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第26巻第4号平成10年12月
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原

容

著
トキソプラズマ原虫由来のhsp70に対する液性免疫（英文）・由井 克之，矢野 明彦 305318
内陸国ラオスで流行した腸炎ビブリオ下痢症の細菌学的検討（英文）山城哲，Sithat Insisiengmay，本馬 恭子，比嘉 直美，
江並 美香，岩永 正明
319−322
研究ノート
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日本医学会だより・・333一罎■■305

Jpn. J. Tro p. Med. Hyg., Vol. 26, No. 4, 1998, pp. 305 318

HUMORAL IMMUNE RESPONSES AGAINSTMEMBERS OF THE HSP70 FAMILYIN TOXOPLASMA GONDIIKATSUYUKI YU11 AND AKIHIKO YAN02
Received May 11, 1998/Accepted September 21, 1998

Abstract: The CDNA for a member of stress induced 70‑kDa protein family (hsp70) from Toxoplasma
gondii was cloned. The deduced amino acid sequence revealed a 667 amino acid protein 70‑80% homologous
to other parasite and mammalian hsp70s. Southern blot analysis suggested that it is encoded by an intronlessgene. A protein of ‑77‑kDa was identified in a lysate of T. gondii tachyzoites by mAbs generated against
the recombinant hsp70 protein. Anti‑human hsp70 mAb also cross‑reacted with a T. gondii protein of theidentical molecular weight. However, immunoprecipitation and Western blot analysis of these proteinsindicated that it was distinct from the cloned hsp70 product, suggesting that T. gondii expresses anotherhsp70‑like protein. Among 22 mice infected with a low virulence Fukaya strain of T. gondii, 6 mice
exhibited significant humoral anti‑hsp70 Ab responses. This Ab responses peaked at 1‑2 weeks of infection,plateaued for 2‑3 weeks and gradually declined to nearly undetectable levels at 6 weeks of infection. In
contrast, the levels of serum Ab specific for soluble tachyzoite Ags continued to increase during the infection
in all mice examined. These features suggest that pathogen‑derived hsp70 may play a unique role in the

induction and maintenance of the host irnmune responses.
Key words: hsp70, Toxoplasma gondii, immune responses, antibody

INTRODUCTION
Members of the stress induced 70‑kDa protein fam‑ily, heat shock protein 70 (hsp70), are expressed inevefy cell type of eukaryotic as well as prokaryotic
cells. They carry out essential functions as molecularchaperones in protein folding, translocation andmultirneric polypeptide assembly (reviewed in Gething
and Sambrook, 1992; Hartl, 1996; Melnick and Argon,
1995). Each eukaryotic cell expresses several different
proteins of the hsp70 family localized in different cellu‑

lar compartments including the constitutively expressedheat shock cognate protein 70 (hsc70) and stress in‑pathogens including Plasmodium falciparum (Yang etal., 1987), Leishmania donovani (MacFarlane et al.,1990), Ttypanosoma cruzi (Engman et al., 1990), Schis‑tosoma mansoni (Hedstrom et al., 1987), andMycobacterium (Young et al., 1985). T cells frompreviously infected individuals have been reported toproliferate in response to native Mycobacterium bovis
hsp70 (Young and Mehara, 1985) as well as recombinant
hsp70 (McKenzie et al., 1991). Those studies indicatethat hsp70s are potent antigens for both B and T cells
during infection with these pathogens. T. gondii is an
obligate intracellular protozoan parasite which can
infect a number of different cell types (Frenkel, 1988;

duced hsp70 in the cytosol, glucose‑regulated protein 78

McCabe and Remington, 1988). Infection of healthy(grp78) in the lumen of the endoplasmic reticulum, andgrp75 in the mitochondrial matrix. Consistent with theiressential functions, the amino acid sequence of hsp70 is

individuals with the parasite induces specific antibody

highly homologous among different species. However,hsp70s are immunodominant antigens of many infec‑
tious microorganisms. Antibodies against hsp70s have

(Aosai et al., 1994; Yang et al., 1995; Yano et al., 1989) .

been described in the serum of patients with a variety of
1

production and both class I and class 11 restricted T cell

responses, culminating in powerful protective immunity

However, the exposed individuals remain chronicallyinfected without apparent symptoms; and the outcome
can be fatal if the host's immune system becomes com‑

Department of Medical Zoology, Nagasaki University School of Medicine

2 Department of Parasitology, Chiba University School of Medicine Correspondence; Akihiko Yano, Department of 'Parasitology,

Chiba University School of Medicine, 1‑8‑1 Inohana, Chuo‑ku, Chiba 260‑8679, Japan306

promised. Considering the importance of the pathogen‑described previously (Komori et al., 1993). DNA

derived hsp70 in the induction of immune responses

extracted from tachyzoites of the T. gondii RH strain
was digested with restriction enzymes, separated on a
1% agarose gel, and transferred to a nylon membrane.

against intracellular organisms, we have cloned a cDNA
homologous to hsp70 from a T. gondii cDNA Iibrary. A
panel of mAbs was generated against the recombinanth'sp70 protein. The humoral immune response against
this hsp70 was investigated in mice infected with a low
virulence Fukaya strain of T. gondii. The kinetics ofthe antibody response against hsp70 was distinct fromthat against other soluble T. gondii antigens, suggestinga unique role of pathogen derived hsp70 in triggeringand maintaining the host immune response.

A Pst I‑Kpn I fragment (nucleotides 430‑935) and a
Pvull‑Hind 111 fragment (nucleotides 1668‑2123) of T.
gondii hsp70 cDNA were used as probes for the 5' and 3'
regions, respectively, of the gene. The blot was prehy‑
bridized and hybridized with 32P‑1abeled probe in 5 x
SSC, 5 x Denhardt's solution, 50 mM Tris (pH 7.5) , 1%
SDS and 50 pglml salmon sperm DNA for 16 hr at 65"C.

The filter was washed twice in 2 x SSC/0.1% SDS at
20'C, twice in 0.2 x SSC/0.1% SDS at 20'C, and twice in

MATERIALS AND METHODS
T. gondii and animals:

Tachyzoites of the T. gondii RH strain weremaintained in vitro using the human B cell line ARH.
The Fukaya strain was maintained in vivo by repeatedoral passages of brain cysts containing bradyzoites into

BIO.A(4R) mice. BALB/C and C57BL/6 (B6) mice

0.1% SSC/0.1% SDS at 65'C. After being rinsed with
2 x SSC, the washed blot was analyzed using a bioimage

analyzer, BAS5000Mac (Fujifilm, Tokyo).

RNA was extracted from the RH strain of T.
gondii by the method described (Chomczynski and
Sacchi, 1987), denatured with formaldehyde, subjected
to electrophoresis through a 1% agarose gel and trans‑ferred to a nylon membrane. The blot was hybridized
and analyzed by the method described for Southernwere originally purchased from SRC (Shizuoka) andwere maintained in the animal facility of Nagasaki
University. BIO.A(4R) mice have been maintained inblotting.

our laboratory for sometime.

Expression and purification of the recombinant hsp70
proteins:

Isolation of T. gondii hsp70 CDNA:

A MOSEloX CDNA Iibrary was constructed fromThe Xho I site was introduced into the 5' region ofhuman hsp70 DNA probe (ATCC, Rockville, MD)

the full length T. gondii hsp70 CDNA (pTH14) and the
ATG start codon was removed by PCR with the oligonu‑
cleotide primers TH5' (5'‑catctcgaggcggactctcctgctgt‑3') and T7 using pTH14 subcloned into pBluescript IISK
as a template. The PCR product was digested with Xho
I and Bam HI and subcloned into the pET15b expression
vector (Novagen, Madison, WI, U.S.A.). This plasmid(Hunt and Morimoto, 1985) in 3 x SSC, 5 xDenhardt's
solution, 50 mM Tris Base (pH 7.5) , I mM EDTA, 0.5%

allows the N‑terminal fusion of a histidine tag sequence
to the T. gondii hsp70 protein. N‑terminal and C‑termi‑

SDS and 20 pglml denatured salmon sperm DNA at

nal fragments of hsp70 were also expressed as fusion
proteins using pET15b. These constructs were generat‑
ed by PCR using pTH14 as a template with TH5' plus
5'‑cgggatccttaaccgagagagagaggcgc‑3' primers, and 5'‑poly(A) + RNA isolated from tachyzoites of the T.
gondii RH strain using a cDNA synthesis module and acDNA rapid cloning module (Amersham, Buckingham‑shire, England) according to the manufacturer's instruc‑tions. The library was screened with the radiolabelled

60'C, and washed in 0.1xSSC and 0.1% SDS at 60"C.
From a total of I x 105 CDNA clones, 15 clones specifi‑

cally hybridizing with human hsp70 cDNA wereselected. The longest CDNA, pTH14, was subcloned

catctcgagctggagacagctggtggt ‑ 3' plus T7 primers,

into Bam HI digested pBluescript SKII (+) (Stratagene,
La Jolla, CA, U.S.A.) , and subjected to DNA sequencing

respectively. After digestion with Xho I and Bam HI,

using a Taq DyeDeoxy Terminator Cycle Sequencing
kit (Applied Biosystems, Foster City, CA, U.S.A.).

expression vector.

Analysis of the nucleotide and amino acid sequences was

genomic human hsp70 gene (Hunt and Morimbto, 1985)
was subcloned into pBluescript SK II. The gene wasamplified by PCR using pH1 primer (5'‑catctcgaggc‑
caaagccgcggcag‑3') and T7 primer. The PCR productperformed using Genetyx‑Mac software (Software
Development Co., Tokyo) .
Southern and Northern blot analysis:
Southern and Northern blottings were performed aseach PCR product was subcloned into the pET15b

A Bam HI‑Hind 111 digested fragment of thewas digested with Xho I (located within the pH1primer) and Hind 111 (located within the multiple clon‑

307

ing site of pBluescript SK ID and was subcloned intoby the sarne method except the fusion was performed

pGEM7Zf ( +) (promega, Madison, WI, U.S.A.). The

using polyethylene glycol. The rat anti‑hsp70 mAb 7.10
(Kurtz et al., 1986) was purchased from Affinity BioR‑
eagents, Inc. (Neshanic Station, NJ, U.S.A.).

DNA fragment containing the hsp70 gene was isolatedby digesting this plasmid' with Xho I and Bam HI and
subcloned into pET15b. The resulting construct can
express human hsp70 protein containing the N‑terminal
fusion of a histidine tag sequence.

