一薗■王

第24巻第4号平成8年！2月
内
原

容

著
タイ国小児におけるロタウイルス感染症の二年間の追跡調査 血清型とRNA
電気泳動型一・山地 幸雄，長谷川斐子，鈴木博，井上Jirapom Supawadee，Somboon Suprasert
Prapasiri

Rangsianond，Boonyong

栄Pongprot・

マニラ在住の邦人におけるデングウイルス感染の血清学的調査
一牧野 芳大，井上 隆一，加根村和美，福永

215−220

利彦

22L223マレーシア産ウエステルマン肺吸虫の動物体内での発育と動物間における伝播・・波部重久，KarenP．F．Lai，吾妻健
C．K．OW−Yang，川島健治郎…………
短

225−232

報
ドミニカ共和国における肝炎ウイルスの血清疫学的研究（英文）II．D，E型肝炎ウイルスの感染状況
・・七條 明久，三舟求眞人，寺尾英夫，力久哲二
糸賀敬，Maria Estela Noman
Aracelis German Rodriguez，Mercedes Castro Bello

Fernando

SolanoFernandez

・…………・・…………

233−236

研究ノート

熱帯熱マラリア原虫47kDおよび29kD抗原分子に対する熱帯熱マラリアおよび三日熱マラリア急性期患者血清の特異的反応性について
・・Mohd−Nor Norazmi，狩野 繁之，Azni Alias
Mohd−Shukri Abdullah，鈴木守…………・… 237−239（裏面に続く）1■

215

Jpn. J. Trop. Med Hyg., Vol. 24, No. 4, 1996, pp. 215 220

TWO‑YEAR FOLLOW UP FROM BIRTH OF THAICHILDREN FOR ROTAVIRUS INFECTION=
SEROTYPES AND RNA ELECTROPHEROTYPESYUKIO YAMAZI*, AYAKO HASEGAWA2, HIROSHI SUZUK13, SAKAE INOUYE2,
JIRAPORN SUPAWADEE4, SOMBOON SUPRASERT",
PRAPASIRI RANGSIYANOND" AND BOONYONG PONGPROT4
Recevied June 24, 1996/Accepted August 13, 1996

Abstract: Twenty‑two group A rotavirus positive stool specimens that had been collected from 19 children
in Chiang Mai. Thailand, followed up for two years from birth in 1988 to 1990 (Supawadee et al.. 1995) , were
examined for subgrouping (1 and ID , and VP7 serotyping (1, 2, 3 and 4) by enzyme‑1inked immunosorbent

assay (ELISA) with specific monoclonal antibodies and RNA electropherotyping by polyacrylamide gelelectrophoresis (PAGE) . Of these, 18 (82%) could be subgrouped (10 subgroup I and eight subgroup ID andserotyped (five serotype 1, 10 serotype 2, one serotype 3 and two serotype 4). Serotype I virus was more
predominant than type 2 during the period from January to June 1989 and type 2 was more predominant thantype I from August 1989 to June 1990. Reinfection with rotavirus was observed in four children during thetwo years of the follow‑up period after birth: one volunteer (No. 11) had an asymptomatic infection withserotype 2‑RNA electropherotype S and a symptomatic infection with serotype 3‑RNA electropherotype L.

Each of the other three had asymptomatic infections twice, i.e.: one (No. 21) with serotype 1‑RNAelectropherotype L and undetermined serotype‑RNA electropherotype L; one (No. 38) with serotype 2‑RNAelectropherotype S and undetermined serotype‑RNA electroherotype L; and the other one (No. 23) with
undetermined serotype‑RNA electropherotype L and serotype 2‑RNA electropherotype S. The results of 18
specimens from asymptomatic infections were compared with those of 202 specimens from symptomatic
infections in the same geographical area in the same period of time and no definite relation was foundbetween the VP7 serotype and severity of the illness.

INTRODUCTION
Group A rotavirus (RV) is known to be a common
cause of severe diarrhea that occurs among children
world wide. The rotavirus particle consists of outer andinner capsid layers and 11 viral RNA segments enclosed
within them. The outer capsid consists of two structuralproteins, VP4 and VP7, which are independent neutrali‑zation antigens and the inner capsid consists of VP6
which is a subgroup specific antigen. By these antigens,otypes of HRV in various parts of the world seems
important not only for understanding the pathology andepidemiology of these infections but also for evaluating
the efficacy of candidate rotavirus vaccines.
Our previous report (Supawadee et al., 1995) de‑
scribed group A RV infection of children in Chiang Mai
followed up for two years after birth, from October 1988

to November 1990. Among the 28 volunteer children,
asymptomatic infection was observed in 15 (54%) andtwo subgroups, seven VP7 serotypes and three VP4

symptomatic infection in four (14%) . We analyzed 202
group A RV‑positive specimens collected from diarrheic

serotypes of human rotavirus (HRV) have so far beenchildren admitted to three hospitals in Chiang Mai

distinguished. Apart from the antigenic analysis, polya‑

during the period December 1988 to June 1990 (Urasawa
et al., 1992 and unpublished data). In this report, 22

crylamide gel electrophoresis of genomic RNA distin‑
guishes long (L) and short (S) RNA electropherotypes
of RV (Kapikian and Chanock, 1990). The prevalenceof particular antigenic types and RNA electropher‑
1

2
3
4

group A RV‑positive specirnens from the 19 volunteer
children in the follow‑up study in Chiang Mai were

examined, and antigenic types of the virus from

Sayama Hakuai Hospital, International Good Will Foundation of Japan, Sayama, Japan
Department of Epidemiology, National Institute of Health, Tokyo, Japan
Institute of Gerontology, Nippon Medical School, Kawasaki, Japan
Departments of Microbiology, Family Medicine and Pediatrics, Chiang Mai University, Chiang Mai, Thailand

216

asyptomatic and symptomatic infections were also

sample. The extracted RNA was analysed by SDS‑

com pared.

PAGE following the method of Rodger et al. (1979);
10% polyacrylamide gels (0.75 mm thick) were usedwith the discontinuous buffer system as described by
Laemli (1970). Electrophoresis was carried out at 20

MATERIALS AND METHODS
Virus specimens
Stool specimens were obtained from 28 volunteers
born in October and November 1988 in Maharaj Nakorn
Chiang Mai Hospital, Chiang Mai University. Speci‑
mens were collected every week during the follow‑up

mA at roorn temperature for four hours. After eletro‑

period of two years after birth and were screened for the

Subgroups and serotypes

phoresis, the gels were stained with silver nitrate.

RESULTS
Subgroups and VP7 serotypes identified withpresence of group A RV as described in the previous
report (Supawadee et al., 1995).

ELISA by using monoclonal antibodies are shown in
Table I and 2. Eighteen out of 22 specimens wereSubgrouping and serotyping of human rotavirus
Direct subgrouping (1 and ID and serotyping (VP7

identified as subgroup I or 11 and VP7 serotype 1, 2, 3 or

1, 2, 3 and 4) of virus in feces were carried out by ELISA

arnounted to 4/6 (67%) , those with subgroup I‑serotype
2 specificity to 2/6 (33%) and neither serotype 3 nor 4

4. Specimens with subgroup II‑serotype I specificity

with monoclonal antibodies developed by Akatani andlkegami (1987). Microplate wells were coated withdilutions of mouse ascites containing innercapsid andoutercapsid‑common monoclonal antibodies. Five to 10

was found during the period from January to April 1989
(Table 1). In contrast, during the period from August
1989 to June 1990, subgroup I‑serotype 2 viruses were
found in 8/17 (47%), accompanied by subgroup II‑ser‑
otype I (1/17, 5.9%), subgroup II‑serotype 3 (1/17, 5.
9%) and subgroup II‑serotype 4 (2/17, 12%) (Table 2).
Five specimens (29%) could not be assigned to either
subgroup (1 and ID or any serotype (1, 2, 3 and 4).

per cent suspensions of fecal specimens were delivered
to each well. After incubation at 4 'C overnight andwashing, biotinylated subgroup‑ and type‑specificmonoclonal antibodies followed by streptavidin con‑jugated with horse radish peroxidase were given as
detection reagents. OD was measured at 492 nm.

Serotypes in asymptomatic and symptomatic infection
Table 3 shows the distribution of subgroups andserotypes in asymptomatic and symptomatic infectionsin the same geographic area of Chiang Mai during thesame period of time. Cases of symptornatic infection
include volunteers No. 6, 7, 8 and 11 examined in thisreport and 198 hospitalized cases examined previously(Urasawa et al.. 1992 and unpublished data). Between
December 1988 and June 1989, serotype I was predomi‑
nant in 4/6 (67%) of asymptomatic and in 761145 (52%)

RNA electrophoresis
The method used to determine the RNA‑pattern of
RV genome by sodium dodecyl sulfate polyacrylamide
gel electrophoresis (SDS‑PAGE) was a minor modifica‑tion of that described by Herring et al. (1982) as de‑scribed previously (Hasegawa et al., 1987). Fecal sus‑

pension diluted in Tris buffer containing EDTA, SDS
and 2‑mercaptoethanol was extracted with phenol andchloroform, and the supernatant was used as an RNA

Table 1 Subgroups, VP7 serotypes and RNA electropherotypes of rotaviruses in stool specimens
collected between January 1989 and April 1989

Volunteer Detection of virus

No. Age of weeks
Date

Symptorn

Virus

Subgroup

Serotype

RNA

II

1

L
L

14

Jan. '89

11

34

Feb. '89

14

21

Feb. '89

15

II

1

11

Feb. '89

19

I

2

40

Apr. '89

18

38

Apr. '89

20

+
+

II

I

S

L

II

2

S

‑ , no diarrheic symptom. :!:, diarhea at the successive week of the detection of rotavirus antigens. L,
long. S, short.

217

Table 2 Subgroups, VP7 serotypes and RNA electropherotypes of rotaviruses in stool specirnens
collected between August 1989 and June 1990

Volunteer

Detection of virus

Virus

Symptorn

No.

Date

Age of weeks

24

Aug. '89

40

16

Sep. '89

44

I

38

Sep. '89

44

5

Dec. '89

60

NC
NC

11

Jan. '90

64

35

Feb. '90

63

23

Feb. '90

66

21

Feb. '90

67

8

Feb . '90

69

10

Feb . '90

70

7

Feb . '90

72

Feb . '90

74

Mar. '90
Mar. '90
Mar. '90

67

Apr. '90

80

June '90

90

39
25
23
6

Subgroup+

Serotype

RNA
S
S

I

++

NC
NC
NC
NC

L
L
L
L
L
L

2

S
S

II

NC
NC

+++

I
I

+

L
L

II

II

2

70

NC

72

I

++

NC

I

S
S
S
S

L

II

‑ , no symptom.
, diarrhea at the succesive week of the rotavirus‑detection. + , mild diarrhea. + +,moderate diarrhea. + + + , hospitalized with severe diarrhea. NC, not clear. L, Iong. S, short.

