ISSN

Japanese

Joumalof

TropicalMedicine

and第15巻 第1号

Hygiene昭和62年3月15日
内原O304−2146

容著
大平肺吸虫の生態学的研究

H

東海地方の揖斐川，長良川，木曽川流域産カイならびにネズミにおける本種の検出成績（英文）…一…………・…一…一……・・……・松尾喜久男，真喜屋

清

1略エクアドル国アンデス斜面の高度差によるリーシュマニア症浸淫（英文）・橋口 義久，EduardoA．GomezLand丘es，VicentaVeraDeCoronel，
三森 龍之，川端 真人
7−15
ファンシダールによるマラリア感染治療後のマウス血液酸素親和性の変動（英文）

日置

敦巳，大友

弘士

17−23

英世

25−28

B型肝炎表層抗原の慢性肝炎，肝硬変および肝硬変に伴った肝細胞癌組織中の陽性率の減少傾向（英文）………・………・……一一…千馬

正敬，中村

剛，板倉

ネッタイシマカおよびヒトスジシマカ（インドネシア株）の唾液腺におけるチクングニァ
ウイルス成熟の電子顕微鏡的研究（英文）……………・…・…一Soedarto

短Soekiman，松村

武男

29−36

報

インドネシア由来のネッタイシマカ，およびヒトスジシマカにおけるデング3型ウイルスとチクングニアウイルス増殖の比較（英文）…一Soedarto Soek㎞an，小西

会

英二，松村

武男 37−41報
昭和61年度第2回幹事会記録………一…
昭和61年度評議員会記録……一………・・

43−45
45−46

第28回総会記録……

46−47

昭和60年度会計決算書一一一
昭和62年度予算書…………一…一………一…・・……

47
47

幹事選挙関係規則改正に至った経過要約……一…………曙一

47−48

斡事選挙関係規則改正案に対する説明一…

48−49

会則…………一
投稿規定・……………一

50−53

54−55
56−86

会員名簿・…一・

日熱医会誌Japan．J．T．M．H．

日

本熱帯医学会Japan. J. Trop. Med. Hyg., Vol. 15, No. 1, 1987, pp. 1 6

1

ECOLOGICAL STUDIES ON THE LUNG FLUKE,PARAGONIMUS OHIRAJ MIYAZAKI, 1939
II. INFECTION RATES WITH P. OHIRAr OF SNAILSAND RODENTS COLLECTED FROM THE IBI,NAGARA AND KISO RIVERS IN THE TOKAI
DISTRICT, CENTRAL JAPANKIKUO MATSUol AND KlYOSHI MAKJYA2
Received September 1 1986/Accepted January 8 1987

Abstract : In the area of the lbi, Nagara and Kiso rivers in the Tokai district, central

Japan, a survey for the detection of Paraglonimus ohirai in snails and rodents was performed

durirrg the period between October 1983 and October 1985. Snails, Assiminea parasitologica,
positive for P. ohirai cercaria were found in 2 of 5 study sites. The over‑all infection rates were

O. 41% at the sites on the lbi and 0.45% at those on the Kiso river. Two species of rodents,
Rattus norvegicus and Apodemus speciosus, were collected, the infection rate for P. ohirai being

50% in the former and 0 fc, in the latter species. The results of the present study and ourprevious report (Matsuo and Makiya, 1985) dlsclosed the life cycle of P. ohirai in the areas along

the lbi, Nagara and Kiso rivers; A. parasitologica is the Ist intermediate host, Sesarma dehaaniand S. intermedia are the 2nd intermediate host, and R. norvegitcus is the final host.

INTRODUCTION
The frst report of this series revealed that 2 species of crabs, Sesarma dehaani and S.
intermedia, with Paragonimus ohirai were widely distributed along 3 rivers, the lbi, Nagara andKiso, which flow into the Ise Bay at the boundary between the north‑eastern part of Mie
Prefecture and the western part of Aichi Prefecture in the Tokai district, central Japan (Matsuo

and Makiya 1985). Metacercariae were detected in the crabs collected at 17 out of 19 study
sites which are situated about 5.5 to 13.0km up from the estuaries of the rivers. The infection

rate was 2.7‑100% for S. dehaani and 0.9‑100% for S. intermedia.
The present report deals for the frst time with the infection rate of P. ohirai in snails androdents serving the Ist intermediate and final hosts, respectively, collected from the study areas

along the lbi, Nagara and Kiso rivers. Although lwata and Nagayoshi (1985) are of the opinion
that P. ohirai is not an independent species, the authors here adopt the current theory that P.

ohirai is an independent species within the genus Paragonimus.

l Departrnent of Medical Zoology, School of Hygiene, Fujita‑Gakuen Health University, Toyoake, Aichi

Prefecture 470‑11, Japan
2 Department of Medical Zoology, School of Medicine, University of Occupational and Enviroumental
Heaith, Kitakyushu 807, Japan

2

SURVEY AREAS AND METHODS
The survey of the Ist intermediate host, Assiminea parasitologica, was carried out at 5 sites

on the lbi, Nagara and Kiso rivers, shown in Figure 1, during 2 years from October 1983. The

snails collected were crushed individually after measuring the shell length. Removing thebroken sheu fragments, the specimens were checked for P. ohirai cercaria under a stereomicro‑
scope.
The final hosts, wild rodents, were captured with live traps set on the river‑banks along thewaterway which are covered with communities of eulalia grass, reed and other grasses. The
traps were set in the afternoon and rodents caught were gathered in the next morning. The
rodents were dissected after identifying species and sexes and measuring body‑ and tail‑length.

Lungs and livers were carefully examiued for P. ohirai worms and feces were also checked forthe eggs.

RESULTS AND DISCUssION
First intermediate host: Two species of snails, A. parasitologica and A. japonica werecollected at 5 study sites shown in Figure I . Assiminea parasitologica inhabits damp areas onriver‑banks covered with eularia grass and other weeds, and was collected from the surface ofsoil, deadwood and fallen leaves. Assiminea japonica, on the other hand, was mainly seen on
the surface of wet soil and on stones on the bottom of the river and the lower part of reed stems,

which were submerged at high tide.
The detection of P. ohirai cercaria was carried out for A. parasitologica which is known as
the principal Ist intermediate host in other endemic areas of P. ohirai in Japan (Yokogawa et al.

1957; Yoshida and Miyamoto 1959; Miyamoto 1961). The results are summarized in Table 1
and Figure I . Parnglonimus ohirai cercariae, as shown in Figure 2, were detected in snails
collected at B and E out of 5 study sites. Site B is located about 8 krn up from the estuary of
the lbi River, where Route I crosses the river. The cercariae were found in 8 of 1,782 snails(infection rate = O. 45%), where the infection rate of the 2nd intermediate host crab with P. ohiraimetacercariae was 90% for S. dehaani and 88. 9% for S. intermedia as reported in the frst part ofthis study. Site E is situated about 6 km up from the estuary of the Kiso River, where 10 out of

2,075 snails were positive for P. ohirai (infection rate =0.48%). The infection rate in crabs
here was 69.3‑85. 7% in the previous report. At the other sites, all the snails examined were
negative for P. ohirai cercariae. In conclusion, a total of 18 out of 5,305 snails examined werepositive for P. ohirai (infection rate = 0.34%).

In addition to the dissection of snails, an experirnent on the cercarial emergence from snails

was carried out with those collected irom Site E. The snails were individually submerged inwater in small petri‑dish for I ‑2 hours and then P. ohirai cercariae emerging from the snails

were counted. The cercarial emergence was detected in 8 out of 2,380 snails (emergence
rate = O. 34%).

Yoshida and Miyamoto (1959) investigated the Ist intermediate host snails in the wide
endemic area of P. ohirai along the Maruyama River, Hyogo Prefecture. The natural infection
rate of P. ohirai cercaria was O. 048% for A. parasitologica, but negative for A. japonica and theexperimental infection rate was 60. 8% for the former and 2.0% for the latter species. The

3

Table 1

Infection rate of snail, Assiminea parasitologica, with Paragonimus ohirai cercariae
collected along the lbi, Nagara and Kiso rivers in Tokai district, central Japan

Collection site

No. of snails

Date of surveyAlbi RiverB

lbi RivercNagara River

Jan. , Jul. , 1984

D

Kiso River

Aug. , 1985

E

Kiso River

Jul. , Oct. , 1984

examined
190

Apr. , 1984
Jan. , Jul. , 1984

Total

(%)
o (o)

1 , 782

8 (0.45)l, 105

o (o)

1 53

O (o)

2,075

10 (O. 48)

5, 30518 (O. 34)

Oct., 1985

Oct. , 1983

No. of snails
infected

1 23
1 : Ibi River̲ 2: Nagara
River
3: Kiso River

Route No. 1D

B c Mehan Highway oeRoute No. 23

Ise Bay

Figure l

Sketch maps showing the lbi, Nagara and Kiso rivers where the survey on Paragonimus ohirai
was carried out.

D

Results of examination of the Ist intermediate host. B and E: Sites where snails positive for the

cercaria were collected. A, C and D: Sites where all snails examined were negative for the
cercaria

ID

Results of examination of the final host. Black dots: Sites where norway rats positive for the

adult worm were collected. White dots: Sites where norway rats negative for the adult worm

were collected. Minus marks: Sites where wood mice negative for the adult worm werecollected

IID

Results of examination of the 2nd intermediate host summarized in the previous report. Black
dots: Sites where crabs positive for the metacercaria were collected. White dots: Sites where
all crabs collected were negative for the metacercaria

4

Figure 2 A: Paragordmus ohirai cercariae recovered from the snail Assiminea para‑

sitologica collected from the lbi River. B: Adult worm of Paragonimus
ohirai recovered from the rat Rattus norvegicus collected from the Nagara
River .

present survey on snails reveaied for the frst time that A. parasitologica serves as the Istintermediate host of P. ohirai in the areas along the lbi and Kiso rivers and that the naturai
infection rate for this snail (O.45( /0 at site B and 0.48% at site E) was 8 to 9 times higher thanthat in the Maruyama River area.
Final host: A total of 190 live traps were set at area8 5‑12, 7‑13 and 4‑8 km up from theestuaries of the lbi, Nagara and Kiso rivers, respectively, during 6 months from December 1983
to May 1984. As showa in Table 2 and Figure 1, a total of 18 norway rats (R . norvegicus) and 6

wood mice (A. speciosus} were captured during this period. Of these, 9 norway rats werepositive for P. ohirai (infection rate = 50%), but all the wood mice were negative. Eight of theinfected norway rats were identified as adults, in the lungs of which adult worms of P, ohirai

were detected. Paragonimus ohirai eggs were also found from the feccs of all the adult
Table 2 Infection rate of rodents, Rattus norvegicus and Apodemus speciosus, with Paragord=

mus ohirai adult worms collected along the lbi, Nagara and Kiso rivers during six

month trom December 1983
R attus norvegicus

Ap(rdemus specios us

Collections ite

No. exarnined

No. positive

No. examined

No. positive

( / )

o
o

lbi River6

4 (66. 7)

4

Nagara River6

2 (33.3)

2

Kiso River6*

3 (50, o)

o

Total

18

9 (50, o)6* including one young rat

(%)

o

5

e

8
g3

D observed*o 6

e calculated

,L)

;

4
2
,

e ' ･ e I . . .

J Lo 2 4 6 8 Io 12 14 25 37
Number of adult worms

Figure 3 Frequency of Parnglonimus ohirai adult in Rattus norvegicus
observed frequehcy agreed well with the negative binomial (k =0.189, x2=1.214, p=0.271).

rodents. One rat was young and an irnmature worm of P. ohirai was obtained from the liver ofthis rat. The number of worms per positive rat was I ‑37, the average being 5. 06. The
frequency distribution of the adult worms among the final host was regarded as overdispersed,
and this agreed well with the negative binomial [(Bliss and Fisher, 1953; degree of overdisper‑sion k=0. 189 and fitness p=0. 271 (Figure 3)]. This meant that many rats harboured a hrnited
number of worms but that only a few individuals were infected with a large number of parasites.

This type of distribution is considered to be advantageous to the parasite, because most of theparasite‑carrying rats can survive longer and disserninate the eggs even if a few heavily‑infected

hosts may die earlier. Further, when live traps were set, crab fragments were found aroundthe lairs of the norway rats in the area. This suggests that the crabs are one of the major foodsources of norway rats in the study area.
In other endemic areas of P. ohirai in Japan, the natural infection rate of the final host wasreported as folbws: 7‑35% in weasels in the Maruyama River area, Hyogo Prefecture, 47% in
weasels, 25 : o in badgers, 14% in wild boars, 8% in Japanese meadow mice (Microtus montebelli)and O% in wood mice in the lzu area, Shizuoka Prefecture.
Comparing our own with these reports, the present infection rate of norway rats (50%) iseven higher than the highest rate for weasels in Shizuoka Prefecture (47%) and much higher thanthose of other final hosts including the Japanese meadow mouse and wood mouse. Norway rats
feed not only on cereals but on fish, birds and insects, while wood mice have a mainly vegetation
diet, such as the bark of young trees. It would therefore seem that the present difference in thenatural infection rate between norway rats and wood mice is due to their differing food habits.
The present survey for rats revealed for the hrst time that the norway rat is one of the final

hosts of P. ohirai in the areas along the 3 rivers, Ibi, Nagara and Kiso.

