ISSN

Japanese Joumalof Tropical

Medicine and Hygiene

第9巻第3，4号

昭和56年12月15日

内原O304−2146

容著

穂庶ど蒲盤鰍9禦離察贈鑓鷺の比較鼎中信雄

151−159丁矧卯初so脚即吻房卿s8免疫マウスの脾細胞あるいは抗血清を移入したマウスの同一原虫感染に対すう抵抗性（英文）一古谷 正人，岡三希生，伊藤義博，岡文雄

161−165

達，高岡 正敏

167−173

実験的バベシア感染犬におけるリンホカイン中の白血球走化性因子に関する基礎的検討（英文）…………一……・…………一・…・……伊藤守，牧村進，鈴木 直義

175−185

DIC症候群類似の症状を呈したクロロキン耐性熱帯熱マラリア症の一例
・・戸田 正夫，石崎好万，尾崎

中米グァテマラの温度条件の異なる種々の標高における0ηohoo670α抄oJむ％」％s幼虫の媒介プユS加％伽卿oo伽σo％卿体内での発育（英文）・高岡弘，Ochoa，J．O．，Juarez，E．L．

187−197

Dゆhグlloゐo∫h吻勉oα泌6プo勉Rausch，1969一カメロン裂頭条虫（新称）一の人体寄生第1例（英文）・・…………………………………・…加茂甫，山根 洋右，川島健治郎

199−205日

熱医会誌Japan．J．T．M．H．

宏行，Hansen，K．M．，高橋

日 本熱帯医学会

Japan. J. Trop. Med. Hyg., Vol. 9,

o. 3, 4, 1981, pp. 151‑159

151

COMPARISON OF BODY SHAPE, BODY COMPOSITION

AND SWEATING REACTION BETWEEN YOUNG
MALE HIGHLANDERS OF PAPUA NEW GUlNEAAND YOUNG MALE JAPANESE
S. HoRI, J. TSUJITA, M. MAYUZUMI AND N. TANAKA
Received for publication 20 April 1981

Abstract : Anthropometric measurements and measurements of sweating rcaction
during exercise were made on I I young male highlanders in Beha Village at altitude
between I ,500 meters and I ,800 meters above sea level in Papua New Guinea in Augustand I I young male Japanese in Nishinomiya City in September. Measurements of local
sweat rate and sodium concentration in local sweat during pedalling a bicycle ergometerat a constant work load of 450 Kg.m/min for 20 min. under a thermoneutral condition
were made. New Guineans were significantly shorter in height, slightly lighter in bodyweight and had a lesser amount of body fat than Japanese. New Guineans showed signifi‑
cantly greater mean values of Rohrer's index and Brugsch's index than Japanese. Skinfold

thickness for New Guineans was significantly thinner than that for Japanese. Physically

New Guineans were more muscular and athletic when compared with Japanese. New
Guineans showed considerably lower local sweat rate and significantly lower Na concen‑
tration in local sweat than Japanese. Differences in anthropometric characteristics andsweating reactions between New Guineans and Japanese might reflect more advanced
acclimatization to hot environments in New Guineans when compared with Japanese.

INTRODUCTION
It is known that body fat content, body composition, and body shape change
according to climate in which individuals live, nutritional conditions as well as
physical activities (Coon, Garn and Birdsell, 1950; Bro ek, 1952; Keys and Bro ek,
1953; Lewis et al., 1960). The subcutaneous layer of fat keeps heat content fromheat dissipation due to low thermal conductance of fat (Burton and Bazett, 1936)
when net heat is transferred from the body into the environment. The decrease in
the subcutaneous fat layer produces such a change in body shape that the ratio ofbody surface area to body weight is increased. Since heat dissipation from the bodyis proportional to body weight, a greater body surface area to body weight is favora‑
ble for heat dissipation from body to the̲ environment. Thus, it is assumed that bodycomposition and body shape are important factors in temperature regulation (Horiet al., 1977). Evaporation of water from the skin surface is the main mechanism ofheat dissipation in a hot environment. It is well known that sweating reaction ofhumans changes adaptively under the influence of climate and the degree of physical
Department of Physiology, Hyogo College of Medicine, Nishinomiya 663, Japan1 52

activities (Dill et al., 1938; Adolph, 1946; Hori, 1977). The Eastern Highlands ofPapua New Guinea at about 1,600 m above sea level , Iocated in a tropical zone, have
hot middays and cool nights throughout the year, while Japan, Iocated in a temperatezone, has not summers and cold winters. Highlanders in Papua New Guinea havea high carbohydrate diet (Hipsley and Clements, 1950) and perform hard muscularexercise such as ascending steep slopes in their daily lives when compared withJapanese (Hori et al., 1980). Thus, it is of interest to compare body composition,body shape and sweating reaction of highlanders in Papua New Guinea with thoseofJapanese. In the present study, an attempt has been made to investigate differ‑ences in physical charac.teristics and sweating reaction between young male high‑landers of Papua New Guinea and young male Japanese and to discuss the differencesfrom the viewpoint of physiology of climatic acclimatization of humans.