Table 3 Distribution of serotypes and subgroupa of human rotavirus in stools
collected in Chiang Mai frorn December 1988 to June 1990
PeriodVirus

Inf ection

Subgrou p

Seroty pe

Asym ptomatic

Symptomatic*

Dec. '88‑

II1

4 (67%)

June '89

NCoI

2 (3.3%)IIoo

29 (51%)1 (1.8%)6 (11%)
3 (5.3%)3 (5.3%)8 (14%)
7 (12%)

6 (10%)

57 (lOO%)

1 (7.7%)5 (38%)

16 (11%)1 (0.7%)8 (5.5%)76 (52%)3 (2.1%)3 (2.1%)1 (0.7%)9 (6.2%)18 (12%)10 (6.9%)

13 (100%)

145 (100%)

NC
NC
NC

II
I

NC
Total

Aug. '89‑

II

June '90

I

1

NC

o
o

O
O

6 (46%)

I

II

3

O

1 (7.7%)

II

NC

O

NC
NC
NC

II
I

NC
Total

O
O

*No. 11, 8, 7 and 6 and 198 cases reported by Urasawe et al. (1992)

218

of symptomatic infections. Between Augu8t 1989 andJune 1990, type 2 was predominant in 6/13 (46%) ofReinf ection

aymptomatic and in 761145 (52%) of symptomatic infec‑

specimens twice during the two years after birth astions. Thus, no significant difference was foundbetween asymptomatic and symptomatic infections onshown in Tables I and 2. No. 21 experienced

the predominant serotype (1 or 2) in either period; i.e.,p > 0,1 during the period from December 1988 to June
1989 and p>0. 95 during the period from August 1989 toFour volunteers excreted RV antigens in their fecal

asymptamatic infectians twice with subgroup II‑ser‑
otype 1‑long RNA in February 1989 and with an uniden‑tified subgroup and serotype10ng RNA of HRV in

RNA electropherotype

February 1990; No. Il suffered an asymptomatic infec‑
tion with subgroup I‑serotype 2short RNA pattern in
February 1989 and a symptomatic infection with sub‑group II‑serotype 3long RNA pattern in January 1990;

Viral RNAS fram group A rotavirus‑positive stool
specimens were examined in PAGE and classified into
two groups: Iong (L) electropherotype, in which RNA

group lserotype 2‑short RNA pattern in April 1989 andwith an unidentified subgroup and serotype‑10ng RNA

segment ll migrates rapidly, and short (S) electropher‑pattern in September 1989; No. 23 had asymptomatic

otype, in which the same segment migrates slowly.
Among the 18 samples whose subgroup and VP7 ser"infectians twice with an unidentified subgroup and ser‑

June 1990 by the x2 test.

otype was known, nine strains of S type were identified

No. 38 had asymptomatic infections twice with sub‑otype‑10ng RNA pattern in February 1990 and with
subgroup I‑serotype 2‑short RNA pattern in Marchas subgroup I‑serotype 2 and eight strains of L typewere subgroup ll‑serotype l, 3 or 4, but a strain fromvolunteer No. 35 with asymptomatic infection was sub‑group Iserotype 2 possessing a long RNA electropher‑l 990.

otype. Fig I shows RNA‑PAGE of RV from three

Twenty‑three HRV‑positive stool specimens col‑lected during a two‑year follow‑up study of 28 Thaichildren in Chiang Mai (Supawadee et al., 1995) were

volunteers.

DISCUSSroN

analyzed for subgrouping (1 and II), serotyping (VP7 l,

1

2

3

2, 3 and 4) and RNA electropherotyping. Among the 23,
six specimens were obtained from children aged fram ll

4

to 20 weeks between January and April 1989 (Ist

1
4
5
‑G

23

4‑78g

period) , and 17 were aged from 40 to 90 weeks between
August 1989 and June 1990 (2nd period) . A11 the volun=teer children were born in October and November 1988,
and they should have had fewer contacts with the virus
and higher levels of maternal immunity against the virus

in the Ist period than in the 2nd period. From many
surveillance reports of rotavirus infection in various
countries (Birch et al., 1988; Flores et al,, 1988; George‑

Courbot et al., 1988; Nakagomi et al., 1988; Akatani etal., 1989; Bishop et al.. 1989; Pongsuwanne et al., 1989;
Unicomb et d., 1989; Urasawa et al., 1989; Gouvea et al.,

1990; Kawamota et aL, 1990; Matson et al., 1990; Na‑kagomi et al., 1990; Sethabutr et al., 1990; Pipittajan et

‑ Io
11

al., 1991; White et al., 1991; Urasawa et aL. 1992; Woods
et al., 1992; Ikegami and Akatani., 1993; Rassool et al.,
1993; Steele, et al., 1995) , it is considered that despite theoverall predominance of serotype I virus, the frequency
of individual serotypes varies cansiderably from place toFigure I Electropharetic migration patterns of double‑8tranded RNAS from stool specimens of volun‑teers No. 24 (1), 34 (2) and 39 (3). RNA pattern of (4)is a contral representing the long pattern.

place and year to year.

We had serotyped 198 group A rotavirus‑positive
fecal specimens collected from diarrheic children
hospitalized in the same area of Chiang Mai from

219

December 1988 to June 1990 (Urasawa et al.. 1992 andunpublished data). In the present report 18 out of 23
rotavirus‑positive specimens were collected from chil‑dren with asymptomatic inapparent infections, and theserotypes of the asymptomatic and symptomatic infec‑
tions were compared. As a result, no significant differ‑ence between asymptomatic and symptomatic infections
was found on the predominant serotype (1 or 2) in two
periods of December 1988 to June 1989 and of August
1989 to June 1990. This result indicates that viral fac‑
tors alone are unlikely to account for attenuated infec‑

tion. Previously, the P6 genotype (with M37‑1ike VP4
allele) was reported to occur only in strains of the virus

recovered from asymptomatically infected neonates(Hoshino, et al.. 1985; Flores et al.. 1988; Gorziglia etal.. 1988). However, strains carrying the P6 genotype
were recovered from symptomatically infected neonatesand older infants (Gerna et al., 1990; Steele et al.. 1993
and 1995) .

Urasawa et al. (1992) isolated three unusual HRV

strains (Mc35, Mc323 and Mc345) by using primary
African green monkey kidney cells from stool specimens
collected in Chiang Mai during the period from 1987 to
1989, assigned them to VP7 serotype 9 and 10 and statedthat they are closely related to bovine and porcine RVs.

They also studied the VP4 gene of strain Mc35 andpresumed that it represents subtypes of type 3 humanrotavus Vp4 type 3B (Urasawa et al.. 1993). Four
specimens (No. 11, 21, 23 and 25) untypable in thisreport could contain unusual RVs, but only direct
ELISA for VP7 serotypes I to 4 was done this time.
It has been demonstrated that there is usually adefinite relationship between the subgroup and serotype

specificities of HRVS and the pattern of segmented

RNA of the virus in PAGE: RVs with subgroup I
specificity usually have serotype 2 or 8 specificity and a"short" or "supershort" RNA pattern, whereas RVs
with subgroup 11 specificity generally have serotype 1, 3,

4 or 9 antigen and a "long" RNA electropherotype(Kapikian and Chanock, 1990) . This was reconfirmed inthis study, except in one strain from volunteer No. 35
with asymptornatic infection, which contained RV with
subgroup I‑serotype 2 antigen and a long RNA patternwhich deviated frorn the correlation. Similar strains
have been reported as Mcl29 in Chiang Mai previously(Urasawa et al., 1992) and some strains with antigenic

and genetic properties different from the usual ones
have been discovered among HRV isolates (Kobayashi
et al.. 1989; Nakagomi et al., 1987).

This work was supported by a grant from Diarr‑

hoeal Disease Control Programme, World Health Orga‑nization (Registry File Number 06/181/231; Project ID
Number 86086) .

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Rorner, H., Flores, J. and Perez‑Schael, I. (1991):
Temporal distribution of human rotavirus serotypes 1, 2,

Chiwakul, M., Susansook, T., Suguanwongse, S.,

3 and 4 in Venezuelan children with gastroenteritis

Jayavasu, C. and Urasawa, S. (1989) : Subgroup andserotype distribution of human, bovine, and porcine

during 1979‑1989, J. Med. Virol., 34, 79‑84
35) Woods. P.A., Gentsch, J., Gouvea, V., Mata, L., Simhon,A., Santosham, M., Bai, Z.‑S., Urasawa, S. and Glass. R.
I. (1992): Distribution of serotypes of human rotavirus
in different populations, J. Clin. Microbiol., 30, 781‑785

rotavirus in Thailand, J. Clin. Microbiol., 27, 1956‑1960

24) Rasool, N.B.G., Green, K.Y. and Kapikian, A.Z. (1993):
Serotype analysis of rotaviruses from different location

221

Jpn. J. Tro p. Med. Hyg., Vol. 24, No. 4, 1996, pp. 221‑223

A SEROLOGICAL STUDY OF DENGUEVIRUS INFECTION AMONG JAPANESERESIDENTS IN MANILA.
YOSHIHIRO MAKlNO1,2, RYUICHI INOUE3, KAZUMI KANEMURA1
AND TOSHIHIKO FUKUNAGAl'2
Received August 27, 1996/Accepted October 11, 1996

Abstract: A total of 84 serum specimens were collected from 67 Japanese residents in Manila, thePhilippines, at a clinic during the periods from September 1992 through February 1993 and November 1993
through March 1994, during which time dengue viruses were actively circulating, and tested for antibodies
to dengue (DEN) viruses. Fifteen (22.4%) individuals were confirmed as recent DEN infection by either thedetection of anti‑DEN IgM or a four‑fold or more rise in neutralizing antibody titer in acute‑convalescentpaired sera. Major serotypes of current DEN epidemic appeared to be DEN type I and type 2 viruses. There
was a case in which all three members of a family had been infected with DEN sirnultaneously, and their

symptoms varied from DEN fever to DEN hemorrhagic fever.