CONCLUSIONSA parasitological survey of P. ohirai was carried out for the Ist intermediate host in the6areas along3hvers，thecon㎞edtosurveyareas．

was

c㎞ed

lhe

lst

Ibi，Nagara

heav丑y㎞ect

and

Kiso

in

the

Tok由（五sthct，centralJapan．

sna丑ノ1．ρσ名硲訪oJo9∫oαand廿1e

Considered together with廿1e previous study

jor

the血』stinte㎜ediate

切nノεμo％s

t上e

as廿1e

t㎞e

that

host，thef血al

P．ohゼ名痂in

crabs

these

S．吻㎞伽ndareas

norway

rat

The

fluke

was

R．no甥磐∫6z硲血廿1e

on廿1e2nd k｝termediate host，it
inhabits

theS．∫n勧％8伽as

snaiL4．加昭s吻Joゆ08as

the2nd

and

the

ratR．

host．

REFERENCES
1）

Bhss，C．1．a皿d

note

on

the

Fisher，R A．（1953）： Fitti119廿1e

ef6ective2）Iwata，S．and

nttingofthenegative

Nagayoshi，K．（1985）：The

negativeb血omialdistribution

tobiological

data

Imd

binomial，Biometrics，9，176−200

species

name

of

P酩㎎o

伽％s

in

Japan，J．Kumme

Med．

on

the

lung

nuk，Pα猶㎎oπ吻螂oみ加∫Miyazaki，brackish

water

crabs

Assoc．，48，383−396（㎞Japanese）
3）Matsuo，K
1939．

in

the

and

Makiya，K（1985）：Ecological

1．1価ection

Tokai

rate

Prefecture．

hl

co皿ectedParton

Pα名㎎i㎝伽πsstudiesandon

Paragonjmiasis

Po猶㎎o

」

in

￠z偲oゐ∫瓶JthenorthemILAn

ecologicalbanks ofthe

Maruyama River，J．Kyoto Pre￡Univ．，69，1669−1683（indisthct，Tokyo

Iji

six

riversDisthct

of

Hyogo

伽％s

its

ende血carea

Japanese）

oh加5inthesouthem

Shinshi，74，403−406（㎞Japanese）

6） Yoshida，Y．and蜥yamoto，M（1959）：
Kuroda，1958（

the

Myazaki，1939hl

5）Yokogawa，M．，Yosh㎞ura，H．，Sano，M and Suzuki，S．（1957）：P幽㎎o
Izu

ffom

district，centraUapan，Jap．J．Trop、Med．Hyg．，13，307−313

4）Miyamoto，M（1961）：Studies

along thestudiesofP．ohf耀∫metacercariae

毛Pロ」％4初εJJα吻σf傭Studies
of

on

a

fkst

hltemediate

host，ノ13s伽z初昭ρα名㏄訪oo蜘8

Yokogawa and Koyamaθ α乙non Gould），of Pα猶㎎o 伽％s o雇π房

Myaz面，1939，JPn．J．ParasitoL，8，122−129（血Japanese）

大平肺吸虫の生態学的研究
II

東海地方の揖斐川，長良川，木曽川流域産カイ

ならびにネズミにおける本種の検出成績
松尾喜久男1・真喜屋

清2

1983年10月から1985年10月の間に，伊勢湾に注ぐ三重，愛知県下の揖斐川，長良川，木曽川の河口周辺に生息するカイ，ネズミについて大平肺吸虫の検出を行った。計5地点で採集したムシヤドリカ
ワザンショウ計5，305個体を剖検し，そのうち，揖斐川，木曽川の各1地点から本種セルカリアを検出した。この2地点における検出率はそれぞれ，0．45％，0．48％であった。ネズミについては，3河川流域からドブネズミ計18頭，アカネズミ計6頭を捕獲して剖検した。ドブネズミでは8頭の成熟ネズ
ミの肺から本種成虫計90個体，1頭の未成熟ネズミの肝から未熟虫体1個体を検出したが，アカネズ
ミはすべて陰性であった。既報のカニの成績ならびに今回のカイ，ネズミの調査結果から，3河川河口流域の広大な大平肺吸虫分布地において，本種の生活史に第1中間宿主としてムシヤドリカワザン
ショウ，第2中問宿主としてクロベンケイ，ベンケイガニ，終宿主としてドブネズミが関与している
ことが初めて明らかになった。

1藤田学園保健衛生大学衛生学部医動物学教室

2産業医科大学医動物学教室

Japan. J. Trop. Med. Hyg. , Vol 15 No 1 1987 pp 7 15

7LEISHMANIASIS IN DIFFERENT ALTITUDES ON

ANDEAN SLOPE OF ECUADORYOSHIHISA HASHIGUCH11, EDUARDO A. GOMEZ LANDIRES2

VICENTA VERA DE CoRONEL2, TATSIJYUKI MIMOR13 AND MASATO KAWABATA4
Received November 1 1 1986 / Accepted February 15 1987

Abstract : An epidemiological survey was performed in a leishrnaniasis‑endemic area aiong

highway which was established about 15 years ago on the Andean slope of Ecuador; the area

ranged from 300 m to 1,500 m above sea level. In general survey, 64 (14.3%) of the 446
subjects examined were positive for leishmanial signs. In order to know leishmanial infections inrelation to the altitudes of dwemng sites of subjects, analysis was made on 224 children with 5 to

15 years of age. At 4 different sites with 500 m, 1,000 m, 1,300 m and 1,500 m above sea
level, the infection rates of the subjects from the individual sites were 17.4, 18. 8, 5. 6 and 8.8%,

respectively. A statistically significant difference was recognized between the altitudes, 00‑

1,000 m and 1,300‑1,500 m (0.01<p<0.05, x2=5.314), but not between 500 m and 1.000 m
and between 1,300 m and 1,500 m. Leishmanial infections of the children who came irom forestand highway areas were compared in each altitude. But no significant difference was foundbetween forest and highway dewellers at any study sites.

INTRODUCTION
In Ecuador, transmission of American cutaneous and mucocutaneous leishmaniasis occurs inrural populations living in bilateral regions of the Andes mountains from the lowlands to highlands

up to the elevation of 2,000 m. The disease is widely spread in most provinces and is aconsiderable public health problem in the country. In the endemic areas, however, little
epidemiological study has been done on the community base, and no control measure has been
applied to reduce or interrupt the transmission of the disease. For a future control, it would benecessary to clarify the epidemiological features in each endemic area of lowlands and highlands.

New World cutaneous or mucocutaneous leishmaniasis is more difficult to control than isthose of Old World, since it is principally a disease of wild mammals in the dense forest, andnumerous reservoir hosts are arboreal; thus, in most endemic areas, reservoir‑vector control isahnost impossible (Marinkelle, 1980). At present, the only alternative measure for the control

1 Department of Parasitology, Kochi Medical School, Nankoku City, Kochi 781‑51, Japan
2 Departamento de Parasitologia, Instituto Nacional de Higiene y Medicina Tropical, Apartado 3961, Gua‑yaquil, Ecuador. S. A.

3 Department of Parasitic Diseases, Kumamoto University School of Medicine, Honjo, Kumamoto 860,
Ja pan4 Department of Clinical Pathology, School of Medicine, Nihon University, Ohtaniguchi, Tokyo 173, Japan8in most parts of the neotropics seems to be evacuation of the entire human population frompotentially dangerous areas, but such measure is inconceivable because of political, socioecono‑

mic and logistic reasons (Marinkelle, 1980). Under such circumstance, it would be worthwhle
to evaluate the effect of environmental changes in relation to the transmission of leishmaniasis.

The present paper deals with the resuslt of an epidemiological survey performed in different

altitudes of leishmaniasis‑endemic areas with 300 m to 1,500 m above sea level. In the area,

migration of inhabitants occurred from forest area to the viciuity of highway which was con‑structed on the Andean slope about 15 years ago. The leishmanial infections, therefore, werealso compared between forest and highway dwellers, in order to know the effect of change in thelife mode of the inhabitants on the transmission.

MATERIALS AND METHODS
Study area
The study site is located in the Departrnent of Cafiar on the south east of Ecuador, 2'30'

West longitude, and located on the Pacific slope of the Andes, ranging irom 300 m to I , 500 m

above sea level. Two vinages, Ocaffa and Javin, in the above area are situated about 70 km
from Guayaquil City and established as agricultural cornmunities along highway to Cuenca City.
A simplified sketch of the area is shown in Figure 1.

The paved highway with 10 meters in width was constructed about 15 years ago in the area
and it changed a mode of viuager's life, including agricultural systems. Before construction of

the highway, bananas and yucas were the main agricultural products in densely forested areas,
but they were replaced by sugar canes cultivated in largely deforested areas after the highway

construction. The highway supported not only the movement of villagers but also transporta‑tion of their agricultural products to major cities, such as Cuenca and Guayaquil. Thus, thehighway construction made a great enviroumental change of the endemic area of leishmaniasis.

Still, however, there were some surviving patches of natural dense forest which would
provide the breeding sites for vector sandflies and reservoir hosts of leishmaniasis. Suchlintited patches are distributed in the one side of the highway, while on the other side there was acontinuous undisturbed dense forest through lowlands to highlands along highway (Photos. I , 2
and 3).

The majority of dweWngs in the study area were built along highway, but the remainings
were in forest areas. There was no livestock, but the people raised the dogs, cats, pigs, guinea

pigs and domestic fowls. Wild mammals (sloths, arrnadilbs, opossums, rats and mice) and 2
species of man‑biting sandflies, Lutzomyia trapidoi and Lu. hartmanni, were found in the area
(Hashiguchi et al., 1985a, b, c).

Epidemiological exam inations

In the inhabitants, the survey was performed by house visit, while it was done in children

with 5 to 15 years of age by visiting 4 study sites (schools), La Delicia (A, 500 m above sea

level), Ocafia (B, 1,000 m), Las Copas (C, 1,300 m) and Javin (D, 1,500 m). All the subjects
were interviewed about their life history, such as occupation, cultivation, migration and contact

history with sandfiies, then examined clirLically by well‑experienced physician (E . A.G.L. and
V. V. C.) to find ulcers (active lesions) and scars (cured lesions) of leishmaniasis. When they had

active lesions, tissue samples were taken from the margin of ulcers for microscopic examina‑

i li

9

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sea level

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'
s "

",,ld::

1

/
,
,

t

////////. ' .

C
,

/

,

/

l
l

/
'

/

"dP

..p ,,

300m above

sea .ievel

,

, ,

1 km
Figure 1

A simplified sketch of the study sites in the Department of Cafiar, Ecuador. A, La Delicia (500 m

above sea level); B, Ocaiia (1,000 m); C, Las Copas (1,300 m); D, Javin (1,500 m).

10

Pho,to . lShowing 4 houses (arrows) along highway in the study site (highway dwellers).

Photo. 2

Showing feldof sugar ca e cultivation and remainiug patchee of natural (primary)Arrows on the above side show 3 hou8es in forest area (farest dwellers).

f ore st .

11

tions. The location, size, onset and duration of lesions were recorded.
With regard to the subjects with scars, the diagnosis was determined only clinically, i.e.,
mainly based on the type and localization of the scars, duration of the lesions and contact history

with sandflies; the chronic leishmanial ulcers resulted in a thin, depigmented scar (Photo. 4).

The viuagers have been calling leishmaniasis as llaga de montaha and sandflies, manta blanca in
Spanish, respectively.

The causative agent of these cutaneous and mucocutaneous leishmaniasis in Ecuador has
been considered Leishmania braziliensis s.1. , based mainly on the clinical manifestations, be‑havior and localization of the organisms in sandfly vectors and growih in cultures.

RESULTSIn the inhabitants from whole area, the positive rates with leishJnanial signs were arrangedby age and sex in Table I . In a total of 446 subjects examined, 64 (14. 3%) were found to bepositive for leishmanial signs. No marked difference was recognized between both sexes in thepositive rates.

In Table 2, Ieishnianial infections of 224 children with age 5 to 15 years in the total ex‑
aminees were arranged by the altitudes of their dweurng sites. The data revealed a statisticallysignificant difference between altitudes, 500‑1,000 m and 1,300‑1,500 m above sea level(0.01<p<0.05, x2=5.314), but not between 500 m and 1,000 m and between 1,300 m and1,500 m. The result, therefore, suggested that the intensity of transmission was markedly
influenced by the altitudes of dweWng sites in the endemic area.

To evaluate the intensity of transmission, the above 224 children were reanalyzed between

forest and highway areas (Table 3). They were divided into the fonowing two groups; 1) forest
dwellers who have or had experience living in forest area, that is, those who settled down orTable I Leishrnanial infections among 446 inhabitants arranged by age and sex in the Depart‑
ment of Caitar, Ecuador

Male

Age

FemaleNo. examined + *Total

No exammed +No exanuned + %

10. 9

1 09

12(3)

ll.O

219

24(7)

11.0

‑10
11‑20
21‑30
31‑

110

12(4)

77

14(3)

18. 2

70

12(1)

17. l

147

26(4)

17.7

16

4(1)

25. O

19

3

15.8

35

7(1)

20. O

19

2

10. 5

26

5

19. 2

45

7

15. 6

Total

222

32(8) 14.4

224

32(4) 14.3

446

64(12) 14.3

* Positives with leishmanial scars or ulcers; the number in parentheses shows positives withactive lesions (ulcers).

