica de Chile,antiago, Chile (DNA sequencer ABI PRISM Model 310 PEpplied Biosystems).The pPSAL12 and pPSAL13 containing the full-ChaPs

ene were, respectively, over-expressed in Caulobacterrescentus PurePro Caulobacter expression system fromnvitrogen and in Escherichia coli BL21-CodonPlus (DE3)-IPL pET expression system from Novagen, according

o manufacturer’s instructions. Full-ChaPs protein over-xpressed in E. coli was further electro-eluted purifiedModel 422 Electro-Eluter, BioRad) for immune analy-is. The pure ChaPs protein was detected using a batteryf specific antibodies against P. salmonis and developedy chemiluminescence (SuperSignal® West Pico Chemilu-iniscent, Pierce).

.8. Immune response against ChaPs

In order to determine the immunologic potential ofhaPs, 70 fish sera collected during 2005 from differentpizooties were used for enzyme-linked immunoabsorbentssay (ELISA). Serum from non-infected fish was used asegative control. The 0.5 �g/ml of purified ChaPs proteinas used to coat Maxisorp microtiter plates (Nunc) at0 �l/well for 20 h at 4 ◦C. Plates were washed three timesn a wash buffer (phosphate-buffered saline with 0.05%ween 20) and blocked with 1% bovine serum albumin inhosphate-buffered saline for 2 h at room temperature andhen incubated with 1:100 dilutions from positive and non-nfected sera for 8 h at 20 ◦C, and washed six times. Finally,he microtiter plates were incubated with 1:5000 dilution ofntibody anti-salmonid Immunoglobulin (Microtek) for 12 ht 4 ◦C. Horseradish peroxidase conjugated goat anti-rabbit

gG (Pierce) was used for detection. After 1 h of incubationt 37 ◦C and a washing step, the specific reactivity wasetermined by the addition of 100 �l/well of an enzymeubstrate (Dako® TM Blue) and incubated for 30 min at

2 accine 25 (2007) 2095–2102

ra

2

sS

3

3

ppoaaedLstbNqtdb

3s

d

Fig. 1. Identification of the highly immunogenic protein from in vivo natu-rally infected salmonid fish organs. Recovery for protein sequencing. PanelA: 12% 2-D PAGE stained with Sypro red (Molecular Probes). Panel B:cti

baltaPirot

3s

3e

Fag

098 S.H. Marshall et al. / V

oom temperature in the dark. The absorbance was measuredt 650 nm on an ELISA Microtiter Synergy HT (BIO-TEK).

.9. Protein sequencing

All procedures were done at the Institute of Marine Bio-ciences, National Research Council Canada, Halifax, Novacotia (NRC-CNRC).

. Results

.1. Recovery of ChaPs from in vivo

In order to select putative membrane bound antigens, therocedure used was oriented to enrich hydrophobic com-onents from crude cell extract of naturally infected fishrgans. The assumption was that the most exposed bacterialntigens might also be the most immunoreactive epitopes,nd hopefully, those rendering immune protection. This cellxtract was screened for immune reactive polypeptides usingifferent sets of antibodies against P. salmonis (Ps-I and Ps-). Fig. 1 shows the stained gel in Panel A, while Panel Bhows the reactivity obtained using anti Ps-I which identifiedwo closely-resolved spots over a reasonably high proteinackground. These spots were isolated and submitted to-terminal sequence analysis. The sequence analysis gaveuite a few peptides which sequence information was usedo search the NCBI non-redundant protein and nucleotideatabases. Some of these peptides corresponded to the mem-er of the bacterial chaperonin-like family.