MATERIALS AND METHODSINTRODUCTION
With a rapid increase in the number of Japanesepeople who visit tropical areas, a problem of contracting

tropical diseases is becoming remarkably increased.
Dengue (DEN) /dengue hemorrhagic fever (DHF) is one

Serum specimens:
A total of 84 serum specimens were collected from
67 Japanese living in Manila, at the Manila JapaneseClub Clinic during the periods from September 1992

of the serious problems in various tropical areas. The
Philippines has been a dengue epidemic area for years.
Mean incidence rate of DEN infection in all ages among

through February 1993, and November 1993 throughMarch 1994, during which time, DEN viruses wereFilipinos is 3.0 per 100,000 population, with the highestwith DEN‑like symptoms, while other 57 came for abeing 10.5 in the children under one year old (Okabe,1993). Manila area has shown the highest incidence inthe country (Piad, 1994). There are many Japanesepeople living in and around Manila. However, theroutine medical checkup or with some ailments or com‑plaints, not available to us right now (non‑DEN). The
information on the individual vaccination history andearly medical history were obtained through question‑

situation of DEN infection among these Japanese resi‑

naire from 37 individuals.

dents was not clear. We, therefore, became interested tofind out the incidence rate among the Japanese living in

Serol ogy :Manila. During or at the end of DEN epidemic seasonfrom 1992 through 1994, we collected sera from thelgM‑capture ELISA (Bundo and lgarashi, 1985). A

Japanese residents and tested for antibodies to DEN in

mixture of DEN‑1, 2, 3 and 4‑infected cell culture fluidorder to give appropriate guide for preventive and thera‑

was used as antigen. Anti‑dengue lgG was purified frompeutic measures. This paper describes the results of thea pool of dengue patient sera and conjugated withhorseradish peroxidase (Wilson and Nakane, 1978).

serological test.

actively circulating. Among them, 10 came to the clinic

lgM‑class antibody to DEN virus was assayed by

Positive ELISA reaction at a serum dilution of 1: 100
was considered lgM‑positive.
Neutralizing antibody was assayed by 50 percent
1 Departrnent of Virology,
2 Research Center of Comprehensive Medicine, Faculty of Medicine, University of the Ryukyus,
207 Uehara, Nishihara, Okinawa, 903‑01 Japan and
3 Embassy of Japan in the Philippines

222

Table 1

Antibody‑positive rate to dengue virus among Japanese residents

Clinical symptom*

No. patients positive by

No. patientslgM‑ELISANT

Dengue *10

6 (60.0) t

6 (60.0)

N on‑dengue *57

8 (14.0)

9 (15.8)

Total

67

14 (20.9)

15 (22.4)

Details are described in text.
t Number in parenthesis indicates percent.

focus‑reduction neutralization (N) test in BHK21 cells

ratio of clinical disease to dengue infection is thought towith the use of peroxidase‑anti‑peroxidase (PAP)be nearly one (Halstead, 1993), these non‑DEN individ‑

staining method (Okuno et al.. 1985). N titers over 10
were considered positive. Individuals showing four‑folduals might have had some symptoms of DEN infection,

or greater rise in titer in acute‑convalescent paired sera

tional symptoms. Unfortunately, the detailed clinicaldata were not available. Fifteen (22.4%) individuals
were positive for DEN N antibody. Among them, eightsuch as undifferentiated fever and non‑specific constitu‑

were considered as recent infection.

had anti‑DEN IgM. Monotypic DEN N antibody was

RESULTS AND DISCUSSION

detected in 8 individuals suggesting that they might
have had primary DEN infection (W.H.O., 1986) . Major

In order to know the incidence of DEN infection

monotypic antibodies were those to DEN‑1 and DEN‑2

among Japanese residents in Manila, sera were collectedfrom Japanese residents who came to the clinic during

viruses. Seven individuals possessed multiple serotypic
N antibodies. Thus, 15 (22.4%) of 67 individuals were

DEN epidemic season and tested for the antibodies to
DEN viruses. Fourteen (20.9%) individuals were posi‑tive for anti‑DEN IgM (Table 1). Among thern, eight
were from non‑DEN individuals. In adults, since theTable 2

Serum Age

code (Year)IA

34

Residence Days

4A
5A

DENl DEN2

DEN3

DEN4

JEV

F

+
+
+
+
+
+

< 10

< 10

< 10

< 10

520

390

110

50

35

560

330

64

17

18

660

12

< 10

< 10

< 10

< 10

< 10

< 10

< 10

< 10

12

200

12

< 10

< 10

13

6/12

F

6/12

37

M

6/12

M

2

F

2

31

3

4

3

77

5C

6A
7A
8A

N titers to

lgM

5

4C

Anti ‑

Sex inthe after
Philippines* onset
82

27

A brief history and serological information of the 10

DEN

23

3C

DEN‑IgM ELISA or N test.

Serological data on patients with dengue‑like symptorn

IC1
IC2

2A
3A

confirmed to have had recent DEN infection by either

4

< 10

< 10

< 10

< 10

60

56

130

70

35

33

56

5

< 10

< 10

< 10

< 10

800

< 10

< 10

700

8

+

180

< 10

13

F

3/12

6

+

105

410

< 10

< 10

2600

39

M

3

1

+

< 10

< 10

< 10

< 10

25

6

F

4112

5

< 10

< 10

< 10

< 10

38

34

< 10

< 10

< 10

< 10

78

< 10 < 10< 10 < 10

< 10

< 10

1350

< 10

< 10

39

8C

9A 9

10A 36

MF

4/12 5
4/12 5*Duration in years of staying in the Philippines. 1, 2 and 3; and 8, 9 and 10 are two families.

223

DEN‑1ike patients are shown in Table 2. Patients # 1, #2, and # 3 were the members of a family (parents and adaughter) . They appeared to have been infected with
DEN (possibly DEN‑1) at the same time. They devel‑
oped the symptoms of DEN such as high fever, positive
Tourniquet test, subcutaneous or gingival bleeding, andthrombocytopenia. The parents developed more severe

symptoms than their child. Mother (# 1) showed DHF
symptom and developed alopecia soon after the acutephase was subsided. It took her about one and a halfyears for a complete recovery. Father (# 3) had altered
taste sensation when a wide area of his oral mucosa hadcome off on the fifth day, while their child (# 2) showed

the symptoms of DEN fever. In the paired sera frompatient # 4, anti‑DEN IgM was not detected on the 4th
and 56th day, although anti‑DEN IgG rose significantly.
Poor response of lgM antibody has also been observed,
especially in the secondary infection (Ruechusatsawat

is being undertaken.

ACKNOWLEDGEMENT
Authors are grateful to the staffs of the JapaneseClub Clinic and the Japanese residents in Manila for
their cooperation in this study. Inoue, R. has moved to

the Embassy of Japan in lran.

REFERENCES
1 ) Bundo, K and lgarashi, A. (1985): Antibody‑capture
ELISA for detection of immunoglobulin M antibodies insera from Japanese encephalitis and dengue hemorr‑
hagic fever patients. J. Virol. Methods., 11, 15‑22

et al .. 1994). Patient # 7 went back to Japan soon after

2 ) Halstead, S.B. (1993): Global epidemiology of dengue:
Health systems in disarray. Trop. Med., 35, 137‑146
3 ) Okabe, N. (1993): Situation on dengue fever and dengue
haemorrhagic fever in the western Pacific region. Trop.
Med., 35, 147‑160

taking the first blood sample on the first day of illness,4 ) Okuno. Y.,Fukunaga, T., Tadano, M., Okamoto, Y.,

which showed positive for anti‑DEN IgM, but not foranti‑DEN N antibody.

Ohnishi, T., and Takagi. M. (1985): Rapid focus reduc‑

It should be noted that patients # 8, # 9, and # 10

were the members of a family (mother and two chil‑dren) and they simultaneously developed DEN‑like
symptoms, such as high fever, pain behind the eyes, joint

pain, sore throat and lymphadenopathy. However, DEN
antibody was not detected. The patient # 9 (9 year oldboy) had relatively high Japanese encephalitis virus(JEV) N antibody (N titer of 1350). This child had ahistory of receiving JEV vaccination twice. Since chi‑kungunya (CHIK) virus, which is also transmitted by
the same vector Ae. aeg pti, induces DEN‑1ike symp‑
tom, we screened N antibody to CHIK virus as well.
Although, one CHIK antibody‑positive case was detect‑ed, this family turned out to be negative. Further study

tion neutralization test of Japanese encephalitis virus in

microtiter system. Arch. Virol., 86, 129‑135

5 ) Piad, J.N.C. (1994): National dengue prevention andcontrol program and Japanese encephalitis in thePhilippines. Trop. Med., 36, 162‑169
6 ) Ruechusatsawat, K., Morita, K., Tanaka, M., Vongcher‑ee, S., Rojanasuphot, S., Warachit, P., Kanai, K., Thon‑
gtradol, P., Nirnnakorn, P., Kanungkid, S. and lgarashi,
A. (1994): Daily observation of antibody levels among
dengue patients detected by enzyme‑linked immunosor‑
bent assay (ELISA) . Jpn. J. Trop. Med. Hyg., 22, 9‑127 ) W.H.O. (1986) : Dengue haemorrhagic fever: diagnosis,
treatment and control. 23‑29, W.H.O., Geneva8 ) Wilson, M.B. and Nakane, P.K. (1978): Immunofluores‑
cence and related staining techniques, 215‑224, Elsevier/North‑Holland Biomedical Press, New YorkJ pn. J. Trop. Med. H yg ., Vol. 24, No. 4, 1996, pp. 225‑232

225

GROWTH OF MALAYSIAN PARAGONIMUS
WESTERMANI IN MAMMALS AND
THE MODE OF TRANSMISSION OF

THE FLUKE AMONG MAMMALSSHIGEHISA HABE1, KAREN P. F. LA12, TAKESHI AGATSUMA3

C. K. OW‑YANG2 AND KENJIRO KAWASHIMA4
Received October 17, 1996/Accepted December 6, 1996

ABSTRACT: The host susceptibility of Malaysian Paragonimus westermani was observed in cats, dogs andrats, infected with metacercariae. In rats, worms were harboured in the muscles and the flukes weremorphologically sirnilar to the excysted metacercariae except for their slightly larger body size. In cats,

about a half number of the flukes were recovered from muscles and 40% from cysts found in the lungs.
Majority of the flukes from these cysts were mature. In dogs, few mature flukes were obtained from cysts
in the lungs and about 90% of flukes were recovered from muscles. The flukes frorn the muscles of cats anddogs were of the same juvenile stage as those from rats. The juvenile flukes recovered from muscles of theanirnals were orally given to dogs and cats. Majority of the flukes were found in cysts in the lungs and mostof thern were fully mature. However, some juvenile flukes still remained in the muscles of cats and dogs.
Therefore, the Malaysian P. westermani has a higher final host susceptibility than those from other loca‑
lities. The present study suggests that many species of animals living in Malaysian jungle serve as paratenic
hosts and may play an important role in the completion of the life cycle of P. westermani in Malaysia.

INTRODUCTION
Japanese Paragonimus westermani (Kerbert, 1878)
usually remains in the muscles of rat, mouse, rabbit, pig

and wild boar and for long period without development,
and such animals could play a role as paratenic hosts ofthe fluke (Habe, 1978, 1983; Miyazaki and Habe, 1976;

done only to get adult worms specifically for identifica‑tion. The growth of Malaysian P. westermani was notclear in the mammalian hosts. The present paper
reports the sites and development of Malaysian P.
westermani in cat, dog and rat and discusses the role ofmammals as paratenic hosts, refering to the presump‑
tive life cycle of this fluke.