Photo. 3

A house constucted in the vicinity of highway (1,000 m above sea leVel), but surrounded by dense

forest (arrow in Figure 1). A considerable number of sandfiy was couected around the house.
All the persons of this family, 8 in total, had already suffered from leishmaniasis, showing thetypical scars.

Photo. 4

A typical leishnranial scar (arrow) found on the foreann of a 34‑year‑old female who lives in thehouse shown in Photo. 3.

12Table 2

Leishrnanial infections of 224 chldren, 5‑15 years old, arranged by altitude of dwell‑ing sites in the Department of Cafiar, Ecuador

Altitude*(in meters)

Schools**

No. examined

Positives with leish‑***manial signs (%)

500

A

46

8 (17.4)

85

16 (18.8)

1, 300C

36

2 (5.6)

1 , 500

D

57

5 (8.8)

1, OOO* Altitude above sea level.

** A, La Delicia; B, Ocafia; C, Las Copas; D, Javin.

*** Statistically significant difference between the altitudes, 500‑ 1,000 m and 1,300‑ 1,500 m(0.01 < p < 0.05, x2= 5.314), but not between 500 m and 1,000 m and between 1,300 m
and 1,500 m.

Table 3

Leishmanial ini;ections of 224 children, 5‑ 15 years old, arranged by their dwellingsites in forest and highway areas in the Departrnent of Cafiar, Ecuador

Childrenfrom

ForestSchools*

No. examined

Positives 'with leish‑
manial signs (%)

A27

6 (22.2)BC

34

5 (14.7)36

2 (5.6)Dll

1 (9.1)

Total

1 08

14 (13.0)

A
Highway19

6 (31.6)51

11 (21.6)

C

O

D

46

4 (8.7)

Total

ll6

21 (18.1)

224

31 (13.8)

Total* A, La Delicia; B, Ocaiia; C, Las Copas;

D, Javin.

lived in the past in forest area, 2) highway dwellers who were born at or imrnigrated fromnon‑endemic area to highway area. No statisticauy significant difference was recognized be‑
tween forest and highway dwellers in each study site of A, B, C and D.
The localization and number of lesions were examined in 52 subjects with leishmanial scars.
Of 1 10 scars found, 40. 9% were in the face, 30.0% in the upper extremities, 26. 4% in the lower

extremities and 2.7% in the trunk. The majority of scars measured less than 15mm in dia‑meter. Only 12 persons (2. 7% of the total examinees), 8 males and 4 females, had I to 8 ulcers

in the cheek, ear and upper or lower extremities. In interviews, the duration of ulcer ranged

from I month to 2 years; the lesions measured between 4 mrn and 32 mm in diameter.

13

DlscussloN
In our previous study in the Pacific slope of the Andes in Ecuador, 15. 8% of the examinees
were positive for active leishmanial lesions and 60%, for leishmanial scars (Hashiguchi et al. ,1984). The present study showed rather low intensity of the transmission, in the Departmentof Cafiar, Ecuador.
In the examination at 4 sites with different altitudes, the prevalences were higher at 500 m(17.4%) and 1,000m (18.8%) above sea level than 1,300 m (5.6%) and 1,500 m (8.8%). The
fact is quite noticeable in connection with the infection of sandfiy vectors with leishmanial pro‑mastigotes. In this area, Hashiguchi et al. (1985c) examined natural irLfections with the parasites

in man‑biting species of sandflies, and reported that the infection rate of Lu. hartmanni was

5. 9% at 350‑600 m, 3. 8% at 950 m, 2.3% at 1,200‑1,500 m and O% at 2,000 m, while the othersandfiy, Lu. trapidoi, was positive for the parasites at only one site of 350‑600 m (8. 1%),
showing a markedly reduced number of fly catches at higher sites. These results indicated thatleishmanial transmission in the Andean slope was greatly infiuenced by the altitudes of dwemng
sites, and also that the intensity of transmission would be very low at higher lands of 1,300 m orover.
By the data analysis of 224 chldren, the prevalence of leishmaniasis was compared between

forest and highway dwellers revealed no marked difference between two groups. The result,
therefore, suggested that there might be no remarkable difference between forest and highway
areas, in terms of the intensity of leishmaniasis transmission in the present endemic area. Thismight be due to the existence of prirnary forest along highway or the remaining patches of naturalforest around dweuings, which would play a role as the breeding sites for the vector andreservoir of the disease. In Panama, an insular effect resulting from clearance of prirnary forest

which surrounded a settlement, guarded properly the community against leishmanial infection
prevalent in the nearby forest (Herrer et al. , 1976). To reduce the transmission in the present

area, ,a iutther clearance of the forested areas would be necessary.

The localization of lesions in the subjects examined mostly agreed with that reported byRodriguez and Aviles (1953) and Hashiguchi et al. (1984) from Pacific coastal regions of Ecuador,but greatiy difiered from that in Amazon regions of the country, where 60% of the lesions
observed were in lower extremities (Amunarriz, 1982). This discrepancy might be caused by
the difference of biting behavior of sandflies or clothing habits of inhabitants between the tworegions.

ACKNOWLEDGEMENTS
We are most grateful to Drs. Hugo H. de Nully. M. Arzube R. , R. Lazo S., E. Gutierrez V.
and F. Parra Gil of the Direction of Instituto Nacional de Higiene y Medicina Tropical (INHMT),

Ecuador for their support throughout this study. We owe a debt of gratitude to our colleagues,
Dr. N. Urdiuares and Messrs. R. Sud and M. Leyion for their assistance in the field phase of thestudy. Thanks are also due to all the members of the Departamento de Parasitologia, INHMT
for their support in laboratory works. We thank Dr. I. Tada of Kumamoto University and Dr.
T. Yoshimura of University of Occupational and Environmental Health for critical reading of themanuscript. Finally, we are much indebted to Drs. N. Ishida (Tohoku University), S. Hayashi(National Institute of Health, Japan), N. Suzuki and H. Osaki (Kochi Medical School) for their

14

encouragement throughout the study. This study was supported by the Ministry of Public
Health, Republic of Ecuador and the Japan International Cooperation Agency (JICA) and Over‑seas Scientific Research Program of the Ministry of Education, Science and Culture, Japan (No.
6104 1059).

REFERENCES
1) Amunarriz, M. U. (1982) : Leishmaniasis, Salud y enfermedad patologia tropical en la region amazonica,

71‑88, Edicion CICAME, Ecuador
2) Hashiguchi, Y. , Gomez, E. A . L. and Coronel, V. V. (1984) : An epidemiological study of leishrnaniasis
in a plantation "Cooperativa 23 de Febrero" newly established in Ecuador, Jpn. J. Parasit., 33, 393‑401

3) Hashiguchi, Y. , Gomez, E. A. L., Coronel, V. V. , Mimori, T. and Kawabata, M. (1985a) : Leishmania
isolated from wild marnmals caught in endemic areas of leishmaniasis in Ecuador, Trans. Roy. Soc. Trop.

Med. Hyg., 79, 120‑121
4) Hashiguchi, Y., Gomez, E. A. L. , Coronel, V. V. , Mimori, T. and Kawabata, M. (1985b) : Biting activ‑
ity of two anthropophilic species of sandfiies, Lutzomyia, in an endemic area of leishmaniasis in Ecuador,

Ann. Trop. Med. Parasit., 79, 533‑538
5) Hashiguchi, Y., Gomez, E. A. L., Coronel, V. V. , Mimori, T. and Kawabata, M. (1985c) : Natural inL
fection with promastigotes in man‑biting species of sandflies in endemic areas of leishrnaniasis in

Ecuador, Arn. J. Trop. Med. Hyg. , 34, 440‑446
6) Herrer, A. , Christensen, H. A. and Beumer, R. J. (1976) : Epidemiological pattems of cutaneous leish‑

maniasis in Panama, II. Incidental occurrence of case in non‑endemic settlements, Ann. Trop. Med.
Parasit., 70, 67‑71

7) Marinkelle, C. J. (1980) : The control of leishmaniasis, Bull. Wld. Hlt. Org. , 58, 807‑818

8) Rodnguez, J. D. M. and Aviles, F. N. (1953) : Algunas observaciones sobre leishmaniasis cutaneo‑
mucosa en el Ecuador, Rev. Ecuat. Hig. Med. Trop., 10, 35‑58

15

エクアドル国アンデス斜面の高度差によるリーシュマニア症浸淫橋口義久1・EDuARDoA．GoMEzLANDIREs2・VIcENTAVERADECoRoNEL2
三森

龍之3・川端 真人4

エクアドル国のアンデス斜面低地から高地（海抜300m−1，500m）にかけて居住する住民について，リーシュマニア症の罹患状況を調べた。この流行地には約15年前にハイウェイ（道幅約10m）が建設され，住民の居住環境ならびに生活様式に大きな変化が認められた。

一般住民446名について検査したところ，64名（14，3％）は本症によると考えられる治癒病変，または皮膚潰瘍を保有していた。

居住地の高度差による住民のリーシュマニア症罹患状況を知るため，被検者のうち5−15歳の学童224名を対象に，4地点（A，海抜500m；B，1，000m；C，1，300m；D，1，500m）において，居住地区

別の罹患を比較した。その結果，学童のリーシュマニア症罹患率は，海抜500m地点で17．4％，
1，000mで18、8％，1，300mで5．6％，1，500mで8，8％となり，500m−1，000mの地域と1，300m−
1，500mの地域との間には，統計学的に有意の差を認めた（0．01＜p＜0．05，Z2＝5．314）。このこと

は，アンデス斜面のリーシュマニア症流行地の比較的低い地域（1，000m以下）では，本症の罹患率が高くなるが，より高い地域では罹患率は低くなることを示唆している。
一方，ハイウェイ沿いと山間部との間で，本症罹患率の差異を検討するため，上記224名の学童を，その居住地の状況によって次の2群に分類した。1）山間部に定住または過去に一時期居住した者，2）ハイウェイ沿いで出生または非流行地から移住した者。上記2群問での学童の罹患率を見る上で，高度差による影響を除去するため，各地区ごとの山間部住民とハイウェイ住民との比較を行った。その結果，4地点のいずれにおいても両群間に有意な差を認めなかった。したがって，本調査地においては，ハイウェイが建設され，環境変化や住民の移動がみられたものの，道路沿いの原生林や人家，および農耕地周辺に原生林の一部が残存し，これがサシチョウバエや保虫宿主の供給源の役割を果たしているものと判断された。

1高知医科大学寄生虫学教室
2エクアドル国熱帯医学研究所寄生虫学部
3熊本大学医学部寄生虫病学教室
4日本大学医学部臨床病理学教室

Japan. J. Trop. Med. Hyg. , Vol. 15, No. 1, 1987, pp. 17‑23

17CHANGES OF BLOOD OXYGEN AFFlNITY IN
FANSIDAR‑TREATED MICE INFECTED

WITH PLASMODIUM BERGHEI
ATSUSHI HIOKI AND HIROSHI OHTOMO
Received November 10 1986/Accepted February 15 1987

Abstract : Changes of blood oxygen affinity in mice infected with Plasmodium berghei andtreated with Fansidar (20 mg/kg body weight sulfadoxine and I mg/kg body weight pyrimetha‑

mine orany) were observed. When 5‑weeks‑old male mice were inoculated intraperitoneallywith l07 of P. berghei‑infected red cells and treated with Fansidar on day 5 after inoculation, 67%of the anirnals sulvived. Parasitemia decreased and abnormal values of glycolytic intermediates

began to recover following Fansidar adrninistration. However, hemoglobin content still de‑creased on day 2 of the treatnent, and methemoglobin iraction increased on days 1, 2, 3 and 4.
Blood oxygen affinity increased markedly on days 2 and 3. These findings suggested aggrava‑
tion of the host's hypoxic state on day 2 of the treatment.

INTRODUCTION
Malaria is relatively easy to cure if treated properly on the basis of early diagnosis.
However, delay in the onset of treaiment, especially for falciparum malaria, causes a higher levelof parasitemia, severe complications and sometirnes death of the host. Furthermore, death ofpatients with markedly decreased parasitemia following antimalarial treatment have been re‑ported (Devakul et al. , 1966; Stone et al. , 1972), but its mechanism is not yet understood.

The pathophysiology of falciparum malaria is considerably complex. Most organs are re‑ported to be involved in this infection, and many factors contributing to dysfunction of theseorgans have been enumerated (WHO, 1980; Bruce‑Chwatt, 1980). Tissue hypoxia is thought
to be one of the main causes of severe complications (Hall, 1977). We have already reported
that the decrease in blood oxygen affinity in the course of Plasmodium berghei infection in mice

may be a compensatory mechanism for tissue hypoxia resulting from malaria (Ohtomo et al. ,l 982).