Escherichia coli BL21 (DE3) cells harboring theexpression plasmid pET15b construct were cultured at

37'C in LB medium supplernented with ampicillin.
Expression was induced by the addition of IPTG at a
final concentration of I mM, and the cells were cultured
for an additional 3 hr. The cells were then suspended in

5 mM imidazole, 0.5 M NaC1 and 20 mM Tris (pH8.0)
and lysed by sonication. The lysate was cleared by

ELISA:The enzyme‑1inked immunoadsorbent assay was
performed as described (Hornbeck, 1996). Briefly, flatbottom microtiter plates (Dynatech, Zug, Switzerland)
were coated with recombinant hsp70 protein and soluble
tachyzoite Ags in PBS at a concentration of 2 and 40
pglml, respectively, at 4'C for more than 24 hr. Plates

were blocked with blocking buffer (borate bufferedsaline containing 0.05% Tween 20, I mM EDTA, 0.25%

was purified from the supernatant using a ProBond

BSA and 0.05% NaN3) , and then incubated with the firstantibody. After washing, each well was incubated withalkaline phosphate conjugated anti‑mouse lgs (y and L

column (Invitrogen, San Diego, CA, U.S.A.) following

chain specific) (Tago, Camarillo, CA, U.S.A.) andstandard procedures. Briefly, cells were homogenized in

washed with distilled water before incubation with p‑binding buffer (5 mM imidazole, 0.5 M NaC1, 20 mM
Tris‑HCl, pH 8.0). After centrifugation, the super‑

nitrophenyl phosphate substrate solution. Soluble tachy‑

centrifugation, and histidine tagged recombinant protein

natant was applied to the ProBond column. The column
was washed with binding buffer followed by wash buffer(60 mM imidazole, 0.5 M NaCl, 20 mM Tris‑HCI pH
8.0). The bound protein was eluted with elution buffer(1 M imidazole, 0.5 M NaC1, 20 mM Tris‑HCl, pH 8.0)

and was dialyzed extensively against PBS. SDS‑PAGE
analysis of the purified tagged proteins from full length,

N‑terminal and C‑terminal T. gondii , hsp70 constructs
indicated single major bands of 76‑kDa, 46‑kDa and 31‑
kDa, respectively, after Coomassie Blue staining. The
yield of the recombinant ptotein was approximately 1
mg from a 1 Iiter culture. The protein was concentrated

with a Centriprep‑30 concentrator (Amiconi Beverly,
MA, U.S:A.).
MAbs specific for hsp70:

BALB/C mice were immunized with purified recom‑
binant T. gondii hsp70 fusion protein in complete
Freund's adjuvant. The booster immunization was

zoite Ags were prepared by extensive dialysis of thelysate of T. gondii RH strain tachyzoites with PBS.
Western blotting:Western blot analysis of hsp70 expression was
performed as previously described with a slight modifi‑
cation (Tamura and Yui, 1995). Briefly, rotein lysatesof T. gondii (7.5xl08 dells/ml) and the human B cellline ARH (2.5xl07/ml) were prepared in lysis buffer
containing 300 mM NaC1, 50 mM Tris'HCI (pH7.5), O.
5% Triton X‑100, 10 pg/ml leupeptin, 10 pglml pep‑
statin A and I mM PMSF, and centrifuged at 10,000 Xg
for 15 min to remove nuclei. Each sample was separated
by 10% SDS‑PAGE and electroblotted onto a nitrocel‑

lulose membrane (NitroBind; MS1, Westboro, MA) .
Blots 'were blocked with 10% milk in Tris‑bufferedsaline (pH 7.6) containing 0.1% Tween 20 (TBST) ,probed with anti‑hsp70 mAbs in TBST for I hr, incubat‑

ed with biotinylated anti‑mouse or anti‑rat lgG Ab(Jackson ImmunoResearch, West Grove, PA, U.S.A.) at

performed using the same antigen in incomplete

1:2,000 for I hr, and incubated with horseradish perox‑

Freund's adjuvant. Spleen cells from the primed mice
were fused with SP2/0‑Agl4 cells using a cell fusion
apparatus type SSH‑1 (Shimadzu Co., Kyoto) accord‑
ing to the manufacturer's protocol. Fused cells wereidase‑conjugated streptavidin (Zymed Laboratories,
San Francisco, CA, U.S.A.) at 1:3,000 for 30 min.
Protein bands were visualized using an ECL detection

system (Amersham, Buckinghamshire, England)

selected in medium containing HAT, and the super‑according to the manufacturer's specifications.

natants of the cultures were tested for the presence ofanti‑hsp70 mAb by ELISA as well as by immuno‑

In some experiments, protein purified by immuno‑
precipitation was used for Western blot analysis. Im‑

precipitation of hsp70 from T. gondii lysate. Positive
hybridomas were cloned by the limiting dilution method.

munoprecipitation was performed as previously de‑scribed with some modifications (Yui et al., 1988).

A mAb specific for human hsp70, IEll, was generated

Briefly, cells were lysed in lysis buffer containing 0.5%30880㏄㏄融GGAGTGTAG鵬A聯欄質CG㈱袖GT耽τC瓢紬G舳G醐G㏄照開㎜GG愚CC㈱㏄τC 160

TGCハAg」嚇丁τGCCムGTGTG TTTCGTCT雪T TTTGGCTTGムACA㏄GハGハ」雪TT㏄ACCGTG鷹TG口C＝博C GCAGCハCGGACA盈GTTGCT口CCA㏄ハICC田ハCGTCCGAGTA CAmACCACAC ACCGCCCTCτTCG麗CC鵬ムτCムTC￠CCコG C鷹鷹㏄G 240
GGTGTGCTGT CCGG田盛CGハG圧℃ハ、田恥GGGTG TGTCCGGCACコ迫㏄CCCGC為、717TC潔1℃丁田丁ぬGGムGムGTGC CGTTCCG魍 320
Gk3魍』短㏄TC 」燃C田駄㏄』㌧ CGGAコ℃GMG G¶臨GNm 熈C￠TCC2』L2聖CCハ」『m¶℃ C質瓢！CILCT CCCCCG㎜C■ 400
420
雪TTTTGTGTC GCGTTGCAGT
1

CGTTTGTCCC

TGCムGムムG盈CハムC

ATG

「10

GCG

MET

GムC

aユa

TC■CC雪GCT

asp8erG『GGGTムTT

pro

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val

g工y

ile

GTG

G軌

1㌧碧C

A「℃

㏄G

val

glu

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ile

ala

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TCT

τGC

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ser

cy5val官lu

ACC

CCG

GTG

GAC

a31》1eu

ACC

T黙T

488

ハ」GG

」』CG

554

C？τGGCムCC
gly

thr

thユr

tyエ・

30

TG矯

1U』G

IU』⊂1

lys

asn

asp

GCG

TTC

ACC

va工trp

G凋T

GCT

ala

40

IUK！

GAC

asn

C」融G

GGllL

袖C

asp

gl篇qユy

asn

arg

thr

GムT㏄T

GCGムAG

A△C

CAA

Gコ℃

50

TCC

TハC

GTC

GAC

ACG

GAG

AGムCTT

GTC

GGT

620

七hr甘ro5er七yrvalalaphe七hra3P七hrgluargユeuva191y＆s津alaalaly8asnglnva1
60

70

80

GCA OGC為ムC CCG GA為ムハC ACC ATT TTC GAT GCCハムG CGC CT蕊ATC GG，C㏄柚G TτT G為T GA響CCC
a工a arg agn pro glu a8n七hr ile phe a3p a工＆ lys arg leu ile qly arg ly8距he ag2agゆ甚》ro90

100

TCG GTC C為G江℃G GAC ハLコX∋」瓶G CAT TGG CCムTTC」眺G GTC AτT GCT GGT CCG GGA GACハAG CCC CTC
8er val gln 3er asp me七 1ys hi8 七rp pro茎》he ly3 val ile ala gly pro gly a3g lys Pro leu
110
A皿

G勘L

G田C

1㌧CG

Tハ己C

C鵠

GGム

鵬

」瞭G

ハLCG

τ¶℃

C」鬼C

CC了

GIU㌧

130

ATG

AハG

752

120

G盃羅

GILG

G宏■

田GC

GCC

1聖τG

GTT

㎜

ilegl廿valthr七yrglnglyglulyslysthrphehispr・qlugl・va19erala鵬tvalleu
GGに：ムAハL

686

818

140

GAハ・ATC

GCG

GムG

GCτ

響』」C

C田C

GGC

AゑG

GムムGTGムハG

GAG

GCC

GTC

盈厘丁ムCC

GTT

884

glylysme七1y891uiユealagユuala七yrleug工ylysgluvally8qlualavalile七hrval
150

160

CCT

GCG

工盛C

T㏄

AムC

Gム江l

pro

ala七yr

phe

asn

asp

TCG

a3r

CAG

CGT

Cハ、G

gln

arg

gln

GCTムCC

ala

170

』噛G

GAT

thrユy3a8p

GCT

ala

GGτ抽CCム、丁田GCC

gly七hr

iユe

ala

GGC

CTC

ムGC

gly

leu

ser

180

950

190

Gコ℃CTC C㏄ATT ATC AムC GムG CCC為、CA GCG GCT㏄Cム了T GCT T為IT GGT CTG Gゑ、C曲GムAG GGC TGC 1016
val 工eu arg ile i工e asa g：しu pro thr ala ala ala ile ala tyr gly leu a5p工y3 1ys gly cys
200

GGT

GハGハITG

gly

glu

AAC

me七

GTC

asn

CTC

val

ハIT（：TTC

leu

ile

GAC

Ehe

210

ATG

GGT

G㏄

asp

me七glyGGTハ・CG

gly

TTC

GAT

gly七hユr

GTG

TCG

1082

CTG

CTTハCAATC

phe皇呈継leu

leu七hrile

GAT

GムC 1148

A

220

GAハGACGG聖ATC

TTT

G肌G1C

230

A融G

GCC

ムCC

GCT

GGT

GACムCC

CAT

CTT

GGT

GG田GAA

TTC

91uaspglyiエephegluvallysalathralaglyasp七hrhisleuglyglygluaspPheasP
240

250

AAC CGT TTG GTG GAC TTC 駅訳】GTC CハG GAC T了CハAG CGCムAG A赴C CGC GGAハAG GハICムTC AGC麹CC 1214
asn arg leu val asp phe cy3 val gln asp pheユys arg lys asn arg gly ly5 asp ile ser七hr
260
CGT

270

納C

AGC

㏄C

CTT

CGT

C（に

C江陽

CGT

asn

3e工」arg

ala

leu

arg

arg

leu

ACT

CハG㏄ハ、ACC

ATC

GAハLム聖T

thr

gユn

ハLCC

CAG

T㏄

Gハ』G

CG（：

gln

cys

glu

arq七hr

280

ハLCC

arg

ハ』鴫

thr

ハ」GA

ユyg

κ田

CTC

a：g七hr

田C田

leu

ala

thr

ile

glu

ile

TCT

asp

CTT

ser

ハIGC

seご

GTG田CTムTC

TCT

CGT

ser

arg

290

GAC

ハ1㏄

ser

1280

ser

300

TTT

GハG

G㏄ATT

ユeu

phe

glu

GAC田ムIC田CT

glyi工e旦一val

3er

ile

1346

A

310

320

GCG C㏄ τTT G2㌧G GAG CTT TGC ATG G趣C T既C TTC C㏄Aハ、C TCC CTG TTG CCC GTC GAG植G GτC CTC 1412
ala arg phe glu glu leu cys me七asp tyr K｝he arq asn ser leu leu pro val glu lys▼al leu
330

AAG

GAC

Figure

TCT

GGT

ATT

GAC

」軌G

C㏄

340

TCG

GTC

ハGC

GAムG田T

GTG

宏TG

GTT

GGT

GGA

TCT

ACC CGTATC1478

l Nucleotide sequence of the T8 on伽hsp70cDNA．Nucleotides are numbered on the righ仁margin and amino acids above the nucleotide sequence．The sequence contains thefollowing

underlined elements：（A）potential glycosylation sites；（B）GGMP repeats；（C）
AATAAT polyadenylation signa1−1ike sequence．Both strands of the subcloned cDNA in
pBluescript SKII（十）were completely sequenced．

309

1ysa5卵erglyiエea3plysarg8ervaエs就glu、valvaUeuvaエ9エygエy3er七hrargne
350

360

CCC

AAG

ATT

C為、G

CハG

OTC

pro

lys

ile

qln

gln工euA碧CムCτGJ』Cile七hr

a8p

TTC

phe

TTC柚C

phe

GGムム瓦G

agぬgly370

GhG

ly3gluCCG

pro

CCC 1544

T㏄ムGG聖CGムTCムハC

cys

arg8er

ile

asn

pro

380

G汎T GAG GCC GTT GCG TAC GGT㏄コ！㏄T GTC CムG GCムGCGハ1τC 雪TG納G GG為、GTτACC 為GC TCT CAG 16工0
asp glu a工a val ala七yr qly ala a工a val gln al＆al旦ile leu lys gly val thr3eτ5er gln
390