Shibahara and Nishida, 1986). Ingestion of raw wildboar meat is an important source of human infection of
P. westermani in Japan (Miyazaki et al., 1978; Norimatu

MATERIALS AND METHODSet al. 1975; Tokudome et al., 1977). In contrast P.

1 ) Experimental Animals and P. westermani Metacer‑

westermani in Philippines does not remain in the muscles

cariaeSeven adult dogs and 11 cats were used for investi‑
gation of site and growth of Malaysian P. westermani inof mammals as a juvenile fluke (Miyazaki and Habe,
1979; Yokogawa et al., 1979). The study of lung flukes

in mammals provide knowledge on the life cycle andthese information would be useful for prevention andmammalian hosts. Before experimental infection, theof lung fluke, P. westermani has been recorded fromvarious animals belonging to the family Felidae andfrom the crab‑eating monkey, and a few experimental

dogs and cats were shown to be free from lung fluke andintestinal parasites by stool examination. Thirty‑two
female albino rats, Rattus norvegicus, SD strain about 9
weeks old were also used.
Malaysian P. westerneani metacercariae were recovered

infections of Malaysian P. westermani in cats have beenfrom fresh water crabs, Parathelphusa maculata andcontrol of paragonimiasis. In Malaysia, only one species

1

2

3
4

Department of Parasitology, School of Medicine, Fukuoka University, Fukuoka 814‑01, Japan
Division of Parasitology, Institute for Medical Research, 50588 Kuala Lumpur, Malaysia
Department of Parasitology, Kochi Medical School, Nankoku 781‑51, Japan
School of Health Sciences, Kyushu University, Fukuoka 812, Japan

226

Parathelphusa malaysiana which were collected from
Kampong Langkap near Kuala Pilah and Sungai Wa at
Taman Negara, Penisular Malaysia,respectively. They
were inoculated into dogs, cats and rats. The metacer‑
cariae from P. maculata collected at Ulu Langat near
Kuala Lumpur were inoculated into cats.

attributable to parasitic invasion were recorded. Vis‑
ceral organs and cavities were examined for lung fluke
infection and worms were recovered. Subsequently, thelungs, Iiver and muscles of the whole body were cut into
slices, 3 to 4 mm thick, and kept in Ringer's solution at

2 ) Inoculation of Metacercariae
The experimental animals were infected with 25 to

36 ‑ 38 'C for 6 ‑ 8 hours to release flukes from thetissues. The sediment was poured into a petri dish andexamined for flukes under a stereomicroscope. Most of
the juvenile flukes recovered were inoculated into dogs

150 metacercariae (Tables 1‑3). Five cats and a dog
were orally infected using a pipette and 29 rats weremorphological observations; they were pressed between

also orally infected with metacercariae in a little water

two slide glasses in 70% alcohol and mounted withusing a syringe connected to a slender tube inserted into

Canada balsam after staining with carmine.

the stomach. Other animals, 6 cats, 6 dogs and 3 rats,
were injected with the metacercariae into the peritoneal
cavity with a little Ringer's solution using a syringe
connected to a slender vinyl tube.

3 ) Recovery of the Flukes
Dogs, cats and rats were bled to death under anes‑
thesia on days 208 ‑ 258, 155 ‑ 249 and 10 ‑ 150 after
infections, respectively, and flukes were recovered.

Postmortem examinations were carried out on eachanimal immediately. Gross lesions which might be

and cats again. The remaining worms were used for4 ) Transfer of Juvenile P. westermani to Cats andDogs
Most of the juvenile worms recovered from themuscles of cats, dogs and rats were fed again to 5 catsand 2 dogs. Numbers and ages of the juvenile flukes andanimals used are shown in Table 4. The animals were
autopsied at 161‑212 days after inoculation. The sameprocedures mentioned above for fluke recovery andexamination were employed.

Table 1 Results of experimental infection of cats with Malaysian Paragonimus westermani metacercariae (Mc.)

Cat
No.

Body weight No. ofat necropsy Sex Mc.

( kg) given

1

2.0

F

30

2

2.9

F

30

3

2.6

F

30

4

2.0

30

5

2.7

M
M

6

2.8

F

50

7

3.7

M

50

8

3.2

F

50

9

2.9

M

40

10

2.9

F

47

11

3.2

M

30

30

MethodofAuto psydays after

inf ectionoral
inf ectionoral
inf ectionoral
inf ectionoral
inf ectionoral
inf ection

peritoneal
inoculation
peritoneal
inoculation
peritoneal
inoculation
peritoneal
inoculation
peritoneal
inoculation
peritoneal
inoculation

No. ofNo. of worms recovered fromlungs

pleuralcavitymuscles

10*

O

3t

It

3 (10.0)

2*

O

O

It

234

5 (16.7)

4*

O

O

It

242

4 (13.3)

2*

O

It

It

249

5 (16.7)

4*

O

155

26 (52.0)

4*

14t

O

8t

192

32 (64.0)

18*

O

It

13t

192

37 (74.0)

16*

O

2t

19t

208

26 (65.0)

14*

O

237

30 (63.8)

10 (2t

O

2t

18t

242

25 (83.3)

12*

O

It

12( It1lt

cyst in

inf ection

w o rmsrecovered ( % )220

14 (46.7)

229

lungs

8*

It

12t

* Adult worm (with eggs in uterus) ,t Immature worm (without eggs in uterus) ,F: female, M: male,$ Juvenile worm (similar to excysted metacercariae) ,Cat Nos. 2, 4, 5, 7 and 8 were infected with metacercariae from Kuala Pilah.
Cat Nos. 1, 3, 6 and 9 from Sungai Wa and Cat Nos. 10 and 11 from Ulu Langet.

227

RESULTS

Development of the flukes:
In cat No. 6, 'autopsied 155 days after infection,

Experimental Infection of Cats with Metacercariae

fourteen flukes recovered from nodules of the lungs
were immature, while four flukes from cysts in the lungs

Distribution of flukes:

The results of the experimental infection in catswith the metacercariae are shown in Table 1. The
average recovery rate of five cats (Nos. 1‑5), orally
infected with metacercariae, was 20.7% ranging fromwere mature (Table 1). The sizes of the mature flukes
were variable, 5.8 ‑ 7.4 mm long and 3.1 ‑ 4.8mm wide,with a few eggs in the uteri. In cats autopsied 192 days

10.0 to 46.7%. Out of 31 flukes recovered, 22 were foundlater, all flukes recovered from cysts in the lung were
fully mature. The sizes were 8.2 ‑ 9.1 mm in length andin cysts in the lungs, 5 in the muscles and 4 in the pleural

5.1 ‑ 6.0 mm in width and the worms had numerous eggs

cavity. A total of 176 worms was recovered from thein the uteri. Flukes recovered from muscles weremorphologically similar to excysted metacercariae

cats (Nos. 6 ‑ 11) inoculated with metacercariae
intraperitoneally and the average recovery rate ofworms was 57.0% (52.0 ‑ 83.3%). Eighty‑two worms
were recovered in the muscles and 88 in lungs or cysts inexcept for their slightly larger body size (0.7 ‑ 1.4 mm inlength and 0.4 ‑ 0.7 mm in width) . The worms had
numerous granules in their excretory bladder. The

Postmortem examinations:flukes from the pleural cavity were immature and theirsizes were about 3.5 mm in length and 2.1 ‑ 3.2 mm inwidth. Their ovary and testes were divided into 5 or 6

On gross examination of the infected cats, in which
1 to 9 cyst formations were recognized in the pulrnonary

lobes but vitelline glands were less developed. No
remarkable differences were observed in the develop‑

parencyma, membraneous rust coloured fibrinous
exudate was present in the cavity, and adhesions

ment of the flukes from the three different localities in

the lungs and a small number of flukes were found in thepleural cavity.

Malaysia.

between the lung lobes were present. In the cats, inwhich migrating flukes were found in the pleural cavity
or lungs, collections of stagnant thoracic fluid were
present and small number of haernorrhagic spots, 4 ‑ 6Experimental Infection of Dogs with Metacercariae

mm in diameter, were recognized in the lungs. In cat
N0.6, a large amount of stagnant thoracic fluid withblood cells was present, and about 30 haemorrhagic
spots or nodules, 1‑16 mm in diameter, were present inof 7 dogs with metacercariae from Kuala Pilah andSungai wa. In dog N0.1, orally infected with 150

Distribution of flukes:

Table 2 shows the results of experimental infection

metacercariae and necropsied 234 days after infection,

only 3 (2%) worms were recovered from muscles. The
remaining six dogs were infected intraperitoneally with80 or 100 metacercariae and were necropsied 208 to 258

the lungs.

Table 2 Results of experimental infection of dogs with Malaysian Paragonimus westermani metacercariae (Mc.)

Dog Body weight No, of Method Autopsy

at necropsy Sex Mc. days afterNo, (kg) given infection infection
of

oral

No. of worms recovered fromNo. ofworms cyst in lungs

recovered ( % ) Iungs

cavityperitoneal
cavitymuscles

pleural234

3 (2,0)

O

o

O

O

3*

peritoneal
inoculation

208

52 (65,0)

2*

o

It

O

49t

100

peritoneal
inoculation

227

29 (29,0)

O

2t

It

O

26( 3t23$

F100

peritoneal
inoculation240

9 (9,0)

O

o

O

O

9t

M
M

100

peritoneal
inoculation245

35 (35.0)

4 (2t

O

O

It

30$

80

peritoneal
inoculation

252

23 (28.8)

O

2t

O

2lt

F

80

peritoneal
inoculation258

23 (28.8)

2t

O

19t

1

17.7

F

150

2

14.7

F

80

3

12.0

F

4

12.2

5

10.3

6

11.2

7

15.6

F: female,inf ection

M: male,2*2 (1lto*, t, t: Cf. the foot in Table 1,

Dog Nos. 1, 3 and 4 were infected with metacercariae from Kuala Pilah.
Dog Nos. 2, 5, 6 and 7 were infected with metacercariae from Sungai Wa.