In this study, we intended to clarify the effect of treatment on blood oxygen affinity in P.

berghei infected mice. We used Fansidar̲ as an antimalarial drug because it is effective in asingle dose and has clear action of only blocking the 2 sequential stages of the pathway leading totetrahydrofolate (Donno, 1974; Weidekamm et al. , 1982).

MATERIALS AND METHODS
Male mice of ddY strain, 5 weeks old, were used in this study.

Plasmodium berghei, strain

NK65, has been maintained in our laboratory by injecting a dilute suspension of infected blood
Department of Parasitology, Gifti University School of Medicine, 40 Tsnkasa‑machi, Gifti 500, Japan18

into mice from the corresponding strain every week. Blood was conected from an infectedmouse by cardiac puncture using heparin as an anticoagulant, diluted with saline to yield I x l08

infected red cells /ml, and O. I ml of this suspension was injected intraperitoneally. The timeinterval between exsanguination and inoculation was less than I hour. Mice were kept at 24‑

26'C and 40‑60% humidity and exposed to a photoperiod of 12 h light and 12 h darkness.
Under these conditions, mice died 6‑7 days after inoculation.
In the initial trials, desigued to evaluate the time of treaiment, 3 groups of 6 mice eachwere infected. Fansidar (F. Hoffrnann‑La Roche, Switzerland) was dissolved in distilled waterand administered orally (1 mg pydmethamine and 20 mg sulfadoxine / kg body weight) on day 4
aiter inoculation to group 1, day 5 to group 2, and day 6 to group 3, respectively (Ferraroni andSpeer, 1982). The course of the infection was followed by determining parasitemia and reticulo‑

cyie count daily frst by new methylene blue‑ followed by Giemsa‑stained blood smears from thetail vein.

In the present study, the mice were inoculated with P. berghei and treated with Fansidar on

day 5 of infection. Eleven mice each were anesthetized with pentobarbital sodium (50 mg/kg
body weight intraperitoneally) on days O, 1, 2, 3, 4, 5 or 6 after Fansidar treatment, and whole

blood was collected into heparinized syringes from the carotid artery withirL 5 minutes. Para‑sitemia was determined on Giemsa‑stained thin blood filrn. Total hemoglobin concentration wasmeasured by the cyanmethemoglobin method and methemoglobin fraction according to the KCN
addition method (International Committee for Standardization in Haematology, 1978; Zwart et al. ,1981). Hematocrit was defermined by means of a microhematocrit centrifuge. Oxygen
equilibrium curve (OEO of whole blood was measured at 37'C on a Hem‑O‑Scan analyzer(Aminco Co. , USA), and blood oxygen affinity was expressed as the half‑saturation pressure
values at actual pH in vivo (P50 act pH). The Hill‑coefficient n was obtained by linear regression
analysis of log [y/(1 ‑ y)] vs. Iog P02, where y is 02 saturation for the data points of the OEC (02saturation=30, 35, 40, 45, 50, 55, 60, 65 and 70 f ). Whole blood pH was measured im‑
mediately at 37'C with a glass electrode. 2, 3‑Diphosphoglycerate concentration was deter‑

mined according to a modified kit (No. 148334; Boehringer Mamheim GmbH, F. R. Germany) of
the enzymatic end‑point method. Blood adenosine triphosphate (ATP) concentration was deter‑
mined by means of the luciferase reaction with a CHEM‑GLOW ( ninco Co.) (Chapman et al. ,1971). Concentrations of blood glucose, pyruvate and lactate were determined according to amodified kit (Nos. 124028, 124982 and 124842; Boehringer Marmheim GmbH).

RESULTS
Fansidar treatment on day 6 of Plasmodium berghei inoculation resulted in 100% mortality inmice 1‑2 days after treatment. Sixty‑seven percent of mice treated on day 5 and 100% of
those on day 4 survived more than 10 days after treaiment (Table 1). Parasitemia decreased
fouowing treatment compared with increasing in untreated subjects. Simultaneously, polychro‑matophilic erythrocyies in the peripheral blood began to increase and significant reticulocylosis

was observed from day 2 after treaiment (p < O. 05). When Fansidar was given on day 5, mostof the parasites appeared to have degenerated on day 2 of treatment, and parasitemia became
undetectable on day 4. Reticulocyie count reached its peak on day 5 after treatment, although
uninfected controls remained low (Table 1).

Parasitemia of mice sacrificed for measurement (Table 2) were much the same as above.

19
Table 1Time course of changes in parasitemia and reticulocylosis of mice treated with Fansidar 4
and 5 days after Plasmodium berghei inoculation

Days after Fansidar treatment

Day of Fansidar
treatment

O 1
6 6 61.7 6O
26. 4 7. 5
3

No. of sutviving mice
4 days after
inoculation

Parasitemia (%)
Reticulocyie (%)

5 days after
inoculation

Unilufected andFansidar treatedcontrols

Values are means

Table 2

6

o

O

O

6
O

+0

O. 5 3. 4:!:0.2 +0.6

6. O lO. O

23. 7

28. 5

19. 6

1.3 +1.3

11.7

d: 6. o:b 4. 2d: 2. 9

15.9
d:1.3

4

4

4

4

O

O

O

d:0.1 0.2

62.9 63.2

Reticulocyle (%)

6
O

+0

6 6

No. of mice

6+0

2.8 2.9

Reticulocyie (%)

60

O. 4 4. 1:1:0.1 0.5

No. of mice

Uninf ected
controls

8+0:1:2.2 +5.8

Reticulocyie (%)

6

0.5 +062. 7 41. 7

Parasitemia (%)

5

5.0 +1.66 4

No. of surviving mice4

0.4 0.3

4 4 4
10. 7 2. 4 O
2.9 +1.0 0

+0

+0

+0

15. 6 17. 9 29. l

35. 2

31.0

1.6

6･ 4

3. 5

20. 9
4. 2

3･3

4.1

2. O

O
d:O
8. 9

O. 2

6 6 6 6 6 6

3. 1

0.2

O. 4

2. 6 2. 7 2. 9 2. 7 2. 6 2. 7

0.3

0.3

0.3

0.2

0.3

6 6 6 6 6 6

2. 7 3. 2 2. 6 2. 9 2. 7 2. 9
0.3 0.3 0.3 0.3 0.2 0.4

6

6
2. 9

O. 4

SE of sulviving mice.

Hematological data of mice infected with P. berghei and treated with Fansidar on day 5 ofinfection

Uninfected controls

Days after Fansidar treatnent

No
treatment
treatm nt O
FansidarParasitemia (%)

1

2

3

4

65. 5

43. 7

12. 6

1.5

O

2.6

+̲5.0

1.4

O. 7

+̲O

5

O

+0

6
O

0

Hb (g/dl)

12. 8
O. 2

12. 7
O. 2

7. 2

7.4

4. 6

5. 7

6･ 5

7. 6

9･ 9

O. 3

O. 4

+ O. 2

O. 7

O･ 3

O. 6

O. 3

Ht (%)

40. 5
O. 7

40. 8
O. 6

25. 7
1. l

25. 4
1. 1

16. 9
O. 8

20. 6
:!:1.8

25. 6

+ 1.4

28.4
1.8

36. 6
+ O. 7

31.7

31.2
O. 3

27. 9
O. 6

29. 2
O. 5

27. 4
+ O. 7

26･ 9

O. 2

+ 1.0

27. l
O. 5

MCHC (%)
Values are means

SE for 1 1 sacrificed mice each.

27. 2

25. 7
O. 5

O. 7

Hb, hemoglobin; Ht, hematocrit;

MCHC, mean corpuscul r hemoglobin concentration.

Hemoglobin content and hematocrit decreased on day 2 of treatrnent and then increased fromday 3 as summarized in Table 2. No significant changes were seen in the parameters of theuninfected controls after the administration of Fansidar. Methemoglobin fraction elevated ondays I (9.3+̲ 1.0%, mean SE, n=6), 2 (13.8 3.2%), 3 (9.6 2.9%) and 4 (13.6 2.3%) after
treatment compared to days O (5.4 0.9%), 5 (5.9+̲ 1.8%) and 6 (4.3 1.5( o), and uninfected
controls and uninfected‑Fansidar‑treated ones showed 0.8 0. 1% and 0.9 0. l% respectively.

20

Mean corpuscular hemoglobin concentration (MCHO remained lower from day O to day 6
compared to uninfected controls (p < 0.01) and reached its lowest level on day 4. P50 act pH,

reflecting blood oxygen aifinity, increased dramatically on day I of treatment compared tounirifected controls and day O (p<0. O1). Then, it diminished on days 2 and 3 (p<0.001) (Table3). Table 3 also presents the Hill's n value reflecting the slope of the OEC, showing an increase

after treaiment (p < O. OO1). Changes in red cell 2, 3‑DPG and ATP concentrations and in blood

pH are also shown in Table 3. Red cell ATP concentration began to increase from day I oftreatment and 2, 3‑DPG from day 2. Although blood pH continued to be decreased on day 1, it
returned abruptly to within normal lirnits on day 2. Concentrations of glycolytic intermediates

are surnmarized in Table 4. Blood glucose concentration, which had decreased on day 5 ofinfection to about one third of uninfected controls, began to increase after Fansidar treatment.

In contrast, blood pymvate and lactate concentrations increased after the infection and remained

high up to day I of treatment but began to decrease from day 2.

Table 3 Mean values and SE (n = 1 1) of parameters measured in mice infected with P. berghei andtreated with Fansidar on day 5 of infection

Days after Fansidar treatment

Uninfected controls

Fansidartreatment treatment

l

2

3

4

5

6

43. 8
O. 6

42. 4

43･ 3

1.0

+ O. 944. 4
d: 1.2

P50 act pH (Torr)

40. O
O. 2

52. 7
1. 1

61.5

47. 8

+̲ O. 5

:1: 2. 6

1.6

Hill's n

3. 02
O. O1

2. 99
O. O1

2. 87
O. 03

3. 03
O. 02

3. 25
O. 03

3. 10
O. 05

3. 13

3. 22

d: O. 04

O. 03

O. 08
23. 1

41.0

3.21

28. 9

30. 2

15.8

22. 2

+ 2. 5

21.8
1.5

23. 9

+ 1.2

13. 8
2. O

26. l

1.0

d: O. 8

1.2

1.0

1.0

ATP (pM IgHb)

5. 8

5. 7

4. 9

6. 4

8. 2

9. 3

9.4

9. O

+ O. 1

O. 1

O. 1

O. 3

O. 4

O. 7

10. 6
O. 4

O. 5

O. 3

Blood pH

7. 43
O. O1

7. 42
O. O1

7.06

7.08

7.39

7.41

7.41

7.41

O. 04

O. 06

O. O1

O. O1

O. O1

O. O1

7. 43
O. O1

2, 3‑DPG (pMlgHb)

P50 act pH, partial pressure of oxygen at 50% oxyhemoglobin saturation (actual pH, temp = 37'C); 2,

3‑DPG, 2, 3‑diphosphoglycerate. Hm's n values are calculated by linear regression analysis of log[y/(1‑y)] vs. Iog P02, where y is 02 Saturation.

Table 4 Concentrations of glycolytic intermediates before and after treatment of P. berghei‑infected
mice with Fansidar on day 5 of infection

Days after Fansidar treatnent

Uninfected controls

No Fansidar O

treatrnent treatnent
Glucose (mM /1)
Pymvate (mM Il )Lactate (mM /1 )Values are means

12. 1

12.9

O. 5

O･ 4

O. 1 1

O. 09

O. O1

+ 0.01

3. 4

4. O+ O. 3

O. 2

2

l

3

4

3. 6 5. O 8. 1 9. O 8. 9
O. 4

1. 3

O. 6

O. 7

O. 5

O. 34 O. 46 O. 24 O. 14 O. 13
0.02 0.04 0.04 0.03 0.01

10. O 12. 1 8. 3 6. 3 7. 2

̲+ O. 4

SE for 6 sacrfficed mice each.

O. 8 + o. 4

O. 9 + 1. 3

56

8. 1 8. 7

0.5

0.4

O. 12 O. 13

0.02 +0.02
5. 9 4. 8

+ 1.8

0.4

21

DrscussloN
Plasmodium falclparum causes severe infection in humans, because it invades red cells of allages and multiplies rapidly. If patients are not provided with rapid diagnosis and treatment,

they may expire due to severe complications.
We have studied the pathophysiological significance of hypoxia in malaria using experimental

animal models. The present study was designed to clarify more accurately the mechanism
leading to death when treatment is delayed. We investigated blood oxygen transport in micetreated with Fansidar on day 5 after P. berghei inoculation, and 67% of the mice treated on day 5

survived compared with a 100% survival rate for those treated on day 4 in the initial study.

