一■■王

第21巻第4号平成5年12月

内

原

容

著

暑熱馴化と運動が暑熱曝露時の生理的反応に及ぼす適応的変化

一堀

清記，田中

信雄

19H99

フィラリア感染好適宿主としてのスナネズミの基礎的検討
一血清生化学的特性に関する被毛色間の比較一
・・七戸

和博，清水

真澄，芳賀

克也，家所

哲夫，

藤田紘一郎

⑳1一獅

マラリア疫学における抗体価分布曲線の有用性

一スーダン・青ナイルの洪水に起因した突発性流行の評価を事例として一一狩野繁之，Salah

Pilarita

H．EI

T．Rivera，Ahmed

Safi，Fakhr

A．EI

EI

Din

M．Omer，Gaddal，鈴木守

207−211

マラリア高度流行地域における血清疫学の有用性
一アマゾン原住一部族の調査を事例として一
・・狩野

Oseas

繁之，宇都宮嘉明，Alfred

Santos，Waldir

MiltonMasatodeHida，松本

Souza

Takeshi

Honda，

Miranda，慶蔵，鈴木守

213−217

ボリビア・サンタクルス総合病院におけるシャーガス病巨大結腸症の外科的治療……一………Edwin

CrespoSimeonMendosa，Freddy

Sengoku，三好（裏面に続く）Udalrico

知明，仲佐Gutierrez

保Velared，

219−224■■

1993年に沖縄で流行した無菌性髄膜炎
・・牧野芳大，玉那覇栄一，宮里松茂良
山田

力，Chorson

直人，新垣

善次，島袋

Yan，石原

順吉，

龍治，町田

孝，

栄，只野

昌之，福永

利彦

225一謝

敏，森

章夫，藤田紘一郎，

長崎市内の飼い犬についての犬糸状虫の疫学的研究
一仔虫保有状況の年次的変動に関する考察一
・・小田
在津

力，末永
誠，黒1．

憲次，西岡

猛，伊藤

達也，

三根真理子

231−237

アフリカ風土病型カポシ肉腫の超微構造………………江藤
症

秀顕，鳥山

寛，板倉

英音

％9一脇

例

ハロファントリンにより治癒した輸入熱帯熱マラリアの4例
・・狩野
斉藤

繁之，増田

正之，鈴木

剛太，山口

剛，久保

譲，

守

245−249

研究ノート

日本産ブユ3種に見いだされた雌雄モザイク個体について…Upik
短Kesumawati

Hadi，高岡

宏行

251一烈

報

ブラジル・レシフェ市周辺に於ける犬姻虫症の血清学的調査
・・岩永

嚢，Jose田辺

將信，建野FelipeGoncalves

正毅，辻

守康，竹内

勤

第21巻平成5年（1993年）総内容………

2弱一蹴
・259一脳会報・記録…・…

265

日本熱帯医学会雑誌

発行所

第21巻第4号

編集者

1993年12月15日

印刷

1993年12月15日

発行

日本熱帯医学会板

倉英喜

印刷所昭和堂印刷
諌早市長野町1007−2（◎854）
容0957−22−6000

本雑誌の刊行にあたりその費用の一部を文部省科学研究費補助金（研究成果公開促進費）によった。

193

Jpn. J. Trop. Med. Hyg., Vol. 21, No. 4, 1993, pp. 193 199

ADAPTIVE CHANGES IN PHYSIOLOGICALRESPONSES OF MEN TO HEAT INDUCEDBY HEAT ACCLIMATIZATION AND
PHYSICAL TRAININGSEIKI HORI

AND NoBUO TANAKA2

Received August 16 1993/Accepted October 7 1993

Abstract: Twelve healthy male nonathletic university students were selected as subjects. Anthropometricmeasurement and a work capacity test using a bicycle ergometer in a climatic chamber of 30'C and 60% R.
H. were carried out in summer and winter. Observations of the physiologic l responses of the subjects weremade while pedalling a bicycle ergometer at a constant work load of 50% V02max and at a cycling rate of
50 rpm for 30 min in a climatic chamber of 30'C and 60% R.H., in summer. The subjects took the same
exercise for 13 successive days except the day after training, for 6 days in a climatic chamber of 30'C and
60% R.H., after the work capacity t.est was tested in winter. Body weight and skinfold thickness showed atendency to decrease in summer. V02max per body weight was considerably greater in summer than in
winter though this difference was statistically not significant. Na concentration in sweat and the intteasein heart rate during exercise were significantly lesser in summer than in winter. Sweat volume induced byexercise increased and rise in rectal temperature during exercise showed a tendency to decrease in summer.
Increase in heart rate during exercise was decreased significantly by physical training. Sweat volume during
e ercise tended to increase and rise in rec̲tal temperature during exercise tended to decrease progressively
during physical training. Decrease in heart rate during exercise induced by physical̲ training was greaterthan that induced by climatic heat acclimatiztion, while decrease in Na concentration of sweat due toclimatic heat acclimatization was greater than that observed during physical training. Indices representing
the magnitude of strain including relative increase in heart rate, relative rise in core temperature andrelative water loss are proposed for the assessment of work capacity in heat.

INTRODUCTION

perature during exercise in heat due to a slower onset ofsweating and a lower sweat rate (Robinson et al.. 1953;

It has been known that the physiological responses
of unacclimatized individuals in heat are changed when
they have been exposed repeatedly to a hot environment(Kuno, 1956; Shvartz et al.. 1979) or they have been

Kuno, 1956; Bass, 1963). After successive exposures toadapted to work in heat (Robinson et al.. 1953;

physical training and a lower rise in core temperature
due to an earlier onset of sweating and an increased
sweat volume with a decreased salt concentration (Lind

Wyndham et al.. 1964). In shortrterm heat acclimation,unacclimatized individuals sweat more readily, anda combination of exercise and environmental heat, alessening of cardiovascular strain has been observed asindicated by a lesser increase in heart rate due tomore profusely, and the rise in their core temperatureduring exercise in heat is lessened due to greater heatand Bass, 1963; Piwonka et al.. 1965; Araki et al., 1981) .

dissipation accompanied by an increased amount ofsweat. These adaptative changes to heat during short‑term heat acclimation are considered to be favorable
for individuals who must work in a hot environment

was much less and the decrease in the heart rate wasmuch greater during successive exposure to a combina‑
tion of exercise and environmental heat when comparedwith those observed in climatic heat acclimatization(Ihzuka et al.. 1986). It is generally agreed that unac‑climatized individuals show a marked rise in core tem‑(Hori, 1977). It can be said, therefore, that there aredifferent characteristics of heat adaptation, with regard

12However, the decrease in the salt concentration in sweat

Department of Physiology, Hyogo College of Medicine, Nishinomiya, Hyogo 663, Japan
Department of Health and Physical Education, Kyoto Sangyo University, Kyoto 603, Japan

194

Table 1 Characteristics of subjects (XiSD)

Season Number

Summer12Winter

12

Age
'(Yr)

Height Body weight(cm) ,. ' (kg)20.1 1.4 170 1+5 3 58.9 3.8
20.6il.4 170 1+5 3 59.8+4.2

to sweating reaction and cardiovascular reaction, during

exercise in heat between climatic heat acclimatizationand heat acclimation caused by physical training in a

hot environment. Thus, an attempt was made to com‑pare, in detail, changes in physiological responses of thesame subjects during exercise in a hot environment
between two types of heat adaptation‑climatic heatacclimatization and heat acclimation induced by exer‑
cise in heat.

Area Mean skinfold
Ar a Bod.y weight
thickness Fat content(m2')(cm2/g)(mm) ( % )1 . 67 i: O . 10 O . 287

O . OIO

10.9 3.1

12.4 3.2

1 . 70 i O . 12 O . 283

O . OIO

12.9+4.2

14 . 4+ 3 . 5

work capacity test, in summer.
Body w̲eight was measured before and immediately
after exercise using a platform balance with an accu‑racy 6f up to d:5g, and net body weight was obtained bysubtracting the weight of the shorts. Rectal tempera‑ture was recorded continuously by a copper‑constantan
thermocouple. Heart rate was taken by a bipolar chest
lead electrocardiogram. Sweat samples from the backwere collected at 10 min intervals using filter paper

method (Ohara, 1966). Na in

weat was eluted with

distilled water from the filter paper and its concentra‑

MATERIALS AND METHODS
Twelve healthy young male nonathletic university
students were selected as subjects after informed con‑sent had been obtained. Anthropometric measurementand determination of the maximal oxygen uptake (V02
max) using the bicycle ergometer in a climatic chamberof 30'C and 60% R.H. were performed in summer andwinter. Experiments were performed at 15:OO h. Sub‑
jects were instructed to fast and remain at rest aftertion was determined by flame phdtometry. In winter,each subject took the same 30 minute exercise for 13
successive days except on the day after training, for 6
days. The subjects rested for one hour in the climaticchamber of 30'C and 60% R.H. on the day without
training, to maintain the state of heat adaptation in‑
duced by exercise in a hot environment. Skinfold thick‑ness was measured on the right side of the body.
Skinfold sites and weighing factors used for calculating

lunch. The subjects, dressed in shorts only, rested on thethe mean skinfold thickness are as follows (Hori et al..

saddle of the Monark bicycle ergometer for 5 min, then1978) .

warmed up for one minute by pedalling at zero load.
The pedal frequency was set at 50 rpm and the work
load was incremented continuously by 15 watts/min

Chest Abdomen Upper arm Sobscapular Waist Thighuntil the subjects reached exhaustion. During exercise,a bipolar chest lead ECG was recorded and the volume
of expired gas was recorded automatically. Expired gas
s.amples were analyzed for oxygen and darbon dioxide.
V02max was determined by averaging the consecutive
15s oxygen uptake value before exhaustion. Each sub‑O . 143 O . 139 O . 141

O . 143 O . 139 O . 295

The body fat (F %) was calculated from the mean
skinfold thickness (X mm), body weight (W Kg) andbody surface area (A m2) by the following equation(Hori et al., 1978)

AXX

F = 28.9 W + 3.67

ject, dressed in shorts only, sat for 30 min, then pedalJed

the cycle ergometer at a constant work load of 50% V02
max at a cycling rate of 50 rpm for 30 min in a climaticchamber of 30"C and 60% R.H., on the 7th day after the

Table 2 All out time, V02max, V02max per body weight and maximal heart rate (XiSD)

MaximalV02rnax
Weight(ml/kg/min)heart rate
(beats/min)O . 3740.6 3.7

182 + 4 . 4

2.29 0 29

37.8 3.6

183 i 4 . 5

SeasonAll out time(min)

Summer

16.7 1.5

2 . 38Winter

16.0il.5

V02max(1/min)195

Table 3 Body weight loss, body weight loss per body weight, mean Na concentration in sweat, rise in rectal
temperature, increase in heart rate, V02 and Vo, per body weight (XiSD)

SeasonTraining (week )Summer

Winter

AW
(kg)

AW W

(%)

C

AH

ATre ('O(mEq/1)(beats/min)

*S

o,

o,/W

(1/min)(ml/kg/min)*So0.52+0 10

o . 89 i o . 22

41.1 8<.3

O . 74 + o . 17

72.1+2.3

1.41+0.17

24.0+3.4oo . 48 + o , 08

0.81+0.21

52 . 90.79 0 19

75.3 2.5

1 . 40+ o . 17

23.3+3.3

10 . 4*t
1

0.53+0 11

o . 89+ o . 22

51 . 010 . 90.74 0 18

70 . 6+ 2 . o

1 . 38O . 7622.9 3.1

2

O . 54 + o . 12

0.9li0.20

50 . 3i 11 . O

0.72+0 19*t69.2+2.1

1.37i:O 15

22.8+3.3* S: Significant differences between summer and winter.
* t: Significant differences between before training and after training.

*: P<0.01
AW: Body weight loss, W: Body weight, C: Mean Na Concentration in sweat,
ATre: Rise in rectal temperature, AH: Increase in heart rate.

RESULTS
The physical characteristics of the subjects are
given in Table 1. The mean values of body weight, bodysurface area, mean skinfold thickness and body fat
percentage were smaller in summer than in winter while

mean Na concentration in sweat and the increase inheart rate. The mean values of body weight loss andratio of body weight loss to body weight indreased

the mean value of the ratio of body surface area to bodyfollowed by a more gradual increase during the last
week of training. The mean values of rise in rectal
temperature decreased during training. The mean val‑

weight was greater in summer than in winter thoughthese differences were statistically not significant. A
greater value of the ratio of body surface area to bodyconsiderably during the first week of training, and wasues of increase in heart rate on the first week and theweight with smaller deposits of subcutaneous fat insummer indicates a body contour changed to a more
slender body shape from winter to summer. The mean

second week were significantly smaller than the meanvalue before training. The mean value of oxygen uptake
and oxygen uptake per body weight decreased graduallyvalues and standard deviations of all out time, V02max,
ratio of 02max to body weight and maximal heart rateduring training. Mean Na concentration in sweat

in both seasons are represented in Table 2. The meanvalues of all out time, V02max and ratio of V02max tobody weight were greater in summer than in winter.
However, these differences were statistically not signifi‑

cant. Changes in body weight‑ Ioss, ratio of body weightloss to body weight, mean Na concentration in sweat,
rise in rectal temperature, increase in heart rate, oxygen

uptake and oxygen uptake per body weight during
exercise in a hot environment induced by climatic heatacclimatization and successive exposures to a combina‑
tion of exercise and environmental heat are shown in
Table 3. The mean values of body weight loss, ratio ofbody weight loss to body weight and oxygen uptake perbody weight were greater in summer than in winter,
while the mean values of mean Na concentration in

showed a tendency to be lower during training.

DISCUSSION
Acclimatization to heat appears to be not only aphysical function concerning body temperature regula‑tion but also a physical characteristics (Coon et al.,1950). It is known that a rise in ambient temperature

results in a decrease of subcutaneous fat due to adecrease in the caloric intake and the body shapeaccordingly becomes more slender by the decrease of
subcutaneous fat (Hori et al., 1982) . Thus it is assumed

that a thinner skinfold thickness and a greater value ofthe ratio of body surface area to body weight in summer

might be caused by a hot climate. The thickness ofsweat, rise in rectal temperature, and increase in heart

subcutaneous fat prevents heat transfer from the bodyto the environment. Heat dissipation from the body torate were smaller in summer thall in winter. Amongthe environment is proportional to the body surface area

these differences, there were significant differences inand metabolic heat produced in the body is proportional

196

55
0.5 5

2.W
"‑ :i W"

.9 50.

, i: Soe'yO‑ "It,

wo

75
e'c

'uJcr

JCE
. .

8 E 45

co

l

'

' 'e'
ulJ:]

5

O‑

L ' 70

cu

CD

,tt

0.50 t

i: ̲

Q'
:;

Wo

CC
/ >

a
<
o

0.4 5

40
0.80

0.85

0.90

Body we ht ioss / Body weight ( '/. )0.70
0.75 0.80
Rise irl rectal tcmperature( 'C )

Figure I Changes in correlation between mean Na

Figure 2 Correlation between increase in heart rate

concentration in sweat and body weight loss per
body weight induced by climatic heat acclimati‑
zation and physical training.

So: Summer, Wo: Winter, IW,2W: Weeks after training

So: Summer, Wo: Winter, IW,2W: Weeks after training
Circle: Drawn around the means with arbitrary radiuses.

‑ Seasonal change‑ Training effectbody weight loss and rise in rectal temperature.

O : Correlation between increase in heart rate and rise inrectal temperature.
Correlation between body weight loss and rise in rectal

temperature.
Circles: Drawn around the means with radiuses of halfvalues of standard errors.

‑ : Seasonal change

to the body weight during movement of the body, forexample, in walking or running. Therefore, the lessen‑
ing of subcutaneous fat and the greater ratio of bodysurface area to body weight caused by a hot climate areconsidered to be convenient for the regulation of bodytemperature in a hot environment. As shown in Table 3,‑ : Training effectweight were greater in summer than in winter thoughthese differences w re not significant. Greater values ofall out time and V02max in summer might reflect alesser increase in he.art rate during exercise in heat, andthe mean value of body weight loss in summer was

greater values of V02max per body weight might becaused by a decrease in body weight and a lesser

greater than that in winter, while the mean value of theincrease in the heart rate during exercise. As shown inmean Na concentration in sweat in summer was signifi‑
cantly smaller than that in winter. These findings werein agreement with the results reported by many investi‑
gators (Ohara 1966; Ih uka et al., 1986). A decrease insalt concentration of sweat increases the difference invapor pressure between the sweat on the skin and thesurrounding air (Hori et al., 1982). It may thus beconsidered that the smaller rise in rectal temperatureduring exercise in summer was caused by a greater heatdissipation due to profuse sweating with lower Na
concentration as well as the physical characteristics
favorable to heat dissipation. Lesser increase in heart
rate during exercise in summer might be caused by alower rise in rectal temperature and an increase in blood

volume with an improvement of the skin circulation(Bass and Henschell, 19.56; Senay 19 2). As shown in
Table 2, all out time, V02max and V02max per body

Table 3, sweat volume increased progressively due tothe imposition of internal and external heat stress dur‑ing successive exposures to exercise in a hot environ‑ment (Ogawa et al.. 1982; Ogawa and Asayama 1986;
Tsujita et al.. 1989). During the first week of physicaltraining, sweat volume increased markedly and theincrease in sweat volume was slight during the next
week while the change in the Na concentration of thesweat was small. In Fig. 1. Seasonal change in correla‑tion between Na concentration in sweat and bodyweight loss (sweat volume) was compared with thechange that was induced by physical training in a hotenvironment. This figure indicates greater differencesin the change of sweating reaction induced by two typesof heat adaptation.

Since the conQentration of sweat Na increases
progressively as the rate of sweating increases (Kuno197

1956), there can be no doubt about decrease in Na

t=0. tconcentrations in sweat at a given sweat rate duringt = 0.63

physical training. However, the decrease in the concen‑

0.64

t = 0.62

tration of the sweat rate from winter to summer was

Womuch greater than that induced by physical training inheat. According to Conn et al.. (1946), reabsorption ofsalt at the duct of the sweat gland from the precursor

t= 0.61

06 2

sweat secreted at the acinus of the sweat gland isenhanced by the increased secretion of mineralocor‑
ticoid in summer, and the Na concentration in sweat

t= 0,60So

t=0.59IW

06 o

t= 0.58

decreases in spite of. the increase in the sweat irate insummer. It is presumed that two weeks were too shorta time span for the effects of mineralocorticoid toappear on the reabsorption of salt at the duct of thesweat gland or that the increase in the secretion ofaldosterone induced by physical training was too small
to decrease the Na concentration in sweat. In heat

2w

t=a57

os8

t=056

018 019 0.20 0.21 0.22 023
S
Figure 3 Changes in correlation between stress index

and value of S induced by climatic heat

adaptation, a reduction in the rise of rectal temperatureacclimatization and training.
I,S,T: The same as in table 4.

is usually accompanied by lessening of the increase in

the heart rate. Changes in the relationship between

So: Summer Wo: Winter IW,2W: Weeks after training

body weight loss, increase in heart rate and rise in rectal

Circles: Drawn around the means with radiuses of stan‑temperature during climatic heat acclimatization anddard errors.

‑: Seasonal change

physical training in heat were shown in Fig. 2. As shownin Fig. 2, changes in the correlation between increase inheart rate and rise in rectal temperature and the correla‑‑= , : Training effectresponses to heat exposure or exercise in a hot environ‑ment. To assess physiological strain induced in the bodyby exercise in heat, we expressed the maguitude of thetion between sweat volume and the rise in rectal temper‑

ature induced by climatic heat acclimatization wereapproximately the same as those induced by physicalstrain by a combination of relative increase in heartrate, relative rise in rectal temperature, and relative

training in heat. Thus it seems certain that there was adiscrepancy with respect to the changes in Na concen‑
tration in sweat and changes in other physiologicalwater loss, using the critical values of these three fac‑

Table 4 Stress index and its components (Xd!:SD)
S asonTraining (week)

Summer

WinterA

C

B

O . 556

O . 018oO . 586 + o . 019

1

O . 543

* *S

* *S

O . 607

O . 0200.220i0.019

O . 592 + o . 019

0.237+0 056

O . 115

O . 019

O . 632

O . 0200.184+0 023

o . 624

O . 213

O . 052

O . 127

O . 025

O . 601

O . 201 i O . 053

O . 130

O . 029

O . 583 + o . 017

* *S

2

O . 531

O . 213

O . 049

A =200‑H
AH 'ATreTre ' C= 0.07
AW W' I = Flr 7, S == 40.6‑C

JA T ' t=F* S: Significant differences between summer and winter.
* t: Significant differences between before training and after training.

*: P<0.05, * *: P<0.01
AH: Increase in heart rate (beats/min)
H: Heart rate before exercise (beats/min)
ATre: Rise in rectal temperature (OTre: Rectal temperature before exercise (C)AW: Body weight loss (kg)
W: Body weight before exercise (kg)O . 017* *t* *t
O . 016

*t

* *t

* *t
O . 016t

O . 126+ o . 022

* *S
o

s

I

O . 213 i O . 029* *to . 228o . 034o . 020* *t
O . 583

O . 017* *t
O . 566

O . 017

198

tors as those which cause all out (heart rate, 200 beats/(C) . Reduction of the magnitude of strain as a whole (1)min) , heat stroke (rectal temperature, 40.6'O andwas brought about by climatic heat acclimatization andphysical training at the expense of the increase in thewater depletion heat exhaustion (body weight loss, 7%of body weight) (Leithead and Lind 1964). The value ofmagnitude of strain C. In Fig. 3, seasonal change instress index I was calculated ̲as follows. I=

relation to the values of I and S was compared with the

JA T Ichange in that induced by physical training in a hot

A=200AH‑ Ho
B‑40.6
AfT‑ ToC=0.07
AWW

where: H0=Heart rate before exercise (beats/min)
AH=1ncrease in heart rate at the end of theexperiment (beats/min)
T0=Rectal temperature before exercise ('C)
AT=Rise in rectal temperature at the end of
the experiment ('O
W= Body weight before exercise (Kg)
AW = Weight loss at the end of the experiment(Kg)
Since the value of I is defined as the magnitude of strain

induced in the body, a smaller value of I during exercise

in heat indicates a superior capacity for prolonged
exercise in a hot environment and we can expect thatheat acclimatization induced by a hot climate and suc‑cessive exposures to a combination of exercise and a hotenvironment. In this figure, iso‑training lines are drawn
by connecting the points of the same value of parameter"t". As shown in Fig. 3, a decrease in the value of "t"

was usually accompanied by an increase in the value ofS during both climatic heat acclimatization and physicaltraining in heat. However, the decrease in the value of"t" was greater in physical training than in climaticheart acclimatization and the reduction of the values of
I and "t" during physical training in heat for I week was

greater than that induced by climatic heat acclimatiza‑tion for 6 months. The increase in the value of S duringphysical training in heat for 2 weeks was greater thanthat caused by seasonal acclimatization to heat. Thus,
it can be said that heat tolerance and the capacity for
prolonged exercise in heat can be improved more rapid‑
ly by successive exposures to exercise and a hot environ‑ment as compared to climatic heat acclimatization.

environment is accompanied by a reduction of the Ivalue.

REFERENCES

By calculation and transformation,
Equation I= t/rlrV can be derived as follows;

I= T

1 ) Araki, T., Matsushita, K., Inoue, Y. and Nakao, M.
Effect of physical training on exercise‑induced sweating
in women. (1981): J. Appl. Physiol. 51, 1526‑1532.

1+ A2 C2+ B2 =t/rT2 ) Bass, D.E. (1963): Thermoregulatory and circulatory
adjustments during acclimation to heat in man. In:
Temperature, Its Measurement and Control in Science

where t= T

S‑FTC :The decrease m the value of parameter "t" was muchgreater in physical training in heat than that in climaticheat acclimatization, while the increase in the value ofparameter "S" was much greater in climatic heat accli‑
matization than in physical training in heat. Thus achange m the value of "t" represents the training effectand change in the value of "S" represents the effect ofclirnatic heat acclimatization. The mean values and thestandard deviation values of A, B, C, I, S and "t" calcu‑
lated using the data obtained in the present experimentare given in Table 4. The mean values of A, I and "t"were significantly smaller in summer than in winterwhile the mean value of S was significantly greater insummer than in winter. The mean values of A, I and "t"decreased significantly and the mean value ofin‑creased significantly during physical training in heat.
The contribution of the relative increase in heart rate(A) to the value of I was greater than the relative risein rectal temperature (B) and relative body weight loss

and Industry, ed. by Herzfeld, C.M. Reinhold, New York,3:299‑305.

3 ) Bass, D.E. and Henschell, A. (1956): Responses of body
fluid compartments to heat and cold. Physiol. Rev., 36:
128‑144.
4 ) Coon, C.S., Garn, S.M. and Birdsell, J.B. (1950): A studyof the problems of race formation in man. Charles C.
Thomas, Springfield, Illinois.

5 ) Conn, J.W., Johnson, M.W. and Louis, L.H. (1946):
Acclimatization to humid heat, A function of adrenal
cortical activity. J. Clin. Invest., 25;912‑913.

6 ) Hori. S. (1977): Changes in physiological reactions to a

hot environment induced by climatic change and physi‑
cal training. Jpn. J. Trop. Med. Hyg., 15(2):93‑104.
7 ) Hori, S., Tsujita, J., Tanaka, N. and Mayuzumi, M.
(1978) : Studies on heat tolerance of subtropical natives
after migration to a temperate zone. Int. J. Biometeor.
22 (2) :82‑93.