228

days after inoculation. The average recovery rates of

average recovery rate of flukes from the rats was 42.8

worms was 32.6% (9.0 ‑ 65.0%) and a total of 171%. Almost all of the worms, 307 out of 310, were

worms were recovered. Majority of the flukes (154 out
of 171; 90.1%) were recovered from muscles and 6 and 8

recovered from the muscles and only a few worms were

flukes were found in cavities and cysts in lungs, respec‑

itoneally inoculated rats with 50 metacercariae eachtively.

from Taman Negara were necropsied 100 days after

Postmortem examinations:
On gross examination, no abnormality was obser‑
ved in any organs of 2 infected dogs (Nos. I and 4) , fromwhich flukes were only recovered from muscles. In theother dogs, some haemorrhagic lesions, I ‑ 10 mm indiameter, were present in the lungs and flukes were
found in the pleural cavity or in the lungs. In these dogs,

stagnant haemorrhagic exudates were present in thepleural cavity, and adhesions of the lungs to the thoracic

wall and between lung lobes were also observed. One or
2 typical worm‑cysts were present in the lungs of dogs
Nos. 2, 5 and 7; and 2 flukes were found in each cyst.
Development of flukes:
A total of 157 flukes were recovered from musclesof the dogs. Three of them measured 1.9 ‑ 3.1 mm inlength, and primordial cells of ovary, testis and uterus

found in the lungs or abdominal cavity. The 3 intraper‑

infection. The recovery rate of the flukes from theserats was 48.7% (46.0 ‑ 52.0%) and all of them wererecovered from muscles.
Postmortem examination:
On the gross examination of the infected rats, nopathological changes were generally observed except
those examined 10 to 20 days after infection. In theserats, haemorrhagic tracks were found in the muscles ofabdominal walls and legs, presumably due to migrationof juvenile flukes.

Development of flukes in rats:
All the flukes recovered frorn rats were juvenile'andmorpho‑logically similar to the excysted metacercariae
with the exception that they were slightly larger in size.

They were 0.7 ‑ 0.9 mm in length and 0.3 to 0.5 mm inwidth.

were recognized. The remaining 154 flukes were very
stunted and they had granules in the excretory bladder,and a small stylet was still visible on the oral sucker.
Their sizes ranged from 0.7 to 1.2mm in length and 0.4 to

0.6 mm in width. Five out of 8 flukes recovered fromcysts of lung were 4.5 ‑ 6.7 mm in length and 2.8 ‑ 3.4
mm in width, and eggs were present in their uteri. Threeother flukes from cysts in lungs and all the flukes fromcavities and lungs were immature. Their sizes were 1.9‑ 4.2mm in length and 1.4 ‑ 3.3 mm in width, though their
ovary and testes were developed slightly.

Experimental Infection of Rats with Metacercariae
Distribution of flukes:

Table 3 shows the results of oral infection of 29
albino rats with metacercariae from Kuala Pilah. The

Transfer of Juvenile P. westermani to Cats and Dogs
The results of oral infection in cats and dogs withjuvenile worms recovered from muscles of cats, dogs
and rats of the previous experiments are shown in Table
4. The average recovery rate of worms in five infectedcats was 68.2%, ranging from 48.0 to 84.0%. Out of 99
flukes obtained, 77, 4, 11 and 7 flukes were recoveredfrom cysts in the lungs, tissues of lungs, pleural cavity

and muscles, respectively. Sixty‑four out of 77 flukes
recovered from cysts in the lungs were mature while 1
out of 11 from pleural cavity and five out of 7 flukes
from muscles were still juvenile worms with granules inthe excretory bladder.
In dog No. 8, 2 and 3 flukes were recovered from thepleural cavity and muscles, respectively. The recovery

Table 3 Results of experimental oral infection of albino rats with 25
Par gonimus westermani metacercariae from Kuala PilahDaysofafter No.
worms*
No. ofNo. of worms recovered fromrats

lungs Peritoneal

inf ecti onused
recovered ( % )cavitymuscles105

48 (38.4)

O

20

5

54 (43.2)

O

30

5

55 (44.0)

O

60

5

62 (49.6)

O

100

4

57 (57.0)

1

o

56

150

5

34 (27.2)

O

o

34

* All worms recovered were juvenile.

o

48
53

o

55
61

229

Table 4 Results of experimental infection of dogs and cats with juvenile Paragonimus westermani
recovered from some paratenic host animals

Host Body weight Juvenile worms reco‑ No. ofat autopsy Sex vered from (days juvenile

animal (kg) after postinfection) worms fed

inf ection

No. of worms recovered fromNo. ofAuto psydays after

worms

cyst inrecovered (% ) Iungs

lungs

pleuralcavitymuscles
3 (2tDog N0.8

14.5

F

Rat

(150)

40

185

5 (12.5)oo2t

Dog N0.9

16.4

F

Rat

(100)

35

209

10 (28.6)

8*

o

2t

not examined

Cat N0.12

2.8

F

Cat

(192)

25

200

12 (48.0)

4*

o

5t

3 (1$

Cat N0.13

4.3

Cat

(237, 242)

23(12.11)

166, 161

19 (82.6)

16( 5to2t1$

Cat N0.14

3.6

Dog

(227)

17

172

12 (70.6)

12*

o

o

o

Cat N0.15

4.3

Dog

(245, 252)50(30, 20)

195, 202

42 (84.0)33( 8t4t

2t

3t

Cat N0.16

3.9

M
M
M
M

Rat

(100)

25

212

14 (56.0)

12*

o

2 (It

F female, M: male,rate was 12.5%. A11 of the flukes recovered from thedog were immature, especially a single fluke frommuscle having the same characteristics of the metacer‑
carial stage. In dog N0.9, typical cyst formation wasrecognized in the lungs, 8 fully matured flukes whichmeasured 7.9 ‑ 8.5 mm long by 3.8 ‑ 4.4 mm wide wererecovered. Two immature flukes were recovered frorn
pleural cavity but the muscles of this dog were notexamined.

DISCUSSION
It is well known that the distribution and develop‑
ment of P. westermani in favorable definitive hosts, Iikecats and dogs and in unfavorable hosts, such as rodents

are quite different. In the former hosts, worms arefound in cysts in the lungs and are mature, but in thelatter hosts, majority of worms remain in the muscle
with little development. In dogs and cats infected with
P. westermani from Japan, the triploid fluke takes about
2 months to reach maturity, and the diploid fluke takes

almost two and a half months (Takizawa, 1964; Habe,
1978; Shibahara, 1983). P. westermani from Korea,
China and the Philippine also shows similar growth indogs and cats (Fan, 1966; Miyazaki and Habe, 1979;
Unpublish data, S. Habe) . In general the worm recovery
rates in cats and dogs infected with metacercariae fromthese countries were about 60 to 80% by oral inoculation

and 90% more by peritoneal inoculation.

In our present study the average worm recovery

11*

25*

1$

2t

It

o

, t, t: Cf. the foot in Table 1,

freshwater crabs, Parathelphusa maculata and Johora
johorense, and recovered 31 worms from 61 to 108 days
after infection. These rates are very low compared tothose of P. westermani from other localities. The resultsof experimental intraperitoneal inoculation also shows asimilar tendency. The Malaysian P. westermani has alower infectivity to mammalian hosts than those fromother localities. Furthermore, in spite of keeping theinfections for 5 or more months after feeding withmetacercariae, about 50% and 90% of the flukes werestill recovered from the muscles of the cats and dogs

respectively. The worms were very stunted and mor‑
phologically similar to the excysted metacercariae withthe exception that their body sizes were slightly larger.
The rates of mature flukes out of total worms recovered
were only 40% from infected cats and 3% from infecteddogs.

All the flukes recovered from albino rats were
juvenile just like those that parasitized the muscles ofdogs and cats. Such stagnated worms have been col‑lected from other animals, such as pig, wild boar, mouse,

hamster, guinea pig, rabbit, monkey and hen infectedwith P. westermani from Japan (Habe, 1978, 1982, 1983;
Shibahara and Nishida, 1986; Takizawa, 1964). This is
the first report where juvenile worms were recoveredfrom cats and dogs infected with P. westermani. The
development and distribution of Malaysian P. wester ‑mani in cat and dog are very different from the flukesof other countries but no significant differences arerecognized in rat. However, Philippine P. westermani

westermani, were 20.7% and 2%, respectively. Kim

does not remain in the muscle of rats and migrates into
the lungs and then mature in relatively high rate. This

(1969) infected six cats with 109 metacercariae fromsuggests that Malaysian P. westermani has a higher

rates of cats and dogs, orally infected with Malaysian P.

230

' '
' ::l

i*
}:/'11!j;;:/ '*:'"' "*‑"**'*̲*="‑*' * **"***'='‑"*'‑^*" :'* 't* * i '

,

'

> ^

:

*=**+

., +

<

..**.

. '* *

;

" '*

'

"

= + { ;*i * : i i"' {' i' i*= >**'* * *'**'*.

=*"'* *=*+=+ '*+*'* '" +‑'*

'

+'

;

2

3

5
Figs. l‑3. Living juvenile worms. Bar=0.2mm.

l. Worm recovered from muscle of rat 150 days after infection with metacercariae.
2. Worm recovered from muscle of dog 240 days after infection with metacercariae.
3. Worm recovered from muscle of cat 237 days after infection with metacercariae:
Fi g .

4 Lungs of dog; 209 days after inoculation with 35 juvenile worms from muscles of rat.
Bar = 3cm.

Fig. 5 Mounted adult worm from worm cyst in lungs of cat; 172 days after inoculation

with juvenile worms from muscles of dog. Bar=2mm.

final host susceptivility than the Japanese. Chinese andKorean flukes while the Philippine P. w stermani has
the lowest.
The final host of P. weslermani is widely distributed

among mammals, especially of the families Felidae andCanidae. In Malaysia, only one species of lung fluke. P.

westermani has been recorded from tiger, Felis tigris,domestic cat, Felis domestica, clouded leopard, Neofelis

pzebulosa diardithe, wild cats, Felis ben;galep2:sis. F.
planiceps and F. temminckii, black panther, Felis par ‑dus, and crab‑eating monkey, Macaca irus irus (Groves
et al., 1967; Lee and Miyazaki, 1965; Lim and Betterton,
1977; Rohde, 1963, 1967; Miyazaki and Kwo, 1969) . Mostof the natural final hosts belong to Felidae. In spite of

the juvenile worms recovered from muscles of experi‑mental, cats in the present study, and it is without doubt

231

that the animals of Felidae are the most important andsuitable final hosts for Malaysian P. westermani. How‑ever, our results showed Canidae to be unsuitable hosts
for Malaysian P. westermani.
It was experimentally proved that juvenile flukes
remaining in the muscles of dogs, cats and albino rats

ACKNOWLEDGMENTS
This study was supported by the research grantNos. 60041052 and 61043047 under the International
Scientific Research Program from the Ministry of Edu‑
cation, Science and Culture, Japan.

were able to reinfect other carnivores. In the case of cat

infections, the recovery rate of flukes was 68.2% on the

average, while in dogs, it was 20.7%. In the animals
inoculated with juvenile flukes, the worm recovery andmaturity rate were also higher than those infected withthe metacercariae. Carnivorous animals are more read‑
ily infected by the Malaysian P. westerneani via feedingof juvenile worms from paratenic hosts than metacer‑
cariae from crabs. Therefore, the present study sug‑gests that many wild mammals living in jungle must
serve as paratenic hosts and they play a very importantrole in the completion of the life cycle of the flukes in

REFERENCES
Fan, P. C. (1966): Comparative studies on some problems of

Paragonimus westermani in experimental animals.
Chinese Med. J., 13, 370‑386

Fan, P. C. and Hsu, J. (1965) : Experimental transfer ofParagonimus infection from srnall to large carnivorous
hosts by feeding. Chinese Med. J., 12, 55‑67
Fan, P. C. and Khaw, O. K. (1964): Can rodents play a rolein infecting domestic carnivorous animals, cats and dogs

with Paragonimus westermani.