Hemoglobin concentration decreased on day 2 of Fansidar treatment although parasitemia
decreased following treatment. Such a reduction in hemoglobin concentration after treatment
against acute malaJial irLfection has been widely accepted (Devakul et al. , 1966; Abdalla et al. ,1980). To make matters worse, in this situation, methemoglobin fraction increased after Fansi‑

dar treatment. It is suggested that the oxygen supply to the tissues decreased due to thepresence of methemoglobin in the red cells which causes not only a loss of oxygen binding
capacity in the blood but also a decrease in oxygen unloading from the bood (Darling andRoughton, 1942). P50 act pH markedly increased on day I of treatment and began to decrease
from day 2, although it remained higher compared to uninfected controls. Such a decrease ofaffinity is an unfavorable change in the blood oxygen supply. In contrast, Hiu's n, reflecting theslope of the OEC, increased after treatment. This change appears to be somewhat advan‑
tageous to blood oxygen transport. Considerisng the available hemoglobin content, however,the blood oxygen‑supplying capacity seems to be lowest on day 2 of treatment. It is suggested
that this might be one explanation for sudden death of falciparum malaria patients after parasite‑

mia has decreased markedly following chemotherapy (Devakul et al. , 1966; Stone et al. , 1972).

Reticulocyie count began to increase following treatment. This may be a reaction of the
body after successful treatment of an anemic situation, and also be due to the decrease in P.
berghei which preferentiahy invades reticulocyles (Bungener, 1979). An increase in hemoglobin

content fouowed this compensatory reticulocylosis, and anemia gradually vanished. A rapid
increase in red cell 2, 3‑DPG concentration after treatment may be due to a higher concentrationof 2, 3‑DPG in the increased younger erythrocyles (Makbaurl et al. , 1983).

Blood glucose concentration increased from day I of treatment, possibly resulting irom
inactivated glycolysis caused by the parasites subsequent to the decrease in parasitemia (Home‑wood, 1977; Homewood and Neame, 1983). However, blood pymvate and lactate concentra‑
tions remained high on day I and began to decrease from day 2. This may have been due to theparasites stiu present in the blood, and also partly to the inabihty of the host to reduce thembecause malaria causes reduction in renal microcirculatory flow, hypoxemia, tissue hypoxia andhepatic dysfunction (WHO, 1980; Hau, 1977). Lactate accumulation in the tissue, especiauy inthe brain which is protected by a blood‑brain balrier (Pardridge and Oldendorf, 1977), may ,persist longer than in the blood following treatment. Although blood lactate concentration stiu

remained high on day 2, blood pH increased to 7.39. It is suggested that the carbonic acid‑bicarbonate buffer system may play an important role in normalizing blood pH. Such an increasein pH may help to account the decrease in P50 act pH (Beuingham et al. , 1971). It should bebome in mind that correction of blood pH of a malaria patient may cause tissue hypoxia byincreasing blood oxygen affinity.

22

REFERENCES
1) Abdalla, S., Weatherall, D. J., Wickramasinghe, ̲ S. N. and Hughes, M. (1980) : The anaemia of P.
falclparum malaria, Br. J. Haematol., 46, 171‑183

2) Bellingham, A. J., Detter, J. C. and Lenfant, C. (1971): Regulatory mechanisms of hemoglobin ox‑ygen affinity in acidosis and alkalosis, J. Clin. Invest. , 50, 700‑706

3) Bruce‑Chwatt, L. J. (1980) : Essential Malariology, Heinemaun, London
4) Btingener, W. (1979) ,: Weitere Beobachtungen tiber den Verlauf der Plasmodium berghei‑Infektion inder Maus, Tropenmed. Parasitol. , 30, 24‑34
5) Chapman, A. G., Fall, L. F. and Atkinson, D. E. (1971): Adenylate energy charge in Escherichia coli
during growih and starvation, J. Bacteriol. , 108, 1072‑1086

6) Darling, R. C. and Roughton, F. J. W. (1942) : The effect of methemoglobin on the equilbrium be‑
tween oxygen and hemoglobin, Arn. J. Physiol. , 137, 56‑68
7) Devakul, K. , Harinasuta, T. and Reid, H. A. (1966) : 1251‑Labelled fibrinogen in cerebral malaria, Lan‑cet, il, 886‑888

8) Domo, L. (1974) : Antifolic combinations in the treatment of malaria, Bull. WHO, 50, 223‑230
9) Ferraroni, J. J. and Speer, C. A. (1982) : Fansidar prophylaxis, therapy, and itnmune response inrodent malaria (Plasmodium berghet), J. Parasitol. , 68, 609‑615lO) Hau, A. P. (1977) : The treatment of severe falciparum malaria. Trans. R. Soc. Trop. Med. Hyg. , 71,

367‑379l D Homewood, C. A. (1977) : Carbohydrate metabolism of malarial parasites, Buu. WHO, 55, 229‑235
12) Homewood, C. A. and Neame, K. D. (1983) : Conversion of glucose to lactate by intraerythrocytic
Plasmodium berghei, Ann. Trop. Med. Parasitol. , 77, 127‑129
13) International Cornmittee for Standardization in Haematology (1978) : Recommendations for reference
method for haemoglobinometry in human blood (ICSH Standard EP 6/2: 1977) and specffications forinternational haemoglobincyanide reference preparation (ICSH Standard EP 6/3: 1977), J. Clin. Pathol. ,31, 139‑143
14) Mairbaurl, H. , Humpeler, E. , Schwaberger, G. and Pessenhofer, H. (1983) : Training‑dependentchanges of red cell density and erythrocytic oxygen transport, J. Appl. Physiol. , 55, 1403‑1407

15) Ohtomo, H. , Hioki, A. and Yoshino, M. (1982) : Pathological influence of hypoxia in mouse malaria,
Jpn. J. Parasitol. , 31 (suppl), 42

16) Pardridge, W. M. and Oldendorf, W. H. (1977) : Transport of metabolic substrates through the blood‑
brain barrier, J. Neurochem. , 28, 5‑1217) Stone, W. J., Hanchett, J. E. and Knepshield, Maj J. H. (1972) : Acute renal insufiiciency due to

falciparum malaria. Review of 42 cases, Arch. Intern. Med., 129, 620‑62818) Weidekamm, E., Plozza‑Nottebrock, H. , Forgo, I. and Dubach, U. C. (1982) : Plasma concentrationsof pyrirnethamine and sulfadoxine and evaluation of pharmacokinetic data by computerized curve fitting,

Bull. WHO, 60, 115‑12219) WHO (1980) : The clinical management of acute malaria, WHO Regional Publications, South‑East Asia
Series, No. 920) Zwart, A.. Buursma, A. , van Kampen, E. J., Oeseburg, B., van der Ploeg, P. H. W. and Zijlstra, W.
G. (1981) : A multi‑wavelength spectrophotometric method for the simultaneous determination of fivehaemoglobin derivatives, J. Clin. Chem. Clin. Biochem. 19, 457‑463

23

ファンシダールによるマラリア感染治療後のマウス血液酸素親和性の変動
日置

敦巳・大友

弘士

抗マラリア薬ファンシダール投与後の，マラリア感染マウスにおける血液酸素親和性の変動について調べた。5週齢，雄のddYマウスにPJ侭郷04ゴ灘加㎎捌NK65感染赤血球107個を腹腔内接種し，その5日後にファンシダール（スルファドキシン20mg／kg体重，ピリメサミン1mg／kg体重）を経口投与したところ，33％のマウスは治療の1〜2日後に死亡したが67％のマウスはファンシダール投与

後7日以上生存した。これらのマウスでは治療によりparasite血aは低下し，低血糖，続いて高乳酸血症も徐々に回復した。しかし，血液中の総ヘモグロビン濃度は治療2日後に低下し，ヘモグロビン中に占めるメトヘモグロビンの割合は逆に増加した。血液の酸素親和性は治療翌日には低下を示した

が，2日後および3日後には著しく上昇した。この上昇は，主として血液pHの回復に起因するものと考えられた。酸素運搬に有効な血液中のヘモグロビン濃度と酸素親和性の変動から推察すると，治療後，特にファンシダール投与2日後の血液酸素供給能はかなり低下しているものと考えられた。

岐阜大学医学部寄生虫学教室

Japan. J. Trop. Med. Hyg. , Vol. 15, No. 1, 1987, pp. 25‑28

25

DECREASE TREND IN PROPORTIONS OF HEPATITISB SURFACE ANTIGEN CARRIER IN CHRONICHEPATITIS, CIRRHOSIS AND CIRRHOSISWITH HEPATOCELLULAR CARClNOMA
MASACHIKA SENBA1, TSUYOSHI NAKAMURA2 AND HIDEYO ITAKURAl
Received September 1 1 1986 /Accepted January 19 1987

Abstract : Histopathological analysis was carried out to determine whether hepatitis B
surface antigen with the autopsy diagnoses of chronic hepatitis, cirrhosis and/or hepatocellular

carcinoma declined between the 1964‑1973 and 1974‑ 1983 decades. In this study, the liver
specimens from 422 autopsy cases at Nagasaki University Hospital were used. All of the 17
cases of acute hepatitis were hepatitis B surface antigen negative. Hepatitis B surface antigen

positive rates in chronic hepatitis and hepatocellular carcinoma showed minor changes during
1964‑1983. On the other hand, proportions of hepatitis B surfce antigen carriers in cirrhosis

and cirrhosis with hepatocellular carcinoma in the recent decade 1974‑1983 were found to
decrease compared to the last decade 1964‑ 1973. However, the difference of the hepatitis B
surface antigen positive rates in cirrhosis with hepatocellular carcinoma between the 2 periods
was marginally significant, but not significant in cirrhosis. A possible explanation may be thatmany cases of posttransfusion hepatitis in the latter decade are not caused by hepatitis B virus.

INTRODUCTION
The most plausible explanation for the increased risk of hepatocellular carcinoma is that theacceleration of cenular replication that occurs in cirrhosis enhances the effects of many carci‑

nogens, including hepatitis B surface antigen. Moreover, the oncogenic potential of hepatitis B
virus in the development of hepatocellular carcinoma has been reported (Sherlock et al. , 1970;
Vogel et al. , 1970; Charinuvati et al. , 1975; Senba et al. , 1984, 1985).

After the discovery of the Australia antigen (hepatitis B surface antigen) by Blumberg et al.

(1967), the subsequent demonstration by Krugman et al. (1967), and Prince (1968) that theAustralia antigen was specifically associated with type B hepatitis, it was widely assumed thatdevelopment of eificient screening tests would detect type B hepatitis infection carriers andniake possible the eradication of the problem of posttransfusion type B hepatitis by preventing

transfusion of hepatitis B virus positive blood. In Nagasaki University Hospital, hepatitis B
surface antigen screening test has been performed since 1974. Therefore, the objective of thisstudy is to see whether hepatitis B surface antigen positive rates in chronic hepatitis, cirrhosis,

and cirrhosis with hepatocenular carcinoma have changed or not between the 1964‑1973 and1974‑1983 decades.

1

Department of Pathology, Institute of Tropical Medicine, Nagasaki University, Nagasaki 852, Japan

2 School of Medical Health Technology, Nagasaki Univesity, Nagasaki 852, Japan

26

MATERIALS AND METHODS
The liver specimens were collected at Nagasaki University Hospital from 422 autopsy casesof various liver diseases, including acute hepatitis (17 cases), chronic hepatitis (40 cases) ,cirrhosis (177 cases), cirrhosis with hepatocellular carcinoma (170 cases), and hepatocellular

carcinoma without cirrhosis (62 cases). The specimens were fixed in formalin, or sometimes in

Zenker's formol and embedded in paraffin for histopathologic study. Sections were cut at 5
micron and stained with hematoxylm‑eosin, resorcin fuchsin method for elastic fibers, Mallory's

method for collagen fibers, silver impregnation method for reticulum fibers, and orcein method

for hepatitis B surface antigen (Senba, 1982). The criteria of Leevy et al. (1979) and Gibson
and Sobin (1978) were applied in assigning the diagnosis of cirrhosis an hepatocellular carcinoma
to the tissue.

Statistical calculation was perforrned using the BMDP (Dixon et al. , 1981) on the IBM 4341

system in the Data Center of A‑bomb Disaster in Nagasaki University. The statistical method
for this study is the Pearson chi‑square test for association in confingency table.

RESULTS
Table I shows the hepatitis B surface antigen positive results obtained by orcein staining.
An of the 1 7 cases of acute hepatitis were not hepatitis B surface antigen positive. On the other

hand, of the 26 chronic hepatitis cases in 1964‑1973 and of the 14 in 1974‑1983, 4 (15%) and 2(14%) were hepatitis B surface antigen positive, respectively. As for the cirrhosis cases, 33(33%) cases were hepatitis B surface antigen positive out of 100 in 1964‑1973 and 8 (24%) out

of 33 in 1974‑1983. As for the cirrhosis with hepatocenular carcinoma cases, 77 (71%) cases
were hepatitis B surface antigen positive out of 108 in 1964‑1973 and 36 (58%) out of 62 in

1974‑1983. Of the 21 and 41 hepatocellular carcinoma cases in 1964‑1973 and 1974‑1983, 5(24%) and 1 1 (27%) were hepatitis B surface antigen positive, respectively.
Results of the statistical analysis on hepatitis B surface antigen, cirrhosis, and cirrhosis with

hepatocellular carcinoma are as follows: The Pearson chi‑square test for the frequencies ofhepatitis B surface antigen positive in cirrhosis between the 2 periods showed Pearson x2=
O. 345; and the Pearson chi‑square test for the frequencies of hepatitis B surface antigen positivein cirrhosis with hepatocellular carcinoma between the 2 periods showed Pearson x2=0.079,
respectively. Thus, the difference of the hepatitis B surface antigen positive rates in cirrhosis
Table I Proportions of hepatitis B surface antigen carriers in hepatitis cases, chrhosis cases,

and hepatocellular carcinoma cases

1964‑1973

1974‑1983

Total

Acute hepatitis

0/10 (O%)
4/26 (15%)

Cirrhosis

33/ 100 (33%)

Cirrhosis with HCC*

77/ 108 (71%)

HCC*

5/21 (24%)

O / 7 (O%)
2/14 (14%)
8/33 (24%)
36/62 (58%)
ll/41 (27%)

0/17 (O%)

Chronic hepatitis

119/265 (45%)

57/ 157 (36%)

Grand total* HCC: Hepatocellular carcinoma

6/40 (15%)
41 / 133 (31%)

113/ 170 (66%)

16/62 (26%)
176/422 (42%)

27with hepatoceuular carcinoma between the 2 periods was marginally significant, but not signi‑
ficant in cirrhosis.