GTG

400

C盈G

GムT

T思G

CTT

C壬τCTG

G蕊T

GTT

GCG

410

CCT

CTC

TCτCTC

GGT

CTG

GムGムCムGCT

GGT

GG：2GTC

1676

va191n＆5Pleuエeuエeロ1eua3pvalaユaproleu3erleuglyleugユu七hralaglyqlyva1
420

ATG

ACC

ムハ・G

CTGムTTme七七hr

lys

ile

GCG

GハJC

ムハζC

ala

asp

asn

gln

AAC

CTC

CTG

GGCム蓋ムTTC

leu

430

Gハハ・ハGA肋Cハ・Cムハ

glu

arg

asn七hr七hr

CGム聖C

CCG

ACC肱G肋GτCτCAG海CC

ile

pro・ヒhr工ys

GTG

TハJC

lyg

8er

440

CAG

CCA

pro

GGA

gly

GTG

val

聖㏄ACC2LCG

gln七hr

CAG

leu

gln

ile

val七yrGムムGGT

GハG

CGT

glu

glu

arg

ala

GGT

GτC

gly

GCG凛TGムCC

Aハハ・GムC

JLAC

me七七hごly3a8p

a3n

C望G

Gム田GGT▲望C

CCC

CCC

480

GCC

CCC

CGT

CCC

CムハムTC

GムA

490

TTC

GAT

ATC

GAC

GCT

AAC

GGTハL田C

盈GC

ムハC

CAA

ser

asn

gln

ile七hrile七hτasu

asp

CA孔GAG

GCA

GAG

TAC

GA瓦GAC

ATG袖C

G田C

ACA

GCG

C曲GムCムAG

ATC

ACC

ATC

ACG

ハハIC

GAC

▲AG

C¶匹｝

G

ils

GGC

gly

C㏄

arg

C田C

AG田

leu3er

GCG

τCC

TCC

AAG

AA八GCC

G瓶ムTC

ala5er

glu

iユe

CハC

CG曽GTG530

ACC

GGAハAGCGC

ATG｝σTG

as茎）aごqGAC

me七val，

GA瓦CねG泌C

A八G

550
ハハC

T顕」C

asn七yr

cyg

TGC

T顕LC

tyr

CムC

Gハ、CムG瓦GAC

2006

GCGハムG

AAT

GGC

2072

TCC

2138

560

A瓢｝

』駈Gハ」CAG

』LCC

蛍TG

GハLT

hi5me七arg9工a七hr

leu

asp

G漉C

GハG

蝕G

asP9：Lu

570

G盈G

工940

540

GAG

ginglualagluly5七yrly5alaglu＆5Pglugエnasnly5hi3argva191ualalygasnrlgly

TCT

1874

ilegluval七hrpheaspileaspalaa㎝91yiエe蹴asnva工七hralagln−thrglylγ5
A
500
510
520h一

leu91u

1808

470

CムC

asnユeuleuglyエyspheh斗sle囲spglyilepr。pr。＆1apr。arqgly▼aエprogl

Gハ

1742

450

CTGムTT460

GTC鳶CTTハC

phe七hr七hr七yr

C口丁

1U鬼G

ly51eu

lyg

G』』C

植G

』配碧C

a8茎）ly3ilLe

ser

CTG

CAA

580

ACτGCCハハJC

AハG

GCC為、TC

CムG

Gム、G

GCC

CTT

GムJC口GGGACムAG

AAC

2204

serglua8pargaspセhralaasnlysalaiエeglngluaユaleロa3ptrpleuaspエy3asngln
590

600

CT五GCA

GハG肱G

GAG

G航TTC

GAG

GCG

leu

ala

glu

lys

gユu

glu

phe

A為G

CTG

TAC

CAG

GCA

GGコ！GCG

glu

AAG

ala

CAG蝕G，G肱GTT

lys・91n

glu

GAG
va1

TCC

91u

G1C

3er

TGC

ACA

CCム駕TC蕊TC

cys

七hr

pro

GGT

ATG

val

GCT

GGT

GGC

ATG

七hrly3ユeu七yrglnaエaglyalaa1＆aエa

l

l

me七ro

CCτGGT

GGT

ATG

GG田Aコ，GCCC

1皿e七ro

l

GGC

l

l

me七1

GGC

GGG

ATG

GGC

l

me七・1

640

l

ATG

GGC

met

GGT

l

l

ATG

CCT

GGT

me七ro1

GGτ・ム響G

2270

ile

630

GCA

GGT

ile

620

610

ACC

lys

工鵬t

CC宏GG■ 2336

ro1

650

GGC

工．me七工GGTハ、TG

CCC

GGT㏄A

1me七ro

l

ala

2402

B

660

G㏄ATG

GGA

GGC

TCT

GGC

GGC

CCCムCCG『GGAG

GAムG田T

2444

GAT−othrva19エugluvaユa叩

丁曲CTくi｝TTGハ、AハCGGAハ」駈Gハ、ハJGTGλACハムA」臨融CCCCムTG聖G恥CGTGハCAG

CGG鵡¶㎜嘔｝・AC質C㏄惚CCTGK≡｝Nτハ工乳㎜G1顯CC」』GT1協丁㏄？G

丁ハC2、LC服｝皿｝■

Cτ㏄CACCτA

田AコXコ㏄1』CAτGT五1コ1LT1曳丁抽」CATC」随CT¶℃¶X｝¶蕊λ鴨ハLG

TCCC「理㏄TG

1（臨麗袖CTT

TTTT田GGGTT

CTTCGGAAG為、盈G』燃G田盛AC 2524

CCGNG舳Gハ」G1鵬剛蝕G』㌧田盛㎜mコ！CGIU噛」GT望LTC

L■㎜LT（：！CCTc

Fig皿e1−2」眺∬！TC」陶（瓢ハGTC 2604

G袖GCATC1顕」C㎜TC㏄㏄A

2684
2739

' ' " " " I " " 'E'GE'WI 'R"S' 'NI I "D'WIDI ItNWt,.DK"‑GTS' I':,TKDru'MQ" 'EAAIXA'IEI ' I IQSTS LP

310

Triton‑X and centrifuged to remove nuclei. Each sam‑ple was incubated with anti‑hsp70 mAb at 4'C for I hr,
and with protein A Sepharbse (Sigma, St. Louis, MO, U.
S.A.) for an additional I hr, washed, boiled with SDS
sample buffer, and separated under reducing conditions

by 10% SDS‑PAGE. After electrophoresis, proteins
were electroblotted onto a nitrocellulose membrane, andthe presence of the specific proteins was detected usinganti‑hsp70 mAbs or mouse serum as described.

The GenBank accession number of the T. gondii
hsp70 cDNA sequence is U82281.

RESULTS
Structure of T. gondii hsp70:

T. gondii hsp70 cDNA was cloned by screening a
cDNA Iibrary derived from the tachyzoites of the T.
goerdii RH strain with the human hsp70 gene probe(Hunt and Morimoto, 1985). The entire nucleotide
sequence (2739 base pairs) of the longest CDNA clone(pTH14) was determined (Fig. 1). The CDNA sequence
contair)ed a single large open reading frame predicting a

667 amino acid protein of 72,292 daltons. This coding
region was flanked by 443 bp on the 5' end and by 295 bp

T･g‑ MA‑‑‑DS‑‑‑‑P‑‑‑iAvGrDLGTTYscvGVWKNDAVE I IANrpGNRTTPsrvAFT DTERLVG
p.f. I 'SIRGl!a'NLIESN:.It I t I " ' t 'RIEN'D' t I t'‑‑'‑"TeEGI:. . . I t I I 'QIERI ' I ' i 'T,c.
M , m.

SRGPI I I ' I " I 'FQEGR" ' I ' t

M･1‑‑‑‑‑‑‑‑R" t " ' I ' INd'VIS ILEeGDPwrvl 'SEI S'

t t ITI 'XNQVAR pENTIFDA L IGRKF DDPSVQS Dl(K 93.ARNG.V. . .QP. . . .‑.‑‑ r‑.Nv.‑‑.T.‑.s‑v!a HMG. .‑T.g. EWpFxVlncPGDl L:EvTYQGE CTFHpErvsAuvLGxM: : IAEAuJG: VKEAVITVPAYFNDSQRQATKDAGTIAGLSVLRI INEPTi :AYGLDX 1 8 8p.f. " t 'T':CS'VDE"M" I " I ' I l 'LI ' I t lllS" 'Q" "N' I 'F' I ISltN" 'N'Mi ' I I I 'Hl
T.c. " " I ' i:XGD" 'V'Q'QFR' IT' I 'NI I I ' 'St "S" I " ' 'S" ' IQI 'XI 'V' I l
M.m. ' I ' IMIV'‑NDAGR'XVQ'E'X"T'S 'Y" I l IS'tlT"'i"""' 'TITN
M.1. '‑ S' 'P‑'
1‑‑ ' I‑‑RI M
' ":,
" tRD
' " " ' IEDITD: D XYTAQ: ‑‑P‑‑‑"‑1‑1‑1‑'‑'"A" l " IE' IQ" " i tVi ""VI " ' I INt l'l

I "T" " N' I

T.g. :cccG‑E:QIVLIFr)ncGeTFDVSL:,TIEDGIFEVXATAGDT!E!GGEDFDNR:,VDFevQDFIWCNRGKDISTNSRALRRLRTQCERT: T:,SSSTQATIE ID 2 8 6p.f. "X'I"R'I""Ll""""""'1ltllt""I'l'l""I'll INltlE'Ii'lllil 'I"X""""'t"t'Atitltll"'1ltll
T.c. VEDlX IR 1'ItlILIII""T""DG"""ttN"'ill"""Ill AEFTDE ' I t"RI I ':,i . ll'i t " " l tA' t 'AI t t l "N 1 1 " " l,,.m. ‑RVIA'R """:,..t"tl' I "'111ltl'e
S ""'t"I'Itl" M'NHF:AE" "EK‑" I I 'E'IC"V" ' "A" IA' I ' " t I I ' 'St ' t 'M. I .
‑GER QTI V l'
T.g‑ S:,FEGID‑‑‑‑YSVS ISRAarEErcMDyFRNSrlLPvsKWJCDSG I D:CRSVSEW:,VGGS TRIPR:QQL :TDFFNGXE?CRS IN?DEAVAyGAA:VQJ IL 3 8 2
P.f.
T.c. A " V' ‑‑‑‑‑'GE:,. .GT'Q" 'R"Q'AXM" 'A'EID" " " "VM"VS" 'R" 'IJ: C"Q" " ...
N･m. "Y" "‑‑‑‑‑FyT"T" " " 'NA':,. .GT.D" ' 'A'R'A:CE"'SQIEDI" ' LNR" " '"."""R'LQ"""'‑‑‑"'"""" R" "H """""""'T"RE'el"'A""""""""""'
TV""""': DT""I' AmS

M. I .

" "M'AVTD'Vl "JTG"'
Yl DS ' pLF:lDEQLI "E 'QRITQ':,1'DRTRQ'FQS'V"A"SVSEIDH'' 'NXev""V"V" 'L"ev'

T.g. RGVTS SQVQDn:iLl!DV7XPLSLGLE TAGG :1CL :ER11lxrlpT!CKS QTFTTYADNQPGVL rQv :GERaMT DNN: GXFHLDGIPPAPRGVPQIEVTFDI 4 8 2P.f. S'DQ'NA' I I ' I ' f I 'CS" " " " " ' 'All'lIIlt'l" ':J. . t I t tK'' e
IPt t l ' ' I 'L
T.c. TIGX':iC'TEGt I " I 'T"TI 'I"
I ' I" '" I I 'ItS' I ll II "S":' I IHI ' tF' I ' " 'CH" 'T'E'S"
l ' t 'V"RI
M.m. S'DRIEN" ' t " '" T t " I 'II" "E'T' t "" " " 'QT"L" 'S'
M.1. ""‑'‑EVKDV" 'D pl"I"Kt. 'I l t t 'RIE' I I IA:)l S Q" ' tQ" 'EIASH'R" 'SIEIT' d "T.g‑ D NG:mfVTAQDRSTGRSNQIT:TNDRGRI'SASE IDRMVQEAEK EDEQNKERVEARNG:,ENycYEMR:Q1:LDD :xtKDKISSEDRDTANZAIQEAI'DW 5 8 2
p.f. " " 'L.". '. .VE" ". ". .Q" "H ' " " " " QD
" . ' . ND
' . . . . . . . . . . . .evRSS.E.Q.r.E.:JQPAEIE.CM.T.TTI.E.
T.c. . . .L. .S.EE.G. . .R. . .V:,. . . . . . . .RA. .E. . .R. .A. .E. . .KDQVRQ:D. . . . . . . .AFS.ruAW.PNVAC. .EEA.: .ITS.VE. . .E .
. . .:,. .S.V. . . . . .E.RL . . . . . ". '. :' ;D" .E . . . . . . . . . . .XQRDR.SS. .S. .S,arN.KA.VE. . . .QG. .ND. .KQXILDRCN. IIS .
M.m.
M.1‑ . . .VH. . .X. .e. . .E .T.R.QEGS .‑. .RE. . . . . .RD. .AEAE . .RK EEADVR.QA.TIN.QTE:Crv QRETENG.RVPE . .L. .V!5AAVAE:A

ERKI RS

T,g. LD!QVQLAE: EFEAROXEVESVCTP:ITRLYQ AGAA AeGMPGGM SG' GGM GMPG

P,f
T,c

M,m

M. l

s‑!MGGts'eGM,

sAGMGGsGeprvEEvD.E. . . . .G.D.Y. . . . . .A. . . .A. .Ms.1. .D‑.AG.‑. . . . . . . .P. . . . . . .Ps.‑‑. .. ‑NF. . .p.A.
. .‑.NAPA. .‑. . . . . .
.