Selected papers,

Malaysia and its neighborhood. In experiments the

National Defence Medical Center, 30‑33
Groves, M. G., Umathevy, T. and Lim, B. L. (1967): Hel‑same mode of infection has been shown in the triploidminth infections in a black panther (Felis pardus Linn) .

and diploid types of P. westermani. P. miyazakii Kamo etal., P. mexicanus Miyazaki et Ishii and P. heterotremus

Chen et Hsia (Fan and Khaw, 1964; Fan and Hsu, 1965;
Habe, 1978, 1983; Miyazaki and Habe, 1976; Shibahara
and Nishida, 1986; Sugiyam

et al., 1990) . For example,

it was proven that the wild boar, Sus scrofa leucomystax,

plays an irnportant role as the paratenic host for humanparagonimiasis westermani (triploid type) in the south‑

ern part of Kyushu, Japan (Norimatsu et al., 1975;
Miyazaki et al., 1978; Tokudome et al., 1977) . Kwo andMiyazaki (1968) reported heavy infections of P. wester‑mani in all the tigers examined in North Sumatra,Indonesia, and Miyazaki and Habe (1976) postulated
that those tigers might have been infected due to feedingon animals harboring immature worms and serving as

Med. J. Malaya,21, 374
Habe, S. (1978) : Experimental studies on the mode of humaninfection with the lung fluke, Paragonimus westermani(Kerbert, 1878) . Jpn. J. Parasitol., 27, 261‑292 (inJapanese with English summary)Habe, S. (1982): Experimental infection of crab‑eatingmonkey with the parthenogenetic type of Paragonimus
westermani and P. w. filipinus. Med. Bull. Fukuoka
Univ., 9, 329‑333

Habe, S. (1983) : A newly recognized para.tenic host of
Paragonimus spp.. Jpn. J. Trop. Med. Hyg., 11, 1‑6Kim, J. S. (1969): Paragonimiasis in Malaysia. Universityof California: International Center for Medical
Research and Training Annual Report, 45‑51
Kwo, E. H. and Miyazaki, I. (1968): Paragonimus wester‑mani (Kerbert, 1878) from tigers in north Sumatra,Indonesia. J. Parasitol., 54, 630

paratenic hosts. Lim and Betterton (1977) reported that

Lee. H.F. and Miyazaki, I. (1965) : Paragonimus westermani

civet cats have the potential of being paratenic hosts
from feeding experiments and suggested that wild boar

infection in wild mammals and crustacean host in

and a number of small marnmals were also potential

paratenic hosts. Although locally acquired par‑agonimiasis has not been observed in man in Malaysia,
P. westermani has been found in a number of animals
and freshwater crabs. Lim and Betterton (1977) gave
the reason that freshwater crab is not generally used asa source of food and wild boar meat is infrequently
eaten. In addition to that, the development of P.
westermani in man could be considered to be similar tothat seen in the present experiments where mature
worms may not be found.

Malaysia. Ame. J. Trop. Med. Hyg. 14, 581‑585.
Lim, B. L. and Betterton, C. (1977): Paragonimus westermani

in Malaysian Felidae and Viverridae: probable modesof transmission in relation to host feeding habits. J.
Helminthology, 51, 295‑299
Miyazaki, I. and Habe, S. (1976): A newly recognized modeof human infection with lung fluke, Paragonimus wester ‑mani (Kerbert, 1878). J. Parasitol., 62, 646‑648
Miyazaki, I. and Habe, S. (1979): Paragonimus westermani
filipinus Miyazaki, 1978, Stat. n. occurring at Jaro,
Leyte, The Philippines. Med. Bull. Fukuoka Univ., 6,
447‑462 (in Japanese with English abstract)
Miyazaki, I. and Kwo, E. H. (1969) . Potamiscus cognatus(Roux, 1936), a new crab host for Paragonimus wester‑mani in Malaysia. J. Parasitol., 55, 459

Miyazaki, I., Terasaki. K. and lwata, K. (1978): Natural

232

infection of muscle of wild boars in Japan by immature
Paragonimus westermani. J. Parasitol., 64, 559‑560

Norimatsu, K., Arikawa. K. and lkehata, M. (1975): (Thepresent situation of paragonimiasis.). Rinsho to Ken‑kyu, 52, 1046‑1051 (in Japanese)

Rohde, K. (1963): Uber einige Malayische Trematoden.
Ztschr. f. Parasitenk., 22, 268‑277

cariae. Jpn. J. Parasitol., 35, 303‑311

Sugiyama, H., Shibahara, T., Katahira. J., Horiuchi, T.,
Tomimura. T., Agatsuma, T., Habe, S., Kawashima, K.,
Ketudat, P. and Thaithong, S. (1990): Rats and miceserved as experimental paratenic hosts of Thai Par ‑agonimus heterotremus. Jpn. J. Trop. Med. Hyg., 18, 295‑300

Rohde, K. (1967): Die Verbreitung von Opisthorchis und

Takizawa, M. (1964): Studies on the deveropment of Par‑

Paragonimus in Malaya und moglische Infektionsquellendes Menschen mit diesen Parasiten. Ztschr. Tropenmed.

agonimus westermani (Kerbert, 1878) Braun, 1899 in thefavorable and unfavorable hosts. Jpn. J. Parasitol., 13,

Parasit., 18, 152‑161

Shibahara, T. (1983) : Studies on the lung fluke, Par‑agonimus westermani ‑diploid type‑ in northern part of
Hyogo Prefecture, Japan. II. Experirnental oral infec‑
tion with metacercariae to dogs and cats, with reference

to morphological characteristics of adult worms andeggs. Jpn. J. Parasitol., 32, 293‑304

181‑199 (in Japanese with English surnmary)
Tokudome, I., Nishizurni, M., Ikeda, M., Kono, S., Jimi, S.,
Nagayama, J., Kuratsune, M., Habe, S., Hayashi, E., andHirose, H., (1977) : Epidemiologic study on a new modeof infection of Paragonimus westermani. Jpn. J. Publ.
Hlth, 24, 31‑36 (in Japanese with English summary)Yokogawa, M., Cabrera, B.D., Kobayashi, M., Hata, H.,

Shibahara. T and Nishida, H. (1986): Studies on the lung

Tazaki. T. and Araki, K. (1979): Comparative studies

fluke; Paragonimus westermani ‑diploid ty el in n0.rth‑
ern part of Jyogo Prefecture: Japan. VI. Experimental

on Philippine Paragonimus westermani and Japaneseoral infection of wild boars and pigs with the metacer‑2, 119‑131

Paragonimus westermani. Act. Med. Philipp., 15, Series

233

J pn. J Trop. Med. Hyg.. Vol. 24, No. 4, 1996, pp. 233‑236

Short communication

SEROEPIDEMIOLOGICAL STUDIES OF HEPATITIS
VIRUSES IN THE DOMINICAN REPUBLIC
II. THE PREVALENCE OF HEPATITIS D
AND E VIRUS INFECTIONSAKEHISA SHICHIJO1, KUMATO MIFUNE1, HIDEO TERA02, TETSUJI RIKIHISA3,

TAKASHI ITOGA2, MARIA ESTELA NoRMAN", ARACELIS GERMAN RODRIGUEZ*,MERCEDES CASTRO BELL04, FERNANDO SOLANO FERNANDEZ4
Received August 13, 1996/Accepted October 17, 1996

Abstract: A total of 841 serum samples collected and stored at the Center of Gastroenterologidal Disease,
Aybar Hospital, Santo Domingo, Dominican Republic were tested for antibodies against hepatitis E virus(anti‑HEV) . Sera of 60 hepatitis B virus (HBV) asymptornatic carriers selected from 2,000 apparently
healthy individuals in the Dorninican Republic were tested for antibodies against hepatitis D virus (anti‑
HDV) . The study demonstrated that the prevalence of anti‑HEV was 19.0% in individuals under 15 yearsof age as compared with the lower prevalence of 4.5% in individuals older than 16 years. The prevalenceof anti‑HDV in HBV carriers was 6.7%, demonstrating that HDV and HEV are prevailing in this country.

Among five hepatitis viruses of hepatitis A (HAV) ,(Rizzetto et al.. 1983, Rizzetto et al.. 1990). In our

B(HBV), C(HCV), D(HDV), and E(HEV), a seroe‑previous study (Shichijo et al.. 1995) , it was found thatpidemiological study of the former three viruses in the
Dominican Republic was described previously (Shichijo

HBV infection is still prevalent in the Dominican Repub‑

lic. The purpose of the present study is to investigate

et al.. 1995).

whether and to what extent HEV and HDV infections

HEV belongs to the Togaviridae farnily and is anenterically transmitted agent that causes epidemic andexist in the Dominican Republic.

sporadic forms of hepatitis in tropical countries. Clini‑

cal symptorns due to HEV infection are nearly the sameas those caused by HAV infection (Bradley, 1990). It

Anti‑HEV antibodies were tested by ELISA. Since
HEV cannot be grown in tissue culture, thirty arninoacids corresponding to from nucleotides (nt) No. 6,965 tofrequently infected with HEV than children; HEV infec‑

7,054 of the open reading frame 2 of the HEV genome
were synthesized and used as a coated antigen. The
synthetic HEV peptide (5pg) was pipetted into eachtion rarely prevails within a family and that infection ofwell of 96 microtiter plates. After incubation for lh at

pregnant women with HEV produces higher maternal
and fetal mortality. HEV is known to distribute widelyin the world, and India, Indonesia, Nepal, Hong Kongand Egypt have been reported to be the areas endemic

37'C or overnight at 4'C, the wells were washed threetimes with phosphate‑buffer saline containing 0.05%
Tween 20 (PBS‑T) . The plates then received 100pl per
well of PBS‑T containing 5% skim milk and 10pl of testfor HEV (Goldsmith et al., 1992, Lok et al., 1992,

serum and incubated for lh at 37'C. The wells were

Corwin et al.. 1994, Longer et al.. 1994).