DlscUssloN
The landmark discovery of the Australia antigen (hepatitis B urface antigen) by Blumberg etal. (1965) and the linking of this antigen to viral hepatitis (Blumberg et al. , 1967) provided thecornerstone for the rapid acceleration on our understanding of viral hepatitis in general, andposttransfusion hepatitis in particular. The hepatitis B surface antigen positive cases wereincreased by such antigen detection, but false‑positive reaction occurred frequently (Honinger et

al. , 1973). Further specific methods were developed for recognition of other hepatits B virus

antigens (hepatitis B core antigen and hepatitis B e antigen). The introduction of sensitive

assays for hepatitis B surface antigen has resulted in marked reduction in the incidence ofposttransfusion hepatitis (Alter et al. , 1975). Therefore, in this study, proportions of hepatitis

B surface antigen carrier in cirrhosis and cirrhosis with hepatocellular carcinorma in the recent

decade 1974‑1983 were found to decrease compared to the last decade 1964‑1973. The
decrease in the hepatitis B surface antigen positive rate from 45% to 36% suggests that infectionof type B virus caused by posttransfusion can be prevented in the latter decade. However, thehepatitis B surface antigen positive rates in chronic hepatitis showed minor changes during 1964‑

1983. A possible explanation may be that high frequency of hepatitis B virus transmission fromthe carrier mother to her infant has occurred.

REFERENCESl)

Alter, H. J., Holland. P. V. and Purcell, R. H. (1975) : The emerging pattern of post‑transfusion hepa‑

2)

titis, Am. J. Med. Sci., 270, 329‑342
Blumberg, B. S. , Alter, H. J. and Visnich, S. (1965) : A "New" antigen in leukemia sera, JAMA, 191,

3)

Blumberg, B. S., Gerstley, B. J. S. and Hungerford, D. A. (1967) : A serum antigen (Australia anti‑

101‑105gen) in Down's syndrome, Ieukemia and hepatitis, Aun. Intem. Med. , 66, 924‑931
4)Chainuvati, T. , Viranuvatti, V. and Pongpipat, D. (1975) : Relationship of hepatitis B antigen in cir‑5)rhosis and hepatoma in Thailand, Gastroenterology, 68, 1261‑1264
Dixon, W. J., Brown, E. L., Frane, J. W., Hill, M. A. , Jennrich, R. I. and Toporek, J. D. (198D:
BMDP statistical software, University of Califomia Press. Berkeley

6)

Gibson, J. B. and Sobin, L. H. (1978) : Histologicai typing of the liver, biliary tract and pancreas:

Intemational histological classification of tumor No. 20 World Heaith Organization, Geneva
7)

Houinger, F. B., Aach, R. D., Gitnick, G. L., Roche, J. K. and Mehaick, J. L. (1973) : Limitation ofsolid‑phase radioirnmunoassay for HB Ag in reducing frequency of post‑transfusion hepatitis, N. Engl. J.

Med. , 289, 385+391
8)Knrgrnan, S., Giles, J. P. and Hammond, J. (1967) : Evidence for two distinctive clinicai, epidemiolo‑

9)gicai and irnmunological types of infection, JAMA, 200, 365‑373
Leevy, C. M., Popper. H. and Sherlock. S. (1979) : Disease of the liver and biliary tract: Standardiza‑tion of nomenclature, diagnostic criteria and diagnostic methodology, Castle House Publications. Guid‑
f ord

10)

Prince, A. M. (1968) : An antigen detected in the blood during the incubation period of serurn hepati‑

1Dtis, Proc. Natl. Acad. Sci. USA, 60, 814‑821
Senba, M. (1982) : Staining method for hepatitis B surface antigen (HBS Ag) and its mechanism, Am28」．C㎞．Pathol．，77，312−31512）Senba，M，Nakamura，T．and

B

surfaceItakura，H，（1984）：Sta血stic

antigen，ckrhosis and13）Senba，M，Nakamura，Taccumulation

and

with

B

surfacepatients

analysis

Scheuer，P．J．（1970）：

hepatitis−associated（Australia）antigen

with

of

relationshipbetweenhepatitisofrelationshipbetweeniron

antigen，Am．J．Cbn．PathoL，84，340−342

A，，Niazi，S・P．and

15）Vogel，C．L，Anthony，P、P．，Mody，N．and
Ugandananalysis

carcinoma，Trop．Med．，26，117−122

Itakura，H、（1985）：Statistical

hepatitis

14） Sherlock，SりFox・，R

Hver−ce皿㎝cerandhepatoce皿ular

Barker，L

hepatoce皿ular carcinoma，LancetinChronic hver

senlm，Lancet

diseases

andp抽maryI，1243−1247

R（1970）：Hepatitis−associatedantigeninII，621−624

B型肝炎表層抗原の慢性肝炎，肝硬変および肝硬変に伴った肝細胞癌組織中の陽性率の減少傾向
千馬 正敬1・中村

剛2・板倉

英世1

1964−1973年と1974−1983年の各年代におけるB型肝炎表層抗原の陽性率を急性肝炎，慢性肝炎，

肝硬変，肝硬変に伴った肝細胞癌および肝細胞癌の組織を長崎大学病院の剖検肝臓422例を使用して検索した。B型肝炎表層抗原は急性肝炎の17例の全て陰性であり，慢性肝炎および肝細胞癌の例では
1964−1983年の差は小さかった。また，B型肝炎表層抗原は肝硬変および肝硬変に伴った肝細胞癌の例で減少していた。しかしながら，2年代におけるB型肝炎表層抗原の減少は肝硬変に伴った肝細胞癌の例では有意であったが，肝硬変では有意ではなかった。このことは最近の10年間ではB型肝炎ウ
イルスによる輸血後肝炎がなくなったためと思われた。

1長崎大学熱帯医学研究所病理学部門

2長崎大学医療技術短期大学部数学科

29
Japan. J. Trop. Med. Hyg. , Vol. 15, No. 1, 1987, pp. 29‑36

MATURATION OF CHIKUNGUNYA VIRUS IN SALIVARY
GLANDS OF INDONESIA STRAINS OF AEDESAEGYPTI AND AEDES ALBOPICTUS
MOSQUITOES= AN ELECTRONMICROSCOPIC STUDYSOEDARTO SOEKIMAN* AND TAKEO MATSUMURA
Received January 12 1987/Accepted February 7 1987

Abstract : Electron micrsocopic studies on the salivary glands of Aedes ae ,ti and Aedes
albopicuts mosquitoes infected with chikungunya virus showed vacuolization in the cells of thisorgans and numerous chikungunya virions as crystalloid structures in intracyloplasmic area,
revealing the spherical particle of 55‑69 nm in diameter and the electron‑dense intemal structureof 41‑48 nm in diameter. Vinons were also found enormously in this apical cavities. No
findings of virus budding from the cell surface membrane of chikungunya infected mosquito'ssalivary glands were obtained.

INTRODUCTION
Chikungunya virus, an alphavirus genus of Togaviridae, is widely distributed in Southeast
Asia and Africa. Aedes ae pti and Aedes albopictus are important vectors of this virus. In thetransmission of the virus, the salivary glands of these mosquitoes play especially an importantrole in producing and maintaining a high titer of viruses throughout their life (Takahashi and

Suzuki, 1979). Experimental studies showed that there were wide variations in susceptibihty
among different species of Aedes mosquitoes and different strains of the same species to transmit

chikungunya virus (Mangiafico, 1971; Tesh et al. , 1976; Mclntosh and Jupp, 1970; Yamanishi etal. , 1983). For the elucidation of the virus susceptibility, the growih of chikungunya virus insalivary glands of Aedes aegrpti and Aedes albopicuts were electron‑microscopically studied.

MATERIALS AND METHODS
Mosquitoes
Aedes ae pti (Surabaya strain) from mosquitoes collected at an urban area of Surabaya, andDepartment of Medical Zoology, Kobe University School of Medlcine, Kobe 650, Japan

This study was supported in part by a 1986‑1987 Japan Society for Promotion of Science (JSPS) andInternational Center for Medical Research (ICMR), Kobe University School of Medicine, Japan, fellowshipfor RONPAKU Program given to the first author (S. S. ).

* Present address: Department of Parasitology, Airlangga University Medical Faculty, Surabaya, In‑
donesia.

30

Aedes albopictus (Malang strain) from mosquitoes collected at a rural area of Malang, Indonesia,colonized in the frst author S.S.'s laboratory since 1983, were used throughout this study.

Eggs of these colonies were transferred to Japan in 1986 and recolonized. Mosquitoes werereared in an insectary maintained at 27' C, with 70‑80% relative hurnidity and a 16 : 8 hr (L : D)photoperiod (Konishi and Yamanishi, 1986).
Virus

Chikungunya virus, African strain passaged in the BHK‑21 monolayer cultures (Konishi and
Hotta, 1979), was used for infection of the mosquitoes.
Infechon of wosquitoes
Eight‑ to 10‑day old mosquitoes were infected by feeding on blood‑virus‑mixture at a titerabout l07. 9 focus forrning units (FFU) per ml (Soekiman et al. , 1986b). Fully‑engorged females

were transferred to another cage and maintained with O. 7% sucrose. Mosquitoes on the 9th‑and 12th‑day postinfection were used for the electron microscopic observation.
E Iectron microscopy

For the transmission electron microscopic observation, each of the mosquitoes of 9th‑ and12th‑day postinfection were anesthetized by chiWng. The salivary glands attached to the head

were dissected in saline solution under a dissecting microscope. After washed with PBS, thespecimens were fixed in 2% glutaraldehyde for 1‑12 hrs at 4'C and postfixed in l% osmium
tetroxide for 2 hr, both in Millonig's phosphate buffered saline. After dehydration in a graded

series of ethanol followed by toluene treatnent, specimens were ernbedded in Epon 812 in
BEEM capsules (Nissin EM Co.) for serial cross sectioning. For easy observation of thedissected mosquito's salivary glands, embedding was performed as previdusly described (Soeki‑

man et al. , 1986b). After thin sectioning were done with the Porter‑Blum MT‑2B ultramicro‑
tome (Sorval), the sections were stained with uranyl magnesium acetate and lead citrate andobserved under a HS‑9 electron microscope.

RESULTS
The proximal portion in the lateral lobe of salivary glands of Aedes ae )pti showed a numberof enveloped chikungunya viruses accumulated in the apical cavity (Figure IA). Microvilli also

protruded into the apical cavities (Figure IB). At a magnification of 48, OOO the diameter ofvirions were between 55‑69 nm with electron‑dense internal structure of 41‑48 nm. Especiallyclose to virion accumulated area (crystalloid structure), a large number of mitochondria wereseen clearly (Figure 2). Observation underneath the surface membrane of the lateral lobe cell inthe salivary glands of Aedes ae pti (Figure 3A) and Aedes albopictus (Figure 3B) showed en‑veloped chikungunya viruses clearly. No finding of the virus budding from the cell surface
membrane in these regions was obtained (Figures 3A, 3B). Precursor particles of chikungunya
virus (Higashi et al., 1967; Matsumura et al.. 1972) were not clearly observed in these sahvary
glands.

31

Figure IAThin section of proximai portion in the lateral iobe of salivary glands of Aedes ae pti

intected with chikungunya virus by oral feeding showed a numerous number of virions
in the apical cavity (Magnification: X 24,000; AC = apical cavity, M =mitochondria,

m =microvil . Arrows show virions).

,A,f.

Figure IB

At a magpification of 48.000 the di4meter of enveloped virions was between 5569
nm, with electron‑dense intemal structure of 41‑48 nm. Arrows show virions (AC =apicai cavity, M=mitochondoria, m = microvilli) .

32<***i.1"+; "*<* *,, i:iii:; +":*::.i'+*** =* ,+ .***;*'+ .:. .. , ..=^.

**'*=** <‑ ‑*"'‑ s:' si'**<'=f*;:"'** ' =**=; (,

Figure 2

Close to the virion's crystalloid structure, a numerous number of mitochondria wereclearly seen (Aedes aegypti salivary glands infected with chikungunya virus. Magnifica‑
tion: X 21,000:, M=mito̲̲chondria. Arrows show virion's crystalioid structure).

Figure 3A

Mature enveloped chikungunya viruses lined along undemeath the cell surface mem‑brane (arrows), without any budding process (Aedes ae pti salivary glands; Magnifica‑
tion: X 28,000).