.2. Identification of ChaPs from in vitro grown P.

almonis

The in vitro integrity of this protein was determined byirect immunodetection using a battery of our own anti-

biC

ig. 2. Direct and indirect immunofluorescence microscopy of P. salmonis-infected cnti-P. salmonis antibodies. (i) CFT; (ii) anti-Ps-C; (iii) anti-Ps-I; (iv) immunoprecipradient-purified P. salmonis exposed to the antibodies mentioned above; (iv) immu

orresponding Western blot profile with anti-Ps-I. The dotted line representshe area shown from the corresponding enlarged area in Panel B. The arrowndicates the sequenced version of the protein.

odies. Fig. 2 shows the strongest response obtained whennti-Ps-I was used in a productive infection in CHSE-214 celline (Panel A) and with the purified iodioxanol gradient bac-eria (Panel B). In order to confirm this observation, as wells to determine how relevant the novel protein was in other. salmonis isolates, we assayed four of the most aggressiven vitro grown strains against the anti-Ps-I, the most immuneeactive antibody. Fig. 3 shows that for equivalent amountsf protein loaded (Panel A), all four strains react equivalentlyo anti-Ps-I (Panel B).

.3. Expression of cloned ChaPs variants in commercialystems

.3.1. Caulobacter crescentus PurePro Caulobacterxpression system

Considering that the novel protein full-length ORF hadeen successfully cloned, we did a detailed analysis of then vitro expressed products of our single clone using theaulobacter Expression System [33]. Fig. 4 clearly shows

ells. (A) Infected CHSE-214 cells 7 days post-infection exposed to differentitated bacteria as a negative control (200×). (B) 1:200 dilution of Iodixanolnoprecipitated bacteria as a negative control (200×).

S.H. Marshall et al. / Vaccine 25 (2007) 2095–2102 2099

F . salmoB ne 3: LFB

tpsatt4e

FCerSba

apwa

ig. 3. Evaluation of the presence of ChaPs in different infective strains of P: corresponding Western blot (anti-Ps-I). Lane 1: EM-90; lane 2; CI-95; la. MW, molecular weight marker.

hat truncated forms of the protein are recovered. The upperanel shows a diagram based upon the amino acid and DNAequence of the ChaPs full-length protein ORF (GeneBankccession number AY256822). Polypeptide 1 corresponds to

he full-length hybrid protein starting at the standard ini-iator methionine residue (Table 1). Polypeptide 2, 3 and

correspond to putative truncated variants expressed byrroneous recognition of internal methionine residues as initi-

ig. 4. Differential expression of alternative forms of the hybrid RsaA-haPs protein in the Caulobacter crescentus system. Upper panel:xplanatory drawing showing the identifiable variants according to theecognition of internal methionine residues as initiators. Lower panel 10%DS-PAGE and Western blot of expressed variants. Lane A: Coomassierilliant blue stained gel; Lane B: anti-Ps-I; Lane C: anti-Ps-C; and Lane D:nti-RsaA.

fwn

3e

pwFICrr

FPim

nis. Panel A: 12% SDS-PAGE stained with Coomassie brilliant blue. Panel-89; lane 4: NOR-92. Dotted area in Panel A corresponds to that shown in

tiors (Table 1). In the lower panel, Lane A shows the full-sizerotein plus four truncated forms of the expressed clone,hile Lanes B to D show the differential recognition of the

lternative forms by different antibodies. The fifth alternativeorm seen as the smallest of all in the stained gel (Lane A)as recognized only by anti-Ps-C (Lane C) as well as othersot visible in the stained gel.

.3.2. E. coli BL21-CodonPlus (DE3)-RIPL pETxpression system

In order to improve the amount of ChaPs recombinantrotein expression not yielded by the Caulobacter system,e over-expressed it in E. coli BL21 CodonPlus System.ig. 5 shows cultures non-induced and induced with 1 mM

PTG (Panel A). Due to the close molecular weight amonghaPs and GroEL, it has been difficult to identify them sepa-

ately, but clearly it show an induced high expression for theecombinant ChaPs.

ig. 5. Expression of ChaPs protein in E. coli BL21-CodonPlus system.anel A: 12% SDS-PAGE Coomassie brilliant blue staining. Lane 1, non-

nduced culture and Lane 2, IPTG induced culture. MW, molecular weightarker. Panel B: Western Blot with anti-Ps-I.