washed 5 times and further incubated at 37'C for lh with

HDV is a defective RNA virus, and it can be trans‑mitted only when HBV infection occurs simultaneously,
or only when a chronic HBV carrier is infected withhuman‑IgG F(ab')2 (diluted to 1/10,000 in PBS‑T‑5%

HDV. HDV and HBV share the same route of infection

2HCI (OPD) was added and the absorbance was read at

has been found, however, that young adults are more

2
3
4100pl of horseradish peroxidase conjugated goat anti‑
skim milk) . After washing 5 times, o‑phenylenediamine'

Department of Microbiology, Oita Medical University, Hazama‑machi, Oita, 879‑55, Japan,
Department of Internal Medicine, Oita Medical University, Hazama‑machi, Oita, 879‑55, Japan,
The Chemo‑Sero Therapeutic Research Institute, Kikuchi Research Center, Kyokushi Kikuchi, Kumamoto, 869‑12, Japan and
The Center of Gastroenterological Diseases, Dr. Luis E. Aybar Hospital, Santo Domingo, Dominican Republic

234

492nm after 30 min incubation at room temperature. In
the experiments, anti‑HEV antibody‑positive and ‑neg‑
ative sera were simultaneously tested as controls.
When the reaction was positive, the presence of anti‑

HEV antibody was confirmed by the following two
methods. One method was a simple repeat of the experi‑
ment to check the reproducibility of positive reaction,and the other was an absorption experiment. In thelatter experiment, 10pg/well of antigen peptide were
simultaneously added with test serum to the plates
which was already coated with antigen peptide. Only

when an anti‑HEV‑positive serum became negative

Chicago, 11linois, USA ) was used for screening anti ‑HDV antibodies.
The prevalence of anti‑HEV antibody by age andsex was shown in Table I and Figure 1. Each age groupconsists of more than 80 samples. The highest antibody
positive rate was 30% for female in an age group from
6 to 10 years and 19.1% for male in an age group from11 to 15 years old. The overall prevalence of anti‑HEV
was much higher (19.0%) in individuals under 15 yearsof age than in individuals older than 16 years (4.5%).
This prevalence by age appears slightly different fromafter the absorption test, the test serum was taken as

the higher infection rates in young‑to‑middle‑age adults
reported in India, Indonesia and Egypt (Goldsmith et al.,antibody‑positive. The RIA kit (Abbott, Lab., North

1992, Corwin et al., 1994, Arankalle et al., 1995). The

Table 1 Prevalence of anti‑HEV by age and sex in the Dominican individuals

Sex

Age groupNo, of

0‑5

6 ‑10

44 7

41 7

tested No. ofMale

11‑15 16‑20

42 45

21‑30

31‑40

41‑50

49 3

65 3

57 2

54 3

6.1

4.6

3.5

9.3

69 2.980 569 560 1.7

6.3

7.2

51‑

Total
397 36 9.1

8 1

anti‑HEV
positive % positive

No. of

15.9

17 . 1

40 7

40 12

tested No. of

19 . 1 2 . 3

40 46

444 40 9.0

6 2

Femaleanti‑HEV
positive % positive

17 . 530 . O

84 14

81 19

15 . O 4 . 3

tested No. of

82 91

anti‑HEV

14 3

No. of

Total

118 5

145 8

126 7

114 4

841 76

positive % positive

16 . 6

23 . 417.1 3.3

4.2

5.5

5.6

5.3

30

Male
25

El

Female

‑ 20
>UJ

I 15
zh

< 10
5

o

0‑5 6 10 11 15 16‑20 21‑30 31‑40 41‑50 51‑
AGE
Figure 1 Prevalence of anti‑HEV by age and sex in the Dominican Republic

9.0

235

Table 2 Prevalence of HBsAg and anti‑HDV in Healthy
Individuals in the Dominican Republic

Anti‑HDV

H BsAgNo. of test

No. (%) of

No of test No. (%) of
Anti‑HDV positive

HBsAg‑ positive
2 , ooo63 (3.1)

60 4 (6.7)

reason for the difference was unknown, however, one
possibility is that HEV may have been introduced to the

Dominican Republic sometime in the past 15 years andsporadic but continuous infection has been taking placesince then. Higher seroprevalence rates under 15 yearsof age could be explained as the consequence of very
active behaviors of the children of these age groups, thatfacilitate frequent infection with HEV via fecallycontaminated water or by person‑to‑person contact.
The HEV infection in the Dominican Republic, however,
was not highly widespread and its incidence was consid‑
erably much lower than that of similarly transrnitted
HAV, indicating that epidemic or sporadic HEV infec‑
tion is possible to occur in this country in the future. To

our knowledge, the present paper is the first descriptionof the existence of HEV infection in the Carribean
countries.

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1 ) Aceti, A., Mohamud, O. M., Paparo, B. S., Mohamed,
O. M., Quaranta, G., Adan, K. M., and Sebastiani, A.
(1991): High prevalence of anti‑hepatitis delta viru
antibody in chronic liver disease in Somalia. Trans. R.

Soc. Trop. Med., Hyg. 85, 541‑542
2 ) Arankalle, V. A., Tsarev. S. A., Chadha, M. S.. Alling,
D. W.. Emerson, S. U., Banerjee. K. and Purcell, R. H.
(1995) : Age‑specific prevalence of antibodies to hepati‑
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4 ) Cenac, A., Pedroso, M‑L., Djibo, A., Develoux. M.,
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lar carcinoma: a comparative study in Niger. Arn. J.
Trop. Med. Hyg., 52, 293‑296
5 ) Corwin, A., Jarot, K., Lubis, I., Nasution, K., Suparm‑awo, S., Sumardiati, A., Widodo, S., Nazir, S.,
Orndorff, G., Choi, Y., Tan. R., Sie, A., Wignall. S.,
Grabam, R., and Hyams, K. (1995): Two years investi‑
gation of epidernic hepatitis E virus transmission inWest Kalimantan (Borneo) , Indonesia. Trans. R. Soc.

Trop. Med. Hyg., 89, 262‑265
6 ) Fonseca, J. C. F., Simonetti, S. R. R., Schatzmayr, H. G.,

Various seroepidemiologic studies on HDV infec‑
tion have demonstrated that the prevalence is high in

Castejon, M. J., Cesario, A. L. O. and Simonetti, J. P.

some parts of Africa, south Americas and southern Italy

virus (HDV) among carriers of hepatitis B surface
antigen in Amazonas State, Brazil. Trans. R. Soc.
Trop. Med. Hyg., 82, 469‑471(Fonseca et al.. 1988, Lalla et al.. 1990, Aceti et al.. 1991,

Sjogren & Colichon, 1991, Hadler et al.. 1992, Sagnelli

et al., 1992, Torres & Machado, 1994, Cenac et al..
1995). On the other hand, in the United States andwestern Europe, it was shown that HDV infection isrestricted to the groups with frequent percutaneus expo‑(1988): Prevalence of infection with hepatitis delta

7 ) Goldsmith, R., Yarbough, P. O., Reyes. G. R., Fry, K.
E., Gabor, K. A., Kamel, M., Zakaria, S., Amer, S. andGaffar, Y. (1992) : Enzyme‑1inked immunosorbent assay
for diagnosis of acute sporadic hepatitis E in Egyptian

sure to blood and its components, primarily i.v. drug

children. Lancet, 339, 328‑331
8 ) Hadler, S. C., de Monzon. M. A, Rivero, D., Perez, M.,

users, hemodialyzed patients (Hoy et al.. 1984, Lettau et

Bracho, A., and Fields, H. (1992): Epidemiology andlong‑term consequences of hepatitis delta virus infection

al.. 1987).

Sixty three out of 2,000 healthy Dorninicans were

HBsAg‑positive as shown in Table 2. Sixty of thosesera were tested for anti‑HDV and four of them (6.7%)
were found to be positive. This rate was higher thanthat observed in the United States or in Japan where it
has been reported to be about 1% or less (Iwanami etal.. 1993), but it was much lower than those of highly
endemic countries as described above.

ACKNOWLEDGMEN TS
This work was performed as one of the research
project in the "Investigation and clinical treatment ofgastroenterological diseases" in the Dominican Republicby the Japan International Cooperation Agency.

in the Yucpa Indians of Venezuela. Am. J. Epidemiol.,
136, 1507‑1516
9 ) Hoy, J. F., Hannson, B. G.. Dimitrakakis, M., Gust, I.

and Lucas, C. R. (1984): Delta agent infection in
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10) Iwanami, E.. Yano, M., Koga. M., Shirahama. S. andTsuda, T. (1993): Local spread of HDV infection
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Jpn. J. Trop. Med. Hyg., Vol. 24, No. 4 1996 pp 237 239

se""

""

REACTIVITY OF SERA FROM PATIENTS WITH
ACUTE PLASMODIUM FALCIPARUM AND P. VIVAXINFECTIONS WITH AN ANTIGEN PREPARATIONFROM A P. FALCIPARUM ISOLATE:MUTUALLY EXCLUSIVE REACTIVITY WITH
A 47kD AND A 29kD BAND RESPECTIVELY.
MOHD‑NoR NoRAZMll, SHIGEYUKI KAN02, AZNl ALIAS3,

MOHD‑SHUKRI ABDULLAH3 AND MAMORU SUZUK12
Received September 2, 1996/Accepted November 12, 1996

Abstract: Western blot analysis was carried out on sera obtained from patients with P. falciparum (Pf) and
P. vivax (Pv) on an antigen preparation of a laboratory propagated isolate of Pf (# 40) cultured in vitro.
The reactivity to a 47kD band was sought based on the results from a previous study (Kano et al., 1990a) .
Pooled Pf sera (n = 15) reacted with the 47kD band whereas pooled Pv sera (n = 13) as well as pooled normal
human sera (n = 45) were not reactive with the 47kD band. An interesting finding was that the pooled Pv
sera reacted with a 29kD band instead, while the pooled Pf sera was non‑reactive to the 29kD band. In

addition, two of four Pf‑and three of four Pv‑ individual sera reacted with the 47kD and 29kD bands
respectively. All of these sera had high IFAT (indirect fluorescent antibody test) titer. One of the sera
obtained reacted with both the 47kD and 29kD bands but was later found to be from a patient with mixed
Pf and Pv infection. The potential of these findings in diagnosis of Pf and Pv infections is discussed.

Key Words: P. falciparum, P. vivax, malaria, diagnosis

The presence of high antibody levels against theuals positive for malaria parasites have antibody titers(against crude malaria antigen preparation) as well asreactivity to the 47kD band, but individuals negative formalaria parasite can be found in the sera of most adultmalaria parasites but with high antibody titers did not

individuals in malaria endemic areas regardless ofThese antibodies react specifically with various

recognize the 47kD band. The latter group probably
represent those with cumulative past malaria infec‑
tions. They concluded that the 47kD molecule may be

antigenic bands prepared from the parasite when anal‑

useful in determining present or recent infections inyzed using Western Blotting. However, Kano et al.

serological surveys of malaria. The present study
attempted to extend above findings to the Malaysian

INTRODUCTION

whether these individuals have clinical malaria or not.