8 ) Hori, S., Ihzuka, H. and Nakamura, M. (1982): Compar‑
ison of physiological responses to heat between subtropi‑

cal and temperate natives. Jpn. J. Trop. Med. Hyg., 10(3,4) :229‑237.

1999 ) Ihzuka, H., Hori, H. and Akamatsu, T. (1986): Seasonalvariations of physiological responses to heat of subtropi‑
cal and temperate natives. Int. J. Biometeor., 30(2) :107
‑113.

10) Kuno, Y. (1956): Human perspiration, Charles C.
Thomas, Springfield, Illinois.

11) Leithead, C.S. and Lind, A.R. (1964): Heat stress andheat disorders. Davis, Philadelphia.
12) Lind, A.R. and Bass, D.E. (1963): The optimal exposure
time for the development of accilmatization to heat.
Federation Proc., 22:704‑708.

13) Ohara K. (1966): Chloride concentration in sweat, its
individual, regional, seasonal and some other variations,and interrelations between them. Jpn. J. Physiol., 16:274‑290.

14) Ogawa, T. and Asayama, M. (1986): Quantitative analy‑
sis of the local effect of skin temperature on sweating.
Jpn. J. Physiol., 36, 417‑422.

15) Ogawa, T., Asayama, M. and Miyagawa, T. (1982):
Effects of sweat gland training by repeated local heat‑ing. Jpn. J. Physiol., 32, 971‑981.

16) Piwonka, R.W., Robinson, S., Gay, V.L. and Manald, R.
S. (1965): Preacclimatization of men to heat by train‑ing. J. Appl. Physiol., 20:379‑384.
17) Robinson, S., Turrell. E.S., Belding, H.S. and Horvath, S.

M. (1953): Rapid acclimatization to work in hot cli‑
mates. Am. J. Physiol., 140:168‑176.
18) Senay, L.C., Jr. (1972): Changes in plasma volume andprotein content during exposures of working men tovarious temperature before and after acclirnatization toheat. Separation of the roles of cutaneous and skeletal
muscle circulation. J. Physiol., 224:61‑81.
19) Shvartz, E.. Bhattacharta, A., Sperinde, S.J., Brock, P.J.,

Sciaraffa, D. and Beaumount, W.V. (1979): Sweating
responses during heat acclimation and moderate condi‑
tioning. J. Appl. Physiol., 46, 675‑680.

20) Tsujita, J., Hori, S., Mayuzumi, M., Meren, J. andTanaka, N. (1989): Effect of ambient temperature on
blood consitituents during prolonged exercise. Proceed‑ing of the 1lth ISB Congress. ed. Driscoll, D. and Box,
E.O. 287‑295.

21) Wyndham, C.H., Strydom, N.B., Morrison, J.F., Wil‑
liams, C.G., Bredell, G.A.G., von Rahden. M.J.E., Hold‑sworth, L.D., van Graan, C.H., van Rensburg, A.J. and

Munro. A. (1964): Heat reactions of Caucasians andBantu in South Africa. J. Appl. Physiol., 19:598‑606.

201

Jpn. J. Trop. Med. Hyg., Vol. 21, No. 4, 1993, pp. 201 206

BASIC STUDIES ON THE MONGOLIAN GERBILAS A SUSCEPTIBLE HOST TO
FILARIAL INFECTION=

COMPARATIVE STUDIES ON SERUM BIOCHEMICALVALUES BETWEEN THE WILD‑COLORED GERBILAND THE COAT COLOR MUTANTS
KAZUHIRO SHICHlNOHE1'2, MASUMI SHIMIZU '2, KATSUYA HAGA2TETSUO IEDOKOR02 AND KOICHIRO FUJITA2
Received August 30 1993/Accepted October 7 1993

Abstract: We made a comparative study on serum biochemical values of mature Mongolian gerbils between
the wild‑colored (agouti) and the coat color mutants of both sexes by autoanalyzers. The coat colors of themutants were white spotted‑agouti, albino, black and white spotted‑black. All of serum biochemical values
we measured of different coat color gerbils were not significantly different each other. We did not find any

lipemic sera and any hyperglycemia in all coat color gerbils. Compare to the other common laboratoryrodents, patterns and values of serum protein fractions of the gerbils were different. Namely, A/G ratios
and rates of y‑globulin fraction of gerbils were much higher than those of mice and rats and were the sameas those of human beings.

INTRODUCTION
We made a comparative study on serum biochemi‑
cal values of Mongolian gerbils between the wild‑col‑ored (called agouti) gerbils and the coat color mutants

kept the closed colonies of the coat color mutants ofMongolian gerbils which have white‑spotted agouti,
albino, black or white‑spotted black coat color andthose coat color mutants are expected as a model tostudy influences of genetic background on responseof both sexes in this report.

against filarial infections. We have studied to charac‑

The Mongolian gerbil (Meriones unguiculatus ) canbe kept and handled so easily in laboratories and is so
readily infected with some filarias (Ash and Riley,terize the coat color mutants of Mongolian gerbils

1970a,b; Dalesandro and Klei, 1976; Matsuda et al.. 1976)

values.

that it has been used as an experimental host of filar‑
iasis. Biological characteristics of this animal werestudied in 1960s when it was started to be used as alaboratory animal (Schwentker, 1963; Ruhren, 1965;

biologically and hematologically (Shimizu et al.. 1990,

1991) and we also need data on serum biochemical

MATERIALS AND METHODS

only as a material, that means as a host only for provid‑
ing parasites used for research, and it's own characteris‑

Mongolian gerbils were fed a commercial pellet forsmall rodents (MF; Oriental Yeast Inc., Tokyo, Japan)
and water ad libitum under a conventional condition andhoused 5 animals of the same sex in one cage. The roomtics have not been studied systematically.
Genetic influences on host reactions against filarial

temperature and the humidity were maintained at 24
2'C and 60 5 %, respectively. The coat color mutants

infections could not be studied because those gerbilsof the gerbils maintained in our laboratory were agouti,

commonly used were only agouti type which was notwhite‑spotted agouti, albino, black and white spotted

controlled genetically at all. But we have raised and‑black and origins of these gerbils were described in ourMays, 1969) . However after that, it has been raised just

1

2

Department of Laboratory Animal Science, Nippon Medical School, 1‑1‑5 Sendagi, Bunkyo‑ku, Tokyo 113, Japan
Department of Medical Zoology, Faculty of Medicine, Tokyo Medical and Dental University, 1‑5‑45 Yushina, Bunkyo‑ku, Tokyo
113, Japan

202

Table I Serum biochemical values of the agouti type and the coat color mutants of Mongolian gerbils (1)Glucose(mg/dl)Cholesterol

Coat color

Sex

Agouti

Male
Female89 + 10

66

89

Male
FemaleMale
FemaleMale
FemaleMale
Female77 + 14

76 9
77i 7

90

91 + 12White spoted‑agouti

AlbinoBlack
White spotted‑black

Triglyceride(mg/dl)(mg/dD

18

18

55i 3*

1037

10

81 17* *
45+ 2
41 9

91

18

74

102

19

39 + 10

88

14

81 16
65+ 8

87 + 1676

23

87 + 14

62

18

44 8
77 26

89

63

9

70

18

15

57

18

18

The values are derived from 14 to 15‑week‑old gerbils and represent means istandard deviations for 10 gerbils of eachgroup. * , * * The difference between sexes is significant by Student's t‑test ( * p<0.05, * * p<0.01). There are no
significant coat color differences in those values.

Table 2 Serum biochemical values of the agouti type and the coat color mutants of Mongolian gerbils (2)Coat color

Sex

Agouti

Male
FemaleWhite spotted‑agouti

Male
FemaleAlbinoMale
FemaleBlack

MaleFemaleWhite spotted‑agouti

Male
FemaleGOT

GPT(IU/1)

388

LDH

(IU/D

152

50

(IU/D

ALP

(IU/D

16

950

452

164 47

61 23
41+22

728

476

172 + 79

910

393

99 21

408 + 120

29

887

433

88 + 22

436

97
103

43i22
43 26

1131 i547

276

363 + 188
355

152

16

422

55

54

30

1128 i 197

403

139

42 23

1012 559

145 53151 25
121 43
129 29

342

143

37 14

389+ 81

34 + 11900 205
950i470

110+27The values are derived from 14 to 15‑week old gerbils and represent means

711 45982

20

standard deviations for 10 gerbils of eachgroup. Glutamate oxaloacetate transaminase, glutamate pyruvate transaminase, Iactate dehydrogenase and alkaline
phosphatase are abbreviated to GOT, GPT, LDH and ALP, respectively. There are no sex differences and no coat color
differences in those values significantly by Student's t‑test.

Table 3 Serum biochemical values of the agouti type and the coat color mutants of Mongolian gerbils (3)Coat color

Uric acid Urea Creatinine

Sex (mg/dl)
nitrogen(mg/dD (mg/dl )Male 1.2:!:0.9 23 8
Female 0.7 0.2 31+ 5
White spotted‑ Male 0.5 0.3 26 6
Agoutiagouti Female 0.8 0.5 25i 2
Male 0.9 0.3 31+ 2
AlbinoFemale I . 2

Black

I . 1 21 :!

13

Male 0.8i0.2 18

3Female 1.4 0.8 20+ 4

White spotted‑ Male O. 7 0.4 18

black Female I .Od:0.7 25

3

6Sodium Potassium Chloride Calcium(mEq/D (mEq/D (mEq/1) (mg/dl)

0.4+0.1
0.5+0.1

157 + 2

0.5 0.1
0.5 0.1
0.5+0.1
0.5 0.1
0.3 0.l
0.4+0.1

156 + 20.3 0.1
0.4 0,l157

6155 + 2149

7.3 1.5
7.0 0.9
6.5 2.1
7.8 1.l3 10 . 7 + I . 2

147i9 9.4i2.3
151 2 9.5 0.7
149

3 10 . 4 + I . 4

149 + 3147 + 1

8.7 0.8
8.8 0.7

121 3 8.8 0.3
123i 6 8.8 0.7122i 2 7.7+0.8
121+ 1 8.1+0.6
119 3 9.0 0.8
113ilO 9.4+0.6
118 1 9.0 0.7
115 3 9.2i0.4
115 4 9.1i0.9
113 2 9.4 0.7

The values are derived from 14 to 15‑week old gerbils and represent means istandard deviations for 10 gerbils of each group.
There are no sex differences and no coat color differences in those values significantly by Student's t‑test.

203

Table 4 Serum biochenucal values of the agouti type and the coat color mutants of Mongolian gerbils (4)Coat color

AgoutiWhite spotted‑agouti

AlbinoBlack
White spotted‑black

A/G ratio

Sex

Total protein (g/dl)

Albumin (g/dl)

MaleFemaleMale
FemaleMale
FemaleMale
FemaleMale
Female5 . 98 i o . 30

3.61 0.16

1 . 566.24d:o. 34

3 . 68O . 22

1 . 44 i O . 09

3.6li0.13

1 . 57+ o . 18

3 . 65

1 . 395. 92

O . 15

6. 32 i O . 25

O . 18O . 29O . 25O . 21

3.49 0.08

1.47+0.11

5.80 0. 32

3.60d 0.13

1'. 68 i O . 37

5. 62 i o . 15

3 . 39dl O . 10

5. 94 d: o . 21

3 . 56

1.53 0.20
1.52i0.27

5.84+0. 39

3 . 50 i O . 13

5 . 76i o . 27

3 . 455 . 86The values are derived from 14 to 15‑week‑old gerbils and represent means

O . 14

O . 081.51 0.20
1.51 0.23

standard deviations for 10 gerbils of eachgroup. There are no sex differences and no coat color differences in those values significantly by Student's t‑test.

previous report (Shimizu et al.. 1990). Ten animals ofeach coat color of both sexes aged 14 to 15 weeks old
were used in the experiment.
Samples of blood were collected from the hearts ofanimals anesthetized with ether in the morning after
overnight fasting. They were centrifugalized and thesera were stored at ‑80'C until assays.

The autoanalyzer, Hitachi 736 (Hitachi co. Ltd.
Japan) or Spotchem (Kyoto Daiichi Kagaku co. Ltd.
Japan) was used to measure the followings: glucose(GOD‑POD) , glutamate oxaloacetate transaminase(GOT) (OAC‑POP‑POD), glutamate pyruvate tran‑
saminase (GPT) (POP‑POD), Iactate dehydrogenase(LDH) (lactate pyruvate method) , alkaline phos‑
phatase .ALP) (p‑nitrophenylphosphate) , cholesterol(CE‑CQD‑POD) , triglyceride (LPL‑GYOD‑POD) , uricacid ('tiricase‑POD), urea nitrogen (urease method),
creatine (Jaffe reaction) , sodium, potassium and chlo‑ride (electrode) , calcium (OCPO , total protein (BCG) .

Protein fractions were measured by using electrophor‑esis. Those values were evaluated statistically by Stu‑
dent's t‑test.

RESULTS

Mongolian gerbils. Mean values of all coat color gerbils
of each fraction were as follows: Albumin 57.8‑62.3 %,al globulin 1.9‑2.5 %, a2‑globulin 8.8‑11.1 %, p‑globulin

4.4‑9.2 %, y‑globulin 18.1‑23.6 %.
All the values we measured were not significantly
different between the agouti gerbil and the coat color

mutants.

DISCUSSION
The present paper is the first report of a compara‑tive study on serum biochernical values between the
agouti type and the coat color mutants of Mongolian
gerbils. In this experiment, significant differences
between the agouti gerbil and the coat color mutants
were not detected in serum biochemical values. But
som values of the agouti gerbils were different fromthose reported by earlier workers.
Mays (1969) reported that uric acid value had maledominance in Mongolian gerbils, but we could not findsuch sex difference in that of each coat color gerbil(Table 3). Activity of y‑glutamyltranspeptidase of
gerbils (data not shown) was under measurable level
similar to that of the other small rodents (Tanimoto,1988). GOT and LDH of gerbils were extremely higher

Table I to 4 show serum biochemical values ofthan those of rats and mice (Table 2) . As those samples

Mongolian gerbils including the coat color mutants after

were not hemolysis and those data were re‑confirmed,

overnight fasting. Means of some biochemical values

we thought that is one of particular characteristics ofwere as follows: glucose 77‑102 mg/dl, cholesterol 62‑81
mg/dl, triglyceride 37‑81 mg/dl (Table 1) , GOT 276‑436

gerbils. The more precise experiments in future areneeded to determine it. A11 the sera from total 100
gerbils we tested were not lipemic at all, although

IU/1, LDH 712‑1131 IU/1 (Table 2), uric acid 0.5‑1.4
mg/dl in males and 0.7‑1.4 mg/dl in females (no sex
difference) (Table 3), total protein 5.62‑6.32 g/dl, A/G
ratio 1.39‑1.68 (Table 4).
Figure I shows patterns of serum protein fractions ofearlier workers pointed out that 30 % of the sera were
lipemic (Ruhren, 1965; Rich; 1968; Mays, 1969). Thisdifference might be caused by that earlier workers did
not establish the suitable way of feeding for gerbils

204
a. ,naldb. female

70

70

60

60

50

so
P

c
o
o
c9

c:

o

‑a 40
co

=

c

b
30
E

o
L
CL

40

o

o.
o
eL

E
=

=
h
o

30

*
o

co

<,,

2o

2o

10

10

coat color

sp‑A9Agsp‑BI
A9 sp‑A9 At sp‑BI BI coat cetor
Al BI

Figure 1 Serum protein fractions of the agouti type and the coat color mutants of male (a) and female (b)Mongolian gerbils. The values are derived from 14 to 15‑week‑old gerbils and represent means
standard deviations for 10 gerbils of each group. The coat colors of agouti, white spotted‑agouti, albino,

black and white spotted‑black are abbreviated to Ag, Sp‑Ag, Al, Bl and Sp‑Bl, respectively. Each bar

represents one of protein fractions as follows; I alburnin, I al globulin,‑globulin and] a2‑globulin, I] p

1 y globulin. There are no sex di‑fferences and no coat color differences in those values

significantly by Student's t‑test.

which were newly used as a laboratory animal in thosedays. Now they are raised in certain condition withnutritionally well‑balanced commercial pellets. In fact,

values of total cholesterol and triglyceride of gerbils

were not so high and similar to those of the othercommon laboratory rodents (Wolford et al., 1986).
Nakama (1977) reported that the Mongolian gerbil washereditary spontaneously diabetic animal. Bonquist(1972) said that there were a few animals which showeddiabetes‑like symptorr!s in a stock of old obese gerbils.

But glucose values we tested were within a normal
range like mice, rats (Wolford et al., 1986) and humans(Ishii et al., 1981) and glucosuria has not been observedat all either (data not shown) . Moreover, food andwater intake of gerbils has been stable (Shimizu et al.,1990). These facts reveal that the Mongolian gerbil isthose of human beings (Ishii et al.. 1981; Tanimoto,1989) . As globulin value changes according to environ‑mental conditions, the breeding conditions of gerbils
might bring the same level of globulin as human beings.
Namely, gerbils in our laboratory have been kept underbacteriologically unsterilized condition called conven‑tional condition and that condition was bacteriologicallythe same as where human beings live. We think that is

one of advantages of the gerbil as an experimental
model of human filariasis as well as their peculiar
sensitivity because the breeding condition of animals
would effect on sensitivity to parasites (Shichinohe etal.. 1990). The Mongolian gerbil has been known to be
the most successful host for experimental filariasis suchas Brugia spp. (Ash and Riley, 1970a,b), Dipetalonema

not a hereditary spontaneously diabetic animal in gen‑vitae (Dalensandro and Klei, 1976) and Litomosoides
carinii (Matsuda et al.. 1976). This character of theeral.

gerbil is really valuable because it is very expensive and

The patterns of serum protein fractions of gerbils

difficult for studies of filariasis to use their own natural

were very different from those of rats and mice.

hosts in laboratories. For example, natural mammalian

Namely, A/G rat"ios (Table 4) and rates of y‑globulin
fraction (Figure 1) of the Mongolian gerbils were muchhigher than those of rats and mice and were the same as

hosts of Brugia pahangi are monkeys, cats and dogs
(Edeson, 1959; Edeson and Wilson, 1964) and numerous
attempts to introduce this worm into commonly avail‑205

able laboratory rodents such as rats and mice have been
failed (Laing et al., 1961; Ahmed, 1967; Ash and Riley,1970b; Sucharit and MacDonald, 1973). However, thereason why gerbils are readily infected with them has
not been fully understood. The earlier studies on experi‑

notes on infections in other rodents, J. Parasitol., 56, 962‑9683 ) Ash, L.R. and Riley, J.M. (1970b) : Development ofsubperiodic Brugia malayi in the jird, Meriones un‑
guiculatus, with notes on infections in other rodents, J.

that the sensitivity of gerbils to filarial worms were

Parasitol., 56, 969‑973
4 ) Bonquist, L. (1972): Obesity and pancreatic islet hyper‑
plasia in the Mongolian gerbil, Diabetologia, 8, 274‑282

brought by less responsivility of cellular immunity

5 ) Dalesandro : D.A. and Klei, T.R. (1976): Evidence formental filarial infections in Mongolian gerbils suggested

against parasites (Lammie and Katz, 1983; Klei et al..

immunodepression̲ of syrian hamster and Mongolian

1990). On humoral immunity of gerbils, they had lgGjirds by Dipetalonema vitae infections, Trans. R. Soc.

antibody (Tomisato et al.. 1983; Farrar et al.. 1991) , IgE

Trop. Med. Hyg., 70, 534‑535
6 ) Edeson, J.F.B. (1959): Studies on filariasis in Malaya:
the periodicity of the microfilariae of Brugia malayi andB. pahangi in animals, Ann. Trop. Med. Parasit., 53, 381‑like antibody in their intact sera, produced it against

worm antigen derived from Dirofilaria immitis (Shi‑chinohe et al.. 1992) and much higher y‑globulin level

than mice and rats in the present experiment. These
facts that gerbils had enough components of humoral
immunity were thought to be interesting in contrast with

their peculiar sensitivity to parasites. They might in‑directly support the statement of earlier workers that
their sensitivity was caused by less cellular immunor‑
eactivity.

Mongolian gerbils commonly used were only agouti

type and their genetic background have not been
controlled at all in contrast with rats and mice whichhave been controlled genetically to establish many in‑bred strains. We have studied characteristics of the coatcolor mutants of gerbils as compared with the agouti
gerbil and until now, we have not found any significant
differences between the agouti gerbil and the coat color

mutants in the serum biochemical values as well as in
biological and hematological values (Shimizu et al..
1990, 1991) . Then, if the different coat color gerbils will

respond differently each other against filarial infection,

it is thought that genetic background of gerbils effects

on response against filarial infection. Consequently,

present study on serum biochemical values of theMongolian gerbil would be useful as a basic data to
analyze mechanisms of host‑parasite relationship using
gerbils and the coat color mutants of Mongolian gerbils
would be expected an useful model to study an influenceof genetic background on reactions against filarial infec‑‑3877 ) Edeson, J.F.B. and Wilson, T. (1964): The epidemiology
of filariasis due to Wuchereria bancrofti and Brugia
malayi. A. Rev. Ent., 9, 245‑268
8 ) Farrar, R.G., Klei, T.R., McVay, C.S. and Coleman, S.U.

(1991): Quantitative characterization of antibody
responses to single and multiple rugia pahangi infec‑
tion in jirds, J. Parasitol., 77, 718‑72=69 ) Ishii, A., Toyama, T. and Sakaguchi, T. (Traslated)(1981): Clinical biochemical and hematological refer‑ence values in normal experimental animals and normal

humans, Mitruka, B.M. and Rawnsley, H.M. 2nd ed.,345pp., Seishi shoin Ltd., Tokyo
10) Klei, T.R., McVay, C.S., Dennis, V.A., Coleman, S.U.,
Enright, F.M. and Cassey, H.W. (1990): Brugia pahangi;
Effects of duration of infection and parasite burden on

lymphatic lesion severity, granulomatous hypersen‑
sitivity and immune responses in jirds (Meriones un‑
guiculatus) . Expl. Parasitol., 71, 393‑405

11) Laing, A.B.G., Edeson, J.F.B. and Wharton, R.H. (1961) :Studies on filariasis in Malaya: further experiments on
the transmission of Brugia malayi and Wuchereria ban‑crofti. Ann. Trop. Med. Parasit., 55, 86‑92
12) Lammie, P.J. and Katz, S. (1983): Immunoregulation inexperimental filariasis II; Responses to parasite and non‑parasite antigen in jirds with rugia pahangi. J.
Immun., 130, 1386‑1389

13) Matsuda, H., Takaoka, M. and Tanaka, H. (1976):
Effect of diethylcarbamazine on microfilaria ofLitomosoides carinii in jird, Meriones unguiculatus. Jpn.
J. Parasitol., 25, 94‑98

14) Mays, A. Jr. (1969): Beseline hema:tological and bio‑chemical parameters of the Mongolian gerbil ( Meriones

tion.

unguiculatus) . Lab. Anim. Care, 19, 838‑842

REFERENCES
1 ) Ahmed, S.S. (1967): Studies on the laboratory transmis‑
sion of sub‑periodic Brugia malayi and B. pahangi. I.
The resistance of guinea‑pigs, rabbits and white mice toinfection, Ann. Trop. Med. Parasit., 61, 93‑1002 ) Ash, L.R. and Riley, J.M. (1970a): Development of
Brugia pahangi in the jird, Meriones unguiculatus. with

15) Nakama, K. (1977): Studies on diabetic syndrome andinfluences of long‑term tolbutamide administration in
Mongolian gerbils (Meriones unguiculatus) . Endocrinol.
Japon., 24, 421‑433

16) Rich, S.T. (1968): The Mongolian gerbil (Meriones
unguiculatus) in research, Lab. Anim. Care, 18, 235‑243

17) Ruhren, R. (1965): Normal values for hemoglobin con‑centration and cellular elements in the blood of Mon‑

206
golian gerbils,, ‑Lab. Anim. Care, 15, 313‑320
18)

Schwentker, V. (1963): The gerbil; A new laboratory

19)

Shichinohe, K:. Shimizu, M.. Asakawa, M. and Ishizaki,

animal. Illinois Vet., 6, 5‑9

M. (1990): Effect of rearing conditions on Ence‑phalomyocarditis (EMO virus‑induced diabetes in
mice, Exp. Anim., 39, 377‑381
20)Shichinohe, IC, Shimizu, M.. Kato, K., Tamura, N.,
Tsukidate, S. and Fjita, K. (1992): Basic studies on the
Mongolian ge, rbil as a susceptible host to filarial infec‑

tion; IgE‑like‑ antibody formation against Dirofilaria
immitis adult.worm antigen, Jpn. J. Trop. Med. Hyg., 20,
271‑28121) Shimizu. M., Shichinohe, K., Tsukidate, S. and Fujita, K.

(1990): Basic studies on the Mongolian gerbil as asusceptible host to filarial infection; Comparative
studies on growth and reproduction among coat color
mutants and genetic analysis of coat colors, Japan J.
Trop. Med. Hyg., 18, 301‑310
22) Shimizu, M., Shichinohe, K.. Tsukidate, S. and Fujita. K.

(1991): Basic studies on the Mongolian gerbil as asusceptible host to filarial infection; Comparative
studies on hematological features between the wild‑colored gerbil and the coat color mutants, Jpn. J. Trop.