MATERIALS AND METHODS
Eleven young male highlanders of Papua New Guinea and I I young male
Japanese were selected as the subjects in the present study. The study on the New

Guineans was made in August 1978 in Beha Village at altitudes between 1,500
meters and 1,800 meters above sea level in the Eastern Highlands of Papua New
Guinea, tropical zone (Fig. 1) and that on the Japanese was made in September in
Nishinomiya City at sea level in Japan, temperate zone. Anthropometric measure‑ments and measurement of sweating reaction during bicycle ergometer exercise were
14F OE

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PA PUAlFig, I situatron of Beha vmage

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Alotau153

made. The skinfold thickness was measured on t.he right side of the body with acaliper 2 sec. after a pressure of 10 g/mm2 of the caliper jaw surface was applied tothe skinfold. The skinfold sites measured and weighing factors used for calculating
mean skinfold thickness are as follows (Hori et al., 1974) :Chest

AbdomenO. 143

O. 1 39

Waist

Subscapular Upper arm

Thigh

0.141

0.295
The body fat (f o/o) was calculated from th.e mean skinfold thickness (X mm), bodyweight (W Kg) and body surface area (S m2) using the following prediction equation
O. 1 39

O. 1 43(Hori et al., 1974).

f 289x SxX +367

' W 'The bicycle ergometer test was performed at around 3 p.m. in a room of about 25'C.
Subjects were instructed not to eat a meal and rest at least two hours prior to theexperiment in order to minimize effects of specific dynamic action and physicalactivities. After sitting in a chair for 30 min. subjects clad in shorts rested on thesaddle of a Monark bicycle ergometer for 10 min. and then pedalled at the constantwork load of 450 Kg'm/min at the cycling rate of 50 rpm for 20 min. Samples ofperspiration from the back were collected at I O min. interval during exercise using

the filter paper method (Ohara, 1966). The Na concentration in the sweat wasdetermined by using eluates with distilled water from the fllter papers by flame
photometry.

RESULTSl . Anthropometric measurements
The physical characteristics of New Guineans and Japanese are shown in Tablel . The mean value of height for New Guineans ( 1 57. I cm) was significantly smaller
than that for Japanese ( 1 69.5 cm) . The mean value of body weight for New Guineans(59.2 Kg) was slightly smaller than that for Japanese. New Guineans showed
greater mean value of chest girth and smaller mean values of upper arm girth andTable I Characteristics of subjects

Number Age Height Weight B.S.A.

(yr) (cm) (Kg) (m2)

25.5
3.9

l 57. 1 59.2

Guineans
11

l.63

I .9

6.7** 5.8

0.09

Circumference (cm)Chest Upper arm Thigh
88.0

3.3

2.8*

86.5
Japanese 1 1 23.6 1 69.5 62.4
27.
1 52. 10.14
1.4
5.2 7.0
I .2 2. l
1 . 74

B.S.A. : Body surface area.
Mean values are given with their standard deviations.
* Significant differences between two groups.
* at 50/0 Ievel ** at lo/o level.

4. 1

154

thigh girth. Among these differences, the mean of thigh girth for New Guineans
was significantly smaller than that for Japanese. The physical status of New
Guineans was characterized by short stature and a stocky body shape.

2. Skinfold thickness

The mean values and their standard deviations of regional skinfold thickness
were shown in Table 2. A11 the mean values of regional skinfold thickness measured
for New Guineans were signiflcantly smaller than those for Japanese. New Guineans
showed much less individual variations in skinfold thickness.
Table 2Skinfold thickness of subjects

AbdomenChest

New Guineans 5. I

0.7*

Japanese I 0.6

6.2

6.0
l 5. I

I .O* *

6.0 I .5**
l 3.6

8.0

Subscapular

Waist

8. I

7.3

Upper arm
3.9 0.7**
8.6 4.3

1.2*

l 2.6 5. 1

Thigh
5.8 I .2**l I .2

4.9

Mean values are given with their standard deviations.
* Significant differences between two groups.
* at 20/0 Ievel ** at lo/o level.