(1990a) reported that in a longitudinal study on theinfection, a 47kD band was strongly reactive only duringsituation and thus to determine whether sera taken frompatients with falciparum malaria infection react with an

the acute phase of the infection with progressively

antigen preparation of a Malaysian Pf isolate. An

decreasing reactivity and was barely detectable in con‑valescence (about 2 months after clinical presentation) .

unexpected finding of the reactivity of Malaysian sera
taken from Pv infection to a 29kD of the same antigen
preparation was noted. This finding provided interest‑
ing information regarding the possible use of both theserum taken from a Japanese man with imported Pf

A pilot study carried out in various endemic locations in

the Sudan by the same researchers showed that individ‑
1

2
3

Department of Immunology, School of Medical Sciences, Universiti Sains Malaysia, 16150 Kubang Kelantan, Malaysia.
Department of Parasitology, Gunma University School of Medicine, 3‑39‑22 Showa‑machi, Maebashi 371, Japan.
Department of Medical Microbiology & Parasitology, School of Medical Sciences, Universiti Sains Malaysia, 16150 Kubang
Kelantan, Malaysia.

238

47kD and 29kD epitopes in diagnosis of acute infections

i<‑ Pf ‑ i<‑PV ‑ I

by both species.

METERIALS AND METHODS
Sera were obtained irom Malaysian Pf‑and Pv‑
infected symptomatic and parasitemic patients prior ta
drug treatments, from the District Hospital of Kuala
Krai, Kelantan, Malaysia. The IFAT was carried out asdescribed by Kana et d. {1990b). Briefly, four‑fold

12 3 4 5 6 7 8 91011

200‑
116 ‑ s94̲ ,';,.,';.'."*.;

67 ‑dilutions of sera (1:4 to l:4096) were incubated for 30

min onto the antigen spots from Pf cultured isoiate

‑47kD

43‑ {

(SGEI strain) and Pv clinical isolate placed onto giass
slides. After washin, g, diluted fluorescein conjugatedrabbit anti‑human lgG (1:30, Behring, Germany) was
then applied and incubated for 30 min. Following final
washing, the fluorescence was read under an incident
light illuminating type fluorescent microscope (Olympus

BH‑RFC, Japan) to determine titers.
The Pf isolate (designated # 40) was obtained froma patient with acute Pf infection during the 1990/92

transmissi,on season and cultured for more than 20
passages i;e vitro using a modified method of Trager(Zolg et al., 1982)in RPMI 1640 supplemented with 5%
washed human red blood cells, lO% heat‑inactivatedhuman serum and maintained in an atmosphere of 5%0=xygen, 5% carbon dioxide, and 90% nitrogen.

Parasite antigen preparation for SDS‑PAGE wascarried as described previously (Kano et al., 1990a) .
Briefly, asynchranous cultured isolate # 40 (at least 5%

parasitemia) was obtained, centrifuged and the super‑
natant discarded. The parasitized packed cells were

resuspended and incubated in 1.5 volume of O.15%
saponin at 37'C ior 20 min, after which the lysed cells

were washed in 10 valume PBS and the dark‑brownparasite pellet was collected. The sediment was thendissolved in the Laemmli's antigen solution 1.aemmli,

1970). The SDS‑PAGE was canducted in lO%
acrylamide gel undet reducing conditions. The electro‑
phoresed proteins were transferred onto a nitrocellulose

mernbrane. The membrane was cut into strips andWestern blotting was carried out under reducing condi‑
tions using standard procedures (Laemmli, 1970) . Brief=
ly l:100 dilution of sera were reacted separately to eachmembrane strip and the reactivity of the sera wasrevealed by reaction with a secondary goat anti‑humanlgG (peroxidase conjugated) (Cappel, USA) and 4‑
chloro‑1‑naphthol as substrate.

‑ooo
‑
5:,
CL 5
$ { $,:;g o ,e,e

Qc *Figure I Western blot analysis of sera obtained frompatients with Pf and Pv infections against a
Malaysian Pf antigen preparation (isolate #40) .
Strip I = pooled normal human sera (n=45) , strip 2= pooled Pf ser (n*15) , strip 3 = pooled Pv sera (n*13) ; and individual sera taken from Pf (strip 4 to7) and Pv (strips 8 to ll) infcct.ions. Individual Pf

sera reacted with the 47kD band but not the 29kD

band whereas individual Pv sera reacted with the291{D band nOt the 47kD band. The serum used instrip 10 wa8 obtained from a patient with mixed
Pf and Pv and infections. (nd = not determined;*= based on Pv titer, the Pf titer was > 4096).

RESULTS AND DISCUSSION
Figure I shows the Western blot profile of the
Malaysian Pf‑isolate (# 40) reacted with pooled andindividual Pf and Pv sera of Malaysian patients. The
rFAT titers are indicated at the bottom of the figure.
Pooled normal human sera (Strip l, n = 45) did not show
any reactivity. Pooled Pf sera (Strip 2, n = 15) reacted

strongly with the 47kD band but not with the 29kD band
while pooled Pv sera (Strip 3, n = 13) reacted with the
29kD band but not the 47kD band. Two of fcur individ‑

ual sera from Pf‑infected patients (Strips 4 and 7)
reacted with the 47kD band, both of which had high
IFAT titers. Three of four individual sera with high
IFAT titers from Pv‑infected patients (strips 9, 10 andll) reacted with the 29kD band. One of these (Strip lO)

reacted with both the 47kD and 29kD bands. This serum
was later found to have been obtained from a mixed Pf=,
Pv‑infected patient. Sera with relatively low IFAT titer(Strip 5, 6 and 8) did not produce any bands following

239

Western blotting. The obtained result employing indi‑vidual sera from Pf infected patients reacted with SDS‑
PAGE antigen from the Malaysian isolate (# 40), wasconsistent with a previous finding (Kano et al., 1990a)that the sera from patients with acute Pf infection

reactivity to the 29kD band could only be seen in acute
Pv infections but not in convalescence as reported forthe 47kD band. Further characterization of the 47kD
and 29kD molecule would prove valuable for the diagno‑
sis or epidemiological survey of Pf and Pv infections.

contain antibodies to a 47kD band. On the other hand, it

was interesting to note that sera taken from patientswith acute Pv infections contain antibodies to a 29kD
band of the same antigen preparation. This pattern ofreactivity was found to be mutually exclusive, in that Pf

sera reacted with the 47kD but not 29kD and Pv sera

reacted with the 29kD but not the 47kD band. The
reactivity towards the 47kD band by Pf not Pv serasuggests that this epitope is unique in Pf. However, thereactivity pattern towards the 29kD was quite un‑
expected. This epitope may share a common antigenic

ACKOWLEDGEMENTS
The authors would like to acknowledge that thesera were provided by Dr. Mary Abraham and Mr.
Abdul Aziz Seman, Kuala Krai District Hospital,
Kelantan, Malaysia. This study was jointly sponsoredby the Malaysian Grant under IRPA (323/0501/4005)
and the Japan Society for the Promotion of Science
Fellowship afforded to the first author.

structure in Pf and Pv. Whereas it may be "hidden" inerythrocytic stages of Pf, and sera taken from patientswith acute Pf infection did not contain measurable
amounts of antibodies towards this masked Pf antigenic
polypeptide. However, the 29kD epitope may have been
exposed in Pv, and patients with acute Pv infections
could be ready to produce antibodies against it. Under

the denaturing and reducing conditions of SDS‑PAGE,
this particular epitope was then exposed, and Pv sera
which contain the corresponding anti‑29kD antibodies
would thus produce a band during Western blotting.
Future work should determine whether the reactivity
was a unique feature of the isolate used or whether thisphenomenon could be substantiated with other isolates
and sera taken from other geographical locations. It

would also be interesting to determine whether the

REFEREN CES1 ) Kano, S., E1 Gaddal, A.A. and Suzuki, M. (1990a) :Clinical and epidemiological studies on a 47kD Plas‑
modium falciparum antigen, Japan.J.Trop. Med. Hyg.
18 (4), 317‑324.

2 ) Kano. S., Waki, S., Igarashi, I., Nakazawa, S., Masuda,

G. and Suzuki, M. (1990b) : Restrospective malaria
diagnosis by indirect flourescent antibody titration on
Japanese patients, Japan.J.Parasitol., 49 (5) , 475‑481.

3 ) Laemmli, U.K. (1970): Cleavage of structural proteinsduring the assembly of the head of bacteriophage T4,
Nature, 227, 680‑685.
4 ) Zolg, J.W., MacLeod. A,J., Dickson, I.H., and Scaife, J.
G., (1982) , Plasmodium falciparum: Modifications of thein vitro culture conditions improving parasite yields. J.
Parasitol., 68, 1072‑1080.

DecemberVOL．24NO．4

l996

CONTENTS
Original

ArticleYamazi，Y．，Hasegawa，A．，Suzuki，H．，Inoue，S．，

Supawadee，J．，Suprasert，S．，Rangsiyanon⊂1，P

Two−Year

FollowThaian（iPongProt，B・

up

from

Birth

of

Infection：Serotypes

and

RNA

Electropherotypes一・

Makino，Y．，Inoue，R．，Kanemura，K，and
A

Serological

Study

of

DengueVims

Childrenfor

Rotavirus
・・215−220

Fukunaga，T．

Infection

among

Japanese

Residentsin・・221−223

Manila．

Habe，S．，Lai，K．P．F．，Agatsuma，T．，OW−Yang，CK

Growth
the

Short

of

Mode

Malaysian勘㎎o％加％s側6s娩脚勉inofTransmission

of

the

Fluke

among

and

Kawashima，K

Mammals

and・・225−232

Mammals…一Communication

Shichijo，A．，Mifune，K．，Terao，H．，Rikihisa，T．，Itoga，T．，

Norman，M．E．，Rodriguez，A．G．，Bello M．A．，Femandez，ES．
Seroepidemiological Studies of Hepatitis Viruses in the Dominican Republic
II．TheResearch

Prevalence

Hepatitis

D

and

E

Vims

・・233−236

Infections一NoteMohd−Nor

Norazmi，Kano，S．，Azni

Reactivity

antigen
a29kD

of

of

sera

preparation

band

from

from

patientsa

Alias，Mohd−shukri
withAbdullahandacute．PZαs魏04伽卿ヵlo吻n槻and

Pヵlo吻鰯郷isolate：一Mutually

exclusive

Suzuki，M．
P∂∫∂砿infections

reactivity

with

with

an

a47kD

and

respectively．・………・………・・……………・…・…一……………・…………一……・……237−239