Med. Hyg., 19, 331‑338
23)

Sucharit, S. and MacDonald, W.W. (1973): Brugia
pahangi in small laboratory animals: attempts toincrease susceptibility of white rats to Brugia pahangi
by host selections, S.E. Asian. J. Trop. Med. Publ. Hlth.,24) Tanimoto, Y. (1988) : Biochemistry of blood and urine

in experimental animals, Laboratory Animal Series, 5,
189pp., Soft Science Inc., Tokyo
25) Tanimoto, Y. (Ed.) (1989): Serum biochemical finding,Tajim , Y. (Supervised) , Biological reference data bookon experimental animals, 151‑201, Soft Science Inc.,Tokyo
26) Tomisato, M., Nakajima. Y., Nariuchi, H. and Nogami,

S. (1983): Detection of lgG antibodies in Mongolian
jirds infected with Brugia pahangi by enzyme‑linked
immunosorbent assay, Jap. J. Parasit., 32, 43‑54
27) Wolford, S.T., Schroer, R.A., Gohs, F.X., Gallo, P.P.,

Brodeck, M., Falk. H.B. and Ruhren, R. (1986) : Refer‑ence range data base for serum chemistry andhematology values in laboratory animals, J. Toxicol.
Environm. Health, 18, 161‑188

Jpn. J. Trop. Med. Hyg., Vol. 21, No. 4, 1993, pp. 207 211

207

ANTIBODY FREQUENCY DISTRIBUTION CURVEFOR RISK ASSESSMENT OF A MALARIAEPIDEMIC IN THE SUDANSHIGEYUKI KANOl, SALAH H. EL SAF12, FAKHR EL DIN M. OMER2PILARITA T. RIVERAl, AHMED A. EL GADDAL2 AND MAMORU SUZUKll
Received September 6 1993/Accepted October 7 1993

Abstract: A seroepidemiological study of the prevalence of malaria, employing the frequency distribution
curve of ELISA titers, was conducted. Comparison of the shape of the curves obtained in the two villages
in the Sudan before and after the flood of the Blue Nile in 1988, clearly demonstrated the existence of certain
malarial foci which might pose a potential risk for development of malaria epidemics in the area, althoughthese malarial foci could not be detected by either slidepositivity rate or seropositivity rate. Thus,
application of this method is of considerable value in identifying latent malarial foci where control measures
should be strengthened.

INTRODUCTION
During the advanced stages of malaria control
programmes, cases in which parasites in the peripheral

blood are in such low numbers that they cannot bereadily detected by microscopical examination becornea majority (Sadun, 1972). On such an occasion, a moresuitable method for epidemiological assessment isrequired, in order to detect latent malarial foci in appar‑

ently controlled areas. In the present study, follow up
epidemiological surveys iv re conducted in two villages

in the Sudan in 1987 and 1989. In 1987, the frequency
distribution curve of ahtib dy titers (Kagan, 1973)
revealed a potential danger of future malaria epidemics
in one of the villages. In 1988, the area was flooded bya heavy rainfall which increased the vector breedingsites and caused the occurrence of malaria epidemics
even in the controlled area. In fact, our survey conduct‑

ed in 1989 clearly confirmed an epidemic in a village
where the potential risk had earlier been presumed. In
the present study, the usefulness of the frequency distri‑bution curve of antibody titers in the identification oflatent malarial foci is discussed.

SUBJECTS, MATERIALS AND METHODS
Study area
The Gezira irrigated are , which covers a total of 2
million acres, Iies between latitude 13'N and 15'N in thecentral part of the Sudan (Fig. 1). It has a populationof about 2 million. The area is irrigated by gravity
through a canal system from Sennar Dam on the Blue
Nile. The area has a hot dry season from April to Junewith an average daily temperature of 32'C and relative
humidity of 20%, and a cool dry season from December
to March, with average daily temperature of 22'C, andrelative humidity of 30%. Average annual rainfall isabout 225 mm occurring between July and September(EI Gaddal et al.. 1985). The Gezira area is covered by
the Sudan Blue Nile Health Project which is responsible
for control of water‑borne diseases including malaria.

In December 1987, two villages were selected for thepresent pilot study. Mobi, a well controlled area with
low incidence of malaria cases. On the other hand,
Sennar, Iocated beside the dam, maintained many prob‑
able vector breeding sites, and was regarded as hyperen‑

demic. A follow up survey was carried out in January
1989 in the same villages. In August 1988, between our
two surveys, the heaviest rainfall in the last 40 years
caused the flooding of the Nile.

1

2

Department of Parasitology, Gunma University School of Medicine, 3‑39‑22 Showa‑machi, Maebashi, Gunma 371, Japan
Blue Nile Health Project, Ministry of Health, Wad Medani, Sudan

208

The Sudan

THE NILE
Khartoum

THE BL UE NILEGEZIRAO Wad Medani

THE WHITE NILE

MObi O
Sennar e

1 OOkmFigure 1 Map of the Sudan Gezira showing study area.

Blood sampling
Sera from the adult were collected by venipuncture.

Blue Nile Health Project at Wad Medani. On the day
before the test was done, the lyophilized antigen was
dissolved to a final protein concentration of 250pg/ml

In children and infants to whom venipuncture was not
acceptable, plasma specimens were obtained by a finger‑prick method (Kano et al., 1989). Thick smears weremade simultaneously from all the donors and subjectedto parasite detection by microscopic observation. Inbottom of the wells. Sera or plasma at 2‑fold dilutions,

Sennar, most of the blood samples were taken from

1:32‑1:1024, were applied. Enzyme reaction was perfor‑

school children 7‑14 years of age, 61 out of 72 of the

med by the VectastainTM ABC kit, which consisted of
biotinylated anti‑human lgG goat serum and avidin
biotin complex horseradish peroxidase (VECTOR Labo‑children tested in 1989 had also been sampled in 1987. In

Mobi, blood samples were obtained at the same healthpost in 1987 and 1989 where donors were gathered fromthe neighbourhood. Seventeen out of 46 in 1987 and 58

and distributed in each well of a 96‑well U‑bottom
microtiter plate (Greiner, Germany) . The plate was left

overnight allowing the antigen to dry and coat theratories Inc., CA) . Addition of the substrate, 4‑chlor0‑1

ABC ‑ ELISA
The measurement of antibodies by the ABC‑ELISA‑naphtol, generated a blue colour in the well with apositive serum, clearly readable with the naked eye.
The final dilution of a serum sample which gave thiscolour was considered as the antibody titer. And thetiter equal to or greater than 1:64 was considered asout of 90 in 1989, were school children 7‑14 years of age.

was carried out according to the method described bypositive according to our previous report (Sato et al..

Sato et al. (1990). This serological method was devised1990) .

particularly for field use. Briefly, the antigen was
obtained from an in vitro cultivation (Trager and'Jen‑

Statistical analysis

sen 1976: Miyagami and Waki, 1985) of a Plasmodium
falciparum (P.f) strain (SGEI strain from Gambie;

The distribution of titers of each group werecompared statistically by the Mann‑Whitney Test (Udonated by Dr. Ambroise‑Thomas in 1979) . The antigen‑test). The seropositivity and slidepositivity rate of thewas lyophilized and brought directly to the laboratory ofgroups were compared by the Chi square test.

209

**1

Table 1 Distribution of malaria titers by the ABC‑ELISA
1h

ABC‑ELISA TITERS< 32 32
1987 Sennar

Mobi
1989 Sennar
Mobi

64

8 30( 1)

128 256 512 1024 Total

+* 26

13
17
15(1) + '.' 3

12
7(4)

63 l

1

2

4

8(4)

2

80(5)
46(o)

2

2

O

O

12(4)

8(5)

12(9)

15(11)

72(34)

7(3)8( I )

3

7(3)

90(7)

( ) Number of people who showed parasitemia

obtained in 1987.

showed only a single peak at low titer of 1:32, whichindicated no occurrence of recent epidemics among the
inhabitants. . Thus the frequency distribution qurves
showed some epidemiological difference between Sen‑nar and Mobi. It was presumed that some potential foci1 ‑ I ) Slide positivity rate:

which might be provoked by some elements into epi‑

RESULTS
1 . Comparison of the epidemiological features ofmaiaria in Sennar and Mobi based on the results

In Sennar, 5 out of 80 examined showed P.f para‑sites in the thick smear, but none out of 46 in Mobi (Tab.

1) . However, no significant difference was recognizedby the Chi square test between the slide positivity rateof Sennar and that of Mobi (P=0.123) .
1 ‑ 2 ) Antibody titration by the A C‑ELISA:
Antibody titers obtained in Sennar and Mobi did notshow normal distribution and the variables of therespective groups differed from each other. Therefore,
instead of comparing the geometric mean of the recipro‑
cal titers (GMRT) of each group by the t‑test, statisti‑
cal difference of the groups was tested by the U‑test.
Significant difference was not convincing between Sen‑

demics, existed in Sennar; whereas, in Mobi, the poten‑
tial risk was minimal if any.
2 . Changes in the' ‑ epidemiological features after theflood of 1988.
2 ‑ I ) Sennar: ‑In January 1989, when the second survey was con‑ducte , every malariometric index dealt with in thepresent study showed a statistically significant risecompared to those in 1987. Parasite rate increased from

6.3% to 47.2% (P<0.001). Seropositivity rate in 1989
was 75.0% versus 52.5% in 1987 (P<0.001). And siguifi‑cant difference in GMRT was also recognized between

nar and Mobi in 1987 (P=0.006). Seropositivity rate

1987 (1:26.0) and 1989 (1:27.3), (P<0.001). Moreover thewas also compared by the Chi square test: 42 out of 80(52.5%) manifested positive titers in Sennar; While in
Mobi, 16 out of 46 (34.8%) were seropositive (Tab. 2).

most remarkable change was observed in the pattern ofHowever significant difference was not recognized

1024, reflecting the epidemic occurring at that time.

between the two groups (P=0.055) .
l ‑ 3 ) The frequency distribution curve of antibody

2 ‑ 2 ) Mobi:

frequency distribution curves of antibody titers. In 1989,

the second peak became higher, at the highest titer of 1:

Seven out of 90 (7.8%) were slidepositive and 26

The frequency distribution curves of the ABC(28.9%) were seropositive in 1989 (Tab. I and 2).
Comparison with the slidepositivity rate and ser‑

‑ELISA titers are shown in Fig. 2. A birnodal distribu‑
tion of titers was obtained for 1987 data in Sennar. It

significant differences (P = 0.052 and 0.066 respectively) .

titers :appeared that the peak at 1:512 was a reflection ofsmall, recent past epidemics which had occurred in thisvillage. While the antibody distribution profile in Mobi
Table 2 Percentage positives by the ABC‑ELISA andmicroscopy

Mobi
Sennar
ABC‑ELISA/microscopy ABC ELISA/nucroscopy
1987 52 . 5
1989 75 . O

6.3

34 . 80.0

47 . 228 . 9

7.8

opositivity rate obtained in 1987 showed no statistically

GMRT in 1989 (1:25.3) was also statistically compared tothat in 1987 (1:25.2), but the difference was not signifi‑

cant (P=0.114). However, the antibody titer distribu‑
tion curve showed a small second mode at 1:256 in 1989
(Fig. 2) , indicating that small scale epidemics had occur‑

red in this village. This finding could only be demon‑
strated by using the frequency distribution curve ofantibody titers.

210

7O
6O

50

70

:h

Gc

h:)>
h5
O

Oa:

L,
co

F'm 19e8

30

40
30

20

20

IO

10

O

O

70

70

60

50

January 198g

50

40
UJ

60

Sennar: December 1987

60

Mobi: Decel7lber 1987

50

40

F'm 19e830nJanuary 1989

40
30

20

20

10

10

O

o

n

(32 32 64 128 256 512 I024 <32 32 64 128 256 512 1024
ABC‑ELISA TITER (1 :n)Figure 2 Comparison of the frequency distribution curves of the antibody titers.

DISCUSSION
Seroepidemiology is important, in that it provides

more informative period prevalence data which cannot
be obtained through microscopy (Draper et al., 1972;

Colins and Skinner, 1972).

done, and they might constitute the probable focus of

succeeding transmission in the community. Thus thesecond mode of the curve was regarded as the siguifi‑cant indicator of potential risk of malaria epidemics
which might occur as a consequence of environmental or

In particular seroe‑
pidemiological assessment is valuable when a malaria

true after the disastrous flood occurred in August 1988,

control programme reaches the more advanced stage

which adversely affected the well implemented malaria

and the endemicity of malaria becomes very low in thecontrolled areas (Sadun, 1972: Kagan, 1972). Usually
the degree of transmission or prevalence of malaria isstudy villages. In the study in 1989, increased malaria

measured by either GMRT or percentage of the sample
population that is serologically positive (Kagan et al.,1969). However it was clearly shown in the present

man‑made changes. This assumption was proven to becontrol programme in the whole area including ourprevalence was actually observed by microscopy. And
the frequency distribution curve of the ABC‑ELISA
titers obtained in Sennar changed into a hyperendemic
pattern, showing the highest peak at the highest anti‑

study that both parameters of assessing malaria preva‑
lence were not adequate in providing detailed informa‑

body titer 1:1024 (Fig. 2). This pattern was the reflec‑tion of the very recent past or current infection in thetion particularly on the potential risk of an epidemic. In

majority of the people at the time of the survey.
On the other hand in Mobi, the frequency distribu‑
tion curve of the ABC‑ELISA titer, characterized by asingle low‑titered peak in 1987, changed into a bimodal

Table 2, statistical significant difference between thepercentage positives of Sennar and Mobi in 1987, was

not convincing by both serology and microscopy.
However, the antibody titer distribution curve of Se nar

pattern suggestive of a small, recent past outbreak ofin 1987 revealed a bimodal pattern showing a Second

malaria in the village in 1989. Comparison of ser‑peak at titer 1:512 (Fig. 2). The subjects who manifest‑

opositivity and slidepositivity rates did not present sig‑
nificant difference between the results before and aftered titer of 1:512 were those considered to have con‑tracted malaria about the time when the survey was

the flood. Indeed, seropositivity rate was lowered in2111989, in contradiction to what had actually happened.
It was thus affirmed in this study that the actual
prevalence of malaria may be more precisely reflectedin the shape of the frequency distribution curve ofantibody titers. Application of this method will be of

considerable value in identification of early or evenpresumptive malaria foci where control measures must
be concentrated.

ACKNOWLEDGEMENTS
This study was supported in part by grants from

Monbusho International Scientific Research Pro‑
gramme (NO. 62045009), and Japan‑U.S. Medical Sci‑
ence programme. We thank Dr. Taiichi Sato, Iecturer ofthe Department of Public Health, Gunma University
School of Medicine, and Prof. Kumiko Sato of theCollege of Medical Care and Technology, Gunma Uni‑
versity, for helpful discussion.

REFEREN CES
1 ) Collins. W.E. and Skinner, J.C. (1972): The indirectflourescent antibody test for malaria, Am. J. Trop. Med.

Hyg., 21, 690‑695
2 ) Draper, C.C., Voller, A. and Carpenter, R.G. (1972) : The

epidemiologic interpretation of serologic data inmalaria. Am. J. Trop. Med. Hyg., 21, 696‑703

3 ) El Gaddal, A.A., Haridi, A.A.M., Hassan, F.T. and
Hussein, H. (1985) : Malaria control in the Gezira‑Managil irrigated scheme of the Sudan, J. Trop. Med.
Hyg., 88, 153‑159
4 ) Kagan, I.G. (1972) : Evaluation of the indirect hemagg‑

lutination test as an epidemiologic technique formalaria, Am. J. Trop. Med. Hyg., 21, 683‑689
5 ) Kagan. I.G. (1973): Parasitic Diseases. In: Serological

Epidemiology, 155‑168, Academic Press, London
6 ) Kagan. I.G., Mathews, H.M., Rogers, W.A. and Fried, J.
(1969) : Seroepidemiological studies by indirect haemag‑
glutination tests for malaria, Bull. World Health Organ.,41, 825‑841

7 ) Kano, S., Takagi, T., E1 Gaddal, A.A. and Suzuki, M.
(1989) : A new method of plasma collection suitable forlarge scale seroepidemiological surveys of malaria in
the tropics, Trans. Roy. Soc. Trop. Med. Hyg., 83, 304
8 ) Miyagami, T. and Waki, S. (1985) : In vitro cultivationof Plasmodium falciparum under aerobic atmosphere ina C02 incubator, J. Parasitol., 71, 262‑263 '9 ) Sato, K., Kano, S., Yamaguchi, H., Omer, F.M., El Safi,

S.H., El Gaddal, A.A. and Suzuki, M. (1990) : An ABC‑ELISA for malaria serology in the field, Am. J. Trop.
Med. Hyg., 42, 24‑27lO) Sadun, E.H. (1972): The research and development ofserologic tests for malaria, Am. J. Trop. Med. Hyg., 2,

677‑682

11) Trager, W. and Jensen, J.B. (1976) : Human malaria
parasites in continuous culture, Science, 193, 673‑675

213

J pn. J. Trop. Med. Hyg., Vol. 21, No. 4, 1993, pp. 213 217

MALARIA SEROLOGY AS AN ALTERNATIVE TO
PARASITE DETECTION IN A HIGHLY ENDEMICAMAZONIAN COMMUNITYSHIGEYUKI KANOl, YOSHIAKI UTSUNOMIYA2, ALFRED TAKESHI HONDA3,
OSEAS SANTOS4, WALDIR DE SOUZA MIRANDA4, MILTON MASATO HIDA5,KEIZO MATSUMOT02 AND MAMORU SUZUKll
Received August 27 1993/Accepted October 22 1993

Abstract: The Indian colony under study is located in relatively inaccessible part of the Amazonian jungle,
restricting frequent visits. Malaria survey in such a community requires methods which would yield reliable
results even from a single visit. Malaria was regarded as a minor disease by the inhabitants who appeared
to be free from malaria. The reported incidence was very low, and in fact, no parasitemia was detected in
the blood smears of a group of inhabitants obtained through our short visit. But on the contrary, an avidin

biotin peroxidase complexed enzyme‑linked immunosorbent assay (ABC‑ELISA) revealed a 100% preva‑
lence of malaria antibodies in the population. Therefore, serological studies which can obtain the periodprevalence are useful for the assessment of malaria situation in the highly endemic community which is notreadily accessible, or whose populations seem to have acquired a certain degree of immunity depressing
parasitemia to submicroscopic levels.

give a truer picture of the endemicity of malaria at the

INTRODUCTION

place.

Immunological methods are regarded as the mostuseful for determining malaria experience of popula‑

MATERIALS AND METHODS

tions especially in the advanced stages toward the eradi‑

cation (Collins et al., 1968; WHO, 1972). Ordinary slide

observation cannot detect latent malaria foci in thecontrolled area because of the lowered possibility ofencountering patent parasitemias (Pampana, 1969).
However, we present another circumstance in which

Study area

The colony was located along the Xingu, about
700km to the south‑west of Bel6m, the capital of thestate of Par (Figure 1). The inhabitants were of the
Amazonian Indian tribe of Kaiap6s with a population ofserological investigation in retrospective diagnosis of

912 people then (Photo 1). They were basically amalaria is of high practical value.

subsistent community, isolated from the rest of theWe had an opportunity to visit an Amazonian In‑

people economically and culturally. However, thedian colony in the state of Par , which per se had been
isolated culturally from the surroundings. We could juststay there for a very short time, during which time we
obtained the blood samples and detailed malarial his‑

life, helping the promotion of community health. A
nurse was being sent who stayed with them and took

tories through a questionnaire. Parasites were not

the state of Par

detected in any individuals examined, and the declaredmalaria histories were rather incidental. However, theABC‑ELISA revealed a high antibody prevalence in thecommunity. The serological profile was considered topositive cases out of a total population of 4,857,199 in

1

2
3
4
5

Brazilian Government started to preserve their way ofcare of their health and needs. The malaria situation of
was one of the worst in Brazil. Annual

report of 1988 of Fundacao Nacional de Satide(National Foundation of Health) described 125,628
Par (Minist6rio da Satide, 1988) . In the nearest city

Department of Parasitology, Gunma University School of Medicine, 3‑39‑22 Showa‑machi, Maebashi, Gunma 371, Japan
Department of Internal Medicine, Institute of Tropical Medicine, Nagasaki University, 1‑12‑4 Sakamoto, Nagasaki 852, Japan
Hospital Amazo^nia, Benefic ncia Nipo‑Brasileira da Amaz6nia, Bel6m, Par , Brasil
Fundacao Nacional de Satide, Ministerio da Satide, Bel m, Par , Brasil
Faculdade de Medicina de Botucatu, Universidade Estadual Paulista, Botucatu, Sao Paulo, Brasil

214

The Amazon
Bel6m

??//

‑

The state of Para¥¥((

BR lZIL¥¥' 7

The indian colOny
Figure I The study area'

Photo. 1 Beautifully painted and dressed indian children in the colony.
The photograph was taken with their permission.

215(50km from the colony) , Sa6 Felix do Xingu, which was

the center of deforestation and gold mining, 24,145
parasite positive cases out of 69,570 people were report‑ed (Ministe rio da Satide, 1993) .

Kit, which consisted of biotinylated, affinity‑purified
anti‑human lgG made in goat, avidin DH and biotinylat‑

ed horseradish peroxidase H (VECTOR Laboratories
Inc., CA) . Addition of the substrate, 4‑chloro‑1‑naph‑
tol, generated a blue color in the well with a positive

serum, clearly readable with the naked eye. This test

Sub jects

Blood smears and sera were collected withcan be carried out without using major electrical equip‑

informed consent from 20 donors, 8‑75 years of age(September 5, 1990). They were asked their age, sex,
and malaria episodes (Table l). The samples werebrought back to Gunma University School of Medicine

ment. The final dilution of a serum sample which gave
this color was considered as the antibody titer.

RESULTSand subjected to microscopical observation and the ABC‑ELISA. The results were made known to them after
the tests were completed.

Malarial incidence
No parasite was detected from microscopical obser‑

vation of Giemsa‑stained thick blood smears which

ABC ‑ELISA
The measurement of antibodies by the ABC‑ELISA
was carried out according to the method described bywere obtained from the 20 donors. Examination of local
malarial records kept by Fundacao Nacional de Satide
revealed 5 parasite‑positives out of 222 feverish cases in

Sato et al. Briefly, the antigen was obtained from an in

the colony for the 2‑week period prior to our visit.

vitro cultivation (Trager and Jensen, 1976; Miyagami
and Waki, 1985) of Plasmodium falciparum (P.f) strain

Inhabitants' response to the questionnaire indicated that

(SGEI strain; donated by Dr. Ambroise‑Thomas inprophylactic measures were taken against the contrac‑

1979). Twenty microliter of the antigen solution at a
protein concentration of 250 pg/ml was placed in eachwell of a 96‑well U‑bottom microtiter plate (Greiner,
Germany) . The plate was left overnight allowing thetion of malaria.

antigen to dry and coat the bottom of the wells. Sera at

ABC‑ELISA titers and declared malaria episodes.

2‑fold dilutions, 1:32‑1:l024, were applied. Enzyme

Examinees numbered 3 and 9 declared the last malaria
episode 5 days before the survey and showed consider‑reaction was performed by the VECTASTAlNTM ABC

malaria was considered as a minor disease and that no

The ABC‑ELISA titers and declared malaria episodes
Table I is the individual records on age, sex, theTable 1 Individual records of a batch of 20 donors

No Declared Sex ABC‑ELISA' age titer

Declared malaria episodeNo. of times / Last episode prior to survey

38

f

64

1

several years

2

26

f

128

2

7 months

3

32

f

l024

2

5 days

4

f

6

44

256
512
64

N one

5

40
73

7

74

512

1

1 month

8

28

mf

32

1

several years

mf

256
128

3
1

5 daysseveral years

mf

64

1

4 months

64

1

1 year

mf

512

N one

64

N one
N one

mf

1
1

1 yearseveral years

9

8

10

73

11

30

12

32

13

56

14

28

15

75

16

25

mf

512
64

2

17

47

f

18

47

f

512
512

N one
N one

19

24

f

64

1

1 year

20

50

f

128

l

l month

5 months

216

able high ABC‑ELISA titers at 1:1024 and 1:256 respec‑tively. Six individuals declared that they had nevercontracted malaria, however, one of them showed thetiter at 1:64, another at 1:256 and the rest 4 at 1:512. This

discrepancy will be explained granting that these 6

people had acquired a certain degree of immunity
against malaria. In fact, there is a correlation between

the ABC‑ELISA titers and ages of the donors (exclud‑ing No. 3 and 9) with a correlation coefficient at 0.73,

a)
O

5

O
CO

4

O
q)

3

E

2

which suggests that one is likely to manifest higher titer1

after the cumulative infectiQn as one grows older in theOcommunity.