2100 S.H. Marshall et al. / Vaccine 25 (2007) 2095–2102

Table 1Aminoacidic sequence of P. salmonis full-ChaPs protein

N on.

3

E

umbers represent methionine residues as alternative initiators for translati

.4. Evaluation of antigenic potential of ChaPs

The purified recombinant protein ChaPs obtained from. coli BL21-CodonPlus was evaluated for immunogenicity

bOi(

Fig. 6. (A and B) ELISA reactivity of 70 fish sera. C corresponds to

y studying its reaction with naturally infected fish sera.f the 70 sera tested by ELISA, 73% were found to be

mmunoreactive in comparison with its negative controlFig. 6A and B).

healthy fish 1–70 correspond to naturally infected fish sera.

accine

3

saw

4

PtA1sfi

sbopccs

isateetpttfo(bmIagc

etitbrtmeg

itaanuttriicaatoba4b

nf(ndC

eepf[tabrmHltct

A

fVAM

S.H. Marshall et al. / V

.5. Nucleotide sequence accession number

The DNA sequence data (P. salmonis HSP 60 family) wasubmitted to the GenBank nucleotide databases under theccession number AY256822. The BLAST2 program [34]as used for searching sequence homologies.

. Discussion

No effective vaccines are currently available to controliscirickettsiosis in the aquaculture world in spite of the fact

hat the aggressiveness of the bacteria continues to expand.s a reference, coho salmon was the only susceptible specie0 years ago while now the disease has spread to all cultivatedalmonids, and remarkably to other commercially differentsh species as well [18].

Although handling procedures like strict managementtrategies have helped, the problem still remains. Chile haseen the most affected geographic area and though a numberf classical vaccines (bacterins included) and other pro-hylactic approaches have been assayed, the appearance ofontinuous epizooties is a clear signal that new and effi-ient strategies are required for the survival of this importantocio-economical activity.

We report, for the first time the isolation of a highlymmunogenic protein ChaPs obtained from naturally infectedalmonid fish reared in Southern Chile (Fig. 1). The protein,lso recovered from an in vitro-infected fish cell line, seemso be a major structural immune domain expressed in a vari-ty of infective P. salmonis isolates obtained from differentpizooties of the world, thus potentially making it an idealarget for vaccine design. We also describe a distinctive novelurification procedure for the protein based upon the utiliza-ion of its hydrophobicity. This was the only way to isolatehe protein, which was not possible through standard cellractionation protocols, demonstrated by parallel evaluationf equivalent macerate organs by immune SDS-PAGE gelsdata not shown). As a result, we correlated the hydropho-icity of our novel protein with the likeliness of being aembrane-associated structural component of the bacteria.

n addition, sequence analysis of ChaPs demonstrates that ismember of the larger family of heat-shock proteins (HSP), aroup of evolutionary conserved macromolecules and pivotalomponents for cell viability [35].

Immunogenic analysis of ChaPs in vivo suggests the pres-nce of isoforms. Notwithstanding, the form expressed inhis report correspond to the most immunogenic to antibod-es elicited against purified intact P. salmonis, suggestinghat at least this form could be partially exposed on theacterial surface. Additionally, since the highest immuneesponse of the protein increases towards its carboxy-end,

his very well might mean that epitopes from this end

ight be the one fully exposed. The fact that antibodieslicited against the carboxy-end of the protein do react withradient-purified intact bacteria (Fig. 2B) tends to favor this

RpPF

25 (2007) 2095–2102 2101

nterpretation. On the other hand, the serendipitous findinghat the Caulobacter crescentus expression system yieldedlternative forms for the ChaPs protein, truncated at themino-terminus through the differential recognition of inter-al methionines as initiators for translation (Table 1), alloweds to map the differential immunogenicity of internal epi-opes of the ChaPs protein. Fig. 4 (Panel B) shows that fromhe complete protein (polypeptide 1) to the shortest immunoeactive one (polypeptide 4), all display different reactiv-ty to different antibodies. To us, the most interesting results that of the shortest form of the protein (polypeptide 4),orrespondingly enriched in the carboxyterminus of ChaPs,nd gives the strongest reaction with anti-Ps-I (Lane B) andnti-RsaA (Lane D). The most reasonable interpretation forhis result is that indeed, the carboxi-terminus of ChaPs isne of the most immunogenic epitopes of the protein. Also,ecause of the preferential recognition by antibodies elicitedgainst the entire bacteria (anti-Ps-I; Lane B-polypeptide), the COOH epitope is most likely on the surface of theacteria.