33

Figure 3B Salivary glands of Aedes albapictus infected with chikungunya virus by oral feeding

showed mature vinons undemeath the cell surface membrane (arrows), No findingsof virus budding from this salivary gi'a:nds were aiso seen. Magnification: x 36, OOO.

DlscUssroN
In Aedes ae pti and Aedes albopictus infected with chikungunya vims by ' oral feeding, thetiters of virus reached the peak on day 9 and 12, respectively (Soekiman et al. , 1986a). Accord‑

ing to these results, electron microscopic observations were carried out in the mosquitoes of
9th‑day postinfection in Aedes ae pti and 12th‑day postinfection in Aedes albapictus,
Under the electron microscopic observation, the infected salivary glands showed increashg
numbers of vacuoles. This vacuolization is a characteristic sigu in mosquitoe's salivary glands
infected with arbovirus by oral feeding, such as Japanese encephalitis virus in Culex tritaerdorhyn‑chus (Takahashi and Suzuki, 1979) and dengue type‑2 virus in Aedes albopictus mosquitoes(Sriurairatna and Bhamarapravati, 1977). Thi8 intensive vacuolization was also found in Aedes
albapictus cell cultures infected with dengue viruses (KO et al. , 1979) and Vero cell cultures
infected with chikungunya virus (Higashi et al. , 1967) ,Several reports have described chikungunya virus morphogenesis in cultured cells. In
mammalian cell cultures such as Vero and BHK 21 cells, irdiected with this virus, the celi surface

membrane was the site of the maturation process of the chikungunya virus (Higashi et al. , 1967;

Matsumura et al.. 1972). Budding process can be seen commonly in this site and precursor
particle of viruscs were found undemeath the cell surface membrane and around the vacuoles in

the cyioplasma of the cells infected with the virus (Matsumura et al, , 1972; Matsumura andHotta, 1978). Similarly cell‑surface budding process was also seen in Vero, J=111 and IMR celi

34

cultures infected with chikungunya or dengue type I viruses (Ohyama et al. , 1977; Matsumura etal.. 1977) but it was not observed in BHK‑21 cell lines infected with dengue type I and type 2

viruses (Hotta and Matsumura, 1979). Our study did not show virus budding from the cellsurface membrane of the salivary glands of Aedes ae )pti (Surabaya strain) and Aedes albopictus(Malang strain) infected with chikungunya virus. The observation in salivary glands of Aedes
albopictus (Oahu strain) infected with chikungunya virus also did not show the budding process(Soekiman et al. , 1986b). Additionauy the crystalloid structures of the vition as seen in Veroand BHK‑21 cell cultures infected with dengue type I and type 2 viruses (Matsumura et al. ,1971; Matsumura et al. , 1977) were observed in the salivary gland of the infected mosquitoes.

The present study also showed that a numerous number of enveloped chikungunya viruses
can be seen well in the intracyioplasmic areas of the salivary glands, and also were densely
concentrated extracellularly in the apical cavities. Many mitochondria close to this crystalloid
structures of the virus indicate that the viral replication may need these intracenular organella as

the energy supplier (Hayashi, 1983). These results so far obtained suggested that in Aedes
ae pti (Surabaya strain) as well as Aedes albopictus (Malang strain) infected with chikungunya
virus, the virus maturation sites of salivary glands were intracytoplasmic membranous structuresuch as cysternae of endoplasmic reticulum and vesicles as already showa in Aedes albopictus(Oahu strain) salivary glands infected with chikungunya virus (Soekiman et al. , 1986b), Vero cells

infected with dengue type I and type 2 viruses (Matsumura et al. , 1971) and Culex tritaeniorhyn‑chus salivary glands infected with Japanese encephalitis virus (Takahashi and Suzuki, 1979).

After maturation of the virus in the cyioplasm of the salivary glands, the virions might bereleased into the apical cavity by the normal secretion mechanism of saliva (Matsumura et al. ,1971; Matsumura and Yamashita, 1978). Electron‑microscopically, there were no critical differ‑ences in the above‑mentioned findings of the saliyary glands infected with chikungunya virus
between Aedes aeg)pti (Surabaya strain) and Aedes albopictus (Malang and Oahu strains). It has

not been clarified yet what does the difference of the maturation site of chikungunya virus
between in vivo salivary glands of mosquitoes and mammalian cell cultures mean in relative to theecology of this virus.

ACKNOWLEDGMENT
The authors thank Dr. Eiji Konishi for supplying chikungunya virus and BHK‑21 cell lines,

Mr. S. Tanioka and Miss S. Matsushita for their technical assistance on this work.

REFERENCESl) Hayashi, K. (1983) : Handbook of Medical Virology, Kindai Shuppan, Co. , Inc. Tokyo

2) Higashi, N.. Matsumoto, A. , Tabata, K. and Nagatomo, Y. (1967) : Electron microscope study ofdevelopment of chikungunya virus in green monkey stable (Vero) cells, Virology, 33, 55‑69
3) Hotta, S. and Matsumura, T. (1979) : Studies on the release of toga virlJses (dengue and chikun‑
gunya), Abstract of the Jknnual Meeting. American Society for Microbiology (Hawai).
4) Ko, K. K., Igarashi, A. and Fukai, K. (1979) : Electron microscopic observation on Aedes albopictus
cells irLfected with dengue virus. Archives of Virology, 62, 41‑52

5) Konishi, E. and Hotta. S. (1979) : Effects of tannic acid and its related compounds upon chikungunya

virus, Microbiol. Immunol. , 23, 659‑667
6) Konishi, E. and Yamanishi, H. (1986) : Titer distribution analysis of chikungunya virus in Aedes albo‑

35
pictus (Diptera: Culicidae), J. Med. Entomol. , 23, 92‑98

7) Mangiafico, J. A. (1971): Chikungunya virus infection and transmission in five species of mosquito,

Am. J. Trop. Med. , 20, 642‑645
8) Matsumura, T. , Stollar, V. and Schlesinger, R. W. (1971): Studies of the nature of dengue viruses.
V. Structure and development of dengue virus in Vero cells, Virology, 46, 344‑355
9) Matsumura, T., Shiraki, K. , Hotta, S. and Sashikata, T. (1972) : Release of arboviruses from cells

cultivated with low ionic strength media, Proc. Soc. Exp. Biol. Med., 141, 599‑605

10) Matsumura, T., Shiraki, K. , Sashikata, T. and Hotta, S. (1977) : Morphogenesis of dengue vims incultures of human leucemic leucocyte (J‑111), Microbiol. Immunol. , 21, 329‑334

1 1) Matsumura, T. and Hotta, S. (1978) : Morphogenesis of togaviruses in cultured cells: an electron

microscopic study, Fourth Intemational Congress for Virology. The Hague, the Netherland, August

30‑September 6, 197812) Matsumura, T. and Yamashita, H. (1978) : Effects of Ca++ ion on the liberation of dengue virus from

BHK‑21 cells in culture, Microbiol. Immunol., 22, 803‑807
13) Mclntosh, B. M. and Jupp, P. G. (1970) : Attempts to transmit chikungunya virus with six species ofmosquito, J. Med. Entomol. , 7, 615‑618
14) Ohyama, A. , Ito, T. , Tanirnura, E., Huang, S‑C., Hsue, J‑Y. and Furu, Y. (1977) : Electron microsco‑

pic observation of the budding maturation of group B arboviruses, Microbiol. Immunol., 21, 535‑5 815) Soekiman, S., Konishi, E. and Matsumura, T. (1986a) : Susceptibility of Indonesia colonies of Aedesaeg)pti and Aedes albopictus mosquitoes to experirnental infection with chikungunya virus, Kobe J.

Med. Sci., 32, 127‑132
16) Soekirnan, S., Matsumura, T. and Yamanishi, H. (1986b) : Multiplication of chikungunya virus in sali‑vary glands of Aedes albopictus (Oahu strain) mosquitoes: an electron microscopic study, Jpn. J. Med.
Sci. Biol. , 39, 207‑211

17) Sriurairatna, S. and Bhamarapravati, N. (1977) : Replication of dengue‑2 virus in Aedes albopictus cell

culture, J. Virol. , 26, 275‑283

18) Takahashi, M. and Suzuki, K. (1979) : Japanese encephalitis virus in mosquito salivary glands, Am.

J. Trop. Med. Hyg. , 28, 122‑135
19) Tesh, R. B., Gubler, D. J. and Rosen, L. (1976) : Variation among infection with chikungunya virus,

Am. J. Trop. Med. Hyg., 25, 326‑334
20) Yamanishi, H. , Konishi, E., Sawayama, T, and Matsumura, T. (1983) : The susceptibility of somemosquitoes to chikungunya virus, Jpn. J. Sanit. Zool., 34, 229‑233

36

ネッタイシマカおよびヒトスジシマカ（インドネシア株）の唾液腺におけるチクングニアウイルス成熟の電子顕微鏡的研究
SoEDARTo SoEKIM州・松村 武男

チクングニアウイルスはトガウイルス科アルファウイルス属で，東南アジアをはじめアフリカなどに広く浸淫している。ネッタイシマカおよびヒトスジシマカは，本ウイルスの重要な媒介蚊である。
ここでは，インドネシアで採取され，実験室で継代飼育されたネッタイシマカ（スラバヤ株），および

ヒトスジシマカ（マラン株）をfeeding法で感染させ，両者の唾液腺における本ウイルスの成熟について電子顕微鏡観察を行った。感染後9日から12日の蚊では，ウイルス力価は最高値に達し，そのとぎの感染蚊の唾液腺細胞では空胞化が認められ，細胞質内には膜構造に囲まれたウイルス結晶構造が多数認められた。それらの粒子は，直径55〜69㎜で，電子密な直径41〜48nmの内部構造を有していた。Virionは，唾液腺管腔内にも無数に遊離している像が観察された。両種の蚊には，特に異なった形態学的所見はなく，いずれも感染蚊唾液腺細胞の細胞表面膜からの出芽所見は認められなかった。

神戸大学医学部医動物学数室

37Japan. J. Trop. Med. Hyg., Vol. 15, No. 1, 1987, pp. 37‑41

Short Communication

A COMPARATIVE STUDY ON GROWTH OF DENGUETYPE 3 AND CHIKUNGUNYA VIRUSES IN
INDONESIAN COLONIES OF AEDES AEGYPTIAND AEDES ALBOPICTUS MOSQUITOESSOEDARTO SOEKIMAN*, EUI KoNISHI AND TAKEO MATSUMURA
Received October 9 1986 /Accepted January 9 1987

Abstract : The growth of dengue type 3 virus in 2 Indonesian colonies of Aedes ae )pti
(Surabaya strain) and Aedes albopictus (Malang strain) was studied in the oral infection system,compared with the growth of chikungunya virus in Aedes ae pti as a control. In all combinationsof the virus and the host mosquito, maxirnum mean titers were as high as about l06 to 108 FFU
per mosquito 8 to 12 days after infection. This result suggests that in addition to Aedes ae ,ti,Aedes albopictus is a possible important vector involved in dengue hemorrhagic fever epidemics inrural areas of Indonesia.

Dengue hemorrhagic fever (DHF) is a major health problem in Indonesia and other South‑
east Asian countries (Hotta, 1978; Braude and Leelarasamee, 1981). Prirnarily DHF was one of
the urban diseases, with most cases reported from larger cities in Java Island such as Surabaya

and Jakarta, Indonesia. Since 1980, however, DHF epidemics have spread over smaller cities
and rural areas in all provinces except Timor Timur. Virological surveillance revealed that
dengue type 3 (DEN‑3) virus, belonging to Flaviviridae, was the predorninant virus isolated irom

the sera of DHF patients (Sunoto et al., 1975; Gubler et al., 1979b). Aedes ae )pti and Aedes
albopictus are common mosquito species in Indonesia. In urban areas, Aedes ae pti is a domes‑
tic mosquito usually found indoors, while Aedes albopictus is widely distributed outdoors in rural

areas (Horsfall, 1955). A recent paper reported many breeding sites for Aedes albopictus out‑
doors, also in urban areas (Oda et al. , 1984). It is wen known that there are wide variations insusceptibility to experimental infection with such mosquito‑bome viruses among different species

of mosquitoes and different strains of the same species (Gubler and Rosen, 1976; Tesh et al. ,1976; Gubler et al. , 1979a; Eshita, 1982; Yamalaishi et al., 1983). Our preliminary studies haveindicated biological and ecological similarities between Aedes ae pti and Aedes albopictus collected

in Indonesia (Soekiman et al. , 1984) and high susceptibility of both species to infection withchikungunya (CHIK) virus, an alphavirus also distributed in this nation (Soekiman et al. , 1986).

In the present paper, the growth of DEN‑3 virus in orauy infected Aedes ae pti and Aedes
Department of Medical Zoology, Kobe University School of Medicine, Kobe 650. Japan.
This study was performed as a part of the 1985 Ronpaku Program activity of the frst author (S. S.) in Kobe

University School of Medicine, partly supported by a 1985 Japan Society for the Promotion of Science(JSPS) and International Center for Medical Research (ICMR) fellowship.