32 64 1 28 256 5 1 2 1 024n

The frequency distribution curve of antibody titers

The frequency distribution curve of the ABC

ABC‑ELISA titer (1 :n)‑ELISA titers is shown in Figure 2. A bimodal distribu‑

Figure 2 The frequency distribution curve oftion of titers was obtained: the first peak which consists

the ABC‑ELISA titers.

of the individuals showing the titer at 1:32 to 1:128 issuggestive of past malaria episodes from months to

big discrepancy between parasite and antibody rates inyears before the survey, and the second one at 1:256 to
1:1024 may be reflecting the recent malaria episodes orcumulative malaria infection. This pattern of frequency
distribution curve suggests that malaria transmission

the present study may probably be due to a certain
degree of acquired immunity among the indians which
depresses parasitemia down to submicroscopic levels(Draper, 1972). Repeated infection with malaria para‑

was actively taking place in the colony (Rivera, 1993).

sites maintaining continuous antigenic stimulation may
have produced adequate antibody levels of the inhabit‑

DISCUSSION

ants which play, in part, a protective role against
malaria (Kuvin and Voller, 1963).
Serological assessment has considerable value inthe advanced stages of a malaria control program whenthe endemicity of malaria has become very low in thetarget area (Kagan, 1972; Voller and Draper, 1982).

Seroepidemiology provides more informative periodprevalence data than can be obtained through micros‑
copy (Collins and Skinner, 1972; Draper, 1972). It isparticularly useful if, Iike in our present survey, the area

where malaria epidemiological studies are to be con‑ducted is not easily accessible, or one cannot stay there

long enough to get standard malariometric indices of the

population. In our present survey, point prevalence dataobtained by microscopical observation of the bloodsmears showed no evidence of malaria transmission.
But the serological method revealed a variety of posi‑tive antibody titers. Indeed, the frequency distribution

curve of the ABC‑ELISA titers with a peak of highDespite this, we stress in this manuscript the importanceof serological surveys of malaria for populations in ahighly endemic area. The value and ease of applicationof a new serological method, the ABC‑ LISA, which
requires no major electrical equipment will also facili‑tate seroepidemiological studies in remote rural commu‑
nities.

antibody prevalence suggested potential risks of malaria

ACKNOWLEDGMENTS

epidemics in the population, or that active transmission

This study was supported in part by grants fromJapan International Cooperation Agency. We express
sincere gratitude to Mr. Tsugio Shishido, secretaryof the disease were actually occurring in the area
(Rivera, 1993) . Thus, we could get a truer picture of theendemicity of malaria in the village by the serologicalmethod even though our visit to the place was short andgeneral, and other staffs of Benefic6ncia Nipo‑Brasileir‑

temporary.

We also thank Dr. B.D. Akanmori of Gunma University
School of Medicine, and Professor Kumiko Sato of theCollege of Medical Care and Technology, Gunma Uni‑

Generally, the absence of patent parasitemia can bemisleading, since patency is influenced by the use of
antimalaria drugs and often occurs only intermittently
during malaria infections (Kagan, 1972). However thisa da Amazo^nia, for providing us with local fascilities.

versity, for helpful discussion.

217

REFERENCES
1 ) Collins, W.E., Skinner, J.C. and Jeffery, G.M. (1968)
Studies on the persistence of malarial antibody response,
Am. J. Epidemiol., 87(3) , 592‑598

2 ) Collins, W.E. and Skinner J.C. (1972): The indirect
fluorescent antibody test for malaria, Am. J. Trop. Med.
Hyg., 21 (5) , 695‑696

3 ) Draper, C.C., Voller, A. and Carpenter, R.G. (1972): The

epidemiologic interpretation of serologic data inmalaria, Am. J. Trop. Med. Hyg., 21(5) , 696‑703
4 ) Kagan, I.G. (1972): Evaluation of the indirect hemagg‑

lutination test as an epidemiologic technique formalaria, Am. J. Trop. Med. Hyg., 21(5) , 683‑689
5 ) Kuvin, S.F. and Voller, A. (1963) : Malaria antibody
titers of west Africans in Britain, Br. Med. J., 477‑479
6 ) Ministe rio da Satide, Superintende^ncia de Campanhas deSatide Ptiblica, Diretoria Regional do Par (1988) :Relat6rio Tecnico Administrativo, Bel6m, 29‑117 (inPortugese)7 ) Miyagami, T. and Waki, S. (1985) : In vitro cultivationof Plasmodium falciparum under aerobic atmosphere ina C02 incubator, J. Parasitol., 71(2) , 262‑263

8 ) Pampana, E.J. (1969) : A text book of malaria eradica‑tion, 2nd ed., Oxford University Press, London
9 ) Rivera, P.T., Kano, S., Miguel, E., Tongol, P. and Suzu‑ki, M. (1993) : Application of seroepidemiology in identi‑

fication of local foci in a malrious community in Pal‑awan, Philippines, Am. J. Trop. Med. Hyg., 49(5) , 608‑61210) Sato, K., Kano, S., Yamaguchi, M., Omer, F.M., El Safi,

S.H., El Gaddal, A.A. and Suzuki, M. (1990): An ABC‑ELISA for malaria serology in the field, Am. J. Trop.
Med. Hyg., 42(1) , 24‑27

11) Trager, W. and Jensen, J.G. (1976) : Human malaria
parasites in continuous culture, Science, 193, 673‑675

12) Voller, A. and Draper, C.C. (1983) : Immunodiagnosis
and seroepidemiology of malaria, Br. Med. Bull., 38 (2) ,173‑177
13) WHO (1972) : Serological testing in malaria, Bull. Wld.
Hlth. Org., 47, 527‑532

Jpn. J. +***' M*d'219**" v*1"*' *' +' *9'3 ** '*""POSSIBLE APPLICATION OF SURGICALTREATMENT OF CHAGASIC MEGACOLON

AT SANTA CRUZ GENERAL HOSPITAL, BOLIVIA
EDWlN CRESPO MENDOSA1, FREDDY UDALRICO GUTIERREZ VELARDEl, SIMEON SENGOKUl,
CHIAKI MIYOSH12 AND TAMOTSU NAKASA2
Received September 6 1993/Accepted October 27 1993

Abstract: Chagas' disease, a chronic parasitic disease caused by the protozoan Tr)panosoma cruzi, is oneof the endemic diseases in Central and South America. In Bolivia its seroprevalence is very high and themost common digestive manifestation of chronic Chagas' disease is megacolon.
A new protocol for the surgical treatment of Chagasic megacolon has been started in Santa Cruz
General Hospital. It consists of a peri‑operative management, a choice of operative technique and a longterm follow‑up.
From July 1989 to August 1992, among 46 Chagasic seropositive megacolons, 37 definitive operations ofcolorectal resection and anastomosis were perfomed. Although five cases developed early postoperativecomplications, there were no deaths in the group which had definitive operations.
The purpose of this study is to establish a standard surgical management for Chagasic megacolon in
Bolivia.

INTRODUCTION
Chagas' disease, a chronic parasitic disease causedby the protozoan Tr)panosoma cruzi, is a disease endmic

to all Central and South American countries. It isestimated that about 16 million people are infected(WHO, 1990; UNDP/WORLD BANK/WHO, 1991).
In Bolivia, Chagas' disease is a serious public healthproblem because its vector ( Triatominae) is found in
83% of the national territory. The seroprevalence of
Chagas' disease is very high (29.8%‑70%) (Foianini,1986) .

There are three stages in Chagas' disease: acute,indeterminate and chronic (WHO Expert Commitee for
Chagas Disease, 1991). After the short acute stage inwhich the symptoms can be very mild and atypical, along asymptomatic phase may last several years. In thisstage, up to 30% of people will suffer damage to theircardiac, digestive or neurological systems. In Bolivia,
the rpost common digestive manifestation of the chronic
Chag s' disease is megacolon.
Many authors have reported on the surgical treat‑ment of'Chagasic megacolon (Joffre and Moreira, 1988;
Milton, 1 85; Moreira et al.. 1988) . However because

12

there is a great difference as for economic situations andlevel of medical care among South American countries,
it is difficult to directly apply these results to Bolivian
cases.

Santa Cruz is the second largest city in Bolivia,

located in the eastern tropical area where Chagas'
disease is also endemic. Currently many people immi‑
grate from the high Andian area to Santa Cruz that theprevalence of Chagas' disease is getting higher.

In Santa Cruz General Hospital (SCGH), a newprotocol for the surgical treatment of Chagasic
megacolon had started. It consists of a pre‑ and post
‑operative management, a choice of operative procedure
and a long term (at least five years) follow‑up.
The purpose of this program is to establish a stan‑

dard surgical management for Chagasic megacolon in
Bolivia.

This report is a preliminary evaluation of the newprotocol.

MATERIALS AND METHODS
Figure I shows the algorithm for the managementof Chagasic megacolon.

Department of Surgery, Santa Cruz General Hospital, Bolivia
Department of Surgery and Bureau of International Cooperation, International Medical Center of Japan, 1‑21‑1 Toyama,
Shinjuku‑ku, Tokyo 162, Japan

220

Clinical Signs & Symptoms
Suspect of Chagasic megacolon(Clinical megacolon)

Fecaloma volvulus ; Other

Clinicaltype type : type

types

Preoperative :' Continuous ene.ma : Rectosigmiodscope : Enema, I tc.

and / or :preparation :Com lementa studies

etc. :

Manual xtraction : mucosal Yecrosis :r no :: ryes no ;yeserum s or agaslDevotvat ion

rLl

IHA

+ ‑

no yes

l

.1'

h d

'

(if6 j.1(Serum Alb.

l FA

>3.5 )‑ +Treatnentyes)Chagasic Megacolon
Climcal findin s

Fecaloma

Volvulus

ther

(yes)

Figure 2 Peri‑operative management of patients with
Chagasic megacolon.

‑operative preparation, surgical treatment and post
‑operative management (Figure 2) .
For example, in the case of fecaloma, a continuous
enema is the first choice, then a manual extraction. If
the preparation is still not completed, first a colostomy

Pre‑operative preparation
Sur ical treatment

Emergent operation
Definitive operation(usually Hartmann's operation)' is made. When thepatient is suspected volvulus, the urgent rectosigmoids‑
copy has to be performed. If the necrosis of the colonalmucosa is found, an emergent Hartmann's operation isindicated. If its mucosa was intact, devolvulation witha rectosigmoidscope or a rectal tube has been the first

Post‑operative management
Follow u
Figure 1 Algorithm for management of patients with

Chagasic megacolon.

All cases diagnosed as megacolon in the Depart‑
ment of Surgery at SCGH were examined by serological
Chagasic tests. The IHA (Indirect hemagglutinationtest) was performed first. When the IHA was negative,
the IFA (Immunofluorecent assay) was examined. All

choice.

All patients with hypoalbuminemia had their serum
albumin levels corrected up to 3.5 mg/dl. Only the casewith normal serurn albumin levels (over 3.5 mg/dD has
to indicate the definitive operation of resection andanastomosis.

All operations listed above were perfomed by
Bolivian surgeons. Colorectal anastomosis was donewith manual sutures usually, while some of cases
required mechanical suture devices (EEA stapler) .

seropositive cases with the IHA or the IFA were includ‑ed as Chagasic megacolon cases in this protocol. TheseRESULTS

Chagasic megacolon cases were categorized into three
types by clinical forms; Fecaloma type, Volvulus type
and Other type. (Fecaloma is a fecal mass impacted bymegacolon cases were admitted to the Department ofSurgery of SCGH. 46 were seropositive for Chagasic

prolonged retension of feces.)

Diagnosis of fecaloma could be made by the historyof a long period without defecation and palpable feces

From July 1989 to August 1992, 61 clinicaltest. These 46 cases included the following studies.
As it is shown Figure 3, the mean age of Chagasic
megacolon patients was 52.7 years old and 31 were male.
There were 25, 16 and 5 case of Fecaloma type, Volvulus

with rectal digital examination. Volvulus was char‑acterized by a sudden onset of severe abdominal pain

type and Other type, respectively (Figure 4).

and usually found no palpable feces in rectum.
Each type has its own protocol consisting of precases (24%), pulmonary tuberculosis in 2 cases (5%)

Associated problems were cardiomyopathy in 9

221

Table 1 Associated problems of 37 patients with defini‑20

tive operationl"o

No. of patients%

Cardiomyopathy

924 . 3(including jabnormal ECG)
Pulmonary tuberculosis
Rectal prolaps

2

5.4
2.7
2.7
2.7

Associated problem

,,

‑e
b
a'

'I

l Female15

El Male31

illIncisional hernia
Acute pancreatitis

10

,
l'
,E
,,
a!
o

1

4.6‑5.0

4.1 ‑4. 5

0‑10 11‑20 21‑30 31‑40 41‑50 51‑60 61‑70 71‑80

3.6‑4,0Figure 3 Distribution by age and sex of 46 patients with

3.1 ‑3.5

Age (yrs)

Chagasic megacolon.
2.6‑3.0

Clinical Megacolon (6 1)

2.1 ‑2.5

Chagasic Megacolon (46)
(Chagasic seropositive)'Number et cases

Figure 5 Pre‑operative serum albumin level of 37 patientswith definitive operation.

Fecaloma Volvulus Other*

type(20) (type1 2)type(5)

Clinical Megacolon(46)Non surgical (7)Surgical (39)*NO complication or recurrence

Figure 4 Clinical types of Chagasic megacolon.

etc. (Table 1).

Preperative serum albumin level is shown in Figure

5. Twenty four cases (65%) were under 3.5 mg/dl.

Arnong 46 Chagasic megacolon cases, sevenreceived only non‑surgical management. Although 6 ofthem were resolved non‑surgically, they refused further

definitive o eration. The other case died of severe
dehydration and acidosis before operation.

Surgical treatment was performed in 39 cases.
Definitive operation of colorectal resection and anas‑

DirectDelayed*colorectal

colorectal

anastomosis

anastomosis(28) **

ffartnaun's
operation
only

(9)

(2)

Defilritive Colostomy

operation (37) (1 1)' First st8ge5 H'rtmalm's operation" I Cecor'ctal and 27 s igmoidorecta] anastolBQsis

Figure 6 Surgical management of 46 patients with
Chagasic megacolon.

tomosis was done in 37 of them. In 2 cases only
Hartmann's operation was performed due to refusal to

The distance of the colorectal anastomosis from thethe definitive operation.
Of the 37 definitive operations, one stage (direct)

anal verge is shown in Figure 7. The largest were
between 5 to 10 cm. In three cases with very low

anastomosis was done in 28 cases and two stages

anastomosis (less than 5 cm) mechanical suture devices(delayed) anastomosis was done in 9 other cases (Fig‑(EEA stapler) were used.
Five cases had developed early post‑operative com‑ure 6) .

222

8.1 %

12
,
l'

: 10

R<5

29.

t'

5.1‑1010.1‑15
(cm)

e
hl

eI 8

S

:

6

4

2

o

11 zo 21 30 31 40 41 50 51‑60 61‑70
Figure 7 Location of colorectal anastomosis of 37 patientswith definitive operation

Table 2 Early post‑operative complication observe in
37 patients with difinitive operation

Complication(days)

Figure 8 Length of hospitalization for 37 patients withdefinitive operation

Table 3 Patient mortality according to management of

Chagasic megacolon

No of patients %Stercoraceous fistula

3

Hemorrhage (intraperitoneal)
Abdominal wall abscess

1
1

8.1
2.7
2.7

plications. Stercoraceous fistulas were developed inonly 3 cases (8.1%) (Table 2).
The mean length of hospitalization was 34.5 days

Number Complications Death
Direct28

4 (3.6)

Fecaloma type3

Volvulus type

1

Delayed9

1 (1.1)

Fecaloma typeo

Volvulus type1

(Figure 8) .

o

o

(%)

There vere no deaths in the group which underwent
definitive operation. Of the two cases which died: one

areas this is true even now where no specialist is avail‑able.

was in the group of Hartmann's operation only and the

The seroprevalence of Chagas' disease is very highother was in non surgical group (Table 3) . The motality

where some authors report it is over 70% in several
areas (Foianini, 1986). Therefore, chronic Chagas'

rate of total Chagasic megacolon was 4.3% (2/46), butthat of the definitive operation group was O% (0/37).

DISCUSSION
Because of the difference of the economic status
and level of medical care among developing countries,
the application of a surgical treatment established in
one country may not be appropriate for another country.

Many studies about the surgical treatment of
Chagasic megacolon have been reported, but there arevery few from Bolivia (Saucedo, 1988) . The prospective

study of Chagasic megacolon was the first trial in
Bolivia.

Usually the surgical treatment of Chagasic
megacolon was only a colostomy for the fecaloma or alaparotomy for the devolvation of volvulus. In ruraldisease is becoming one of the most serious and common
public health problems in Bolivia where the megacolon
is more often encountered than the megaesophagus.
In such circumustance, the surgical management for
Chagasic megacolon is indispensable for public health.
The protocol, developed by Bolivian doctors, is intended

to establish a standard surgical management for Char‑gasic megacolon in Bolivia.
Below are some points to be noted for information.
1 ) Operative technique
An operative technique should be determined by thephysiopathology of the disease. Chagasic megacolon
originates in the aganglionic portion of the intestine anda decreased number of ganglion cells are found through‑out the colon (Earman, 1972; Todd et al.. 1969). Fromthe point of the physiopathology, resection of the dilatat‑

223

ed colon is inessential for the operation of themegacolon.
The enlarged intestine, usually rectosigmoid colon,
is likely to be the most common locus of the fecaloma or

volvulus. Therefore the resection of rectosigmoid isdone for these complications (Earman, 1972; Todd et al.,1969) .

recommended.
When the delayed anastomosis indicated, thepatient generally went back home to the rural village
with a colostomy. After that, most never came back to
the hospital for the definitive operation unless any
urgent problems appeared.
Therefore this protocol applied the direct anas‑

Some authors recommend Duhamel's operation

tomasis as first priority and the delayed anastomosis in(Milton, 1976; Joffre and Moreira, 1988). From thisthe same hospitalization as second.
There were no deaths in either groups of the direct

point of view, it is ideal for the relapse of complications

should be minimal. However this technique reports thehigh rate of morbidity and mortality.

and delayed anastomosis. Morbidity was also low in

In developing countries, there are not many special‑both of them.
In this study, covering colostomy was performed forists in digestive surgery. Moreover very few patients

just a single case. Covering colostomy was worth

can receive TPN (total parenteral nutrition) . For these

considering but the result verifies it is not always neces‑reasons, the operative technique should be as straight assary if the pre‑operative preparation is completed.

possible.

One of the problems with this protocol was theMoreira et al. have operated 624 patients with
Chagasic megacolon using Duhamel's technique. Mor‑

lengthy hospital stay required when they received thedefinitive operation during the same hospitalization.

tality rate was 6.6% while early post‑operative compli‑
cations such as into‑anal suppration (7.6%) and lower

cases financially. Without such assistance, they would

SCGH and the Japanese project team supported someOn the other hand, the abdominal rectosig‑

not have been able to continue further treatments.
ldeally, the protocol will remain sustained by them‑moidectomy is easier and the rate of complication is notselves, but financial condition of the hospital is quite

so high. Most general surgeons in Bolivia can perform

inadquate.
Another problem is the difficulty of the follow‑up ofcolon necrosis (2.4%) were developed.

it. This is the reason why the abdominal rectosig‑
moidectomy was chosen as a standard technique in thisprotocol.

the patients. Most patients do not return to hospital
unless they face severe problems. Alternative incen‑

2 ) Location of anastomosis
Another important point is the location of color‑ectal anastomosis from the anal verge. If it is lower,recurrence of fecaloma or volvulus is fewer, because
more of the aganglionic portion of the rectum would betives, such as free charge of examination etc. should beresected. However, the lower anastomosis makes it

and the course should be continued.

considered.
This is a preliminary report and we regret it is too

short to assess the study more in detail. However the
early results of this are considered fairly satisfactory

more difficult for the operative techniques and therefore

is recognized higher rate of post‑operative complica‑
tions. Milton reported that the anastomosis should be
performed within 2‑4 cm from the pectinate line.
Mechanical suture devices are used for lower color‑ectal anastomosis. They can also be used also for casesof Chagasic megacolon (Cuitait, 1980), but in manycases the wall of the colon is too thick to be anas‑
tomosed with them.
Moreover it is so expensive that they can not be
used for most patients in Bolivia.

In this study, most anastomoses were performed
between 5 to 10 cm from the anal verge. Only 3 caseswith lower anastomosis used mechanical suture devices.
3 ) Direct or delayed anastomosis

For decreasing complications, the delayed anas‑

tomosis (two or three stages operation) was

ACKNOWLEDGEMENTS
This study was supported in part by Japan Inter‑national Cooperation Agency (JICA) .
We express our heartfelt thanks to all members of
the Department of Surgery, Santa Cruz General Hospi‑
tal and the Japanese technical cooperation team, allinvolved in Santa Cruz General Hospital project.

REFERENCES
1 ) Cutait, D.E. (1980):

Anastmosis mecanica por

grampeamento cirugia colorectal, Pren. med. argent., 67,

658‑6622 ) Earlam, R.J. (1972): Gastrointestinal aspect of Chagas'
disease, Digestive Disease, 17, 559‑571

2243 ) Foianini. J.E. (1986) : Enfermedad de Chagas‑Revision
Bibliografica, Santa Cruz (Bolivia)
4 ) Joffre M. de Rezende, and Moreira, H. (1988): Chagasic

megaesophagus and megacolon. Historical view andpresent concepts, Arq. Gastroenterol. Sao Paulo, 25(special issue) , 32‑43

5 ) Milton da Silveira, G. (1976) : Chagas' disease of thecolon, Br. J. Surg., 63, 831‑835
6 ) Moreira. H., de Rezende, J.M.. Sebba. F., de Azevedo, I.

F.. Leite, A.C.A. and Soares, E.P. (1985): Chagasic
megacolon, colo‑proctology, 5, 260‑267

7 ) Saucedo, E. (1988): 15 anos de experiencia en el
tratamiento de megacolon chagasico, Anurario delAteneo de Medicina, 11‑16, Santa Cruz de la Sierra
8 ) Todd, I.P., Porter, N.H., Morson, B.C., Smith, B.. Fried‑mann, C.A. and Neal, R.A. (1969) : Chagas disease of thecolon and rectum, Gut, 10, 1009‑1014

9 ) UNDP/WORLD BANKIWHO Special Programme for
Research and Training in Tropical Diseases (TDR)(1991): Tropical Diseases Progress in Research 1989
‑1990, 69‑77, WHO, Geneva
10) WHO (1990): Chagas Disease, Frequency and geogra‑
phycal distribution, Weekly Epidemiological Record, 65,
257‑264.

11) WHO Expert Committee on the Control of Chagas
Disease (1991): WHO Technical Report Series 811,
Control of Chagas Disease, WHO, Geneva

225

Jpn. J. Trop. Med. Hyg., Vol. 21, No. 4, 1993, pp. 225 229

AN OUTBREAK OF ASEPTIC MENlNGITISIN OKlNAWA IN 1993

YOSHIHIRO MAKlNO ' 2, EIICHI TAMANAHA3, ZENJI MIYAZAT03,

JUNKICHI SHIMABUKUR03, CHIKARA MATSUMORA3, CHORSON YAN3,RYUJI ISHIHARA3, TAKASHI MACHIDA3, NAOTO YAMADA3,SAKAE ARAKAK11, MASAYUKI TADANOI AND TOSHIHIKO FUKUNAGAl' 2
Received October 1 1993/Accepted November 5 1993

Abstract: In the summer of 1993, an epidemic of aseptic meningitis had broken out in Okinawa island, thesouthernmost area of Japan. It started in June (or probably in May) , reached a peak early in July andsubsided in August. A total of 216 specimens (cerebrospinal fluids, throat swabs and sera) was collectedfrom the patients with aseptic meningitis and viruses were recovered from 83 specimens (38.4%). Fiveisolates randomly selected were examined for the identification of the virus. The virus was identified asechovirus type 30 (ech0‑30) by plaque reduction neutralization test using enterovirus antiserum pools andmonospecific antiserum. Large‑plaque and small‑plaque phenotypes of ech0‑30 were isolated. The twowere neutralized by anti‑ech0‑30 sera. For the plaque assay of ech0‑30 in RD‑18S cells, the use of
tragacanth gum for the overlay medium could reduce the incubation period to 25‑30 hr.

MATERIALS AND METHODSINTRODUCTION
Aseptic meningitis is a disease of central nervous
system, mainly caused by viral infection. Most of theSpecimens

epidemics of aseptic meningitis in summertime arein Okinawa city, Okinawa Prefecture, during Junecaused by enterovirus infection (7, 9) . Echovirus type 30

through August 1993. Cerebrospinal fluid, serum andthroat swab were collected from the children with theclinical symptoms of aseptic meningitis. They were

(ech0‑30) was first isolated from a patient with aseptic
meningitis in 1959 (3). In Japan, since the first report of

The specimens were collected at a private hospitalech0‑30 infection in 1978, a number of epidemics of
aseptic meningitis were reported to be due to ech0‑30

stored at ‑70'C, then transferred to our laboratory.

infection (4, 10, 13). In Okinawa island, the southern‑most area of Japan, we had experienced a big outbreak

Ce I lsof aseptic meningitis in the summer of 1989. Many

‑18S) (6) cells were kindly supplied by Dr. Hara,

strains of ech0‑30 were recovered from cerebrospinal

National Institute of Health, Japan. They were grownin Dulbecco's modification of Eagle's medium (DMEM)

fluids (CSF) and throat swabs (TS) taken from thepatients. In the summer of 1993, we encounteredanother outbreak of aseptic meningitis in Okinawa
island. We collected specimens at a hospital and tried to

A stable line of human rhabdomyosarcoma (RDsupplemented with 8% fetal calf serum (FCS) andantibiotics, and maintained in the same medium but FCS

was reduced to 2% (MM).

isolate the etiological agent of the disease. In this paper

we describe the results of the virological investigationsof the outbreak of aseptic meningitis occurred in thesummer of 1993.