.NN. .E.s. . .Y.!E . . .L.NL. . . .M.NM. .GM. . .GMP. . . . . . .P. . .. .. .GMP.G,G).
. .P. . .. . . .P.‑. . .G. .NPss.s. .E. . .

...FP*.
‑‑ .Ass.‑‑. . . .T. . . . EQ... . .L.x. .N.
.‑‑..APPs.
. s‑.
. MP. . . ‑‑. . I. . .

: TALGGTDISAI SAMEXLGQDSQA:,GQAI . EATQA , sxv . . ‑EASAP . . ‑SNSTDDV:J‑TRRWSTTN . spR‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑

Figure 2 Alignment of arnino acid sequences of T. gondii hsp70 (T.g.), P. falciparum hsp70 (P.f.)(Yang and Tan‑Ariya, 1987) , T. cruzi hsp70 (T.c.) (Engman and Dragon, 1990), L.
donovani hsp70 (L.d.) (MacFarlane and Blaxter, 1990), mouse hsc70 (M.m.) (Giebel and
Dworniczak, 1988), and M. Ieprae (M.1.) (McKenzie and Adams, 1991). The amino acidsequence of T. goudii hsp70 was predicted from the cDNA sequence of clone pTH14(GenBank accession number U82281) . Identical arnino acids are indicated by asterisks.

Gaps were introduced to maximize homology. Amino acids of T. gondii hsp70 are
numbered on the right margin.

667

311

terminal signal sequence. There were three patential
Nlinked glycosylation sites in the deduced amino acidsequence. At the C‑terminal end, there were four com‑

s:'
*

*b

plete and one incomplete GGMPGGM repeatedsequences. Similar GGIV!:P repeats are present in the
majarity of parasite hsp70 proteins as well as humanand rodent hsc70 (Fig. 2). T. . cruzi and P. falcl:parum

S

:

;;:j

!!.!'i,f

hsp70 contain 9 and 5 GGMP repeats, respectively.
Human and mouse hsc70 contain 2 repeats. The C‑
'

S

'

.

s s

terminal regulatory motif, EEVD, was conserved as inhsp70s of other eukaryotic cells (Freeman et al., 1995) .

The 'I'. go;rdii gene encoding hsp70 was analyzed bySouthern blotting (Fig. 3A) . The DNA was digested
with restriction enzymes and probed with two cDNA
probes; one spanning nucleotides 430‑935 (correspond‑
ing to amino acids Met‑1 to Thr‑164) an,d the other
1668‑2123 (Thr‑407 to Leu‑560). These probes hybrid‑･( " sized with the identical restriction fragments of the hsp70

hybridized with a *'P‑1abeled Pst I‑Kpn I frag‑gene. Only single bands of approximately 4.9, 4.5, 7.0
and l.9 kb were detected in Bam Hl, Eco Rl, Hind 111,
and Pst I fragments of the DNA after high stringency
washing (0.1><SSC, 0.1% SDS at 65'O. The size of thePst I fragment was identical to the distance between the
twa Pst I sites of the cDNA sequence (nucleotide 431‑

ment (nucleatides 430935) (14) or a Pvu II‑
Hind 111 fragment (nucleotides 1668‑2123) (5sites. The blot was also washed at low stringency (O.2 x

Figure 3 Southern and Northern blot analysis of T. gondii
hsp70 gene.
A. Analysis of T, gondii hsp70 gene by Southernblotting. DNA (3 pg) was cleaved with therestriction enzymes, Bam Hl (1, 5), ECoRl (2,6) , Hind 111 (3, 7) and Pst I (4, 8) . The blot was2300) , implying that there is no intron between these two8) of pTH14 CDNA. The blot was washed

SSC, 0.1% SDS at 20'O after hybridization, but nounder stringent conditions (0,2 x SSC/0.1% SDS
at 65"O . Kilobases shown at left.
B. Northern blot analysis of hsp70 gene expression

additional signals were detected. The expression of thisin cultured T. gop2:dii tachyzoites. Total tachy‑zaite RNA (5 ,lg) extracted from cultured
tachyzoites (1) and tachyzoites heat shocked at
42 C for 2 hr (2) , was analyzed by blotting. The

hsp70 probe was a rst I‑Kpn I fragment spanning the nucleotides 430935 of pTH14. The
positions of rRNA are indicated at left.

on the 3'‑untranslated end. The higher eukaryotic
polyadenylation signal AATAAA was absent in the 3'

untranslated region of hsp70 CDNA. Instead, the
AATAAT sequence was found 10 nucleotides upstreamof the poly (A) tail. The deduced primary sequence was
80 8%, 72.3%, 72.3% and 74.1.0/0 homologous to P. .fal‑

ciparum hsp70 <Yang and Tan‑Ariya, 1987), T. cruzi
hsp70, L. dovtova ei hsp70 (MacFarlane and Blaxter1990), and human and rodent heat shock cognate pro‑teins (hsc70) (Dworniczak and Mirault 1987; Giebel etal., 1988), respectively, indicating that this CDNA en‑
codes T. gordii hsp70̲ The homology to M. leprae was
46.1%. Inspection of the amino acid sequence revealed
the absence of long hydrophobic stretches and the N‑

Figure 4 ldcntification of the native T. gondii hsp70 bymAbs .
A. Lysate of T. gordii RH strain tachyzoites was

separated by 10% SDSPAGE, blotted, andprobed with mAbs TXDll (1), TxA5 (2) and7.lO (3). IYlolecular weights are indicated on
the left.

B. Lysate of T. govrdii tachyzoites was immuno‑
precipitated with no Ab (1) , T x Dll (2) , TX G3(3), TxB12 (4). TxH2 (5), TxA5 (6) and
IEll (7); separated by 10% SDSPAGE; blot‑ted; and probed with anti‑hsp70 mAb 7,10.

31 2

Table I ELISA of anti‑‑hsp70 mAbs produced in this study
Binding of mAb with recombinant proteinsprepared as*

Immum2 a‑ N ame

Isotypetion * *hsp70 T >< D11y2b

T x G3ylyl

T >< B12

hruTlanT x H2T x A5

y2a

IEll

y2b

T, g0 rdii

N‑terminal

C‑terminalhuman

hs p70f ragment

fragITLent

hs p70

yl

+
+
+

+
+
+
+
+

+
+
N.D. * * *

N.D.

+
+

hs p70*The specificity of each mAb w as tested by ELISA using total 'J'. gondii hsp70, N‑ as well as C‑
terminal fragments of the. hsp70, and human hsp70 recombinant proteins.
The results were measured using plate reader with a 405 nlT1 filter.
+indicates positive results. ‑ indicates the value is within the mean value of the cOutrol (withoutA,b) tstandard error of the mean.
* *BALB/C mice were immunized with recombinant 'J', g ondii hsp70 cr htmlan hsp70 protein. After
fv:sion of spleen cells with SP2, hybridoma supernatants were screened by ELISA using the same
antigen.

* *not done

gene was deten l ined by Northern blot ar,,1alysis (Fig.
le.vel of hsp70 RNA after treatuent of the tachyzoites at

detected by anti‑hsp70 mAb 7.10, T. gordii lysate was
immunoprecipitated with the panel of mAbs, separated
on SDS‑PAGE, blotted, and was probed with 7.lO. A11 of

42 C for 2 hr.

them specifically precipitated the ‑77‑kDa protein

ldentificatiOn of T. goerdii hsp70 by mAbs:

from T. gondii lysate that can bind to 7.lO, although theintensities of the bands were not equivalent (Fig. 4B) .

3B) . There was no significant increase in the expression

cDNA (pTl･‑I14) was expressed in E, coli as a tag‑ged protein carrying six histidine residues at its N‑
terminal, and was purified by immobilized nickel affin‑
ity cl,1romatography. Mice were immunized with thisrecombinant protein and spleen cells wer, e fused with
SP2 cells to produce hybridomas. The initial screeningof the hybridoma supernatant wa perfomled by ELISA
using the recambinant protein. The hybridomas secret‑Since this was the only band that could be detected by

TXDll and TxA5, we concluded that these mAbs
specificaily bind ta the natural hsp70 expressed iu T.
gondii. The expression of this protein was not signifi‑
D :1 a s 4 ,:::s:::::. ,:e ::, :i7l.･OO :
t;in, g specific antibodies were further screened for theirability to bind to natural< ;i t ;*>‑ ? :piro;. :ei :1'. gordii protein. I'. gordii

lysate was immunoprecipitated with each hybridoma
superl,latant, separated on SDS‑PA('.E, blotted, and4

44: 'irprobed with antisera of the immunized mice. Fivehybridomas producing mAb that can bind recombinant
as well as natural T. golrdii protein were cloned bylimiting dilution (Table l). Western blot analysis was
performed to determine the T, go;rdii protein detectedby these mAbs (Fig. 4) . T XDll and TXA5 bound only
an ‑77 kDa protem in the T govrdu lysate. Anti‑hsp70
mAb 7.lO which is widely cross‑reactive with members
af the hsp70 family including hsp70, hsc72, grp78 andheat inducible hsp72 (Kurtz and Rossi, 1986) detected
the ‑77‑ and ‑72‑kDa proteins expressed in T. go;rdii
tachyzoites. To determine whether ‑77‑kDa T. goprdii
protein identified by our mAbs is identical to that

SS e "̲"*,j27; ,'*".S;j

NLtermjn ;lf ragmente* tcrmiu?: f ragin nt

Figure 5 Western blot analysis of the specificity af anti‑

hsp70 mAbs to recombinant hsp70 fragments. The
mixture of purified recornbinant hsp70, N

and C‑

tcrminal fragments was separated by lO% SDS‑
PAGE. After blotting, Ianes were cut and each

strip was probed with mAb TXD11 (1), TxG3(2). TxB12 (3), TXH2 (4), TxA5 (5), IE11 (6)and 7.10 (7) . Molecular weights are indicated on
the left.

313
BALB f c

C57BL/6

o
0.6

O.4
!

{)4

t
e

.{,ll

1

5 : o.2

02
,)

O

b

o

' ･*
e Q

06
0

'v* 's

e

O.4
s
tp

04

' s]

:

e

0.
0.2

Figure 6 Immunoprecipitation of T. g(,Pidii hsp70s by T X
Dll and by IEll. Cell lysates of T. gordii tach‑yzoites (1, 2) and the human B cell line ARH (3, 4)

were immunoprecipitated with T XD11 (1, 3) or
IEll (2, 4) . Each precipitated sample was split

into 4 groups and separated by lO% SDSPAGE
generating 4 identical gels (A‑D) . Each gel was
blatted onta a nitrocellulose membrane and probedwith TXDll (A), IE11 (B). TxA5 (O and 7.10
(D) . The position of the 77‑kDa molecular size
marker is indicated as ( ‑ ) .

w*k* atte. infectton with T.g""diiFigure 7 ELISA af anti‑hsp70 Ab in the serum of mice
infected with T. goPrdii. Serum samples wereobtained from each of 11 BALB/C and C57BL/6
mice 1, 2, 3, and 6 weeks after oral infection with
20 cysts of T, gopidii Fukaya strain 2is indl'cated.