The use of E. coli BL21 CodonPlus System as an alter-ative expression system allowed us to obtain high yield ofull-ChaPs recombinant protein to be used on ELISA testsFig. 6A and B). The immunoreactive differences among theegative control sera and infected fish sera were stronglyifferentiated, demonstrating high immunogenic potential ofhaPs.

Members of the chaperones family of proteins, which arexpressed constitutively in all cells, are essential for sev-ral important cellular processes, such as protein folding,rotection of proteins from denaturation or aggregation andacilitation of protein transport through membrane channels35]. “Stressful” stimuli like microbial infections enhancehe intracellular synthesis of Cpn-60 which act as potentntigens in a number of infection including those causedy Helicobacter pylori, Mycobacteria tuberculosis, Erhlichiauminantum, Histoplasma capsulatum, Streptococcus pneu-oniae and Legionella pneumophila [36–44]. Since bacterialSPs are immunodominant targets of both humoral and cel-

ular immune responses in infected organisms [35], ChaPsurns out to be a good candidate as a vaccine component toontrol outbreaks caused by P. salmonis who still is affectinghe Chilean aquaculture.

cknowledgments

This research was supported by grant 122.761-02 to SMrom Direccion de Investigacion-Universidad Catolica dealparaıso-Chile and by a special grant from Alpharma,quatic Animal Health Division-Oslo-Norway also to SM.Z was supported by a post-doctoral position from Vice

ectorıa de Investigacion-Universidad Catolica de Val-araıso-Chile. VH was supported by Alpharma, Aquaticroducts. Animal Health Division-Oslo-Norway. JO, PC andG were supported by FONDEF grant DO3I 11 37.

2 accine

R

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[Immunolocalization of Hsp60 in Legionella pneumophila. J Bacteriol

102 S.H. Marshall et al. / V

eferences

[1] Woese CR. Bacterial evolution. Microbiol Rev 1987;51:221–71.[2] Winkler HH. Rickettsia species (as organisms). Ann Rev Microbiol

1990;44:131–53.[3] Mohamed Z. The discovery of rickettsia in a fish. Ministry of Agricul-

ture Egypt, Technical and Scientific Service, Veterinary Section 1939.Bulletin No 214.

[4] Fryer JL, Mauel MJ. The rickettsia: an emerging group of pathogensin fish. Emerg Infect Dis 1997;3:137–44.

[5] Bravo S, Campos M. Coho salmon syndrome in Chile. Am Fish SocFHS Newslett 1989;17(3):3.

[6] Cubillos V, Farıas C, Alberdi A, Alvarado V, Schaffer JW, Enrıquez R.Caracterısticas anatomopatologicas del Sındrome del Salmon Coho,una nueva enfermedad de los salmonıdeos. Patol Anim 1990;4:14–7.

[7] Graggero A, Castro H, Sandino AM. Fish isolation of Piscirickettsiasalmonis from coho salmon, Oncorhynchus kisutch (Walbaum), andrainbow trout, Oncorhynchus mykiss (Walbaum), during the freshwaterstage on their life cycle. J Fish Dis 1995;18:277–9.

[8] Fryer JL, Lannan CN, Garces LH, Larenas JJ, Smith PA. Isolation of arickettsiales-like organism from diseased coho salmon (Oncorhynchuskisutch) in Chile. Fish Pathol 1990;25:107–14.