Virus isoiationTS, obtained using cotton swab, was dissolved in 2

ml of DMEM containing antibiotics. The specimens(CSF, TS and serum) were centrifuged at 15,000 rpm for
5 min and the supernatant was used for the recovery ofvirus.

1. Department
2.

of Virology, Faculty of Medicine, University of the Ryukyus, 207 Uehara, Nishihara, Okinawa 903‑01, Japan

Research Center of Comprehensive Medicine, Faculty of Medicine, University of the Ryukyus, 207 Uehara, Nishihara, Okinawa 903‑01, Japan

3. Departmentof Pediatrics, Nakagami Hospital, 723. Chibana, Okinawa city, Japan.

226

Confluent monolayer of RD‑18S cells was preparedin 25‑cm2 flask or 24‑well plate. The culture medium
was replaced with MM (Iml for flask or 0.5 ml per wellfor 24‑well plate) and the monolayer was inoculatedwith 0.1 or 0.05 ml of the specimen. The cells wereincubated at 37'C for 1.5 hr in a 5% C02 atmosphere.
Then MM was added to the cultured cells (4 ml into
flask or 0.5 ml per well to 24‑well plate) , and the cells

were cultured at 37'C for one week and observed forcytopathic effect (CPE). When the cells showed maxi‑

mum CPE, the cultured fluid was harvested. The
centrifuge supernatant of the cultured fluid was

No. of patients

45

40

35
30

25
20
15
IO
5
O

7!J.‑ 7/lS ‑ 7/25 ‑ 81' ‑ 818‑d/lp d/20dl27‑. 7nl
d / 2d 7/3 7110
7l 1 7 rl24 7/31 8 1 f

Figure I Number of patients specimens were taken by

inoculated into the new RD‑18S cells (second passage) .
The virus stock was prepared at the second passage andweek. The specimens (CSF, TS and Serum)
were collected at a hospital in Okinawa city,
Okinawa, during June through August 1993.

was used for the identification of the virus. All the virus

preparations were stored at ‑70'C until used.

was prepared. General procedures were described else‑taken from 123 children with the clinical symptoms ofmeningitis and brought into our laboratory. The num‑
ber of meningitis patients from whom the specimens
were taken are surnmed up by week and shown in Fig.
1. The epidemic of meningitis appeared to have occur‑

where (15). For the overlay medium, 1% ethanol

red since June, reached a peak early in July, and subsid‑‑washed tragacanth gum (Wako Pure Chemical, Japan)
in Eagle's medium (5, 11) containing 100 pglml of
DEAE‑dextran and antibiotics was used. The infected

ed in August 1993.

Plaque assay
Confluent monolayer of RD‑18S cells in 6‑well plate

cell culture was incubated at 37'C for 25‑30 hr. Afterthe incubation period, the overlay medium was decanted
and the cells were washed 3 times with phosphate‑buf‑
fed saline (PBS), and fixed with methanol for 5 min.

The cells were washed 3 times with PBS and stainedwith 0.2% crystal violet solution for 5 min. Afterwashing the cells 3 times with deionized water, theplaques were counted.
ldentlfication of recovered viruses

Virus isolation

A total of 101 CSF specimens was inoculated into
RD‑18S cells in 25‑cm2 flasks or 24‑well plates. In caseof 24‑wells, duplicate wells were used for one specimen.
Characteristic CPE (rounding up, shrinking and nuclear
pyknosis) was usually observed in 4‑5 days after inocu‑
lation. The viruses were recovered from 29 specimens(isolation rate : 28.7%) (Table 1). The isolated viruses

were further inoculated into new RD‑18S cells andconfirmed the appearance of the same CPE.
Table I Results of virus isolation

The recovered viruses were identified by 50%
plaque reduction neutralization test (NT) on RD‑18S
cells using pooled antisera against enteroviruses (Denka

Specimens

Virus isolation

Positive

Seiken Co. group E‑O). The serotype of the virus was
further confirmed by NT using monospecific antiserum.

Cerebrospinal fluidSerumo ( o%)

Three different preparations of anti‑echo 30 serum wereThroat swab

used : anti‑Bastianni serum was purchased from Denka
Seiken Co.: anti‑ech0‑30 (5353) was a kind gift fromTotal

54 (69.2%)
83 (38.4%)

29 (28.7%)

Negative Total

72 (71.3%)
37 ( 100%)
24 (30.8%)133 (61.6%)

101

37
78

216

Kagawa Prefectural Institute of Public Health; and anti

A total of 78 TS specimens was examined for virus‑8914 serum was prepared by inoculating mice with 8914
strain of ech0‑30 virus, which was isolated in Okinawa

isolation as described above. Same type of CPE as
above was appeared in 2‑4 days after inoculation.

in 1989.

Viruses were recovered from 54 TS specimens (isolation

rate : 69.2%). Thirty‑seven serum specimens were

RESULTS
Number of specimens brought into laboratory by week
A total of 216 specimens of CSF, sera and TS was

inoculated into RD‑18S cells. No virus was recovered.
Plaque assay described below was performed on the
12 clinical specimens to know the infectivity titers of thespecimens. The titers of these specimens were varied

227

Figure 2 Plaques of Okinawan strain of echo30. Confluent monolayersof RD‑18 cells were infected with Okinawan strain of ech0‑30(upper column) or mock‑infected (down column) and incubat‑
ed for 25 (A), 30 (B), and 45 hr (O, under tragacanth gum overlay

medium. After the incubation period, the overlay medium wasdecanted and the cells were stained as described in the text.

CSF and 4.0 X 10' ‑5.4 x 104 PFU per swab for TS.

the serotype of the isolates, NT was performed using
three different preparations of monospecific antisera

ldent fication of the recovered virus

ized by the anti‑echo30 (5353) and anti‑8914 sera

The viruses recovered from CSF were identified byplaque reduction NT. For the infectivity assay of thevirus, plaque titration in RD‑18S cells was performed.

(Table 2). The 8914 strain of echo‑30 was isolated in
Okinawa in 1989. Since all the strains were neutralized

between O250 plaque forming unit (PFU) per ml for(anti‑ech0‑30) . A11 of the isolates tested were neutral‑by antiserum to 8914 strain, the etiological agent of themeningitis appeared to be quite similar strain as that of

We used l% tragacanth gum containing DEAEdextran

four years ago. It should be noted that prototype echo
‑30 Bastianni strain) was neutralized effectively by 10

for the overlay medium. The incubation period of theinfected cells appeared to be enough for 25‑30 hr (Fig.

‑20 units of anti‑Bastianni serum, while the Okinawan
isolates were partially neutralized (less than 50%)2). In the beginning, 11 sets of antiserum pools wereused. These sets of antiserum pools contained the(Table 2) by this antibody concentration, indicating that

antibodies to Coxackie A9, Coxackie B1‑6, and Echo l
30 viruses. Five isolates were randomly selected andexamined. These isolates were neutralized effectively
by the antiserum pools that contained anti‑echo‑30
serum (antiserum pool ‑N and O). In order to confirm

the isolates were antigenically slightly different fromthe prototype echo‑30 virus.

Table 2 Neutralizing activities of various antisera on the echo‑30 strains
Echovirus type 30#93001#93117#93002

Bastianni(39 . 7)22 (47.6)

51 (38.6)

1 (>99)O ( 100)

O ( 100)

O ( 100)

O ( 100)

O ( 100)O ( lOa)O ( 100)

1 (97.6)

O ( 100)

Pooled sera‑O

3 (96.3)

18 (69,0)

13

ND
ND
ND

Diluent

81

58

58

#93090#9311048") (40 . 7)

36 (37.9)

35

O ( 100)

Anti‑891 4

Anti ‑ Bastianni

Anti‑5353(77 , 6)2 (95.2)

23 (72.3)

4283a ) No. of plaques. Numbers in parentheses represent the reduction rates in per cent.

> 100

228

Plaque morphology of the isolates
The plaques of the isolates showed varieties in size
(Fig. 2). Direct plaquing of CSF specimens also showedof ech0‑30, inhibition of CPE in microplate culture has

been used (9). It takes longer observation time andvarious plaque sizes. Plaque purification of the isolates

frequently encountered break‑through phenomenon,
which made the results complicated. The plaque

was performed and the large‑plaque and small‑plaquephenotypes were isolated. The two phenotypes were

method we used was more simple and the results were

neutralized by antisera to ech0‑30 (5353) , anti‑8914 andwith CSF usually showed maximum CPE in 4 days,

antiserum pool ‑O (data not shown) .

obtained in a short incubation period. The cells infected
while it usually took 2 days for those with TS, indicatingthat more infective viruses were present in TS than in

DISCUSSION
The epidemic of aseptic meningitis had broken out
since June (or probably May) , reached a peak early in
July and subsided in August, 1993. During this period,
not only children but also adults were hospitalized withaseptic meningitis. Viruses were recovered more fre‑CSF. In order to know the infectivity titers of theclinical specimens, direct plaque assay was performedon the virus‑positive specimens. The geometric mean
titers of the randomly selected specimens were 42.9

PFU/ml for CSF and over 2.8xlO' PFU/swab for TS.
In the study of ech0‑12, 50% human infectious dose (forquently from TS (69.2%) than from CSF (28.7%). No

adult volunteers) was 919 PFU (14). High infectivity
titer of TS observed in this experiment may indicate

virus was recovered from sera. Five isolates, randomlythat the droplets and aerosols from coughing or sneezing

selected, were examined by antiserum pools and allturned out to be ech0‑30 viruses. In Okinawa island,
there had a big outbreak of aseptic meningitis in thesummer of 1989. We recovered many strains of ech0‑30

can be the important sources of infection together withthe fecal contamination.
In this experiment, we could not recover any virus
from serum specimens. Neutralization test was perfor‑

from CSF and TS. Since the first report of ech0‑30

med on the sera, whose CSF and/or TS were positive

infection in Japan in 1978, a number of similar outbreaks

for ech0‑30. All the sera were taken during the acutewere observed in several areas in Japan in 1983, 1989 and1991 (4, 10, 13). High communicability of enteroviruses,

phase of the illness. Neutralizing antibody (titer > 1:5)

including ech0‑30, has been documented (9) . It has beenreported that preexisting serum antibody did not protect

was detected in 25 of 26 sera (96.2%) . The viruses in theserum might be inactivated by the antibody, althoughviruses were recovered from CSF and/or TS. Protec‑

against the same serotype of echovirus infection (14).

tive role of serum antibody against poliovirus illness has

These natures of the echoviruses may cause repeatedbeen reported (8). While, the presence or absence ofserum antibody to echo‑12 caused no siguificant infec‑

outbreaks in the same area.

The isolates were neutralized bY anti‑ech0‑30
serum. They were fully neutralized by the antiserum to
the Okinawan strain, isolated in 1989, while they werepartially neutralized by the 10‑20 units of antiserum tothe prototype ech0‑30 (Bastianni strain) , indicating thatthe isolates might be antigenically slightly different
from the prototype strain (prime strain) . We isolated

large‑plaque and small‑plaque phenotypes. The twowere neutralized by anti‑ech0‑30. It has been reported

that a single mutation in 5' non‑cording region ofpoliovirus genome resulted in the production of small‑plaque phenotype (12). Characterization of these
phenotypes are being under way.
For the plaque assay of ech0‑30 virus in RD‑18S

cells, we found 1% tragacanth gum containing DEAE

tion rate among volunteers infected with the same virus(14) . The presence of serum antibody to Norwalk virus,
another enteric virus, failed to protect against the chal‑lenge with the same virus (1). High titer of serum
antibody to rotavirus ( > 1:128) was protective against
homotypic reinfection, while titers less than that failed
to protect (2). Ech0‑30 causes high incidence of aseptic

meningitis (10) and sometimes fatal infection to im‑
munodeficient person (16) . It may be important toknow how circulating serum antibody affects theappearance of illness. At the same time, seroe‑
pidemiological study of this virus will be necessary tocope with the possible outbreak in the future.

‑dextran to. be a good overlay medium. After an incuba‑

REFERENCES

tion period of 25‑30 hr in the stationary culture, the cells

1 ) Blacklow, N.R., Cukor, G., Bedigian M.K., Echeverria,
P, Greenberg, H.B., Schreiber, D.S., and Trier. J.S.
(1979) : Immune response and prevalence of antibody to

infected with the virus formed visible foci, which were
fixed and stained. When agar overlay medium was used,
it took 5 days to form foci. For the neutralization test

Norwalk enteritisw virus as determined by radioim‑

229
munoassay, J. Clin. Microbiol., lO, 903‑909
2 ) Chiba, S., Yokoyama, T., Nakata, S. (1986) : Protective
effect of naturally acquired homotypic and heterotypicrotavirus antibodies, Lancet, 2, 417‑421
3 ) Ducan, I.B.R. (1960) : Aseptic meningitis associated witha previously unrecoguized virus, Lancet, 2, 470‑471

4 ) Fujita, N., Sasaki, N., Ookita, K., Takeda, M., andTakao, N. (1991): Receent outbreak of echovirus 30
aseptic meningitis in Onomichi area in 1989, Jap. J.
Pediatr., 44, 484‑490

5 ) Kedmi, S., and Katzenelson, J. (1978): A rapid
quantative fluoresces antibody assay of poliovirus singtragacanth gum, Arch. Virol., 56: 337‑340
6 ) McAllister, RM, Melnick, J.L., Finkelstein J.Z., Adams
E.C. Jr., and Gardner M.B. (1969) : Cultivation in vitroof cells derived from a human habdomyosarcoma, Can‑cer, 24, 520‑5267 ) Melnick, J.L. (1965): Echoviruses, 513‑545, In Horsfall
F.L., J., and Tamm, I. (eds) , Viral and rickettsial infec‑

tions of man, 4th ed, Lippncott Co., Philadelphia8 ) Melnick, J.L. (1978): Advantages and disadvantages ofkilled and live poliomyelitis vaccines, Bull. WHO., 56, 21‑389 ) Melnick, J.L., Wenner, H.A. and Phillips, C.A. (1979) :Enteroviruses, 471‑534, In Lennette, E.H. and Schmidt,
N.J. (eds) , Diagnostic Procedures for Viral, Rdckettsial

and Chlamydial Infections. American Public Health
Association, Inc., Washington, D.C.
10) Miki, K., Fujii, K., Ikejiri, K., Yamanishi, S., and Okade,T. (1993): Epidemic of aseptic meningitis due to echo
virus 30 and its nosocomial infection in neonates, Clin.
Virol., 21, 22‑27

11) Okuno. Y., fukunaga, T., Tadano, M., Okamoto, Y.,
Ohnishi, T., and Takagi, M. (1985): Rapid focus reduc‑tion neutralization test of Japanese encephalitis virus in

microtiter system, Arch. Virol., 86, 129‑135
12) Pelletier, J., Flynn, E.E., Kaplan, G., Racaniello, V., andSonenberg, N. (1988) : Mutational analysis of upstream
AUG codons of poliovirus RNA, J. Virol., 62, 4486‑4492
13) Sato, H., Saito H., and Morita, M. (1993) : An outbreakof dcho virus 30 and it's seroepidemiology, Clin. Virol.,21, 13‑16.
14) Schiff, G.M., Stefanovic, G.M., Young, E.C., Sander, D.S.,

Pennekamp, J.K., and Ward, R.L. (1984): Studies ofechovirus‑12 in volunteers: Deterrnination of Minimal
infectious dose and the effect of previous infection on
infectious dose, J. Inf. Dis., 150, 858‑866

15) Schmidt, N.J. (1979): Cell culture techniques for diag‑nostic procedures for viral, rickettsial and chlamydial
infections, 5th ed, Americal Public Health association,

washington, D.C.
16) Ziegler, J.B., and Penny, R. (1975): Fatal echo 30 virus

infection and amyloidosis in X‑linked hypogammag‑
10bulinemia, Clin. Immunol. Immunopathol., 3, 347‑352

231

Jpn. J. Trop. Med. Hyg., Vol. 21, No. 4, 1993, pp. 231 237

STUDIES ON EPIDEMIOLOGY OF DIROFILARIAIMMITIS IN HOUSE DOGS IN NAGASAKI CITY,
JAPAN, WITH CONSIDERATIONS ON YEARLY
CHANGES IN MICROFILARIAL PREVALENCETSUTOMU ODAl, OSAMU SUENAGA2, AKIO MOR13,
KOICHIRO FUJITA4, MAKOTO ZAITSU5, KENJI KUROKAWA5,
TAKESHI NISHIOKA7, TATSUYA ITOH7 AND MARIKO MlNE8
Received October 6 1993/Accepted November 8 1993

Abstract: The positive rate of Dirofilaria immitis infection was studied in the eastern, western, southern andnorthern parts of Nagasaki City 3 times between 1968 and 1983. Chronological changes in the positive rateof dogs for microfilariae in these 4 parts and the roles of epidemic factors in the changes of the positive rate

were evaluated. The positive rate decreased in the northern part, where houses and buildings increased

annually, whereas the number of dog owners and the density of dogs decreased. Improvements in thedrainage such as side ditches, which are primary breeding sites of vector mosquitoes ( Culex p. pallens) , areassumed to have led to a decrease in the mosquito density, resulting in a decrease in the positive rate of dogs.

However, the positive rate increased in the eastem, western, and southern parts. The density of dogs
decreased also in these parts, but it remained higher than in the northern part as the number of houses was
smaller, and more people kept dogs. Also, as the sewage system did not seem to be so well developed as in
the northern part, more mosquitoes might be present. The high positive rates in these parts may be ascribedto these conditions.

INTRODUCTION

PLACES AND METHODS

The adult worm of Dirofilaria immitis transmitted
by mosquitoes usually lives in the heart of dogs. Human

As Suenaga et al. (1971) reported, Nagasaki City is
divided into 44 elementary school districts, or into 4

infections are also known in Japan (e.g., Yoshimura

parts, eastern, western, southern and northern. To

1989; Suzumiya and Nawa 1990). In Nagasaki City,

examine the microfilariae of D. immitis in each division,

house dogs were infected with this worm in high rates
and the main vector was Culex pipiens pallens (Suenaga
et al., 1971; Suenaga and Itoh, 1973; Suenaga et al.,1980). However, the relative importance of the factors
actually related to the prevalence remains unknown.
Therefore we conducted an epidemiological study of D.

we sampled blood from an earlobe of a dog in April andMay, 1983 when the City Health Center was conducting
rabies vaccination to registered dogs. A drop of blood
was taken, smeared on a slide glass, and stained with
10% Giemsa's solution. The number of microfilariae in

immitis. Herein, the changes in microfilarial prevalencefrom 1968 to 1983 are reported with a consideration of
the factors responsible for these changes.

the blood specimen was counted with a stereomicros‑cope. The data on sex and age of examined dogs weretaken from the record of Nagasaki City Health Center.
Cx. p. pallens, the main mosquito vector, werecollected by a light trap with black light (20 watts) from

2.
3.
4.
5.
6.
7.
8.

Department of General Education, the School of Allied Medical Sciences, Nagasaki University, 1‑7‑1 Sakamoto, Nagasaki 852, Japan
Reference Center, Institute of Tropical Medicine, Nagasaki University, 1‑12‑4 Sakamoto, Nagasaki 852, Japan

Department of Medical Zoology, Nagasaki University School of Medicine, 1‑12‑4 Sakamoto, Nagasaki 852, Japan
Department of Medical Zoology, Faculty of Medicine, Tokyo Medical and Dental University, 1‑5‑45 Yushima, Bunkyo‑ku 113, Japan
Sasebo City Health Center, 1‑10 Yahata‑machi, Sasebo 857, Japan
Department of Bacteriology, Nagasaki University School of Medicine, 1‑12‑4 Sakamoto, Nagasaki 852, Japan
Nagasaki City International Culture Hall, 7‑8 Hirano‑machi, Nagasaki 852, Japan
Scientific Data Center of Atomic Bomb Disaster, Nagasaki University School of Medicine, 1‑12‑4 Sakamoto, Nagasaki 852, Japan

232

Table 1 Positive rates for microfilariae of D. immitis in dogs with the numberof registered dogs per house in the districts of Nagasaki City in 1983
Part

District

N ame

No.

positive

%

(A)

(B)

418
69

O . 0273l08

O . 0361(B/A)

lrabayashi

3

o

0.0

9072

2

Togiya

l

O

0.0

3

Katsuyama

3

3

100 . O

2524
2993

4

Shinkozen
Kaminagasaki

1

o

0.0

1938

27

O . 0139

4

2

50 . o4939

250

o . 0506

6528 2459

352 74

O . 0539

16

32 . 7

3280

163

6 7

Koshima Nishizaka

Easterno49

O . 0461

O . 0301

8

Himi

o

9

80

28

35 . O

3448

122

O . 0354

10

Yagami
Koga

68

28

41 . 2

1849

103

O . 0557

11

Toishi56

22

39 . 31113

72

o , 0647

265

99

37.4*

40143

1758

O . 0438

216
96
116

o . 0378

Total
12

Inasa

69

20

29 . O

13

Asahi

56

13

23 . 2

5715
2571

14

89

24

27 . O

2787

17

Akunoura
Tategami
Kosakaki
Fukuda74

29

39 . 22489

18

Teguma

4

o

0.0

19

Shikimi

1

o

15

16

Total

o

989
1381

o . 0497

O . 0373
O . 0416

66

O . 0667

58
101

o . 0420111

o . 0538

0.0

2063
1482

66

O . 0445

19477

830

O . 0426

82

O . 0261

84

o . 0400oo . 0406

293

86

29 . 4

20

Sako

19

l

5.3

21

Nita
Kitaohura

112

18 . 2

3139
2098

6

2

33 . 3

4668

248

O . 0531

Minamiohura
Naminohira
Tomachi
Kogakura

17

8

47. 1

2491

O . 0510

22

23
24
25
26 27

Southem
28

Doinokubi
Fukahori

24

3

12 . 5

1463

127
71

149

57

38 . 35044

232

o . 0460

46 49

22 29

47 . 81779

64

o , 0360

59 . 24574

27

42 . 2

3545

221
104

O . 029364

O . 0485

o . 048313

5

38 . 5244

16

o , 06565712

21 . 1

O . 0469

O

2049
1955

96

31

Minami
Mogi
Hayasaka131

o . 0670

32

H iyoshi

o

343

18

o . 0525

O . 0447

29
30

Total

455

1 68

36 . 9 *

33392

1494

2563
4051
6628

75

O . 029379

O . 0195

227
110
186
86

o . 0342

33

Zenza

53

11

20 . 83432

515 . 6134

34

25 . 436

Sakamoto
Takao
Yamazato30

620 . o37

Nishishiroyama

7

1

14 . 338

Shiroyama

92

22 . 2

39

207

56

27 . 112

4

33 . 341

N ishiurakami
N ameshi
Nishikita

42

14

33 . 342

Nishimachi

227

31 . 843

Kawabira
Ohzono7

4

57 . 1o35

N orthem

examined

No. of houses No. of dogs registered No, of dogs per house

1

5

WestemNo. of dogs

40

44

Total
S um total* Significant (P<0.01)

5079
4387
2232
8692
9255
4273

0.0217o . 0424
o . 0385

387

O . 0445

420

O . 0454

166

O . 0388

6476
2573
2950

299

o , 0462

115

o . 0447

61

O . 0207

555

144

25 . 9 *

59159

2211

O . 0374

1568

497

31 . 71521716293

O . 0421

233C) : Eastern

1972 to 1983 in 4 points in residential area: Irabayashi(Eastern part) , Inasa (Western part) , Tomachi (South‑

ern part) and Sakamoto (Northern part) . In addition,
the collection was made in Sakamoto‑machi also from1967 to 1971. The light trap was regularly operated
from 6 PM to 7 AM (one night) , once a week from May
to October.

e : westernC.̲I IVO : southern

O : NOrthern@d)1)C O) ;i Co o

O. 5

te

J

q,

IJ
c:'

h

Q,
:'

6)

co

RESULTSe@l . Microfilarial prevalence in house dogs in Nagasaki

City
Table I shows the results of the blood examinationof dogs in Nagasaki City in 1983. The positive rate formicrofilariae was 31.7% for the whole city. The positive
rate in the eastern or southern part was significantly
higher than that in the northern part (x' ‑ test, p < 0.01) .

We thought that the difference in the positive rate might

be related with the dog density (the number of dogs perhouse) . To make clear this hypothesis, the numbers ofo .CC
Dog density

Figure I Relation between dog density and positive rateof dogs in districts in Nagasaki City in 1983.
* Points were plotted only when the number ofdogs examined was over 10.

(Table 1). As explained later, the positive rate isnorthern part, the total positive rate of 40.5% in 1968
clearly decreased to 25.9% in 1983, while the rate in‑creased in the eastern, western and southern parts.

correlated with the dog density, which was lowest in thenorthern part with the lowest positive rate among the 4

4 parts were obtained in spring, but in 1968 the resultsparts.

were composed of data taken in spring and autumn.

2 . Comparison of positive rates in dogs by sex and age
in the 4 parts of the city

The positive rate calculated only for spring was 34.6%,
19.5%, 21.6% and 41.8% in the eastern, western, south‑
ern and northern parts, respectively, there being notdogs per house were also examined in these parts

The positive rates were compared according to sex

and age in the 4 parts in Table 2. The rates were
calculated only for dogs with an exact record of age.