The levels of antibody against hsp70 and soluble
tachyzoite antigens were determined by ELISA as
described in Materials and Methods. Serum sam=ples were used at l:50 dilution. The means of
triplicate or duplicate experiments are shown.

Five C57BL/6 mice died between I and 2 weeks
after infection.

cantly altered after treatment of T. gordii for 2, 4, or 6

hrs at 42'C (data not shown) . MAblEll, which was
originally generated using recombinant human hsp70,
also precipit ted the protein of the same molecular

N‑terminal ATPase domain. Interestingly, T X A5 mAb
cross‑reacted with recombinant human hsp70, while

weight that can bind to 7.lO (Fig. 4B). This protein,however, was not identical> to hsp70 detected by T.

none of the other 4 mAbs bound to it (Table 1). Anti=human hsp70 mAb, IE11, did not cross‑react with therecombinant T. golrdii hsp70 protein. The 7.lO mAb
bound to the C‑terminal lragment of the recombinantgordii hsp70 specific mAb T X Dll, as will be discussed

hsp70 protein (Fig. 5).

later.

Proteins of the hsp70 family consist of two domain

structures: an N‑termi,nal ATPase and a C‑terminal
peptide‑binding domain (Chappell et al., 1987). To
determine the domain to which these mAb.s bind, we
created recombinant Nterminal and C‑terminal hsp70
peptides both tagged with six histidine residues. The
N‑terminal fragment spanned from amino acid Ala‑2 to
Gly‑404, and the C‑terminal fragment from Leu‑405 to
Asp667. These peptides were affinity purified using

Distinction of T. govrdii hsp70s precipitated by T >< Dll

and IE11 mAbs:
Although Western blot analysis of T. govrdii hsp70
indicated that IEll can bind to the hsp70‑1ike molecule
expressed in T. gordii (Fig. 4) , IEll did not bind to therecombinant hsp70 (Fig. 5) . To detenrrine that themolecule detected by IEll mAbs is distinct from thecloned hsp70 gene product, each protein was purified byimmunoprecipitation with IE11 and T X Dll mAb. The

nickel chromatography and were used for ELISA andWestern blot assays (Table 1. Fig. 5 . Of 5 mAbs

precipitated materials were split inta four groups, separ‑

specific for the hsp,70, 4 mAbs bound to the C‑terminal

distinct anti‑hsp70 mAbs, T XDll, l･Ell, T x A5 and 7.lO

peptide binding domain and I mAb, T >< A5, bound to the(Fig. 6) . Human hsp70 was used as a control for theated by SDS‑PAGE, blotted, and probed with four314

3 :.4. $ . . 7... 1.8

A. T :n i;, #‑;' :::s: "'" e e8i;<;+ :.: ; ,>::;:;;gj.>;:. ;4;::':̲: ̲"S ; :: {):t< .t‑ i S pSl*

N... 'S
f
st ¥

SSi

gn.;

S# !:‑Si;'St

lae'e ･*s ses
Figure 8 Western blot analysis of anti‑hsp70 Ab in the
scrum of mice infected with T. gordii. Lysate ofT. g(; adtii tachyzoites vas immunaprecipitatedwith TXD11 (A.) or IEl.1 (B, and separ'ated on
lO; ; SD

S ･PAGE. After blotting, lanes were separ‑

ated and each strip was probed with mAb T x Dll1), IE11 (2), sera fron i individual BAI̲1'), /c (3‑=5)or C,') 7BL/6 (6 8) mice infected for l. week, anci

e :teSntm ; ‑f igmcn ‑. . 'Figure 9 Western blot analysis of the specificity of anti‑
hsp70 Serum from infected mice. The exp, eriment
was perfauned as described hl the legend of Fig. 5,
The blat of total T. jordii recombinant hSP70 as

well as N=terminal and C‑terminal fragments af
hsp70 Was cut, and each striP was l:)robed withmAb TXDll (1) , TxA.5 (2) , serum from individ‑
ual l))ALB/c (3‑5) or B6 (68) mouse infected for

13AL13/c mice (9lO) irlfected for G weeks. SermTi

l week. The serum samples used herc'* were identi

samples from m. ice which praduced detectable

cal to those used in Fig. 8.

levels of antihsp70 Ab by ELISA were used in thisex periment.

imrnun(.)precipitation. The protein precipitated by T x

detected in the serum of mice hlfected with T. gordit;.

Dll was detected by T XA5 and 7.lO mAb, but not by

BALB/C and C57BL/6 mice were infected with the low

IE11. IEll precipitated a T, go 4dii protein as detec'.ted
by itself (r}2) an,d 7.lO mAb (D‑2) , althr:.)ugh the inten=

virulence Fukaya strain of T. grjrdii, and serum samples

sity af the band was* weak, This malecule is like]yanother member of the hsp70 family, because it can bindto anti‑hsp70 mAbs IEll and 7.lO, and because it has amolecular weight eimilar to the cloned hsp70. This
hsp7Olike molecule is not human hsp70 that could bave
contaminated during the propagation of 'f'. gondii invilro, because the m, olecular weight af the T. gordii
protein precipitated by IE11 was larger than that of thehuman hsp70 (B‑4) . MAbs T XDll and T >< A5 did notbind to this protein (A‑2, C‑2). These reslts suggestthat 'r. golrdii tachyzoites express another hsp70‑likewere collected l, 2, 3, and 6 weeks after infection,
El̲1SA of these samples was perfOrmed using plates
coated with the recon"ibinant hsp7O (Fig. 7). Antibody
to soluble t.achyz(̲)ite antigens was also examined as acontrol. In bath BALB/C and C57r: . L/6 mice, significant
levels of anti‑‑hsp70 antibody were detected in the seraof 3 out of ll mice infected. The antibody levels weremaintained for 23 weeks and gradually declined there‑
after. Six weeks after infection, the serum Ab levels
became nearly undetectable in C5/ BL/6 mice. In con‑trast, the levels of serum antibody specific for solublet.achyz:oite antigens continued to increase during theprotein in addition to the cloned hsp70. These two

infection in all mice we have examined. During themolecules can be specifically identified by T x D11 andcourse of the study, 5 C57BL/6 mice died between I and

IEll mAb.

2 weel{s of h･･ifection. Interestingly, all 3 mice whichdeveloped anti‑hsp70 antibody survived while only 3 out

Detection of anti‑hsp70 Ab in the serum af T. gcndii

of 8 mice which did not developed anti‑hsp70 antibody

infected mice:

did. Therefare, to determine whether there is any

Patho en‑derived hsp70s have been shown to bestrongly immunogenic to the host immune system incorrelation between the ability to produce anti=hsp70 Ab

several infectious diseases. To determine whether T.

these C57BL/6 mice 7 weeks after infection and deter‑
mined the number of brain cysts. There was, however,gordii hsp / O can be a target of the host immune
responses, we examined whether anti‑hsp70 Ab can be

and protection against 'r. gondii infection, we sacrificed

no significant difference in the cyst number between the315

mice which. developed an anti‑hsp70 antibody response(803 153 cysts/brain) and those which did not (790̲+

their close ,evolutionary relationship (Kohler et al. ,1997). This T. gondii hsp70 may be a cytosolic protein

361 cysts/brain) .

because it lacks a signal sequence and ER‑retention
signal. There were three possible N‑glycosylation sites.

Characterization of the anti‑hsp70 antibody in T. gondii‑

It is unclear whether these =sites are glycosylated, but

infected mouse serum:
To determine that the anti‑hsp70 antibody detectedin the serum frorn infected mice by ELISA did indeed

recombinant hsp70s (Figs. 4, 5) shggests that there is nocomparison of the molecular size of the native andextensive glycosylation in these sites. An interestingbind to the natural T. gondii protein, we purified native

feature of the deduced amino acid sequence was the

hsp70 by immunoprecipitation with T X D11, separated it

presence of multiple repeated sequences; four complete

on SDS‑PAGE, blotted and probed with the serum fromand one incomplete GGMPGGM sequence near the C‑
terminus. Similar GGMP repeated sequences were

the infected mice (Fig. 8A) . As expected, the antisera

obtained frorn mice one week after infection bound to
the natural T. gondii hsp70. The antisera from the same
mice 6 weeks after infection barely bound to this pro‑tein. In a parallel study, we also determined whether
these sera contained antibody specific for the hsp70‑1ike

observed in all the members of the parasite cytosolic
hsp70s that have been cloned to date as well as humanand mouse hsc70s. The number of repeats in T. gondii
hsp70 is particularly high among these proteins and is at

protein purified by IEll mAb. The protein isolated
from T. gondii by immunoprecipitation with IE11 mAb

a similar level to other protozoan parasites such as P.
falciparum and T. cruzi. The function of this repeatedsequence is unknown but may have a role in adaptation

was probed with the same sera as used for the detectionof hsp70 (Fig. 8B). These sera did not show any signifi‑
cant binding to hsp70‑1ike protein purified by IE11 mAb.
Finally, to determine whether anti‑hsp70 antibody in thetheir developmental stages within hosts and vectors.
Western blot analysis of the T. gondii lysate withanti‑hsp70 mAb, T X D11, indicated that T. gondii tachy‑of the parasites to the changing envirbnment during

for the Western blot assay (Fig. 9) . A11 6 serum samples

zoites cultured in vitro expre, ss this protein. The
anti‑human hsp70 mAb IE11 also identified a protein of
the same molecular weight. The reciprocal binding ofthese two mAbs, however, indicated that they are dis‑from both BALB/C and C57BL/6 mice bound to the C‑

tinct proteins. Although we were unable to detectterminal but not to the N‑terminal fragment of hsp70,

multiple signals in Southern blot analysis of the T.
gondii genome with the hsp70 probe under a low strin‑

sera of the infected mice bound to the ATPase or
peptide binding domain of the protein, recombinant N‑
terminal and C‑terminal fragments of hsp70 were usedindicating that the major antigenic determinant exists in
the C‑terminal portion of hsp70.

gency condition, the protein identified by IE11 is likely
a mernber of the hsp70 family because it can specifically

DISCUSSION
Full length CDNA encoding T. gondii hsp70 was
cloned by screening the T. gondii cDNA Iibrary with thehuman hsp70 probe. Southern blot analysis suggested
that the gene is located at a single locus in the T. gondii

genome. This gene appears to lack an intron between
nucleotides 431‑2300, which covers 92.8% of the coding
sequence, suggesting that the T. gondii hsp70 gene may
be encoded by one continuous open reading frame, simi‑

bind to two anti‑hsp70 mAbs (1E11 and 7.10) andbecause of its molecular size. The nature of this protein

is unclear and its furth r understanding may require
molecular cloning of the corresponding gene. Lyons andJohnson identified 65‑ and 70‑kDa proteins expressed inT. gondii using polyclonal antiserum against the hsp70
of P. falciparum (Lyons and Johnson 1995). The hsp70s
identified by our mAbs are clearly distinct from their
70‑kDa protein which was expressed in tachyzoites onlyin vivo and not in vitro. Their 65‑kDa protein was

lar to the human hsp70 gene (Hunt and Morimoto,similar to the hsp70s identified here in that it is con‑1985). In the 3' untranslated region, the canonical poly(A) signal AATAAA is not present, although a sirnilar

stitutively expressed, although their molecular weights
are clearly different. The side by side comparison of the

AATAAT sequence is present 10 nucleotides upstream

molecular size or the sequential immunoprecipitationof the poly (A) . This signal is also absent in transcripts

with the two Abs will determine whether they are indeedof other T. gondii genes including that of the major

dif f erent .

surface antigen P30 (Burg et al., 1988).