3. Physical and nutritional indices

The physical and nutritional indices for New Guinean and Japanese are shownin Table 3. New Guineans showed significantly greater mean values of Rohrer's
index and Brugsch's index. Greater values of Rohrer's index and Brugsch's index
for New Guineans reflect their more stocky body shape compared with Japanese.
The mean values of body fat content (4.92 Kg) and body fat percentage (8.31 O/o) for
New Guineans were significantly smaller than those (8.24 Kg and 13.20/0 respec‑tively) for Japanese. The mean value of mean skinfold thickness for New Guineans(5.8 mm) was significantly smaller than that for Japanese (1 1.8 mm). Thus it canbe said that the body shape and body composition of New Guineans are an athletic
typ e .

Table 3 Physical and nutritional indices

Rohrer's index

New Guineans 152.8
Japanese

128.2

index

13.3**

56. I

14.8

51.0 3.8

2.3*

M.S.T.

Body fat

Brugsch's

(Kg)
4.92

0.58*

8.24 3.01

(mm)

("/･)

8.3 1
l 3.2

0.48* *
3.4

5.8 :1: 0.8*

l 1.8 5.4

M.S.T. : Mean skinfold thickness.
Mean values are given with their standard deviations.
* Significant differences between two groups.
* at lo/o level ** at 0,lo/o level.

4. Local sweat rate and sodium concentration in sweat
In Table 4, the peak local sweat rate, peak Na concentration in local sweat and1 55

Table 4Comparison of peak local sweat rate, peak Na concentration in local
sweat and mean Na concentration in local sweat between young male
highlanders of Papua Newguinea and young male JapaneseVp (mg/cm2/min)

Cp ( EqjL)

New Guineans

0.644 0.292

26.9

Japanese

0.72 143.6 1 1 .6

0.337

1 4.0*

C (*EqjL)
24. I
40. I

13.5*
1 1 .3

Vp : Peak sweat rate, Cp : Peak Na concentration,
C : Mean Na concentration.
Mean values are given with their standard deviations.
* Signiflcant difference between two groups at 5% Ievel.

mean Na concentration in local sweat for New Guineans are compared with thosefor Japanese. New Guineans showed considerably smaller mean value of peak local
sweat rate (0.644 mgjcm2/min) than Japanese (0.721 mg/cm2/min) though thisdifference was not statistically significant. The mean values of peak Na concen‑
tration in local sweat (26.9 mEqjL) and mean Na concentration in local sweat(24.1 mEq/L) for New Guineans were signiflcantly smaller than those for Japanese(43.6 mEqjL and 40.1 mEqjL respectively). Sweating reaction of New Guineans
was characterized by lower sweat rate and lower Na concentration in sweat com‑pared with that ofJapanese.
DISCUSSION
It has generally been believed that nutritional conditions, physical activities
and climate affect physical status of men (Hipsley and Clements, 1950; Coon et al.,1950; Lewis et al., 1960). The growth of children is retarded by low caloric intake
and low protein intake. Since most of the energy and protein in the diet for high‑landers in Papua New Guinea comes from the sweet potato, the intake of energy
always seems low and. intake of protein, especially animal protein, is low when com‑pared with Japanese (Hipsley and Clements, 1950; Norgan et al., 1974; Hori et al.,1980). As was pointed out by Coon et al., (1950), tropical natives tend to havesmaller stature as a result of long‑term exposure to a hot clirnate than temperate
natives. Thus, it can be said that the shorter stature of New Guineans is due inpart to their smaller intake of energy and protein and due in part to their long resi‑
dence in a tropical climate. It is known that a rise in ambient temperature results
in a decrease in the energy intake, and a decrease of body fat content is caused by low

energy intake and hard muscular exercise (Pascale et al., 1955; Parizkova, 1959).
Consequently, the thinner subcutaneous fat layer and less body fat content for New
Guineans might be induced by the lower energy intake due to higher ambient tem‑
perature and walking long distances and ascending steep slopes often loaded withheavy baggage in their daily lives. Since a thicker deposit of subcutaneous fat
prevents heat dissipation from body into the environment when ambient temperature
is lower than skin temperature, a thinner layer of subcutaneous fat is favorable fortemperature regulation of the body in hot climates. In an attempt to estimate