* Present address: Department of Parasitology, Airlangga University Medical Faculty, Surabaya, In‑
donesia.

38

albopictus was studied, in comparison with the CHIK virus growth in Aedes ae pti.

The mosquitoes employed were a colony of Aedes ae pti established in 1983 from speci‑
mens couected in Surabaya, an urban area of Indonesia, and a colony of Aedes albopictus originat‑

ing from specimens collected in Malang rural area. Eggs of these colonies were taken to Japan
in 1985 and recolonized. Mosquitoes were reared in an insectary maintained at 27'C, with 70‑
80% relative huntidity and a 16 : 8 hr (L : D) photoperiod. Larvae were given a diet of mousepellets and adult mosquitoes were provided with O. 7% sucrose. DEN‑3 virus. H87 strain, wasprovided from the Department of Microbioldgy of our University at the 30th suckling mousebrain passage. After passed twice more in this laboratory, the seed virus was obtained from
10% homogenate of infected brain specimens in phosphate‑buffered sahne (PBS). CHIK virus,
African strairL (Konishi and Hotta, 1979), was also used in which the seed virus was the culture

fluid obtained from infected BHK‑21 monolayer cultures.

Feeding experiments and subsequent preparation of mosquito samples were performed aspreviously described (Konishi and Yamanishi, 1986). Four to 5 day‑old adult female mosquitoes

were allowed to feed on a mixture of the seed virus and defibrinated sheep blood with aninfective titer of 107. 7 FFU (focus forming unit) per ml in DEN‑3 virus and l08.2 FFU per ml in

CHIK virus. At 2‑day intervals, 5 to 12 mosquitoes of each species were homogenized in 2.0
ml of culture medium, centritilged at 700 xg for 5 min, and then filtered through a 0.45 pm
membrane to remove contaminating bacteria. The filtrate was assayed for viral infectivity by
the indrect fluorescent antibody technique essentially based on the original focus‑counting

method by lgarashi and Mantani (1974). The frst antibody was the DEN‑3 monoclonal anti‑
body obtained from culture fluid of the 5D4 hybridoma cell line (Henchal et al. . 1982) that wasprovided from the Department of Microbiology of our University, and the mouse anti‑CHIK virus

9

hO
O
U)
OE
:)

:D
LL
LL

OO

.J ‑

7

..A, Jl.

5

A.
'A . .

t ll' . "'

l

I;

3

;d;

1

o

6

12

18

DAYS POST‑INFECTION
Figure 1

Growth of DEN‑3 virus in Indonesia colonies of Aedes aeg)pti (e) and Aedes albopictus (O) after
feeding on virus at 107‑ 7 FFU per ml, compared with growth of CHIK virus in Aedes ae pti (A)after feeding on virus at 108. 2 FFU per ml. Mean virus titer obtained from 5‑12 mosquitoes perpool was represented as FFU per mosquito.

39

antiserum obtained as described elsewhere (Konishi and Takahashi, 1985). , The titer was ex‑pressed as FFU per mosquito.
The results are shown in Figure I , where growih curves were obtained untjl 14 or 18 days
after infection. The virus titer in Aedes ae )pti infected with DEN‑3 virus was l04. 6 FFU on

day O (the datum immediately after feeding) and l03. 9 FFU on day 2, whle in Aedes albopictus

infected with DEN‑3 virus the titer decreased from l04.6 FFU on day O to 102.6 FFU on day 2.
The initial drop was remarkable in CHIK virus‑infected Aedes ae pti in which titers on days O

and 2 were l05.4FFU and 101.7 FFU, respectively. The titer of DEN‑3 virus reached a max‑
imum of l08.2 FFU in Aedes ae pti and l07.5 FFU in Aedes albopictus both on day 12, in contrast

to CHIK virus infection where the peak titer was 106. 9 FFU on day 9.
The initial drop observed in this study is consistent with other reports (Gubler et al. , 1979a;

Yamanishi et al. , 1983). On the 2nd day of oral infection, no virus was recovered fromhemolymph in CHIK virus‑infected Aedes ae )pti (Lanzen et al. , 1970), and DEN‑2 virus antigen
was limited to the posterior midgut cells of Aedes albopictus (Kuberski, 1979), suggesting thepresence of "gut barrier" in the mosquito midgut. Subsequent virus growth indicates that these

species were highly susceptible to experirnental infection with DEN‑3 and CHIK viruses. HighDEN‑3 virus titers observed in Aedes ae pti and Aedes albopictus were similar to those reported

by Gubler and Rosen (1976) and Gubler et al. (1979a) with several geographic strains. Althoughsusceptibility of Aedes ae pti to oral infection with CHIK virus was relatively low in general(Yamanishi and Konishi, data unpublished), the maximum titer obtained in this study was compa‑

rable to those of DEN‑3 virus in Aedes ae )pti and Aedes albopictus. The present data strongly
support the opmion that Aedes ae pti is very important in the epidemiology of DHF as a potential

vector of DEN‑3 virus (Gubler et al., 1979b; Tan et al. , 1981). However, it is also suggested
from the population distribution of Aedes mosquitoes in Indonesia that Aedes albopictus is anotherimportant vector in DHF epidemics spreading over rural areas. Field studies including virus
isolation from this species and its feeding pattern should be performed to verify the epidemiolo‑
gical importance of Aedes albopictus. Further studies to confirm the ability to transmit the virus

are now in progress incltiding the electron microscopic observation of the virus maturation
process in the salivary gland of Aedes aegrpti and Aedes albopictus.

ACKNOWLEDGMENTS
Our special thanks are due to Dr. Hiroshi Yamanishi for his technical advice on mosquito
infection and to Dr. Soewignjo Adipoetro for his helpful comments on this work.

REFERENCESl) Braude, A. I. and Leelarasamee, A. (1981): Dengue and other hemorrhagic fevers, p. 1534‑1544.
In : A. I. Braude, ed. , Medical nticrobiology and infectious diseases. W. B. Saunders Company, Phi‑
ladel phia2) Eshita. Y. (1982) : Experimental studies on the transmission of dengue virus by Japanese mosquitoes,

Teikyo Medical Journal, 5, 17‑27
3) Gubler, D. J, and Rosen, L. (1976) : Variation among geographic strains of Aedes albopictus in suscep‑tibility to infection with dengue vinlses, Am. J. Trop. Med. Hyg. , 25, 318‑325
4) Gubler. D. J. , Nalim, S., Tan, R. , Saipan, H. and Saroso. J. S. (1979a) : Variation in susceptibility to40oral iniection with dengue viruses among geographic strains of Aedes ae rpti, Am. J. Trop. Med.

Hyg., 28, 1045‑1052
5) . Gubler, D. J., Suhmjono, W., Lubis, I. , Eram, S. and Saroso, J. S. (1979b) : Epidemic dengue hemor‑
rhagic fever in rural Indonesia. I. Virological and epidemiological studies, Am. J. Trop. Med. Hyg. ,28, 701‑710
6) Henchal, E. A., Gentry, M. K., McCown, J. M. and Brandt, W. E. (1982) : Dengue virus specific andflavivirus group determinants identified with monoclonal antibodies by indirect irnmunofluorescence,

Am. J. Trop. Med. Hyg. , 31, 830‑836
7) Horsfall, W. R. (1955) : Mosquitoes. Their bionomics and relation to disease, The Ronald Press

Company, New York8) Hotta, S. (1978) : Dengue and related tropical viruses, Yukosha Printing House, Kobe, Japan9) Igarashi, A. and Mantani, M. (1974) : Rapid titration of dengue virus type 4 infectivity by counting
fluorescent foci, Biken J., 17, 87‑93
10) Janzen, H. G. , Rhodes, A. J. and Doane, F. W. (1970) : Chikungunya virus in salivary glands of Aedes
ae pti (L. ): an electron microscope study, Can. J. Microbiol. , 16, 581‑586
1 l) Konishi, E. and Hotta, S. (1979) : Effects of tannic acid and its related compounds upon chikungunya

virus, Microbiol. Imrnunol. , 23, 659‑667

12) Konishi, E. and Takahashi, J. (1985) : Detection of chikungunya virus antigen in Aedes albopictus

mosquitoes by enzyme‑linked irnmunosorbent assay, J. Virol. Meth. , 12, 279‑285
13) Konishi, E. and Yamanishi, H. (1986) : Titer distribution analysis of chikungunya virus in Aedes
albopictus (Diptera: Culicidae), J. Med. Entomol. , 23, 92‑98

14) Kuberski, T. (1979) : Fluorescent antibody studies on the development of dengue‑2 virus in Aedes
albupictus (Diptera: Culicidae), J. Med. Entomol. , 16, 343‑349
15) Oda, T., Igarashi, A., Hotta, S., Fujita, N., Funahara, Y., Djakaria, S., Hudojo, R., Isfarin, A. andDjohor, D. (1984) : Studies on bionomics of Aedes aeg)pti and Aedes albapictus and dengue virus
isolation in Jakarta, Indonesia, 1983, ICMR Annals, 3, 31‑35
16) Soekirnan, S., Konishi, E. and Matsumura, T. (1986) : Susceptibility of Indonesia colonies of Aedes

ae pti and Aedes albopictus mosquitoes to experitnental infection with chikungunya virus, Kobe J.

Med. Sci. 32, 127‑132
17) Soekiman. S. , Machfudz, Subagyo, Adipoetro, S., Yamanishi, H. and Matsumura, T. (1984) : Com‑
parative studies on the biology of Aedes ae pti (Linnaeus, 1762) and Aedes albopictus (Skuse, 1895),

ICMR Annals, 4, 143‑151
18) Sunoto, van Peenen, P. F. D., Sumarno, Sarno Sinto and Joseph, P. L. (1975) : Studies on dengue inchildren at Dr. Cipto Mangunkusumo General Hospital, Jakarta (May 1973‑January 1974), Paed.
Indonesiana, 15, 143‑150
19) Tan, R. , Abdin, M. , Maroef, C. and Gubler, D. J. (198D: Comparative growih of dengue viruses in
Aedes ae )pti and Aedes albopictus after parenteral intection, Mosquito News., 4, 71‑73
20) Tesh, R. B., Gubler, D. J. and Rosen, L. (1976) : Variation among geographic strains of Aedes albopic‑

tus in susceptibility to infection with chikungunya virus, Am. J. Trop. Med. Hyg. , 25, 326‑334

21) Yamanishi, H. , Konishi, E., Sawayama, T. and Matsumura, T. (1983) : The susceptibility of somemosquitoes to chikungunya virus, Jpn. J. Sanit. Zool. , 34, 229‑233 (in Japanese)

41

短

報
インドネシア由来のネッタイシマカおよびヒトスジシマカにおけるデング3型ウイルスとチクングニアウイルス増殖の比較
SoEDARTo

SoEKIM州・小西

英二・松村

武男

スラバヤ系ネッタイシマカにおけるチクングニアウイルス増殖を対照として，同蚊およびマラン系

ヒトスジシマカにおけるデング3型ウイルスの増殖を経口感染により調べた。蚊の平均保有ウイルス量の最高値はいずれの組合せにおいても高く，感染後8日から12日目に約106から108FFUを示した。この結果からインドネシアの農村部におけるデング出血熱の流行に関与する媒介蚊として，ネッ
タイシマカの他にヒトスジシマカも重要であることが示唆された。

神戸大学医学部医動物学教室

JAPANESE JOURNALOF
TROPICAL MEDICINE AND HYGIENE
March, 1987

Vol. 15 No. 1

CONTENTS
Original article
Matsuo, K. and Makiya, K.
Ecological Studies on the Lung Fluke, Parnglonimus ohirai Miyazaki, 1939
II Infection Rates with P. ohirai of Snans and Rodents Collected from thelbi, Nagara and Kiso Rivers in the Tokai District, Central Japan . . . . . .

1 ‑6Hashiguchi, Y., Landres, E. A. G., Colonel, V. V., Mirnori, T. and Kawabata, M.
Laishnraniasis in Different Altitudes on Andean Slope of Ecuador. . . . . . . . . . . . . .

7‑ 1 5

Hioki, A. and Ohtomo, H.
Changes of Blood Oxygen Affinity in Fansidar‑treated Mice Infected

17‑23with Plasmodium berghei. . . . . . . . . . . . .

Senba, M. , Nakamura. T. and Itakura, H.
Decrease Trend in Proportions of Hepatitis B Surface Antigen Carrier in
Chronic Hepatitis, CitThosis and CitThosis with Hapatocellular Carcinoma . . .

25‑28

Soekiman, S. and Matsumura, T.
Maturation of Chikungunya Vims in Salivary Glands of Indonesia Strains ofAedes ae pti and Aedes albopictus Mosquitoes: an Electoron Microscopic Study . .

29‑36

Short communication
Soekiman, S. , Konishi, E. and Matsumura, T.

A Comparative Study on Growth of Dengue Type 3 and Chikungunya Vimses inIndonesian Colonies of Aedes ae pti and Aedes albopictus Mosquitoes . . . . . . . . .

Published byJAPANESE SOCIETY OF TROPICAL MEDIClNE
c/o Institute of Tropical Medicine, Nagasaki University

12‑4 Sakamoto‑machi, Nagasaki 852, Japan37‑41