In an attempt to understand the role of T. gondii‑
derived hsp70 in stimulating host immune responses, weinvestigated, the ability of mice infected with T. gondii

The overall homology of the deduced amino acidsequence was highest with P. falciparum. consistent with316

to generate humoral immune responses against path‑

More detailed study will be required to determineogen‑derived hsp70. Two distinct features were obser‑

whether there is any correlation between the ability ofved when compared to the response against soluble

mice to produce anti‑hsp70 antibody and protection

tachyzoite antigens. First, the kinetics of the antibody

against T. gondii infection.

response was quite different. Serurn antibody against
hsp70 reached peak levels in the acute phase of theinfection (1‑2 weeks after infection) and then gradually
declined. In the chronic phase, the antibody declined tolevels barely or not detectable by ELISA or Western
blotting. In contrast, the levels of seruril antibody
against soluble tachyzoite antigen continued to riseduring the acute and chronic phases of infection. Sev‑

The majority of the antibodies produced during
acute infection with T. gondii appear specific for the
C‑terminal domain of hsp70, suggesting that the C‑ter‑minal peptide binding domain contains the dominant B‑
cell epitope. In agreement with this possibility, arnong
our panel of 5 mAbs specific for T. gondii hsp70, 4 were
specific for the C‑terminal and only one was specific for

the N‑terminal portion of hsp70. Furthermore, antibody

eral possibilities may account for these kinetic differ‑

responses against M. Ieprae hsp70 were previously

ences. Antibodies to pathogen‑derived hsp70 is often

shown to be directed mainly to the C‑terminal sequences(Davenport et al., 1992) . The majority of the antibodies

cross‑reactive to hsp70 of other species including self
proteins. The early anti‑hsp70 response could be due tothe cross‑priming of the reactive lymphocytes by hsp70
of other species prior to infection by T. gondii. The
early clearance of the Ab may also be explained by theabsorption of crossreactive Abs by host‑derived hsp70.
Alternatively, the kinetic differences might be due to thespecific for pathogen‑derived hsp70 appear directed
towards non‑conserved sequences of the protein (Eng‑man and Dragon, 1990) . However, differences in overall

homology in the C‑ and N‑terminal sequences may not
be sufficient to explain the immunodominancy of the
C‑terminal portion. The homology of the amino acidmolecular mechanisms by which hsp70 may be presented
to lymphocytes in a manner distinct from other soluble

sequence between mouse and T. gondii hsp70 is 76.7% intachyzoite antigens. Proteins of the hsp70 family appear

C‑terminus (Leu+405 to Asp‑667). When the GGMP

to have unique roles in the induction of immune

repeated region is excluded, the homology in the C‑
terminal sequence (Leu‑405 to Gln‑614) is 73.3%. It
would be crucial to determine the dominant epitope tounderstand the molecular basis underlying the im‑munodominancy of this protein.

responses such as carrier effects for its associatedmolecules (Barrios et al., 1992; Udono and Srivastava,1993). Finally, T. gondii becorne encysted bradyzoites
with their growth limited in the chronic phase of infec‑

tion. Our preliminary study suggests that the brady‑zoites express hsp70. However, this antigen may
become inaccessible to immune recognition leading to
failure to maintain the antibody level.

The second difference of the antibody response to
T. gondii hsp70 was that only 3 out of a total of 11
infected mice showed significant anti‑hsp70 antibody
responses in both BALB/C and C57BL/6 mice, while all
mice exhibited similar responses against soluble tachy‑zoite antigens. Age and sex matched mice were used in
the experiments, so that it is unlikely to explain thesedifferences. Mice were housed in the same environment
and were infected with the sarne group of cysts. We
currently do not know why only some of the infected

mice generated significant anti‑hsp70 antibody
responses. During the course of this study, 5 C57BL/6
mice died between I and 2 weeks of infection. Interest‑
ingly, all 3 mice that exhibited anti‑hsp70 antibody

responses survived. However, we did not find any
differences in the number of brain cysts between themice which produced anti‑hsp70 antibody during theacute phase of the infection and those that did not.

the N‑terminus (Ala‑2 to Gly‑404) and 70.5% in the

Finally, it has been proposed that mammalian hsp70
participates in the processing of peptide antigens within

antigen presenting cells (DeNagel and Pierce, 1992;
Srivastava, 1993). The members of the hsp70 family of
infectious organisms have a conserved structure. They
may not only become targets of the immune response
but also modulate the presentation of pathogen‑derived
antigens to specific T cells. With molecular probes now
being available, it would be of interest to learn themolecular and cellular mechanisms underlying the stim‑
ulation and modulation of the host immune response bya member of the pathogen‑derived hsp70s.

ACKNOWLEDGMENTS
This work was supported in part by Grant‑in‑Aids
for Scientific Research from the Ministry of Education,
Science, Sports, and Culture, Japan.

We greatly appreciate for the critical comments
and discussions from Drs. K. Yamashita and F. Aosai.
We also thank Mr. M. Ueda for his excellent technicalhelp, and Dr. G. Massey for correcting English.

317

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319

Trop. Med. Hyg. Vol 26 No 4 1998 pp 319 322

BACTERIOLOGICAL STUDY ON VIBRIOPARAHAEMOL YTICUS ISOLATED FROMTHE OUTBREAKS OF DIARRHEA IN LAOS,AN INLAND COUNTRYTETSU YAMASHIRol, SITHAT INSISIENGMAY2, YASUKO HONMAl,
NAOMI HIGA1, MIKA ENAM11 AND MASAAKI IWANAGA1
Received August 24, 1998/Accepted October 21, 1998

Abstract: An epidemic of Vibrio parahaemolyticus gastroenteritis with several outbreaks occurred inVientiane, People's Democratic Republic of Lao (Laos) , an inland country in August and September in 1997.
Serotypes of the 36 isolated V. parahaemolyticus strains frorn the patients were all 03:K6. The organisms
grew in peptone water supplemented with 10% NaCl.. A11 isolates were positive for Kanagawa phenomenon,
and they were positive for tdh, negative for trh and urease. Genomic patterns were not completely identical,
as determined by pulsed‑field g.el electrophoresis. The causative food was suspected to be a papaya saladcontaining minced and salted fresh water crab. Although a Kanagawa phenomenon positive V. para ‑haemolyticus strain was isolated from the suspicious food, the serotype was 02:K28, tdh and trh were
negative.

Vibrio parahaemolyticus is a halophilic gram‑nega‑from a nearby fresh water river. Considering that V.
parahaemolyticus 03: K6 may have become prevalent inSoutheast Asia recently (Okuda et al., 1997), it is notive rod, and is commonly present in coastal waters

wonder the infection occurred in Lao PDR. In thisthroughout the world if the water is not too cold. It is

communication, the details of the isolates in Lao PDR

an important causative agent of food‑born infection
characterized by severe cramping, abdominal pain, andexplosive watery diarrhea. The illness is closely as‑

are described.

INTRODUCTION

sociated with the ingestion of seafood, and the illness
has rarely been identified in area distant from the coast

where sea food is not readily available. However, as
proceeding the borderless era, seafood has become avail‑able to people in areas far from the sea where this illness

had been formerly uncommon. People's Democratic
Republic of Lao (Lao PDR, or Laos) is an inland coun‑try sharing borders with 5 countries. In August and
September, 1997, a few outbreaks of diarrhea due to V.
parahaemolyticus occurred in Vientiane city. V. para‑
haemolyticus was isolated from 36 diarrhea specimens
out of 53 examined. Some patients had ingested salted
minced fresh‑water crab with papaya salad on the day
before developing the illness. V. parahaemolyticus was

isolated from the crab which was a domestic product

MATERIALS AND METHOD
Patients and bacterial strains: Several outbreaks ofdiarrhea occurred in Vientiane. Lao PDR, in August and
September, 1997. The total number of patients is un‑
clear, but the patients were treated in four hospitals.
One hospital (Chanthabory) accepted 63 patients, 31 ofthem were examined for stool culture and 22 werepositive for V. parahaemolyticus. Another hospital(Sikhotabong) accepted 41 patients, 8 of them were
examined and 4 were positive. The number of patientsat the other 2 hospitals, Sethatirath and Mahosot, were
not reported, but 8 of 8 and 2 of 6 stool samples fromthese hospitals were positive, respectively. Among a
total of 53 stools examined, 36 were positive for V.
parahaemolyticus. Age distribution of the patients with1

Department of Bacteriology, Faculty of Medicine, University of the Ryukyus, 207 Uehara, Nishihara, Okinawa 903‑0215, Japan

2

National Institute of Hygiene and Epidemiology, Km3 Thadeua Road, Vientiane, Laos
Corresponding author: Masaaki lwanaga, Department of Bacteriology, Faculty of Medicine, University of the Ryukyus, 207
Uehara, Nishihara, Okinawa 903‑0215, Japan

320

positive V. parahaemolyticus was 5 to 70 years old withan average of 25.6 years old. The male to female ratio
was 19 to 17. Other than these 36 isolates of V. para‑
haemolyticus, one isolate from the suspected food whichwasubmitted by a patient at Sikhotabong Hospital,and one stocked strain isolated from a patient in Laosin 1993 were also examined.
Biological characterization: Biological behaviors of
the organisms to determine the species were examinedby routine laboratory techniques. Additionally, produc‑

tetracycline (TC, Wako Pure Chemicals) , ofloxacin(OFLX, Daiichi Pharmaceuticals) , erythromycin (EM,Dainihon Pharmaceuticals) , and cefdinir (CFDN,
Fujisawa Pharmaceuticals) . Minimum inhibitoty con‑centrations (MICs) of the drugs were examined by aplate dilution technique. The plates of heart infusion
agar supplemented with 3% NaC1 and I pg/ I of crystal
violet, containing the drug at the serial 2‑fold concentra‑

tioh from 0.2 pg/ml to 100 pglml were prepared. The
organisms in HIB supplemented with 3% NaC1 cultured

tivity of urease was examined using urea mediurn(Eiken) . Hemolytic activity was examined using
Wagatsuma medium (peptone 10 g, yeast extract 5 g,overnight were diluted 1:10 with normal saline solution
for the inoculum. The organisms were inoculated using

mannitol 5 g, K2HP04 5 g, NaC1 70 g, crystal violet I pg,

determination of MIC was made after incubation at 37'C

agar 15 g, Aq. dist. 1,000 ml, pH 7.5) with human andsheep erythrocytes.

for 24 hr.

a micro planter (Sakuma Co. MIT#00257), and theSerot ipe: Commercialized diagnostic sera (Denka
Seiken Co.) were used to determine O and K serogroup.

The organisms cultured overnight on nutrient agar

RESULTS
Phenotypes of the isolates: All isolates from thepatients revealed the same phenotype, showing the typi‑
cal behaviors of V. parahaemolyticus, except for weak

supplemented with 3% NaCl were examined by the slide
agglutination method.
Gene detection: The presence or absence of the tdh

growth in 10% NaCl‑peptone water. Swarming wasand trh genes of the isolates were determined by

Table I Characterization of the isolates

polymerase chain reaction (PCR) . The prirners for tdh,

Vibrio parahaemolyticus strains from

VP21 (5'‑ TGGTTGACATCCTAGATGACTGTG ‑ 3')

and VP22 (5'‑GGGGATCCCTCAGTACAAAGCCTT‑3') were used for amplifying 396 bp of tdh with the PCR
condition described by Lee and Pan (Lee and Pan, 1993) .

outbreaks suspicious food patient in 1993

Growth dn TCBS

Briefly, whole cells were embedded in 1.5% Iow‑melting

+

+

+

+
+

+
+

+

+

+

+

+

+

+

+
+
+

+
+
+

+
+
+

O%
6%

+
(+)*

+

+

10%

(+)**
02:K2804:K63

lactose

Decarboxylation
lysineornithine

Analysis of chromosome: The chromosomal type ofphoresis (PFGE) as described by Wong et al. (1996).