1 56changes in body composition and body shape as induced by different climates, weplotted body weight and body fat content against height in Fig. 2. In this figure,r.CL4 r.02

70
L･ O･S22H‑102･4

F= 0･1 50H‑16･67

Votkybatt Eesketbalt

60

10

r=‑Q2
at

x

e Guinr Japa e
Okina:wa OJapanese
50

Q

88u

Thai

fo

O Basketbatl
O Volteybatl

,

>

r=‑Q4

40
O Thai6

O OkinaiW30

O Ncw Gui

1 50 1 60 1 70 1 80
Height (cm )Fig. 2 Comparison of height, lean body mass and fat content among New Guineans. Than andJapanese.

anthropometric data on I 19 young male .Iapanese were used as a reference (Horiet al., 1977) and those on residents in Okinawa (a subtropical zone in Japan) andJapanese athletes (volleyball players and basketball players) were used for the sakeof comparison (Hori et al., 1974; Tanaka et al., 1977). Mean values of height, Iean
body mass and body fat content in the reference group were 1 71.0 cm, 55.3 Kg and
9.1 Kg respectively. Standard lean body mass (L Kg) and standard body fat content(F Kg) IA'ere obtained from height (H cm) by using the following prediction
equations :L 0992 H 1024 and F=0.150H‑16.67.
In this figure, the degree of overweight of lean body mass or underweight of leanbody mass (fatness or leanness) is expressed as a percentage deviation of the actuallean body mass (body fat content) from the standard lean body mass (body fat
content), "r". As shown in this figure, New Guineans were shorter in height andheavier in lean body mass. than Japanese; natives in Thai and Okinawa had less body

1 57

fat content, while Japanese athletes were taller, heavier, and had less body fat content.

WkLen these groups were compared with a reference group with relative values
as different from standard lean body mass and standard body fat content, New
Guineans showed heavier lean body mass and smaller body fat content, while nativesofThai and Okinawa and Japanese̲ athletes showed smaller body fat content. Thus,
plotting of lean body mass and body fat content against height is a feasible method of

estimating changes in physique, body composition and body shape as induced bydifferent climates in which individuals live. It is known that, sweating reactionchanges with acclimatization to heat (Adolph, 1946; Dill et al., 1938). The
inhabitants acclimatized to hot environments from childhood sweat less in spite of thesame rise in core temperature in heat, and Na concentration in their sweat is lower

when compared with individuals unacclimatized to heat (Kuno, 1956; Hori et al.,1976). The results presented in Table 4 that show New Guineans had less local
sweat rate and lower Na concentration in sweat are consistent with the reports
described above. The amount of evaporation of sweat is in proportion to sweat rate
when s weat rate is low, but an increase in sweat rate represents wasted sweat which
is not used for heat dissipation after the skin surface becomes a completely wettedarea. The amount of' evaporation of sweat from skin surface is in proportion to thevapor pressure difference between sweat and surrounding air when the wetted areais the same and this vapor pressure difference is increased by a decrease in Na
concentration of sweat (Hori et al., 1976). Thus, the evaporation of sweat for New
Guineans appears to be more effective for cooling the body than that for Japanese.
Since evaporation of sweat from the skin surface is the mechanism of heat dissipation
for man in hot environments, better efficiency of sweating of New Guineans for heat.
dissipation can be considered as an important form of physiological adaptation totropical climate.

ACKNOWLEDGEMENT
Authors express their thanks to Prof. H. Koishi for his help in carrying out theexperiment.

REFERENCES
Adolph, E. F. ( 1946) : The initiation of sweating in response to heat, Am. J. Physiol., 145, 7 l0‑715.

BroZek, J. ( 1952) : Changes of body composition in man during maturity and their nutritional im‑
plications. Federation Proc., I l, 784 793.
Burton, A. C. and Bazett, H. C. ( 1936) : A study of the average temperature of the tissues, of theexchanges of heat and vasomotor responses in man by means of a bath calorimeter, Am.
J. Physiol., I 1 7, 36‑54.

Coon. C. S., Garn, S. M. and Birdsell, .J. B. (1950) : A Study of the Problems of Race Formation inMan, Charles Thomas, Springfield, Illinois.
Dill, D. B., F. G. and Edwards, H. T. (1938) : Changes in composition of sweat during acclimation
to heat, Am. J. Physiol., 123, 412‑419.