In 1977 and 1983, the results of positive rates in themuch difference between the results obtained only inspring and those obtained in spring and autumn com‑bined (Table 1).

The positive rate is generally lower at any age for either

sex in the northern part of the city than in the other 3

5 . Yearly changes in the number of the registered dogs

parts. This means that the lower prevalence in the

3 . Correlation between positive rate and dog density
We examined the correlation between positive rateand dog density in districts in Figure 1, where points

in Nagasaki City
Figure 2 shows the yearly change in the total num‑
ber of dogs registered in whole Nagasaki City. The
number of these dogs decreased gradually from 1968 to
1983, with some variations. Changes in numbers of dogs
and houses in each of 4 parts of Nagasaki City are
shown in Table 4. In each part, the decrease in thewere plotted only when the number of dogs examined

number of dogs was apparent, while the number of

was over 10. The microfilaria positive rate was posi‑houses increased.

northern part was not due to different age distribution

nor sex ratio in the dogs examined.

tively correlated with dog density in the city (correla‑tion coefficient 0.4163, 0.01 <p<0.05) .

6 . Yearly changes of the relation between positive rateand dog density in the 4 parts
4 . Yearly changes in positive rates of the dogs in the 4parts of the city

The yearly changes in positive rates in the 4 parts

were examined by comparing the results reported by
Suenaga et al. (1971; 1980) with the present data by thelinear trend test of Armitage (1955) (Table 3). In the

As the numbers of houses and registered dogs ineach district were available for 1968 and 1983, the dog
density (number of dogs per house) was calculated. The
relation between the microfilaria positive rate in dogs
and the dog density in each district in 1968 was compar‑

ed with that in 1983 in Figure 3. The positive rate

234

Table 2 Number and percentage of dogs positive for D. immitis microf ilariae
by age and sex in 4 parts in Nagasaki City, 1983

Part

Sex

Male
Eastern

FemaleMale
Western

FemaleMale
Southern

FemaleMale
N orthern

FemaleAge

No. and %

Total

o

1‑2

3‑4

5‑6

Dogs examined

o

65

40

28

18

9

7

Dogs positive% positive

o

17

12

11

10

6

6

62

o

26 . 230 . o39 . 355 . 6

66 . 785 . 737 . 1

Dogs examined

o

31

19

16

10

7

12

95

Dogs positive% positive

o

7

7

8

4

4

7

37

o

22 . 636 . 850 . o40 . o57 . 158 . 538 . 9Dogs examined

1

51

13

15

16

1 74

o

8

43
21

35

Dogs positive% positive

15

7

4

6

61

o

15 . 748 . 842 . 953 . 826 . 737 . 535 . 1

Dogs examined

o

37

33

15

12

11

8

116

Dogs positive% positive

o

7

6

5

l

4

2

25

o

18 . 918 . 233 . 38.3

36 . 425 . o21 . 6

272

9‑10

7‑8

11

167

Dogs examined

3

99

72

43

30

11

14

Dogs positive% positive

o

25

36

18

16

7

9

111

o

25 . 350 . o41 . 953 . 363 . 664 . 340 . 8

Dogs examined

1

54

15

19

8

171

o

10

42
16

32

Dogs positive% positive

14

3

7

3

53

o

18 . 538 . 143 . 820 . O

36 . 837 . 531 . O

Dogs examined

3

95

72

65

42

1

15

22

25

17

29
10

29
9

335

Dogs positive% positive

33 . 315 . 830 . 638 . 540 . 534 . 531 . O

29 . 6

Dogs examined

1

76

41

41

24

217

o

5

6

13

6

20
6

16

Dogs positive% positive

7

43

o

6.6

14 . 631 . 725 . o30 . o43 . 819 . 899

Table 3 Yearly changes in D. immitis positive rates of dogs in 4 parts in Nagasaki City from 1968 to 1983
1968*

Increase ( + ) or

No. of dogs Positive

No. of dogs Positive

No. of dogs Positivein prevalence

examined positive rates (%)

examined positive rates (%)

1968 to 1983

328 34 . 5
334 29 . 3
504 35 . 3
555 31 . 4

265 37. 4
86
293 29. 4
455 36. 9
153
555 25. 9

675 28 . 7
337 20. 2
137
639 21
.
4
291
719
40
.
5
Cited from Suenaga

Eastem*1983

examined positive rates (%)

Western Southern N orthern

**

1977*

decrease ( ‑ )

Part

194

68

et al. (1971, 1980) .

113
98

1 78

174

99

+** +** +** **166

Significant P<0.001

decreased in parallel with the decrease of the dog den‑sity in the northern part, but in the other parts the
opposite tendency was generally observed.

7 . Yearly changes in number of Cx. p. pallens females
at 4 points

In Figure 4, the mean number of Cx. p. pallens
females per night was shown as an annual populationindex. The mosquito population was not large from235

: Blood examination

12, 500

O : No. of Registered dogs

10, OOO= 7, 500,e'' 5, OOOz
2, 500O

1966 I970 rs75 1980
Y***

Figure 2 Yearly changes in the total number of registered dogs in Nagasaki City.

1972 to 1983, in the eastern, western and southern parts,

though population varied with year. In Sakamoto Eastern part
o. 5

C 1
O : ‑. ̲‑ ‑(northern part) where the mosquitoes were collected

Cfrom 1967 to 1983, the density was also low since 1971. .̲ ‑‑‑:C‑‑‑C ̲̲C : Ig68

We studied the infections of D. immitis in registered western partdogs in the eastern, western, southern and northern o. 5

parts of Nagasaki City in 1968, 1977 and 1983, and found o̲̲̲̲̲̲̲̲̲̲

a significant
betweennorthernpart and ‑‑‑C̲C
theother 3 parts. difference
In the northernpart, thethe
positive
rate t‑̲‑‑‑‑‑‑‑̲̲‑‑‑‑‑‑‑

had decreased, whereas in the other 3 parts, it had‑

Cincreased markedly. As one of the factors causing thisphenomenon, introduction of lvermectin, an effective
O‑̲ southern part'* 0.5 OL‑ .̲̲
Table 4 Changes in numbers of dogs and houses OL̲̲O:: '̲:: : ‑'･( ̲̲̲i )in 4 parts between 1968 and 1983 *$1¥*

Part

Dog and House
No. registered dogs (A)Eastern

Dog density (A/B )No. houses (B)No, registered dogs (A)Western

Dog density ( A/B)
No. houses (B)No. registered dogs (A)Southern Dog density (A/B )No. houses (B)1968*

CP: lll]1 ' .i@ o

1983

3 , 666

1 , 758

O . 1212

O . 0438

30 , 243

40 , 143

1 , 428830

O . 0932

O . 0426

15 , 316

19 , 477

2 , 491

1 , 494

O . 1034

O . 0447

24 , 085

33 , 392

3 , 733

2,211

O . 1050

O . 0374

35 , 566

59 , 159

O‑‑‑‑‑‑ ‑‑ o
Northern parto. 5 J ! . J@ ̲
'f!

8

.̲ ‑

i

i/*' :: :̲:‑

!‑

'

'@

:C

‑‑‑

o. l

No, registered dogs (A)N orthern Dog density (A/B )No. houses (B)Dog densi ty

Figure 3 Relation between the dog density and thepositive rate for D. immitis in the districts of*Cited from Suenaga et al. (1971, 1980) Nagasaki City in 1968 and 1983.

236
ro

lrabayashi (E)l972 I975 I O IsE3
lo

Inasa CU)

:LplthlLlu

ro'I972 I975 I o I 8Tomachiomac l (S)l972 I9 5 180 l 3

owners became to take measures to prevent exposure of
their dogs to mosquitoes by, for example, Ietting themwithin the house at night and providing mosquito repel‑
lents. In the northern part, the increase in houses andoffice buildings was associated with the development of

the sewage system thus decreasing drainages such asside ditches, according to the Department of Sewage
System in Nagasaki City. Such improvements in thesanitary 'environment are considered to have been afactor in the decrease of the Cx. p. pallens. These
conditions are assumed to relate to the decrease in thepositive rate in the northern part.
On the other hand, the positive rate increased ineastern, western and southern parts. The density ofdogs increased also in these parts, but it remained higher

Sakamotorothan in northern part as the number of houses was
smaller, and more people kept dogs. In addition, it
seems that the sewage system was poorly developed inthese parts, because the number of houses was smaller**

Isl lg72 ' I9i5 ' 1 0 I 3

than in northern part. From this we assumed that as thedrainage has not been so well developed as in the north‑Y,**

ern part, more mosquitoes would be present in the 3

Figure 4 Yearly changes in the number of Cx. p. pallens
females per night in Nagasaki City. E, W, S andN show eastern, western, southern and northernparts, though this result was not clearly shown by thedata of light trap. According to dog owners, more dogs
seem to be bred outdoors. Therefore it is suspected thatpart respectively.

dogs had more chance to be attacked by mosquitoes* The collection by light trap was not made.

even if mosquito density was low in these parts.
As for the role of other mosquitoes in the transmis‑

preventive drug for D. immitis, was considered, but it

Dog density, which is an important factor respon‑sion of D. immitis, Suenaga and Itoh (1973) reported
that Aedes albopictus may be the secondary important
vector, and Cx. tritaeniorhynchus may also be somebearing on the transmission in Nagasaki City. How‑ever, there is no evidence that these mosquitoes in‑creased in the 3 parts in particular, and they are notconsidered to have contributed much to the increase of
positive rate. The number of stray dogs may be anotherfactor influencing the transmission. Their number, as

sible for infection (Wada et al., 1989) , decreased appar‑

given by the number of arrested dogs (unpublishedently in all 4 parts, but the positive rate decreased in thedata) , decreased with years, therefore stray dogs are notnorthern part and increased in the other 3 parts. As
shown in Table 4, the number of houses in the northernpart, which was the largest among the parts, had in‑creased. This implies that many new residential areas

an important factor for the increase of positive rate.

can not be a reason for the decrease in the positive ratein the northern part since 1968 because its introduction

was 1988. The present result showed that mosquito
density was scarcely different in all the parts after 1972.

But the density decreased in the northern part from 1968

to 1972, and thus, this decrease in mosquito density may
be related to the decrease of the rate.

ACKNOWLEDGMENTS

had developed in this part, where it is assumed that theProfessor Y. Wada, Department of Medical Ento‑

space to keep dogs outside was limited and there wassome environmental complaint against dogs. Therefore,
many dogs may have been kept within the house, andprevented from attack of the vector mosquito, Cx. p.

mology, Institute of Tropical Medicine, Nagasaki Uni‑

versity kindly read this manuscript and provided
detailed suggestions. Dr. T. Fukuda, President of theSociety of Veterinarian, in Nagasaki City and theirpallens, which occurs in the breeding sites such as side

member, veterinarians really cooperated with ourditches. Furthermore, when we took dog's blood, we
had an impression that in the northern part more dog

these persons.

research. We wish to express our sincere gratitude to237

REFERENCES
1 ) Armitage, P. (1955): Test for linear trends in propor‑
tions and frequencies. Biometrics 11, 375‑386.
2 ) Suenaga, O., Itoh, T. and Nishioka, T. (1971): Studies

on the filarial prevalence among house dogs and themosquito vectors in Nagasaki City, Western Japan. 1.
On the microfilarial prevalence among house dogs in
Nagasaki City. Trop. Med., 12, 169‑178. (in Japanesewith English summary) .
3 ) Suenaga, O., Itoh, T. and Nishioka, T. (1973) : Studies

on the filarial prevalence among dogs and the mosquito

vectors in Nagasaki City, Western Japan. 5. On thevector mosquitoes of Dirofilaria immitis in Nagasaki
City. Trop. Med., 15, 131‑140. (in Japanese with English

summary) .
4 ) Suenaga, O., Nishioka, T. and Itoh, T. (1980) : On thetransition of canine heartworm prevalence among dogs
in Nagasaki City, Western Japan. 1. The filarial preva‑
lence among dogs in the city. Trop. Med., 22, 35‑45.

5 ) Suzumiya, J. and Nawa, Y. (1990) : Three cases of
pulmonary Dirofilariasis found in Miyazaki prefecture.
Jpn. J. Parasit., 39, 67‑71.

6 ) Wada, Y., Tsuda, Y. and Suenaga, O. (1984): Transmis‑
sion dynamics of Dirofilaria immitis in a Southwesternpart of Japan. Trop. Med., 31, 35‑47.

Jpn J Trop Med Hyg Vol 21 No 4 1993, pp. 239‑243

239

AN ULTRASTRUCTURAL STUDY OF AFRICAN
ENDEMIC KAPOSI'S SARCOMAHIDEAKI ETO, KAN TORIYAMA and HIDEYO ITAKURA
Received May 12 1993/Accepted November 9 1993

Abstract: An ultrastructural study of African endemic Kaposi's sarcoma (KS) of the skin and lymph nodes
revealed an irregularity of blood vascular tissues and prominent diapiresis of erythrocytes in the early stageof the disease. In the advanced stage, incomplete blood capillary structures covered with primitivemesenchymal cells were observed. Erythrophagia was frequently seen. These results suggest that KS cells
are derived from primitive mesenchymal cells which may differentiate to immature endothelial cells.

INTRODUCTION
KS is now thought to be a reversible hyperplasia at
least at its inception and gradually progresses towards atrue neoplasm at an advanced stage (Brooks, 1986;
Itakura et al., 1986). KS cells are characterized byspindle‑shaped cells forming slit‑1ike structures. On thehistogenesis of KS cells, several hypotheses about origi‑

Histological growth pattern and cellularity were deter‑

mined.
Ultrastructural studies:

Small pieces of formalin‑fixed, paraffin embedded spec‑
imens were refixed in 2.5% glutaraldehyde, post‑fixedin 2% osmium tetroxide, dehydrated in graded ethanol
solutions, and embedded in Epon 812. Sections were

nal cells have been suggested by ultrastructural studies;observed with a JEM‑100CX transmission electron

vascular endothelial cell (Hashimoto and Lever, 1964;

microscope.

Ramos et al., 1975) , Iymphatic endothelial cell (McNutt,

et al., 1983), Schwann cell (Pepler and Theron, 1962)and mesenchymal cell (Harrison and Kahn, 1978; Leu

RESULTSand Odermatt, 1985; Weich et al., 1991; Kostianovsky etal., 1992). However, the histogenesis of KS cells still

The clinical aspects and histological features of the four

remains obscure.
We performed an ultrastructural study of African
endemic KS and discussed the histogenesis of KS cells.

MATERIALS AND METHODS
Materials:

Four cases of KS were obtained at Rift Valley Provin‑cial General Hospital in Nakuru and Nyanza Provincial

General Hospital in Kisumu, in Republic of Kenya.
Clinical data and relevant information were recorded as
accurately as possible.

Light microscopy:
cases are shown in Table 1. According to the predomi‑
nant histological features, two types of growth patterns;

a granulation tissue type and a spindel cell/fibrosar‑
coma type were recoguized. The former was character‑ized by an angioproliferative process with edema and an
inflammatory cell infiltration, but less cellular (Figure 1) .

This type was seen in Case 1, at an early stage of thedisease. The latter (in Case 2‑4, at an advanced stage)
was classified as a spindle cell/fibrosarcoma type. This
type showed a high cellularity, but its cellular atypism
and mitotic figures were not prominent (Figure 2) .

Electron microscopy:
Light microscopy:

In the case of granulation tissue type, endothelial cells

Each specimen was prepared with hematoxylin‑eosin

were arranged with no continuity and basal lamina were

stain (H. E.) , periodic acid‑Schiff reaction, Azan‑Mal‑fragmented. Diapedeses of erythrocytes were promi‑

lory's stain, and silver impregnation for reticulin fibers.

nent. Fibroblastic cells appeared between these capil‑

Department of Pathology, Institute of Troprcal Medicme Nagasakl University, 1‑12‑4 Sakamoto, Nagasaki 852, Japan

240

Table l Clinicopathological findings of African Endemic Kaposi's sarcoma
Case

Age (yr)

S ex

Site of Location

Histological type

Cellularity

138

skin of leg

G

+

2

53

skin of leg

s

+++

3

lO

scrotum

S

++

4

4

M
M
M
M

axillary lymph nodes

S+++

G: granulation tissue‑like type
S: spindle cell/fibrosarcoma‑like typelaries (Figure 3) . In the case of spindle cell/fibrosarcoma

type, incomplete blood capillary structures were covered(powdery chromatin and small nucleoli. These cells
had abundant cytoplasm with few organelles and nowith the enlarged and irregular‑shaped cells. The nuclei

Weibel‑Palade bodies. Diapedeses of erythrocytes andof the cells had irregular membrane with fungating

erythrophagia were also observed (Figure 4) .

s # :! si i

+ ' t;# : #':+'' ‑' * i''" " st; 's!!̲" ' t̲ 't ' :i{'̲';;{ '^'̲. .̲7';̲̲:̲I i;? 'ii"; "! ;!;
"r!/
, ...
il::
i;
: #'
''‑‑
'
1
"
. S.̲ = .,^.
̲,.
̲ '/ ''̲
'^' ‑'‑' ^ '‑‑'
̲/:! .'+‑''i '"' '; ‑' # ' r' t*;':ssi;; ;;‑ji$ ':s#̲ ̲ ' ‑ : ' r{' .i ̲ ' !j..
^ ̲!
̲‑ ::!
...:;'' i':"
'=
' ' '
*'‑
.>
.̲.,}j '..
; . ‑#.i ,i; r;; . # #'t‑"!
̲'{i :j ' " =1: ;; ; :;... . i :
i!

:

:

f:;:;

" '̲ " '

:#

'

"{ j

;

is w{

'i

; ' * '!!

'

:^;

' :

*' ;

x ^' *; ;'" : ':;sS r *i'
i; !i '

; t ':':#'si (s ':i;

'; F

i"

i!i

'̲;i i''{ss t;:i:;:;{;i#)' Fi'/::;': ( i;{:::::;:L 'f:;: ,̲
l! ; ‑ 7j' :i;'.i' #s‑‑ ‑‑^ . .,.} ̲ ̲ ' " ;'t{ :(:;{j;;:f;;F'itj " ;i:(} :/i:;i"i';'!̲ i;"' r '#ji#j.̲ ;{',' ,# ># ', ' .^,j '. ̲ ̲ ^ .. ̲." s l';',̲^,;j {:..
{i'"
̲.i
{̲.̲
. . . ,f' "'' 'j'= "i{'.'' :=
Ff^
;
;ii
'
'
'
̲ ":
i̲i
̲ ' ' ;f!j" **=* '̲''* '. ‑F̲t:;;! " ': '..i;.̲ ̲ j ̲̲
: ' ' j‑‑
̲" i'
'I'{
'i!̲K
̲i ;
"' ;'" :;;;j";'f*‑' "*̲*';' ^̲':*+ ' '‑''‑ ;j::1=
: ‑"
{/ ̲
/̲'
;;;
'{{
: :+'
:(
! lj
}‑{'
.* ..
' t'̲*̲'̲'
̲' '*';"'1ss:‑
i ''x; i;s
i:
/t's
i!
;;
';
'; ^{i"';xt'' ̲‑ '‑.‑̲‑'
̲̲
+
"'t
;+
*
;'
'
{
̲+̲.'
'.
;'^s 'j'
'
;‑‑
;
+‑̲
;.
jii
:I
t:
;i'
,
‑
'
‑‑
‑
‑'
' ';Il
t:*{
>,ji
j̲;
'
:'̲'
i
̲
.
.
̲
:
'
i j 'Ii' ' ' ":I{;S';'1; 1 ";;iI '; '
j ''
̲ 't
' '}
' '. ' '^‑'
" t"r
' '//̲:{;:̲ '̲,.
̲̲; '> ‑
'
‑
"+
'
.
̲̲̲.
.
̲̲*
̲
,
.
.
̲"
..
̲
̲'
̲
'
'x;;:
';!F;:;i:" (! ;;f ;sf r'; 'j ; . ̲ . .̲ = .

.̲..
,;;' ;{'"**',! "':i‑ !S'‑.̲. ..
̲ ,̲....
'#ix'' *>"'
'̲‑‑ss{' s l'‑. ^'i '̲;'‑‑ ‑ ̲ ̲ ̲ ̲ :. * .̲̲̲ ̲̲̲ '*‑

";

. . .
'::'

x

i

&s#

:' ‑'

: ‑ T '̲ ' ' * ?

j s: j ' 'ji'^"' ' ' '"

:ji: :#' j 'ix '';"';
;! '?
;"'
̲
;s ‑‑
"j "(';' f' "‑s'i'i'**‑"t=' ‑i!!;‑ ';T ''; * j'{';= ; t;# isx':'' *̲''=̲'';̲
' '^'
'̲''
̲' +!:
.,̲
.,
x
i
;f
'
5'$x"
#' ""='ji'̲#';'F' ,̲'
':i
̲.'
̲ ̲..
'‑‑
i;x
̲ ‑
K'
'^^
'‑'
'‑
'
‑
‑
‑‑
'
'
̲"'
‑̲:
'
‑/'i
̲
;
̲'
"' ": ̲‑ i'‑:{ :̲‑ '' ' " #!‑ 'i!‑' ;‑^''':̲'‑‑‑'T ':'‑''' '‑ '
‑
‑ ‑‑x
;i'
‑
:;'
'
‑' ' ' :‑ ' ''‑: l i' ‑‑ ‑‑ ;r‑'=' * ; "'
' '‑i /^‑' s;'" ‑ :#s'
; ; '
::
' ;
;<'s:;::i‑=' '‑^*"*i'‑‑̲:' ''‑'{^''" :" " ' f‑
'' /
‑'
'{*
'':̲‑‑''''"'‑' ‑'+̲̲
s'̲̲'̲=
'
'
'
'
'‑
'
'
;!
:/
'‑
‑x*
‑
' ‑ '‑‑‑‑' '‑s'‑4'"'+''''‑ '̲''‑̲'^‑̲̲"'''* ‑‑ ' ‑' ^ ; '‑ ‑'" ' s̲'̲̲"''''' ' =* '' *" '+'"'+'‑^"+*+x{* '+st#"'''^^
̲ "'*': "'‑‑'‑ji" '‑‑ *'>
̲ s"'' ;'* ^
' ‑"'
‑*"
'
'‑
;'‑‑
s
'
'
i;
i :
; = i
:
' ' :
S '# ;'is; :i'' 'b* ' S
'
#

' ̲'

: ' : i 1 :i"'!/r ' ' $#s'

i ;‑'

F7' '

*

i '1

::

'

r ;/

̲̲; ;+ ::ii{;; f ; ' ' { ':;b ' i ' ' ;^s':x' "" : ' ̲ i '' ' '+" ';{T # ' *#' i'b'i;' ' ;'i ; {̲ s "‑'*
' '(:#'‑ i: +‑ t : '#i '‑' '' ‑{ '‑s'+{ /;I

'L

̲ ' i

:= ;

' ‑ 8 *' ‑'>‑:#!i >1 ' :#' = "' '$‑ ̲ ' x ': ‑': s ;'k#^i ': ;f't' ‑ 1 "'" # ' ‑ T L‑‑' ' " ‑ ='""ii#: !''t:"; :;t F‑'=#t' '' :#:''; # ‑"
i;"':ii:' ;'? ‑‑'
*FF: ?'+:#;''i':ts'‑ '" x) ;i' :‑7: '' ':stt '‑'*' ;$'/ ' :'' t' t‑i' ';'>"''"‑:i ': is"::iTt
' ''i ':'#'
ti i'J{:T;'""!"' ': ' '':#: : ‑''"^> ':‑‑'' i‑' :'
s

: :'

= '

'

4'

:‑""

i '

;

' ' i

: *

i '

' '* t̲c { '*$:'}: i{x; t =F'fi' ; ;:#{"' ;;{';; +' ' ‑ '* '' s;'

s

;

' i:

;i

'

;

'

:
*

''

' ;'

'x :

" ‑+i4*"= ' ; 7

s : ;

Is

'

'i**

t *i'*'T+* i' #i's ' '* ::: T

:'

' <

';+'

* t 's' '';'( 't:' ; ' :'i' '# :; :;' T;:"' :" 't
; ':'
:; i:
;##:;
;/'
; :;
i‑
‑=': ' ;;#t;'::::}:";}:: :k i::+' ;:ti'L:: :'':' '':

::' :

'i

':‑ ‑

‑#i
‑ :" : ' {t; ‑ ‑ t'
x :!"'t '' #F: *';t;': #:' ! i; {s
"
; '
i " "
' i' x'if};f i'=*̲ ' "'‑"!::# '#1' 1 tt ';;'‑ :t;+": ;:;: if!"'=;'i;:si ; ;^J s# :^ ;;;{ i‑ E's ' >:

I

'

' ' i

s'* " '1
l:' /r' ' ';' ';; 'F" :/r;'i i; r!:{ {i"' 'tri" :' ' ;r i=#'/ i: i{x : '# '^ :; : i;'; i';''*"'"; ir! r'

"

s

: }

+

/

' '

is

'

t::7'
c4;:r*:!
' *

i

'

^ '

'

‑
;

: ; {s/ ̲"* ' *'

Figure I case ll Granulation tissue type of KSt The lesion iS char cterized
by an angioprohferatiVe process with an inflammatory ceu intntra
tionl (H'EI ><100);1e: : *' :