+

sucroseconditions described by Tada et al. (1992).

the strains were examined by pulsed‑field gel electro‑+

glucosePrimers for trh, trh‑s (5'‑GGCTCAAAATGGTTAAG‑
CG‑3') and trh‑r (5'‑CATTTCCGCTCTCATATGC‑3')
were used for amplifying 250 bp of trh with the PCR+

Acid fromarginine

Production ofcytochrome oxidase

Ureasepoint agarose gel blocks for PFGE. The blocks wereIndol

treated with lysis solution containing lysozyme (1 mgl

H,Sml) and N‑sodium lauryl sarcosine (1 mg/ml) at 37'C

VP

for 48 hr. Then, the blocks were treated with proteinase

Utilize citrate

K solution containing 0.5 mg/ml of proteinase K, 0.5 M
EDTA and 10 mg/ml of sodium lauryl sarcosine at 45'C

Growth in NaCl

for 48 hr. The blocks were washed 3 times with TE
buffer (10 mM Tris‑HCl, I mM EDTA, pH 8.0). Finally

Motility

the blocks were immersed in a solution containing 10 U

Kanagawa phenomenonof Sfil restriction enzyme at 4'C for 16 hr and then 37'C

Serotypefor 48 hr. Electrophoresis was carried out using a CHEF‑
DRIII system (Bio‑Rad) in gels of 1% agarose for 20 hr
at 6 V/cm at 14"C with a ramp time of 3 to 80 sec.
Drug susceptibilities: A11 isolates were examined forsusceptibility to ampicillin (ABPC, Meiji Seika) ,

+

+
03:K6

+

All 36 isolates from the outbreaks grew in peptone watersupplemented with lO% NaCl. That growth abi'lity wasmaintained for about 6 months after isolation, and then it
disap peared.

** Hemolysis on Wagatsuma medium by the strain isolated
from the suspected food was weaker than the other strains.

321
1

2 3 4 5 6 7 8 9 10 11121314 15

Table 2 Distribution of drug susceptibilities

MIC( glm!) OFLX Aispc TC EM CFDN
> 100

o

o

O

l OO

o

o

o

O

o

50

o

3

o

O

o

25

o

27

o

O

o

6

o

l 7

o

o

o

18

30

12 . 56 . 253 . 1 333ol . 562

9

O

O . 78

o

o

27

O

O . 39

o

o

o

O

o

;0.2

o

o

O

O

o

6

o

Tatal‑･36 strains

Among the patient strains, Il strains were identicai
Figure I PFGE banding pattern of Sfil‑digested total cellu‑
lar DNAS from V. parahaemolyticus isolates.

lane l: Molecular mass maker (bacteriophage

while one strain from Sikhotabong Hospital had adifferent digestion pattern (Figure l) .

Drug suscaptibilities.' The MICs of each drug against

lamda DNA Iadder) : Ianes 2, 3, 4: the strains

the isolates from the patients were distributed in a

97LVPl, 97LVP2, 97LVP8 (Chanthabory Hospi‑

narrow range, 3.13 ;eeg/ml of OFLX, 25 ,eeg/ml of ABPC,O.8 fcg/ml of TC, 6.25 pg/ml of EM, and 6.25 feeg/ml oftaD; Ianes 5, 6: the strains 97LVP23, 97LVP32(Mahosot Hospital); Ianes 7, 8, 9: the strains
97LVP24, 97LVP25, 97LVp28 (Sethatilath Hospi‑tal); Ianes 10, Il, 12, 13: the strains 97LVP33,

97LVP34, 97LVP35, 97LVP36 (Sikhotabong Hospi‑tal); Iane 14: the strain isolated from suspicious
food (crab) ; Iane 15: the strain isolated from adiarrhea patient in Laos in 1993.

CFDN within the variation of one tube (Table 2). An
isolate from the suspicious food and another isolated in
1993 were also included in this sensitivity pattern.

DISCUSSION

growth in lO% NaC1peptone water and strong swarm‑

Although there have been a few sporadic cases of V.
paraheemolyticus enteritis in Laos (Insisiengmay d al.,1997), this is the first recorded outbreak. The respon‑ing disappeared within 6 months after isolation. The

sible pathogens may belong to the saiue clone withorganisms were hemolytic on Wagatsuma medium withhuman erythrocytes, but hemolysis was variable when
sheep erythrocytes were used. Serotypes were all 03:
K6. However, an isolate from a minced crab which was

Calcutta strain from the viewpoint of recent

observed to be very strong. However, the abilities ofsuspected to be the causative agent did not grow in lO%
NaCl‑peptone water from the beginning, had a serotypeof 02:K28, and was hemolytic. One isolate from apatient in 1993 did not grow in lO% NaClpeptonewater, the serotype was 04:K63, and was hemolytic.
None of the isolates produced urease (Tabie l) .

epidemiological features. However, a strain out of 12

examined had a different PFGE pattern, and strains
with various serotypes have been isolated in the past
few years. These facts suggest that illness due to V.
parahoemolyticus is not uncommon in Laos. Since theCalcutta strain seems to have high epidemic potential
and according to Okuda et al.. it is genetically distinct

from the V. parahaemolyticus 03:K6 strains isolated
before 1994. What ive have found as the phenotypicalGene analysis: As examined by PCR, tdh was

distinction from the other V. parahaewolyticus clones is

detected in all isolates from the patients but not in thethe weak growth in lO% NaCl containing peptone waterand production of a novel filamentous phage (unpub‑

isolate from the suspicious food, and trh was totally

negative. Genomic DNA of the 12 strains from thelished data). However, these phenotypes can notpatient isolates (2 to 4 strains from each hospital), a

explain the epidemic potential af the Calcutta strain.

strain from the suspicious food and a strain from a

An isolate from the suspicious food was 02:K28
serotype, idh negative, trh negative, hemolytic onpatient in 1993 were analyzed by PFGE after Sfil diges‑tion. The latter 2 strains exhibited different digestionp, atterns from each other and from the patient strains. .

Wagatsuma medium, and did not grow in 10% NaCl‑
peptone water. Thus this food can not be identified as322

the outbreak source. The reason why this isolate ishemolytic on Wagaisuma medium in spite of negative
tdh and trh is unclear. Thermostable direct hemolysin(TDH) is believed to be a major catise of hemolysis on

Wagatsuma medium (Kanagawa phenomenon) . TRH(TDH‑related hemolysin) producing strains are nega‑tive for the Kanagawa phenomenon (Honda et al..
1990). Therefore, it is possible that an isolate from the2 ) Honda, T., Ni, Y.. Hata, A., Yoh, M., Miwatani, T.,Okamoto, T., Goshima, K., Takakura. H., Tsunasawa,
S. and Sakiyama, F. (1990): Properties ofhemolysin
related to the thermostable direc, t hemolysin produced

by a Kanagawa phenomenon negative, clinical isolate ofVibrio parahaemolyticus. Can. J. Microbiol., 36, 395‑399
3 ) Okuda, J., Ishibashi, M., Hayakawa, E., Nishino, T.,
Takeda, Y., Mukhopadhyay, A. K., Garg, S., Bhattachar‑ya S. K., Nair, G.B. and Nishibuchi, M. (1997): Em r‑

suspicious food produces a novel hemolysin.

gence of a unique 03:K6 clone of Vibrio para ‑The results of PFGE revealed that the present
outbreaks were not caused by a single clone of V.

from the same clonal group from Southeast Asian trav‑

parahaemolyticus, therefore, it is possible that the causa‑

tive food is not of a single origin. Although the genetic
homology of the present strains to Calcutta strains andto V. parahaemolyticus 03:K6 isolated before 1994 were
not examined in the present study, most of the isolated

pathogens may belong to the same clone with Calcutta
strain from the viewpoint of recent epidemiologicalfeatures. The isolates from the present outbreaks were
not distinguishable via drug susceptibilities because ofnearly identical susceptibility level.

REFERENCES
1 ) Lee, C. and Pan, S.F. (1993): Rapid and specific detec‑tion of the thermostable direct hemolysin gene in Vibrio
parahaemolyticus by the polymeras chain reaction. J.
Gen. Microbiol., 139, 3225‑3231

haemolyticus in Calcutta, India, and isolation of strains
elers arriving in Japan. J. Clin. Microbiol., 35, 3150‑3155
4 ) Insisiengmay, S., Munnalath, K., Sithivong, N., Sisavath,L., Panthouamas, B., Chomlasak, K.. Higa, N., Yama‑

shiro, T., Nakasone, N. and lwanaga, M. (1997):
Etiology of diarrheal diseases and the healthy carrier ofenteropathogens in Vientiane, People's Democratic
Republic of Lao. Jpn. J. Trop. Med. Hyg., 25, 89‑93
5 ) Tada, J., Ohashi, T., Nishimura, N., Shirasaki, Y.,
Ozaki, H., Fukushima, S., Takano. J, Nishibuchi, M. and
Takeda, Y. (1992): Detection of the thermostable direct

hemolysin gene (tdh) and the thermostable direct
hemolysin‑related hemolysin gene (trh) of Vibrio para ‑haemolyticus by polymerase chain reaction. Mol. Cell
Probes, 6, 477‑487
6 ) Wong, H., Lu, K., Pan, T., Lee, C. and Shih, D.Y. (1996):
Subspecies typing of Vibrio parahaemolyticus by pulsed‑
field gel electrophoresis. J. Clin. Microbiol., 34, 1535‑
1539

J pn. J. Tro p. Med.

323

H yg ., Vol. 26, No. 4, 1998, pp. 323 326

Research "'*=

INCIDENCE OF FILARIASIS AS A C0‑INFECTION

IN MALARIA PATIENTS COMlNG FROM
THAI‑MYANMAR BORDER BETWEEN 1995‑1997
SOMBAT TREEPRASERTSUK1, DUANGRUDEE CHlNDANOND2, POLRAT WILAIRATANA3, SRIVICHA KRUDSOOD1
VALAI BUSSARATID', RATCHANIDA GLANARONGRAN2, SOMPAN SRlNUKHAM',ROBERT HUTAGALUNG2 AND SORNCHAI LOOAREESUWAN3
Received June 23, 1998/Accepted October 26, 1998

Abstract: Concomitant infection with malaria and filariasis is known to occur in animals and the co‑infection appears to lessen the severity of malaria. We report here the incidence of co‑infection withfilariasis among 4,201 malaria patients admitted to the Bangkok Hospital for Tropical Diseases. Mahidol
University, Thailand, between 1995 and 19g7. There were eight patients (0.2%) with microfilariae (allWuchereria bancrofti) in the peripheral blood smear. Four of the 8 patients had falciparutn malaria and twopatients among this group had cerebral malaria which resPonded to treatment without any long term
sequelae. The rest four patients, three had vivax malaria while the last one had uncomplicated mixed
infection of falciparum and vivax malaria. Filariasis was asymptomatic in all patients and cured with
diethylcarbamazine. The eight patients resided along the Thai‑Myanmar border, which is known to beendemic for both diseas s. Our findings indicate the existence of co‑infection of malaria and filariasis in

Thailand, especially among patients from the Thai‑Myanmar border. From this small number of patients,
it is difficult to conclude that filariasis affects the severity of malaria. However, the data does emphasizethat early diagnosis and early treatment of both infections is possible.

Key words: Filariasis, Malaria, Thailand

INTRODUCTION
Malaria and filariasis are mosquito‑borne diseases.

There are epidemiological studies in Southeast Asia
about concomitant infection of malaria and filariasis in

Public Health, Mae‑Hong‑Sorn, Tak, Kanchanaburi,
Suratthani and Sarathiwat are five provinces inThailand, w̲hich are endemic for both malaria and filar‑
iasis (Triteerapraparp, 1997) . There are several r portsman (Kan et al., 1985; Schuurkamp et al., 1987). In

on the effect of filariasis on malaria in animal models.

Thailand, despite recent advances in vector control andIn monkeys,. Schmidt and Esslinger found that the co‑infected group developed less severe malaria than thegroup with malaria infection alone (Schmidt et al.,1981). Recently Yan and colleagues foimd that the co‑chemotherapy, both of these infections remain major

public health problems. The increasing number ofMyanmar immigrants and the high incidence of thevector may lead to an epidemic of filariasis (WHO,1997) .