Hipsley, E. H. and Clements, F. W. ( 1950) : Report of the New Guinea Nutrition Expedition,
Canberra : Department of External Territories,1 58

Hori, S., Ihzuka, H. and Nakamura, M. ( 1974) : The comparison ofskinfold and fat content between
residents born and raised in Okinawa and those born and raised in the Main Island of Japan,
J. Jpn. Soc. Food Nutrition, 27, 335‑339 (in Japanese).
Hori, S., Ihzuka, H. and Nakamura, M. ( 1976) : Studies on physiological responses of residents in
Okinawa to a hot environment, Jpn. J. Physiol., 26, 235‑244.
Hori, S., Tslnjita, J. and Yoshimura, H. ( 1977) : A certain consideration of methods for evaluation ofphysical characteristics with special reference to physical characteristics of athletes, J. Jpn. Soc.

Food Nutrition, 30, 79‑85 (in Japanese).

Hori, S., Tsujita, J., Mayuzumi, M. and Tanaka, N. ( 1980) : Comparative studies on physicalcharacteristics and resting metabolism between young male highlanders of Papua New Guinea
and young male Japanese, Int. J. Biometeor., 24, 253‑261.
Keys, A. and Br0 ek, J. (1953) : Body fat in adult man, Physiol. Rev., 33, 245‑325.
Kuno, Y. ( 1956) : Human perspiration, Charles C. Thomas, Springfield, Il],inois.

Lewis, H. E., Masterton.J. P. and Rosenbaum, S. ( 1960) : Body weight and skinfold thickness ofmen on a po]ar expedition, Clin. Sci., 19, 551‑561.
Norgan, N. G., Ferro‑Luzzi, A. and Durnin, J. V. G. A. ( 1974) : The energy and nutrient intake andthe energy expenditure of 204 New Guinean adults, Phil. Thons. Roy. Soc. (1.0nd.) B., 268,
309‑348.
Ohara, K. ( 1966) : Chloride concentration in sweat : its individual, regional, seasonal and some othervariations, and interrelations between them, Jpn. J. Physiol., 16, 274r290.

Parizkova, J. ( 1959) : The development of subcutaneous fat in adolescents and the effect of physicaltraining and sport, J. Physiol. Bohemia, 8, I 12‑1 1 7.

Pascale, L. R.; Frankel, T., Grossman, M. I., Freeman, S., Falier, I. L. and Bond, E. E. (1955) :Report of changes in body composition of soldiers during paratrooper training, Army Med.
Nutrition Lab. Denver Col. Rep., No. 156, 1‑14.

Tanaka, N., Tsujita. J., Hori, S., Senga, Y., Otsuki, T. and Yamazaki, T. (1977) : Studies on
physique and body shape of athletes with special reference to differences in physique among
athletes of various kinds of sports, .J. Physical Fitness Jpn., 26, I 1 il23 (in Japanese) .

159

パプアニューギニア高地人と日本人の体型，体構成および発汗反応の比較
堀清記・辻田純三・黛

誠・田中信雄海抜1500m−1800mに住んでいるパプアニューギニア高地人の成人男子11名と西宮市在住の日本
人成人男子11名について，身体計測と運動中の発汗反応の測定を行った。パプアニューギニア人の測

定は8月に，日本人の測定は9月に行った。発汗反応の測定は25。Cの室内で午後3時より開始した。

仕事量450kg・m／minの自転車労作計を用いたペダル踏み運動を20分間行わせ，背部の局所発汗速度と汗のNa濃度を発汗カプセル炉紙法で測定した。パプアニューギニア高地人は日本人と比較して，身長が低く，体重は軽く，皮下脂肪厚が薄く，体脂肪含有量が少なかった。これらの差はいずれも統計学的に有意であった。パプアニューギニア高地人のローレル指数および比胸囲は日本人のそれらより有意に大きかった。パプアニューギニア高地人の局所発汗速度は日本人のそれより可なり低く，又前者の汗のNa濃度は日本人のそれより有意に低かった。パプアニューギニア高地人の身体的特徴および彼等の発汗反応が日本人のそれと異なっているのは，彼等が日本人より高温環境によく馴化したことによると思われる。

（兵庫医大第一生理） 663西宮市武庫川町1−1

Japan. J. Trop. Med. Hyg., Vol. 9, No. 3, 4, 1981, pp. 161‑165

161

RESISTANCE TO CHALLENGES WITH HOMOLOGOUS
TRYPANOSOMA GAMBIEN,SE IN MICE TRANSFERRED
MOUSE IMMUNE SPLEEN CELLS OR IMMUNE SERAMASATO FURUYA, MIKIO OKA, YOSHIHIRO ITO,YOSHIKAZU OKA AND HUMIO OSAKI
Received for publication 18 I¥/Iay 1981