241

離

灘
灘︑

灘

灘．…

騨
9

灘懸

Figure3Case1．Granu1；．離．。繋

peofKS．Endot蓋elialcellsl

co

｝w）．

prominent〈D）、（

Oμm）

ged

with

no

雛灘灘灘灘灘灘

D量apedesasoferythro242

Figure 4 Case 2. Spindle cell/fibrosarcoma type of KS. Incomplete capillary
structures (O covered with enlarged and irregular‑shaped cells.
Erythrophagia (arrow) is characteristic. ( x 4000, Bar: lOpm)

DISCUSSION‑Palade bodies, as ocassionally seen in primitive mesen‑chymal cells. Erythrophagia was frequently seen. Using

KS shows a wide spectrum of histological changes

immunohistochemical methods we have observed thatin the course of the disease. At an early stage, similar
lesion to non‑specific granulation tissue, with cutaneous

KS cells show a positive reaction for vimentin, which isedema and perivascular inflammatory cell infiltration,
are characteristic. Then spindle‑shaped cells appears
gradually in the course of the disease. At an advanced
stage, slit‑like structures with characteristic spindle

partially positive for factor Vlll‑related antigen, which isshaped cells, similar structure to angiosarcoma andmay diffcrentiate to immature endothelial cells.

a marker for mesenchymal cells, and that KS cells area marker for vascular endothelial cells (Komuro andToriyama, 1991). These results suggest that KS cells
are derived from primitive mesenchymal ceils which

fibrosarcoma, are observed (Itakura et al., 1986; Enzin‑

ger and Weis8, 1988) . According to McNutt et al.
(1983) , ultrastructural findings of KS at an early stage

ACKNOWLEDGMENTS

revealed more prominent irregularity of vascular struc‑ture than other vascular proliferative diseases. In thisThe authors are grateful to Dr. A. Makatta for
helpful comments and advice on the manuscript instudy, the capillaries of KS showed fragmented basal
lamina and no continuity of the endothelial arrange‑
ment. Diapedeses of erythrocytes were also prominent

English, and are also indebted to Mr. A. Ichinose for his
help of electron microscope.

at an early stage. Some immune factors related toangiogenic activity> such as thymosin, interferon,

REFEREN CES

lymphokines and prostaglandin, are thought to be cru‑

1 ) Brooks, J,J. (1986}: Kapasi's sarcoma: A reversible

cial in the development of KS (Levy and Ziegler, 1983) .

hyperplasia, Lancet, 2, 1309‑1310

The lesions mentioned above may be caused by someimmune factors. At an advanced stage, incomplete

2 ) Enzinger, F.M. and Weiss, S.W. (eds) . (1988): Softtissue tumors. 2nd. ed. pp. 533‑544. C.V. Mosby Co.

blood capillary structures covered with enlarged andirregularshaped cells were observed. These cells had
abundant cytoplasm with few organelles and no Weibel3 ) Harrison, A.C., and Kahn,.L.B. (1978): Myogenic cells
in Kaposi's sarcoma: An ultrastructural study. J. Path‑

Washington

243ol., 124, 157‑171

4 ) Hashimoto, K. and Lever, W.F. (1964): Kaposi's sar‑coma: Histochemical and electron microscopic studies,
J. Invest. Dermatol., 43, 539‑549
5 ) Itakura, H., Toriyama, K. and Uzuta, F. (1986): Kaposi's sarcoma: Pathology and local epidemiology in Kenya,
Trop. Med., 28 (suppl.), 3‑86 ) Komuro, S. and Toriyama, K. (1991): Geopathology ofendemic pediatric lymph node Kaposi's sarcoma inwestern Kenya, Jpn. J. Trop. Med. Hyg., 19, 251‑263

7 ) Kostianovsky, M., Lamy. MAJ. Y. and Greco, M.A.
(1992) : Immunohistochemical and electron microscopic
profile of cutaneous Kaposi's sarcoma and bacillary
angiomatosis, Ultrastruct. Pathol., 16, 629‑640

8 ) Leu. H.J. and Odermatt, B. (1985): Multicentric an‑
giosarcoma (Kaposi's sarcoma) : Light and electron

microscopic and immunohistological findings of
idiopathic cases in Europe and Africa and of cases
associated with AIDS, Virchows. Archiv. A., 408, 29‑41

9 ) Levy, J.A. and Ziegler, J.L. (1983): Acquired im‑
munodeficiency syndrome is an opportunistic infection

and Kaposi's sarcoma results from secondary immune
stimulation, Lancet., 1, 78‑80

10) McNutt, N.S., Fletcher, V. and Conant, M.A. (1983):
Early lesion of Kaposi's sarcoma in homosexal men: An
ultrastructural comparison with other vascular prolifer‑ations in skin, Am. J. Pathol., 111, 62‑77

11) Pepler, W.J. and Theron, J.J. (1962): An electron
microscope study of Kaposi's haemangiosarcoma, J.
Path. Bact., 83, 521‑525

12) Ramos, C.V., Taylor, H.B., Hernandez, B.A. andTucker, E.F. (1975): Primary Kaposi's sarcoma oflymph nodes. Am. J. Clin. Pathol., 66, 998‑1003
13) Weich, H.A., Salahuddin, S.Z., Gill. P.. Nakamura, S.,
Gallo, R.C. and Folkmann, J. (1991): AIDS‑associatedKaposi's sarcoma‑derived cells in long‑term culture
express and synthesize smooth muscle alpha‑actin. Am.
J. Pathol., 139, 1251‑1258

Jpn. J. Trop. Med. Hyg., Vol. 21, No. 4, 1993, pp. 245‑249

245Case Report

FOUR IMPORTED CASES OF FALCIPARUM MALARIASUCCESSFULLY TREATED WITH HALOFANTRINESHIGEYUKI KANO , GOHTA MASUDA2, YUZURU KUB03
MASAYUKI SAIT03, TSUYOSHI YAMAGUCH12 AND MAMORU SUZUKll
Received September 29 1993/Accepted October 15 1993

Abstract: Halofantrine, a phenanthrene derivative, has been used for the treatment of human malaria since
1984, and reported to be effective against all parasite species without any major side effects. The chemical
structure of the drug is unique and not closely related to any other antimalarials, therefore, it is particularly

effective for the treatment of the wide‑spread drug resistant malaria. Four cases of imported falciparum
malaria, including chloroquine and pyrimethamine resistant malaria, were treated with halofantrine andreported in this paper. Halofantrine cleared parasites and fever of all the cases rapidly and produced

salutary symptomatic improvement of the patients. No adverse reactions were recognized after theadministration of the drug, nor was there recrudescence. Fansidar is the only antimalarial drug commer‑
cially available in Japan, and imported drug resistant malaria not only against chloroquine but also Fansidar

has been on the increase. The general use of halofantrine is thus expected in Japan.

INTRODUCTION

years old, who stayed in Uganda froin December 24,
1991 to January 15, 1992. He was taking chloroquine

In 1992, the total number of Japanese who went

once a week during this period. In spite of the prophy‑

abroad amounted to as many as 11,790,699, and of for‑eigners who entered Japan to 3,251,753 (Annual record

laxis, on his way back to Japan (Jan. 16), he showedof the office of statistics and information, Division of
Immigration Service, Ministry of Justice) . Consequent‑

Medical Association Hospital on Jan. 20, he manifested

ly the reported annual number of imported malaria
cases increased to not less than 100 (Ohtomo et al..

fever of over 40'C. On admission to Isesaki‑Sawa
chills, a headache, slight splenomegaly, and throm‑
bocytopenia, but anemia. The following morning, Plas‑
modium falciparum (P.f) parasites were detected from1993) , and therefore, malaria is no longer recognized asa thin blood smear at the density of 0.5% of red blooda very rare disease in Japan. Nevertheless effective

cells, but antibody titer against malaria was negative bydrugs against malaria are not easily available and somesevere cases have been reported because of the delay inthe onset of treatment (Kano et al.. 1998; Hosaka et al..

the indirect fluorescent antibody test (IFAT). Two

1992). Particular attention has to be focused on quick
and proper treatment with safe and effective antimalar‑ials. Four cases of falciparum malaria which weresuccessfully treated with HalfanTM (halofantrine hydro‑
chloride, 233mg base/tablet, SmithKline Beecham Phar‑maceuticals) are reported in this paper.

CASE 1The patient was a Japanese T.V. cameraman, 39
1

2
3

tablets each of HalfanTM were administered orally at 19:
30 on Jan. 21, 2:30 and 8:30 on Jan. 22. Body temperaturefell to normal at 10:OO on Jan. 24 (36.9'C) and parasites

were cleared at 9:OO on Jan. 25. No adverse reaction wasrecognized after the administration of the drug, nor was
there recrudescence of P..f malaria. (Fig. 1).

CASE 2The patient was a Japanese male, 37 years old, whostayed at Vangunu in Solomon Islands during April 11‑25, 1992. He was on regular weekly chloroquine pro‑

Department of Parasitology, Gunma University School of Medicine, 3‑39‑22 Showa‑machi, Maebashi, Gunma 371, Japan
Department of Infections Diseases, Tokyo Metropolitan Komagome Hospital, 3‑18‑22 Honkomagome, Bunkyo ku Tokyo 113
Japan
Isesaki‑Sawa Medical Association Hospital, 481 Shimoueki, Isesaki, Gunma 372. Japan

246

Date 1992January

ZO Zl 22 23

Admission
Halofantrine( )24

25 26 27 28 29 30
Discd.. 1'3 ' I 500mq x 3

harge

0,5

Parasitemia(oc) 4 O

39
BodyTemp. 3 8

37
36
35

WBC (/mm3) 3600 3ZOO 4100 5800 5500 5000 5200 7200
517 497 467 465 497 466 450

7300
458

Hb (g /dl ) I

14.0

RBC(xl04/mma) 518

916,3 1 ,9 13.6 1 LO 1 .9

14.1 Ia6

H t (% ) 45 46 43 41 41 44 41 41
Plts (xlo /mm3) a3

a2 3.5 4.8 7.8 1 1.3 18.1 2a8

4Z
33.4

Figure I Chnical course of Case 1' 39y.. M*1*.

phylaxis. He was admitted to Tokyo Metropolitan

that period. He manifested fever in March, 1992 andKomagome Hospital on April 28, complaining of fever

was diagnosed as having malaria by a local doctor, andof 39.3'C, a headache, and general fatigue. On examina‑
tion, there was hepatomegaly, 2 fingers in width, but thetreated with Fansidar. Despite the treatment, hespleen was not palpable. Microscopic observation of athin blood smear revealed P.f parasites at 0.4% of red

then seen at Tokyo Metropolitan Komagome Hospitalon May 15. Peripheral blood smear showed P.f par‑blood cells. He was treated with a total of 6 tablets of
HalfanTM, because he was clinically suspected of suffer‑asitemia at 0.08% of RBCS and he was hospitalized on
the following day. Treatment with 1500mg of HalfanTM

ing from chloroquine resistant P.f malaria. However,in vitro drug susceptibility tests indicated that theparasites were susceptible to chloroquine (IC=0=84.5

cleared the parasites in 72 hours and fever in 63 hours.
On admission, the IFAT titer was 1:64 and increased tonM) , and highly resistant to pyrimethamine (IC50 > 1000

recrudescence of the parasites was recognized after thenM). His clinical course after the drug administration
is illustrated in Figure 2: parasitemia was cleared by
April 30 and body temperature fell to normal on May 2.

drug administration. Besides malaria, examination of

showed fever on April 25 and May 13 (38.6 'C) , and was

1:1024 on May 18 (Fig. 3). No adverse reaction norstool on admission revealed Shigella flexneri infection.
The patient was also successfully treated with a total of

Antibody levels of P.f and P. v. titers are also shown in

2000mg (400mgx5days) of lomefloxacin (LFLX), a

the figure. The case was successfully treated withquinolone derivative, and discharged from the hospitalon June 3.

HalfanTM and showed no adverse reaction nor recrudes‑
cence.

CASE 4
CASE 3The patient was a Japanese male student, 23 years
old, who visited China, Pakistan, India, some Africancountries (Kenya, Tanzania, Zaire, Zambia, Uganda andMalawi), and Thailand, between April 1991 and April
24, 1992. He was taking chloroquine irregularly during

The patient was a Japanese female and a dentist, 53

years old, who stayed in Mali from September 1989 to
May 3, 1993. She suffered from several episodes ofmalaria in September‑November 1991 and was treatedwith chloroquine, Fansidar, or quinine. She was nottaking any prophylactic measures against malaria dur‑

247

Date

1 992 April

28

Ma y

29 30 1 2 3

4 5 c

Admission

Disc{:

IP

September

126 rJIJIC
harge

I 500 q x 3

Halofantrine(%) 0,5

Parasitemia‑ ‑O(oc) 40' 39
Bod yTemp, 3 83736
IFATtiter l024

lz5c

P.f, ‑F c4

P,v. ‑o‑ Ic
<44

e

; a‑

WBC(/mm3) 6800 5800 5000 4400 5200 6400 6500

7100 7300

RBC(xl04/mrn3) 582 593 613 630 585 5Z7
Hb ( g/dl)
17.1 17.3 17.3 1 a2 t G.C 1 5.0

5G6 568

Ht ( )

49.7 51.2 53.3 53.7
Plts (xlOa/mm3) I 90 14B 8.0
4.4

534
1 5.5

IG.2 Ia2

4 9.7

44.5

4 5L9

49.5 48.8

4

8.2

21.1

l 9 17.3

Fig"*. 2 Chnical course ot Case 2. 37yr. Male.

Date 1993 June
8

g

IO 1]

AdmissionIP

12 13 14 ,5

DiscJ harge

Halofantrine 1 500*q x 3( ) 0.5

Parasitemia
O
(oc)

38
Bod yTemp. 3 736
35
IFATtiter l02a

25c

P.f. ‑ 84

P.v, ‑ 164<a

WBC (/mm3) 4900 4500 3900 4200
RBC(xl04/mm3) 373 3G9 347 357

4300
379

Hb (g/dl) 11.2 11.1 Iu4 1 0.6

1 1.3

Ht ( %) 32.8 334 31.0

31.9

33.6

Plts (xl04/mm3) 8.5 11.5 11.4

1 4.0

20.9

Fig"re 3 ai*i.*1 '*urse of Case 3. 23yr. Male.

248

1 992 May

AugustDate 15 16 17 18 I9 20 21 22 f‑=
25 29
28
F ! 17‑ :fL2J
AdmissionT h e rap y Jtl Halfan,500.q x 3

LFLX,400mex5days(%) 0.2

Parasitemia
0.1

r c tci+!

O(oc) 38

BodyTemp.3 73G

H5IFATtiter loz4

25G

P.f, ‑e‑ 8
P.v. ‑o‑ 16
<44

r (/

e

5

f

5000 5400
4800 450045005000
3G5
332 3G5 11.6
4 1410O 451
luO 10.6 9JB 10J 10.3
Hb ( g/dl)
IZ8
1
u5
1
l.
I
Z8
1 5
1 37.9Ht (%) Z C 31.5 Z 6 32.3 3aL8 31.2 34 134 37.9
40.4
Plts (xl04/mm3) 1 a2
14J 133 16.1 17.4
27.3 2a3
WBC ( /mm3)

31 OO

3900 4300 4200 4000

RBC ( xl04/mm3) 3l9

340 318 346 328

22 27.3 2a3 35.I

Fig"** 4 ch i.*1 .*urse *f Case 4. 53yr. Female.

ing her stay in Mali. After she came back to Japan, sheshowed fever of 39.2'C on May 16, 1993, and had been
feverish since then. On June 8, she was admitted toTokyo Metropolitan Komagome Hospital complainingof fever, chills, a headache and nausea. However, her
general condition looked apparently milder than that ofother Japanese patients with malaria. On adrnission, shemalaria cases which were successfully treated with
halofantrine. Halofantrine is an antimalarial drug,
unrelated to existing antimalarials, effective against all
species, effective in drug resistance, very well tolerated,

and with a simple dosage regimen. Treatment withof RBCs. Antibody titer against P.f antigen was 1:64 byhalofantrine cleared parasitemia within 7 days in morethan 99% of 1315 patients with falciparum malaria, andrecrudescences occurred in only 78 patients (6%) . Meanparasite clearance time was 57.9 hours in P.f malaria
patients, and mean fever clearance time was 50.2 hours(SB, 1992). Parasite clearance times of the reported

the IFAT (Fig. 4). Two tablets of HalfanTM werecases 1, 2, 3, and 4 in this paper were 85.5h, 48h, 63h, andadministered orally three times at 6 hourly intervals,
which cleared parasites by June 10. No adverse reaction

and 3 were 62.5h, 87h, 72h, which are well within thewas recognized after the drug administration.

ranges seen for the drug. Case 4 was a patient who

had mild anemia and hepatomegaly but nosplenomegaly. Falciparum malaria parasites weredetected from a thin blood smear at the density of 0.34%

48h respectively, and fever clearance times of Cases 1, 2,

involved more than 2500 patients in 30 countries (Srhith‑suffered from malaria several times before, and conse‑
quently, she was clinically semi‑immune; her symptoms
were rather mild, parasites were cleared more rapidly,
and her body temperature was only slightly elevated.
Therefore, changes in her body temperature following
the administration of halofantrine may not in itself be

Kline Beecham Pharmaceuticals (SB), 1992), but inindicative of the effectiveness of the drug.

Japan, few cases have been reported (Kano et al.. 1992;

Halofantrine is also reported to produce rapid
symptomatic improvement, and is very well tolerated

DISCUSSION
Halfan has been used for the treatment of humanmalaria since 1984. To date, clinical studies have

Masuda et al., 1992; Yodonawa et al.. 1992). This ispractically the first report of imported falciparum

without showing major side effects. One of the typical249

findings in the 4 cases in this paper was throm‑

We obtained the informed consent from every

bocytopenia, which is commonly seen in acute malaria.
Halfan product data (1992) shows that mean platelet

patient reported in this paper prior to the treatment
with halofantrine.

counts steadily improved from day 3 and reached aplateau between days 7 and 14 after onset of treatment.
Platelets count of the 4 cases in this paper increased tonormal in 4‑7 days after the drug administration (Fig. 1‑4). No apparent side effect was recognized after thetreatment with halofantrine in any of the 4 cases.
The IFAT for the detection of the malaria anti‑

ACKNOWLEDGEMENTS
The authors are grateful to Mrs. Rumiko Kosugi forher excellent technical assistance. We also thank Dr. B.
D. Akanmori for helpful discussion.

bodies is useful for retrospective diagnosis of malaria.

We can also distinguish the species of the parasites
which a patient has contracted (Kano et al.. 1990).
Cases I and 2 represent patients who suffered frommalaria for the first time, who were thus seronegativeon admission although showing parasitemia. However,Cases 3 and 4 had earlier malaria episodes and showed

REFERENCES
l . Kano, S., Tsuji, M., Hosoya, J. and Suzuki, M. (1988): A

case of severe malaria treated with qinghaosu. Jpn. J.
Trop. Med. Hyg., 16(4) , 301‑307
2 . Kano. S., Waki, S., Igarashi, I.. Nakazawa, S., Masuda,
G. and Suzuki, M. (1990) : Retrospective malaria diagno‑

considerable levels of antibody titer on admission to thesis by indirect fluorescent antibody titration on Japanesepatients. Jpn. J. Parasitol., 39(5), 475‑481

hospital. Both manifested P.f titers as high as 1:1024 in

3 . Kano, S., Yodonawa, S. and Suzuki. M. (1992): Twoa very short time. In Cases 2, 3, and 4, the IFAT titers

cases of vivax malaria treated with halofantrine. Jpn. J.

against P.f antigen were always higher than those

Trop. Med. Hyg., 20, 72
4 . Hosaka, S., Ishikawa. A.. Hosono, T., Iikuni, Y., Kondo,

against P. v. antigen throughout the clinical courses of

H.. Kobayashi, Y., Nakamura, T., Ito. Y.. Ohtomo, H.,
Kano, S. and Suzuki, M. (1992) : A case of severe mixed
Plasmodium falciparum and P. vivax infection success‑fully treated with artemether. Jpn. J. Trop. Med. Hyg.,

the cases. Although the IFAT is not always useful forthe diagnosis of malaria at the early stages of the infec‑

tion, we can picture the episodes and course of theinfection retrospectively through the changes in thetiters.

Halofantrine belongs to a class of compounds‑thephenanthrene‑methanols‑, which does not share chemi‑
cal structure with any other animalarials, and is there‑

fore particularly effective in the treatment of drug
resistant malaria (SB, 1992). Elimination half‑life of
halofantrine is 1.3‑6.6 days (SB, 1992) which is longer
than that of quinine but considerably shorter than thatfor mefloquine or chloroquine, and the therapeuticcourse of treatment is completed within 12 hours.
Because of these properties the parasite is not exposed
to sub‑optimal levels of the drug for an unnecessarily
10ng time, thus the risk of P.f strains emerging which
are resistant to halofantrine is thought to be minimized.

WHO data for 1993 shows that there is a widespread
distribution of chloroquine‑resistant P.f, while resis‑
tance to Fansidar is also on the increase (WHO, 1992) .

However, Fansidar is the only antimalarial drug com‑
mercially available in Japan so far despite the fact thatthe number of imported malaria cases is increasing,including drug resistant malaria. In fact, cases I and 2

in this paper were considered as chloroquine resistantclinically, and case 2 as in vitro pyrimethamine resis‑
tance. The general use of halofantrine in Japan is thus
expected.

20(4) , 305‑312

5 . Masuda, G., Ajisawa, A., Negishi, M.. Yamaguchi, T.,
Kano. S. and Suzuki, M. (1992): A case of mix infection

with Plasmodium falciparum and P. malariae treatedwith halofantrine. Clin. Parasitol., 3, 38‑40

6 . Ohtomo, H., Takeuchi, T., Masuda, G., Akao, M., Ta‑kada, S., Araki, T., Tsuji. M. and Tanaka, H. (1993):
Malaria incidence in Japan for past 5 years. Jpn. J.
Parasitol., 42, (suppl.) , 137

7. SmithKline Beecham Pharmaceuticals (1992): Halfan

product data, SB House, Middlesex, UK
8. WHO (1993) : Weekly Epidemiological Redord, 68(34) ,245‑252, and 68(35) , 253‑260
9. WHO expert committee on malaria (1992): Nineteenth
Report, 8
10.

Yodonawa, S., Kano, S., Saito, T.. Yokoyama, Y.,
Kawai, S. and Suzuki, M. (1992) : An imported case ofvivax malaria treated with halofantrine. Jpn. J. Trop.
Med. Hyg., 20(4) , 299‑303

251

Jpn. J. Trop. Med. Hyg., Vol. 21, No. 4 1993 pp 251 254

Research NoteSEXUAL MOSAICS IN THREE BLACKFLY SPECIES(DIPTERA= SIMULIIDAE) IN JAPANUplK KESUMAWATI HADI AND HIROYUKI TAKAOKA
Received October 7 1993/Accepted October 29 1993

Abstract: Sexual mosaics were found in eight adult Japanese blackflies, i.e., two Simulium arahawae
Matsumura, 1921, four S. bidentatum (Shiraki, 1935) , all collected from cattle sheds while biting, and two S.
aokii (Takahasi, 1941) which emerged from pupae. They were distinguished into three types, i.e., bilateral,antero‑posterior and other types of gynandromorph.

Sexual mosaicism, the appearance of morphological

species mentioned below were evident, being chiefly(phenotypic) features of both sexes in the same individ‑

recognized in the eye facets, frons, a tennae, mandibles
and maxillary lacinia on the head; in the scutal patternof the thorax; in the subcosta of the wings; in the clawsof the legs; in the tergites and sternites of the abdomen;uals, is known to occur in insects. Such sexually mosaic

individuals can be classified either as gynandromorphs(a mixture of genetically male and female cells) orintersexes (only cells of a single genotype) (Brust,and in the genitalia. Wing length represents the distancefrom arculus to the wing tip.

1966) .

In family Simuliidae (Diptera) , sexual mosaicism
has been reported to occur in several different species,

i.e., Simulium grisescens var. palmatum Puri, 1932 in
India (Puri, 1933), S. venustum Say, 1823 in Canada(Wolfe and Peterson, 1959) , S. arcticum Malloch, 1914 in

DESCRIPTION OF THE SEXUAL MOSAICS
Table I shows the descriptions of the eight sexuallymosaic individuals.

Canada (Fredeen, 1970), S. soubrense Vajime and Dun‑bar, 1975 in lvory Coast (Dang and Peterson, 1979) , S.

No. I Simulium arahawae Matsumura, 1921

damnosum Theobald, 1903 (complex) in West Africa

The body' Iength was about 3.2 mm and wing length(Cheke and Garms, 1985) and S. aokii (Takahasi, 1941) ,S. bidentatum (Shiraki, 1935) , S. takahasii (Rubtsov,1962), and S. iwatense (Shiraki, 1935) in Japan (Saito

was about 2.5 mm. Collected on May 5, 1989 fromand Kanayama, 1986). We report here eight cases ofgynandromorph, showing a male phenotype on the leftsexual mosaics found in three Japanese blackfly species
collected from Oita in Kyushu island, southwest Japan.

side and a female phenotype on the right side (Figs. l, 2,

MATERIALS AND METHODS
Fly numbers 1‑6 were collected at cattle sheds in
Oita City, and the remaining two (fly numbers 7 and 8)

Kokubu cattle shed in Oita.
This appeared to be an almost complete bilateraland 3a) . With an exception, Ieft mandible was serrated

like the right mandible. The epipharyax and cibarium
were of female phenotype. The genitalia was of female
phenotype on the right side, although the paraproct (Fig.