In 1997 the incidence tate of filariasis in Thailahd is

2.08 per 100,000 (Surannadabba, 1996). The vector forfilariasis is Culex quinquefasciatus which is foundinfection of malaria and Brugia pahangi in mice protect‑

ed them from developing severe malaria (Yan et al.,1997) . To our knowledge there has been no report fromThailand about coLinfection of malaria and filariasis inman.

throughout Thailand. According to the Ministry of

3
4

Department of Tropical Hygiene
Bangkok Hospital for Tropical Diseases
Department of Clinical Tropical Medicine
Department of Tropical Pediatrics, Faculty of Tropical Medicine, Mahidol University, Bangkok 10400 Thailand
Address for correspondence: Dr. Somba't Treeprasertsuk, Faculty of Tropical Medicine, Mahidol University,
420/6 Rajavithee Road Bangkok 10400, Thailand, Fax (66‑2)247‑1688 E‑mail: tmsip @ mahidol.ac.th324

male and 24.5% were female. 9.9% (418/4,201) werediagnosed as severe malaria (PF) and the rest were
uncomplicated malaria both PV, PF. Eight of 4,201

MATERIALS AND METHODS
The present study was conducted prospectively in
the Bangkok Hospital for Tropical Diseases, Thailand
between January 1995 and December 1997. Blood sam‑malaria patients (0.2%) had co‑infection with filarial

parasites (Table 1). The m'ean (iSD) age of theples wer collected from the malaria patients. Complete
blood count, blood urea nitrogen, creatinine and liverfunction test evaluations were performed at the time ofpatients was 27.4i5.6 years. A11 of them were male andadmission and repeated on day 7, 14, 21 and 28 after
admission. Thick and thin blood films were sent for

Four of the eight patients with filariasis. had P. falcipar ‑lived along the Thai‑Myanmar border. Wuchereriabancrofti was the only species found in blood smears.
um, three had P. vivax and one had mixed P. falciparumand P. vivax. Six of the eight patients had uncomplicat‑

Field's stain for the detection of malaria parasite andmicrofilariae (Chularerk et al., 1970)J The patientswere examined every 12 hr for both malaria parasites
and microfilariae until the parasitemia became negativeed malaria infections and two patients had cerebral
malaria. The clinical findings of the 8 patients areshown in Table 2.

and then once daily tmtil discharge. The patients who
had filariasis were treated with diethylcarbamazine(DEO 6 mg/kg/day for 6‑12 day.

All eight patients responded to treatment. Parasite
clearance time (PCT, ) =and fever clearance time (FCT)of P. vivax‑infected patients were 45i 19.3 and 18 14 hr

respectively. PCT and FCT of P. falciparum‑infectedpatients were 125.5

RESULTS

123.7 and 39.5

42.9 hr respectively.

The initial geometric mean parasite counts of P. vivax
and P. falciparum were 31,405 and 54,701/pl respective‑

There are 4,201 malaria patients admitted to the
Bangkok Hospital for Tropical Diseases; 75.5% werely. Patients no. 7 and 8 had cerebral malaria with

Table I Incidence of concomitant infections of malaria and filari,asis of Bangkok Hospital for TropicalDrseases Mahidol Umversity, Thailand (1995‑1997)
No. ofP. falciparum

Year malaria(Pf )inf ectionP, vivax(Pv)

P. malariae

P. ovale
(Po)

Mixed

Mixed

(Pm)

inf ection(malaria +(Pf + pv)

f ilariasis)

646

9

o

52

3

1995

1 , 363656

1996

1 , 655

870

688

15

1

81

4

1997

1 , 183

583

551

6

1

42

1

Total 4 , 201
3 years (lOO%)

2 , 109

1 , 885

30

2

175

8

(50 . 2%)(44 . 9%)(o . 7%)(0.05%)(4.2%)(0.2%)

Table 2 Details of 8 patients with co‑infection of malaria and filariasis (1995‑1997)

Patient Sex Age

Previ ous Ty peofNo. (years) malaria(No, of malaria

Severityof

Initialmalariacounte pisodes)
1

2
3
4
5
6
7
8

M
M
M
M
M
M
M
M

23

o

30

7

pv + pf

Uncorn plicatedo

35

FCT Result

Other(hours) of

co‑inf ection

treatment( / p I )

Pv
Pv
Pv

30

PCT

parasites (hours)

Uncomplicated

10 , 100

67

34

Cure

Al, Hw, Tt

Uncom plicated

10 , 680

31

12

Cure

Ov, Ss, Tt

Uncom plicated288 , OOO *37

8

9 , 480

65

298

Cure
Mix Pf Dll

Tuberculosis

N‑one

23

o

Pf

Uncomplic ted

4 , 020

309

O

R2

24

o

Pf

Uncomplicated81 , 600

38

22

RID18

Tt

35

o

Pf

Cerebral malaria

249 , 600*

79

100

RID24

Hw, Ss, Tt

19

o

Pf

Cerebral malaria

109 , 200

76

36

RID21

None

Al, TtM = MaleRl = Resistant type 1

Ss= Strongyloides stercoralis

Pv = Plasmodium vivax
Pf = Plasmodium falciparum

R2 = Resistant type 2

Tt = Trichuris trichiura

A1 =Ascaris lumbricoides

PCT=Parasite clearance time
FCT=Fever clearance time

Hw = Hookwarm* = Hyperparasitemia
D11 =Day 11 after admission

Ov= Q isthorchis viverrini

325

Table 3Clinical and laboratory findings of concomitant infections of malaria and filariasis of

Bangkok Hospital for Tropical Diseases, Mahidol University, Thailand (1995‑1997)
Concomitant infections(malaria+filariasis) (n= 8)

Sex M/F

8/0

Age (years)

Mean i SD

27.3 5.6

Hematocrit (Mean SD) (%)

29 . 7 :8 . 6

Total eosinophil count Day O (Mean SD) (/mm3)
G6PD normal/deficiency
Parasite clearance time (mean‑P. vivax group

168 . 8236 . 78/0SD) hr‑P. falciparum group45

19 . 3125 . 5 : 123 . 7

Fever clearance time (mean SD) hr‑P. vivax group‑P. falciparum group18 i 1439.5i42.9

Geometric mean parasites count/pl (range)‑'P. vivax group

31 , 405 (10 , 092‑288 , 740)

‑P. falciparum group

54 , 701 (4 , 018‑249 , 459)

G6PD=glucose 6 phosphate dehydrogenase

Glasgow coma scores of 7 and 8 respectively. Both ofthern were initially treated with a standard dosage ofarteether but both recrudesced on day 24 and 21. One
patient responded to further treatment with artesunate

both studies came from a highly endemic area of filar‑
iasis and malaria. The differences of our study fromprevious work was that our data collected the patients
who were admitted to the hospital. The clinical picture

followed by mefloquine, and the other patient respondedof our malaria infection was more severe than thecommunity‑based population.

to quinine followed by tetracycline. One patient had
hyperparasitemia and acute renal failure which required

hemodialysis. Both patients recovered with no detect‑able sequelae. The three uncomplicated cases of P.
vivax (patient no 1‑3) were treated with chloroquineand primaquine as standard regimen and the threeuncomplicated cases of P. falciparum were treated withquinine and tetracycline.
A11 patients with co‑infection of malaria and filar‑

The characteristic findings of the patients in ourstudy confirm the results of a report by the Ministry of
Public Health (Suvannadabba et d., 1998) with respect

to age, sex and geographical location. The eightpatients of this study came from Kanchanaburi and Tak
provinces where the endemicities of both malaria andfilariasis, especially W. bancrofti infection, are still high.

The PCT and FCT of the co‑infection group were notiasis were asymptomatic for filariasis. The average

different from the results of patients with malaria infec‑

date of the positive smear for filariasis was the 9th daytion alone. However, the results may not be conclusive
because of the small number of cases.
The majority of the patients with co‑infection had

after admission and all patients were treated with
diethylcarbamazine. The mean hematocrit and eosino‑phil count (iISD) on the day of admission were 29.7 8.

uncomplicated malaria. There were two patients who

6% and 168.8 236.7/mm3 respectively (Table 3). Cost

had cerebral malaria. It seemed that our data did not goof the patients had other parasitic infections; such asalong with the previous data which demonstrated that

hookworm and trichuriasis.

the co‑infection had protected the patients from devel‑

oping severe malaria.

DISCUSSION
In our study, of co‑infection of malaria and filar‑

iasis was 0.2%. It was lower than the Indian study by
Ravindran and colleagues which found a incidence of
0.33% (Ravindran et al., 1998). Most of the patients in

However we cannot conclude the reason of thisfinding because we did not study in the detail about theimmune response of both cerebral malaria patients.

The hematocrit was low in the majority of thepatients due to poor nutritional status and other para‑

sitic infections such as hookworm. Most of the cases

326

had iron deficiency anemia. The average eosinoph. ilcount on the d :y of admission was 168 236.7/mm3
which was lower than average counts of 3,000lmm3
reported previously for filariasis (Triteerapraparp,
1997). However, all of our patients were asymtomatic
for filariasis while the patients of previous reports ofisolated filariasis were symptomatic with cough, fever

and abnormal lung infiltration, which were related to
hypersensitivity to microfilarial antigens.

The results of our study emphasize the existence of
co‑infection of malaria and filariasis in our patient

population. Despite the low incidence we should beaware of this possibility. This finding should alert
physicians to early diagnosis and early treatment of theco‑infection in order to reduce disease transmission.

REFERENCES
1 ) Chularerk, P. and Desowitz. R.S. (1970): A simplified'membrane filtration technique for the diagnosis of
microfilaremia. J. Parasitol., 56, 623‑624
2 ) Kan N. et al. (1985): Bancroftian filariasis and malaria
in island and hinterland populations in Sabah, Malaysia.

Trop. Geog. Med., 37, 93‑1013･ ) Ravindran, B. et al. (1998) : Lymphatic filariasis andmalaria: concomitant parasitism in Orissa, India. Trans.
R. Soc. Trbp. Med. Hyg., 92, 21‑23

4 ) Schmidt, L.H. and Esslinger, J.H. (1981): Course of
infections with Plasmodium falciparum in owl monkeys
displaying microfilaremia. Am. J. Trop. Med. Hyg., 30,
5‑115 ) Schuurkamp, G.J. et al. (1987) : Malaria, splenomegaly
and filariasis in the Ok Tedi area of the Star Mountains,

Papua New Guinea; three years after residual DDT
spray. Papua New Guinea Med. J., 30, 219‑300
6 ) Suvannadabba. S. (1996): Current status of filariasis in

Thailand. Annual Reports Filariasis Division Ministryof Public Health, Thailand, 6‑52
7 ) Triteerapraparp, S. (1997): Update in Filariasis. Chula.
Med. J., 41, 611‑622
8 ) Yan, Y. et al. (1997) : Down regulation of murine sus‑
ceptibility to cerebral malaria by inoculation with third
stage larvae of the filarial parasite. Brugia pahangi
Parasitol., 114, 333‑338

9 ) World Health Organization (1997) : Control of Lym‑
phatic Filariasis by Division of Control of Tropical
Diseases, 1‑13

VOL．26NO．4

0ECEMBER1998

CONTENTS
Original

ArticleYui，K．，and

HumoralYano，A．

ImmunelTo％oμαs

Responceszαgonゴ万AgainstMembersofthe

HSP70Familyin一305−318…

Yamashiro，T．，Insisiengmay，S．，Honma，Y．，Higa，N．，Enami，M and Iwanaga，M
Bacteriological Study onγあ吻勿昭hα翻o加歪6粥Isolated from the Outbreaks of
Diarrhea

ResearchinLaos，anInland一319−322

Country…

Note

Treeprasertsuk，S．，Chindanond，D．，Wilairatana，P．，Krudsood，S．，Bussaratid，V、，

Glanarongran，R．，Srinukham，S．，Hutagalung，R
Incidenceof

Filariasis

Thai−Myanmar

BorderasaCo−infection

in

and

Looareesuwan，S．

Malaria

Patients

Comingfrom・・323−326

between1995−1997一0o■