[9] Mauel MJ, Giovannoni SJ, Fryer JL. Phylogenetic analysis of Piscir-ickettsia salmonis by 16S, internal transcribed spacer (ITS) and 23Sribosomal DNA sequencing. Dis Aquat Org 1999;35:115–23.

10] Cvitanich JD, Garate O, Smith CE. The isolation of a rickettsia-likeorganism causing disease and mortality in Chilean salmonids and itsconfirmation by Koch’s postulate. J Fish Dis 1991;14:121–45.

11] Fryer JL, Lannan CN, Giovannoni J, Wood ND. Piscirickettsia salmo-nis gen. nov., sp.nov., the causative agent of an epizootic disease insalmonid fishes. Int J Syst Bacteriol 1992;42:120–6.

12] Lannan CN, Fryer JL. Extracellular survival of Piscirickettsia salmonis.J Fish Dis 1994;17:545–8.

13] Fryer J, Hedrick R. Piscirickettsia salmonis: a Gram-negative intracel-lular bacterial pathogen of fish. J Fish Dis 2003;26:251–62.

14] Almendras FE, Fuentealba IC, Jones SR, Markman F, Spangler E.Experimental infection and horizontal transmission of Piscirickettsiasalmonis in freswater-raised Atlantic salmon Salmo salar L. J Fish Dis1997;20:409–18.

15] Olsen AB, Evensen O, Speilberg L, Melby HP, Hastein T. Ny laksesyk-dom forarsaket av rickettsie. Norsk Fiskeoppdrett 1993;12:40–1.

16] Rodger HD, Drinan EM. Observation of a rickettsia-like organismin Atlantic salmon Salmo salar L., in Ireland. J Fish Dis 1993;16:361–9.

17] Grant AN, Brown AG, Cox DI, Birkbeck TH, Griffen AA. Rickettsia-like organism in farmed salmon. Vet Rec 1996;138:423.

18] Arkush KD, McBride AM, Mendonca HL, Okihiro MS, Andree KB,Marshall S, et al. Genetic characterization and experimental pathogene-sis of Piscirickettsia salmonis isolated from white seabass Atractoscionnobilis. Dis Aquat Org 2005;63:139–49.

19] Larenas JJ, Contreras J, Oyanedel S, Morales MA, Smith P. Effectof loading density and temperature on rainbow trout (Oncorhynchusmykiss) inoculated with Piscirickettsia salmonis. Arch Med Vet1997;29:1.

20] Smith PA, Lannan CN, Garces LH, Jarpa M, Larenas J, Caswell-Reno P, et al. Piscirickettsiosis: a bacterin field trial in coho salmon(Oncorhynchus kisutch). Bull Eur Assoc Fish Pathol 1995;15:137–41.

21] Smith PA, Contreras JR, Larenas JJ, Aguillon JC, Garces LH, Perez B,et al. Immunization with bacterial antigens: piscirickettsiosis. Dev BiolStand 1997;90:161–6.

22] Kuzyk MA, Thorton JC, Kay WW. Antigenic characterizationof the salmonid pathogen Piscirickettsia salmonis. Infect Immun

1996;64(12):5205–10.

23] Barnes MN, Landolt ML, Powell DB, Winton JR. Purification of Pis-crickettsia salmonis and partial characterization of antigens. Dis AquatOrg 1998;33:33–41.

[

25 (2007) 2095–2102

24] Jammet A, Aguayo J, Miquel A, Muller I, Arriagada R, Becker MI,et al. Characteristics of monoclonal antibodies against Piscirickettsiasalmonis. J Fish Dis 2001;24(4):205–15.

25] Wilhelm V, Huaracan B, Martinez R, Rosemblatt M, Burzio LO, Valen-zuela PD. Cloning and expression of the coding regions of the heatshock proteins HSP10 and HSP16 from Piscirickettsia salmonis. BiolRes 2003;36:421–8.

26] Wilhelm V, Soza C, Martinez R, Rosemblatt M, Burzio L, Valen-zuela P. Producction and immune response of recombinant Hsp60 andHsp70 from the salmon pathogen Piscirickettsia salmonis. Biol Res2005;38:69–82.