2e) was incompletely formed and the cercus (Fig. 2f)
was small like that of a male (Fig. 2g) . The genital fork

were found among those that emerged from pupae(Fig. 2b) was incOmplete, Iacking a left arm. The

collected from a stream in Yufuin, Oita. All specimens
were identified according to descriptions given by Ta‑
kaoka (1976, 1977). As in most other blackfly species,

ventral plate (Fig. 2j) of the male genitalia was shaped
only on the left side. The median sclerite (Fig. 2k) waspresent but looked narrower than a normal one.

sexually dimorphic characters in the three blackfly
Dmsron of Medical Zoology, Oita Medical University, Hasama, Oita 879‑55, Japan

252

Table l Descriptions of eight sexual mosaics of blackflies collected from Oita, Japan

No.

Head Thorax

S pecies

L R L RFM F

S. arahawae
S. arahawae
S. bidentatum
S. bidentatum
S. bidentatum
S. bidentatum

F X
F F
F F
F X
F F

2.
3.
4.
5.
6.
7. S. aohii
8. S. aokii

M M
F

F

M
F
F

M
M

X X
X
X

X F
X F

M
F

F

M

Abdomen

LR MLRF

M F
F M

X X
.X
X

F
F

Wing

Leg

M M

F
F
F
F

M

F

M

M
M

M

Genitalia

LR

L R
M F

M F
M M
M M
M M
F F
M M
F F
M M

X X
X X
X X

F
F
F
F
F

F

F

M M
F X
M M

* L, Ieft; R, right; M, male; F, female; X, mixed phenotype(see detailed descriptions in the text) .

.. ;: 7"j{'̲l4"'*} :L" r;･ :!; : ; i:i .'h; a"i'*:‑"'" :**'j;"';s"{"':i ";'}"/ /i:: "'‑'t ;"f "*+'
?̲+ :{{ :i*:;:;
i i'=:':"*' .','., j;:: ; :' *{ I f:f!!:'. : iJ" :j;::it̲;.>^';

.

, ¥ ¥

f :*<

r ‑1 * >.H"j ^?i"A ¥"''i .･ ･; ':;:i" *L r .

,,"t"' 'l". *hb.$'* ,: {/""'p''{/'J ;Jr:.... .:"s '' r='j'=:';il;"'̲: *':!'s""i::'...'<"..
,..̲.' ';"'' '; ･ ":"'"'"t ;‑'u; ', ':j'r;;1:'t:i' :,'e'‑'1 t !i'! ' "

f/jr/ /:;'/

1'$'h' 'J'::"" '/.. /.J. 'I' """/''

' """":*F" "i"if/1'L;"' 'If="f' .

.v[f:k' :'.'f'',';'tlt"'n"' '..
'',. """';'
i'. .* ,,:' ; " """"t‑ " ';: ::" i !"t' 'k "j';J '.':1d :'}1". ,,, ,ie t .' '" J'"'' "‑"'1'
5'1""" ' J".. *,, '"'. I : i

" ' *:(:'

1; t" ' r

j. ' :.:;: :f . s ';/

' p,': '

r̲':

N"'.

:i'::: ;.

'::/'iii)"'$"'':" If:' ;/':iFigure I Head, thorax and abdomen (dorsal view) of S.
arahawae N o I , showing a bilateral gynandromor‑
phism, with a female character on the right anda male one on the left side.

No. 2 Simulium arahawae
The body length was about 2.7 mm and wing length

was about 2.3 mm. Collected on September lO, 1989

:h *;" "'/;;;;' h/1 1 i:P:.'..:"

g 1 1'
Frgure 2 Termmalia (ventral vrew) of S arakawae No 1'
a' spermatheca; b' genital fOrk; c' 8th Sternite; d'anterior gonapophysiS; e' paraproct; f' cercus(female) ; g' cercus (male) ; h' style; i' coxite; j'ventral plate; k' median sclerite; l' paramere'from Imanaga cattle shed in Oita.
This fly possessed a female character on the head
except for the eye facets near the right front‑occularNo. 3 Simulium bidentatumarea which were somewhat enlarged. The thorax (Fig.

was 2.4 mm. Collected on October 29, 1989 from3b) , Iegs and wings were bilaterally divided, with female

Imanaga cattle shed.

phenotype on the left side and male one on the right.
Abdominal segments 2 to 5 showed a female phenotype

The scutal pattern on the thorax showed a female

whereas segments 8 and 9, and genitalia were of anorngal male. Abdominal segments 6 and 7 were mosaic,each with a female phenotype on the left and a malephenotype on the right side.

The body length was about 3.2 mm and wing length

This fly possessed a female head and male genitalia.

phenotype on the left and a male one on the right as inNo. 2. The fore legs showed a mixed phenotype, i.e., amale character on the left side, and a female one on theright, while mid and hind legs were of female and malecharacters, respectively. The wings were of female

253

c

b

Figure 3 Scutal patterns of sexually mosaic blackflies. a, S arakawae No I b S
arahawae No 2; c, S. bidentatum No 4; d, S. bidentatum No 6; e. S. aokii No
7; and f, S. aokii No 8.

phenotype on both sides. The ventral surface of the phenotype, except for all the legs and the small area ofabdomen showed a male phenotype. However, dorsal the right eye which were of male character. This fly
surface of the abdomen was mosaic as in No. 2. was infected by mermithid.
No. 4 Simulium bideniatum
The body length was about 3.9 mm and wing length

No. 6 Simulium bidentatum
The body length was about 3.4 mm and wing length

was about 3.0 mm. Collected on April 26, 1990 from was about 2.4 mm. Collected on April 10, 1990 from
Imanaga cattle shed.

Imanaga cattle shed.

This fly had a female head and a male abdomenThis specimen possessed a female head and male
genitalia as in No. 3. The thorax showed an irregular including the genitalia. The scutum showed an unusual
scutal pattern having a dark and wide longitudinal line phenotype on the right side, but had a normal female
medially (Fig. 3c) . The hind legs were of female pattern on the left side (Fig. 3d) . The legs also had aphenotype on both sides whereas fore and mid legs female phenotype except the left midleg being of male
showed a mixed phenotype with a male character on the phenotype. The wings were bilaterally divided, a maleleft side and a female character on the right side. The phenotype on the left side and a female phenotype on thewings were of female phenotype on both sides. The right side.
ventral surface of the abdominal segments 2 to 4 showed

a mixed phenotype with a female phenotype on the right No. 7 Simulium aokii
The body length was about 3.8 mm and wing lengthand a male one on the left side, whereas that of theremaining segments was of normal male. The dorsal was about 2.9 mm. Collected as a pupa on March 21,
surface of the abdomen showed a male phenotype. This 1989 in Yufuin and emerged in the laboratory.
This specimen possessed a male head and female
fly contained a blood meal in the midgut.
genitalia. The thorax showed a normal male scutalNo. 5 Simulium bidentatum pattern on the left side, but had a slightly aberrant
The body length was about 4.2 mm and wing length pattern on the right side (Fig. 3e) . The legs were of
was about 3.0 mm. Collected on March 10, 1990 from female character, except for the fore and mid legs on the

Imanaga cattle shed. Ieft side being of male character. The wings were as inThis fly was, for the most part, of female No. 6. The abdomen was of female phenotype except

254

for the basal scale and segment 2 on the right side, whichwere of male character.

No. 8 Simulium aokii
The body length was about 3.0 mm and wing length
was about 2.8 mm. Collected as pupa on April 7, 1990
and emerged in the laboratory.
This specimen exhibited a female head and a maleabdomen including the genitalia as in No. 6. The scutal
pattern on the thorax (Fig. 3f) , Iegs and wings weredivided bilaterally, bearing a male phenotype on theright side and a female phenotype on the left side.

REMARKS. As previously reported in other blackfly
species (Wolfe and Peterson, 1959; Dang and Peterson,
1979 and Puri, 1933), the eight sexually mosaic speci‑
mens reported here are distinguished into three types ofgynandromorph: (1) bilateral type, as found in one S.
arahawae (No. 1); (2) antero‑posterior type, as found insix specimens i.e., one S. arahawae (No. 2), three S.
bidentatum (No. 3, 4, and 6) , and one S. aokii (No. 8) , allhaving a female phenotype on the anterior part and amale phenotype on the posterior part, while other bodyareas showed a mixed phenotype; and one more speci‑men, S. aokii (No. 7) , with reverse character; (3) anothertype, as found in one S. bidentatum (No. 5), mainly
having a female phenotype and sexually mosaic areas of
the body being distributed in an irregular pattern. Noneof these flies was interpreted as intersexes.

REFERENCES
1 ) Brust, R.A. (1966): Gynandromorphs and intersexes inmosquitoes (Diptera: Culicidae) , Can. J. Zool., 44, 911‑921.

2 ) Cheke, R.A. and Garms, R. (1985): Sexual mosaics inSimulium damnosum species complex (Dipt,
Simuliidae) in West Africa, Entomologist's Monthly
Magazine, 121, 137‑141.
3 ) Dang, P.T. and Peterson, B.V. (1979): A case of bilat‑eral gynandromorphism in Simulium soubrense Vajime
and Dunbar (Diptera: Simuliidae) , Tropenmedizin und
Parasitologie, 30, 548‑550.
4 ) Fredeen, F.J.H. (1970): Sexual mosaics in the black fly

Simulium arcticum (Diptera: Simuliidae) , Canadian
Entomologist, 102, 1585‑1592.

5 ) Puri, I.M. (1933): A case of gynandromorphism inSimulium. Indian J. Med. Res., 20, 801‑803.

6 ) Saito, I and Kanayama, A. (1986): Gynandromorphismof Japanese blackflies, Jpn. J. Sanit. Zool., 37, (3) , 292(In Japanese) .

7 ) Takaoka, H. (1976) : Studies on black flies of the
Nansei Islands, Japan (Simuliidae; Diptera) . II. On six

species of the subgenera, Gomphostilbia Enderlein,
Morops Enderlein, Odagmia Enderlein and Gnus Rubzov,
with a description of Simulium ( Gomphostilbia) oki‑
nawense sp. nov., Jpn. J. Sanit. Zool., 27, (4), 385‑398.

8 ) Takaoka, H. (1977): Studies on black flies of the
Nansei Islands, Japan (Simuliidae; Diptera). 111. On six
species of the subgenus Simulium Latreille. Jpn. J. Sanit.
Zool., 28, (2), 193‑217.

9 ) Wolfe, L.S. and Peterson, D.G. (1959): Blackflies(Diptera: Simuliidae) of the forests of Quebec, Can. J.
Zool.,

7, 137‑159.

Jpn. J. Trop. Med. Hyg., Vol. 21, No. 4, 1993, pp. 255 258

Short c*255**i***i**

SERO‑EPIDEMIOLOGICAL STUDY ON HUMAN
TOXOCARIASIS IN THE RURAL SECTORAROUND RECIFE, NORTHEAST BRAZILYUZURU IWANAGA1,2, JOSE FELIPE GONCALVES3, MASANOBU TANABE2,+
SEIKI TATEN02,4, MORIYASU TsUJ15 AND TSUTOMU TAKEUCHI'
Received August 23 1993/Accepted October 7 1993

Until 1981, over 1,900 hurnan cases with toxocar‑

and immunoelectrophoresis were performed according

iasis had been reported in the world (Glickman and
Shantz, 1981). Prevalence and epidemiology of humantoxocariasis have been extensively studied in manyto the methods of Tsuji (1974, 1975). Crude extracted

countries (Brunello et al.. 1986; Salinas et al.. 1987;

Lynch et al.. 1988) . In Brazil, two cases of infant
toxocariasis (Kawakami et al.. 1984) and prevalence ofantigens were prepared according to the previous
method (Tsuji, 1974) from the adult worms of T. canis.
Ascaria suum. Dirofilaria immitis Schistosoma mansoni
and Taenia saginata, and from the larvae of Anisakis sp.,

Two hundreds and eighty‑eight sera were primarily

human toxocariasis (Chieffi et al., 1988) have beenreportod in Sao Paulo. Recently, two human toxocar‑

examined by Ouchterlony using T. canis antigen. Seven

iasis of inpatient and outpatient have been reported in
two hospitals located in the urban area of Recife, north‑antigen as shown in Table 1. However, these seven sera
also revealed positive reaction with other helminthous
antigens: all sera were positive for S. mansoni and D.

east Brazil (Virginia et al.. 1991). However, to oursera (2.4%) showed positive reaction with T. canis

knowledge, there is no reliable datum on the incidence,

immitis antigens, and one reacted with both A. suum

prevalence and epidemiology of human toxocariasis in

and Anisakis sp, antigens. Stool examination of all ofthese seven cases showed positive for S. mansoni eggs.
Six subjects were also positive for hookworm eggs, two

the rural sector around Recife. According to par‑asitological examinations in Sao Lourenco da Mata,northeast Brazil (Goncalves et al.. 1990) , four out of 485

inhabitants were positive for unembryonated eggs ofwere positive for T. canis eggs, and one was positive for
Ascaris lumbricoides eggs (Table 1).

Therefore, a sero

To evaluate the specificity of precipitation reaction‑epidemiological study was conducted to obtain prelimi‑

on Ouchterlony test, these seven sera were further
analyzed by immunoelectrophoresis. All sera did notshow any precipitin band with the antigens prepared

Toxocara canis in stools.

naty data on the prevalence of human toxocariasis in
Sao Lourenco da Mata.
Serum samples were collected from 288 inhabitantsin four villages (Ceramica Bicopeba, Engenho Pitan‑
gueira, Engenho Camurim and Engenho GeneraD in Sao

Lourenco da Mata during July to December, 1989.
Prevalence of intestinal parasitic infections of thesesubjects has been reported previously (Goncalves et al..
1990) .

The serological examinations using Ouchterlony

from A. suum. T. saginata and Anisakis sp. But all sera
revealed positive reaction with S. mansoni antigen, an
one reacted with D. immitis antigen. On the other hand,all of those specimens showed plural precipitation lines
with T. canis as demonstrated in Figs. I and 2, and thespecies‑specific precipitin band for T. canis (Tsuji,1975) were detected in four serum samples.
Although definitive diagnosis of the toxocariasis is

Department of Parasitology, Hiroshima University School of Medicine, 1‑2‑3 Kasumi, Minami‑ku, Hiroshima City, Hiroshima 734, Japan
2. Laborat6rio de Imunopatologia Keizo Asami, Universidade Federal de Pernambuco, Cidade Universitaria, Recife, Pemambuco
3

CEP=50,730, Brazil
Centro de Pesquisas Aggeu Magalhaes, Cidade Universitaria, Recife, Pernambuco CEP=50,730, Brazil

4

Department of Tropical Medicine and Parasitology, School of Medicine, Keio University, 35 Shinanomachi, Shinjnku‑ku, Tokyo 160, Japan

5

Department of Parasitology, Kyorin University School of Medicine, 6‑20‑2 Shinkawa, Mitaka City, Tokyo 181, Japan

256

Table l The results of Ouchterlony test and stool examination for inhabitants in Sao Lourenco daMata, Brazil

No.

Sex

Age

T,c

s.m

D.i

101 9

F
F

7

+
+
+
+
+
+
+

+
+
+
+
+

l028
3009

3053
3075
4008

4185

Stool examination

Ouchterl ony test

Sub jects

17

+
+
+

36
54

+
+

8

+
+

32

M
F
F
F
F

19

A,s Ani+ +T.c: 'roxocara canis

S,m: Schistosoma manso;ei

D,i: Dirofilaria im mitis

A,s: Ascaris suum

Ani: Aleisakis larvae
A.1: Ascaris tu, bricoides

H.w: Heok worm

diagnosis of human toxocariasis has been usually perfor‑
med by variaus serological tests using different types ofantigens (Cypess et al.. 1977; Savigny and Tizard, 1977;

+
+
+
+
+
+

+

based upon the larvae in biopsy specimens, demonstra‑tion of larvae appears to be difficult. Therefore, the

s .m

H,w
T.c
+
A.l

+
+
+
+

+

Kawamura, Ig83; Clemett et al.. 1985; Inoue and Tsuji,1989), However, in the case of toxocariasis as well asother nematoda infections, there exist many crossreac‑
tions with other parasitic antigens on any kind of ser‑ological methods. In contrast. Tsuji (1975) has demon‑
strated that toxocariasis can be diagnosed by the immu‑
noelectrophoresis according to the presence of species‑
specific precipitin band. In the present study, such a
species=specific precipitin band was identified in four
serum samples by immunoelectrophoresis. It is, there‑
fore, Iikely that at least four cases may considered to bepositive cases of human toxocariasis serologically.
*****

'

i =

**

* *

Goncalves et al. (1990) suggest that the circumferential"= """+'i ";" ";:;i;, "' ,, j:;:'""*;"*"

;'

' "

;

:***' '.""

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

environments around their housings may be heavily
contaminated with 'r. canis eggs. So, a high incidence of
toxocariasis is expected in this area. But, in the present""="'i="' "' ';i' '{, j:i;j;:j:;i!:,:1:;;%

study, toxocariasis were a few cases. This finding
suggested that some of ingested T, canis eggs poten‑
tially developed in humans. As mentioned above, noreliable data on human toxocariasis in the rural sector

around Recife, northeast Brazil have been reported
ti I.= .

"';" i" = i #*!'":' :' *i! ' ; ' '‑ '=before so that the present communication may be firstreport on the occurrence defined cases of human tox‑
ocariasis in this area by serological approach.

ACKNOWLEDGEMEN T
The authors are indebted to Dr. Luis Carvalho,
Universidade Federal de Pernambuco, Brazil, for his
suggestion,

Fig. lImmunoelectrophoresis against Toxocara canis
antigen for inhabitants in Sa o Lourenco da Mata,
BrazilT. can: Toxocara captis adult worrn.

This study was supported by Research Grants fromthe Japan International Cooperation Agency (JICA) .

257

3075

701 9

=O

=i
O

T. can

T can

1028

O

4008

O

T. cant

T. canx09

O

4 1 c5.

T can

T. canO 'r3053*: Specific band for
Toxocara canis

r‑O
T can

Fig. 2

Immunoelectrophregrams against Toxocara canis antigen forinhabitants in Sao Lourenco da Mata, Brazil

REFEREN CES
1 ) Brunello, F., Falagiani, P. and Genchi, C. (1986) :Enzyme linked immunosorbent assay (ELISA) for thedetection of specific lgG antibodies to Toxocara canis ES
antigens, Boll. Ist. Sieroter Milam., 65, 54‑60
2 ) Chieffi, P.P., Ueda, M., Camargo, E.D., Souza, A.M.C.,

Silva, C.L., Nova, A.V. e Guedes, M.L.S. (1988) :Contacto domiciliar e profissional com caes como fator‑
es de risco para infeccao humana por larvas de Toxocar‑
a. Rev. Inst. Med. Trop. Sao Paulo., 30, 379‑382
3 ) Clemett, R.S., Hidajat, R.R., Allardyce, R.A. and Stewar‑t, A.C. (1985) : Toxocaral infection in hydatid control

officers; diagnosis by enzyme immunoassay, New
Zealand Med. J., 98, 737‑739
4 ) Cypess, R.H., Karol, M.N., Zidian, J.L., Glickman, L.T.,
Gitlin, D. (1977): Larva‑specific antibodies in patientswith visceral larva migrans, J. Infect. Dis., 135, 633‑640

5 ) Glickman, L.T. and Shantz, P.M. (1981): Epidemiology
and pathogenesis of zoonotic toxocariasis, Epidemiol.
Rev., 230‑2506 ) Goncalves, J.F., Tanabe, M., Melo, F.P.M., Goncalves, F.
J., Aca, I.S., Motta, S.R.N., Tateno, S. and Takeuchi, T.

(1990): Parasitological and serological studies onamoebiasis and other intestinal parasitic infections in
the runal sector around Recife, northwest, Brazil, Rev.
Inst. Med. Trop. Sao Paulo., 32, 428‑435

7 ) Inoue, H. and Tsuji, M. (1989): Studies on visceral larva

migrans: Detection of anti‑Toxocara canis lgG anti‑
bodies by ELISA in human and rat sera, Jap. J. Par‑
asitol., 38, 68‑76

8 ) Kawakami, E., Fagundes, N.U., Wehba, J. and Patricio,
F.R. (1984): Visceral larva migrans in infancy; report of2 cases, Arg. Gastroent., 21, 83‑87
9 ) Kawamura, H. (1983) : Detection of circulating antigen

and antibody of Toxocara canis by radioimmunoassay
and antigen‑binding assay with a purified Toxocara
canis antigen, Hiroshima Med. J., 31, 265‑274lO) Lynch, N.R., Wilkes, L.K., Hodgen, A.N. and Turner, K.
J. (1988) : Specificity of Toxocara ELISA in tropical
populations, Parasit. Immun. 10, 323‑337
11) Salinas, P., Reyes, L., Sotomayor, M.T. e Letonja, T.
(1987): Prevalencia de huevos de Toxocara sp. en al‑

gunas pazas publicas de la region metropolitana deSantiago, Chili Boletin Chileno de Parasitol., 42, 33‑36

12) Savigny, D.H. and Tizard, I.R. (1977): Toxocaral larva
migrans: The use of larval secretory antigens in haemag‑
glutination and soluble antigen fluorescent antibody
tests, Trans. R. Soc. Med. Hyg., 71, 501‑507

13) Tsuji, M. (1974): On the immunoelectrophoresis forhelminthological researches, Jap. J. Parasitol., 23, 335‑34514) Tsuji, M. (1975): Comparative studies on the antigenic
structures of several helminths by immunoelectrophor‑258
esis, Jap. J. Parasitol., 24, 227‑236

15) Virginia, P., Nagakura, K., Ferreira, O. and Tatemo, S.
(1991) : Serologic evidence of Toxocariasis in northeast
Brazil, Jpn. J. Med. Sci. Biol., 44, 1‑6

Mendosa，E．C．，Velared，F．U

G、，Sengoku，S．，Miyoshi，C．and

Possible

Application

of

at

Cruz

Hospital，Bolivia…………一・…・…・…………・…．．．．…・．．…・…・

Santa

GeneralSurgicalTreatmeptofChagasic

Nakasa，T．

Megacolon
・・219−224

Makino，YりTamanaha，E，Miyazato，Z．，Shimabukuro，J．，Matsumora，C．，
YanンC。，Ishihara，RりMachida，T，，Yamada，N．，Arakaki，S．，Tadano，M．and
Fukunaga，T．
An

Outbreakof

Aseptic

Meningitis

inOkinawa

in1993…・………・……………．．．．．．．

一225−229

Oda，T．，Suenaga，0．，Mori，AりFujita，K，Zaitsu，M．，Kurokawa，K．，Nishioka，T．，

Itoh，T．and

Mine，M．

Studies

EpidemiologyonJapan，withofConsiderations

Eto，H。，Toriyama，K．and
An

Case

Ultrastructural

Z万zφ吻蛎α1窺吻燃inon

YearlyHouse

ChangesinEndemic

KaposiDogs

inMicrofilarial

Nagasaki

City，

Prevalence＿．．＿・・231−237

Itakura，H．

Study

of

Africans

Sarcoma…一一……一・・…

・・239−243

reportKano，S．，Masuda，G、，Kubo，Y．，Saito，M．，Yamaguchi，T．

andSuzuki，M．

Four

Imported

TreatedwithResearch

note

Short

of

Falciparum

Malaria

Successfully

Halofantrine………・……………一……．．…・…・．．．…・一・．…・一．……卓…．

Hadi，U、K．and
SexualCases

Mosaics

一245−249

Takaoka，H．
in

Three

BlackflySpecies（Diptera：Simuliidae）inJapan………・・・251−2瓢

communication

Iwanaga，Y・，Goncalves，J．F．，Tanabe，M．，Tateno，S．，

Tsuji，M．and

Takeuchi，T．

Sero−epidemiologicalStudy

AroundBrazil・・一………・………一・………．…・．．．一…・．．．一…・．．

Recife，Northeast

on

Human

Toxocariasis

in

the

Rural

Sector・・255−258

OECEMBER1993

VO■．21NO．4

CONTENTS
Original

articleHori，S．and

Tanaka，N．

Adaptive

Changes。Induced

by

in

Heat

Physiological

Acclimatization

Responses

and

of

Men

PhysicaltoTraining…一…

Shichinohe，K．，Shimizu，M．，Haga，K．，Iedokoro，T．and
BasicStudies

Filarial
Values

Betweenthe

MongolianFrequency

Malaria

Gerbilas

StudiesWild−Coloredaon

Gerbil

Fujita，K．

Susceptible

Serum

and

一193−199

Host

to

Biochemical

the

Coat

Color

Mutants一…

・・201−206

Safi，S．H．，Omer，F．E．D．M．，Rivera，P．T．，

Gadda1，A．A．and

AntibodyatheInfection：Comparative

Kano，S．，EI

El

on

Heat

Epidemic

Suzuki，M．

Distribution
in

the

Curve

for

Risk

Assessmentof

Sudan……・…・…………………………・…一…

一207−211

Kano，S．，Utsunomiya，Y．，Honda，A．T．，Santos，O．，Miranda，W．S．，

Hida，M．M．，Matsumoto，K．and
Malaria

SerologyinHighlyaasSuzuki，M．

anAltemativeto

Endemic

AmazonianCommmity……………・…・（Contents

Parasite

continued

on

Detection

reverseof

thiscover）

・・213−217