27] Kuzyk MA, Burian J, Thornton JC, Kay WW. OspA, a lipoproteinantigen of the obligate intracellular bacterial pathogen Piscirickettsiasalmonis. J Mol Micro Biotechnol 2001;3(1):83–93.

28] Kuzyk MA, Burian J, Machander D, Dolhaine D, Cameron S, Thorn-ton JC, et al. An efficacious recombinant subunit vaccine againstthe salmonid rickettsial pathogen Piscirickettsia salmonis. Vaccine2001;19(17–19):2337–44.

29] Sanchez-Ferrer T, Bru A, Garcıa-Carmona F. Phase separation ofbiomolecules in polyoxyethylene glycol nonionic detergents. Crit RevBiochem Mol Biol 1994;29(4):275–313.

30] Henriquez V, Rojas MV, Marshall SH. An alternative efficient proce-dure for purification of the obligate intracellular fish bacterial pathogenPiscirickettsia salmonis. Appl Environ Microbiol 2003;69(10):6268–71.

31] Sambrook J, Russel DW. Molecular cloning: a laboratory manual. 3rded. Cold Spring Harbor, New York: Cold Spring Harbor LaboratoryPress; 2001.

32] O’Farrell PH. High resolution two-dimensional electrophoresis of pro-teins. J Biol Chem 1975;250:4007–21.

33] Simon BJ, Nomellini P, Chiou W, Bingle J, Thornton J, Smit J, et al.Recombinant vaccines against infectious hematopoietic necrosis virus:production by the Caulobacter crescentus S-layer protein secretion sys-tem and evaluation in laboratory trials. Dis Aquat Org 2001;44:17–27.

34] Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W,et al. Gapped BLAST and PSI-BLAST: a new generation of proteindatabase search programs. Nucleic Acids Res 1997;25(17):3389–402.

35] Zugel U, Kaufmann SHE. Role of heat shock proteins in protectionfrom and pathogenesis of infectious diseases. Clin Microbiol Rev1999;12:19–39.

36] Kamiya S, Osaki T, Taguchi H, Yamaguchi H. Immune response toheat shock protein of Helicobacter pylori—a candidate as a vaccinecomponent. Keio J Med 2002;51(2):24–5.

37] Mustafa AS. Development of new vaccines and diagnostic reagentsagainst tuberculosis. Mol Immunol 2002;39(1–2):113–9.

38] Pretorius A, Van Strijp F, Brayton KA, Collins NE, Allsopp BA. Geneticimmunization with Ehrlichia ruminantium GroEL and GroES homo-logues. Ann NY Acad Sci 2002;969:151–4.

39] Deepe Jr GS, Gibbons R, Brunner GD, Gomez FJ. A protective domainof heat-shock protein 60 from Histoplasma capsulatum. J Infect Dis1996;174(4):828–34.

40] Deepe Jr GS, Gibbons RS. Cellular and molecular regulation of vacci-nation with heat shock protein 60 from Histoplasma capsulatum. InfectImmun 2002;70(7):3759–67.

41] Amir-Kroll H, Nussbaum G, Cohen IR. Proteins and their derivedpeptides as carriers in a conjugate vaccine for Streptococcus pneu-moniae: self-heat shock protein 60 and tetanus toxoid. J Immunol2003;170(12):6165–71.

42] Bai Y, Li LR, Wang JD, Chen Y, Jin JF, Zhang ZS, et al. Expres-sion of Helicobacter pylori Hsp60 protein and its immunogenicity. JGastroenterol 2003;9(12):2711–4.

43] Garduno RA, Faulkner G, Trevors MA, Vats N, Hoffman PS.

1997;180:505–13.44] Garduno RA, Garduno E, Hoffman PS. Surface-Associated Hsp60

Cahaperonin of Legionella pneumophila mediates invasion in a HeLacell model. Infect Immun 1998;66:4602–10.