Abstract: To know the mechanism of protection from the homologous Trypanosoma
gambiense infections in mice, the incidence of passive transfer of specific resistance to chal‑lenges with the parasites was examined in mice inoculated with immune spleen cclls andimmune sera separated on days 3 to 2 1 (day 0=immunization with microsomal fraction,
144,000 x g sediment of T. gambicnse homogenate, in complete Freund's adjuvant) .
Passive transfer of specific resistance was successful only when immune sp]een cells

separated on days 3 and 5 wcre employed and the resistance was extinguished by treating
the recipients with dextran sulfate 500 (2 mgjmouse) , a macrophage‑dysfunctional agent.
In case of immune sera, a powerful rdsistanc.e to the infection was inducible by trans‑

ferring immune serum separated on study day 7. About one‑third of respective micetransferred immune sera separated on days 5, 14, and 2 1 were able to conquer the infection

with 3 >< 103 parasites but none of the mice transferred immune serum separated on day 3

overcame the challenge.

INTRODUCTION
Protective immunity has been successfully induced in animals immunized withhomogenate antigen oftrypanosomes (Weitz, 1960; Seed, 1963 ; Miller, 1965 ; Furuya,

1977; Osaki and Furuya, 1978) but the defensive mechanism has been discussed
mainly on humoral immunity (Seed and C.am, 1966; Takayanagi and Enriquez,
1973; Seed, 1977).

In our serial immunological study in mice (Furuya, 1977; Osaki and Furuya,1978), animals received homogenate or microsomal antigen of Trypanosoma gambiense
together with adjuvant were able to overcome challenges with the homologous
parasites given on and after day 3 of immunization irrespective of the agglutination
antibody titer of their sera. This might suggest that factors other than humoral
ones are also involved in the elimination of the parasites in these animals. This
report aims to define the cellular and humoral factors controlling the protection
from T. gambiense infections in mice by transferring immune spleen cells and immune

sera in combination with dextran sulfate 500, a macrophage‑dysfunctional agent(DS 500).
Department of Parasitology, School of Medicine, The University of Tokushima, Tokushima, JAPAN162

MATERIALS AND METHODS
Animal and antigen : Forty to sixty days old female ddY mice and T. gambiense, strain
Wellcome, obtained from the Department of Protozoology and Parasitology, ResearchInstitute for Microbial Diseases, Osaka University and maintained for years in ourlaboratory by serial passages in mice, were used for the experiment.
Microsomal fraction was prepared from the homogenate of the parasites isolated

from the blood of heavily infected mice following Lanham and Godfrey's method(1970) by differential centrifugation (Furuya, 1977), and used as antigen.
Transfer of immune spleen cells and immune sera : Antigens were administered intra‑

peritoneally at a dose level of 2 mg protein per mouse with an equal volume ofFreund's complete adjuvant. Immune spleen cells and immune sera were takenfrom donor mice on study days 3, 5, 7, 14, and 21 (day 0=immunization).
Spleens removed from. the donors were washed in cold RPMI 1640 medium,pH 7.2 containing 25 mM HEPES and 0.020/0 2Na‑EDTA, and the tissues wereminced into small pieces and pressed gently between two pieces of glasses to give
single cell suspensions. The spleen cells were filtered through cotton wool to remove
large clots before they were washed twice with the above cold medium by centrifuging
at 1 70 x g for 10 min and adjusted to a final concentration of I x 108 cells/ml with thesame medium. Immediately after transferring 0.5 or I ml of the suspension intra‑
peritoneally, recipient mice were challenged with 50 parasites by the same route.
A part of the recipients transferred spleen cells taken from the donor mice on
day 3 were treated with DS 500 (2 mg/head) intraperitoneally just before challenges.
Normal or immune serum (0.25 ml) was injected into the recipient mice intra‑
peritoneally 5 hours after challenges with 3 x 103 parasites by the same route.

RESULTS
A11 of the untreated and those mice transferred normal spleen cells died 4 to

6 days after challenges with 50 parasites. The recipients transferred immune
spleen cells separated from the donors on days 3 and 5 offered a strong resistance tothe challenge. Per cent survival and mean survival days of the dead mice in therecipients transferred immune spleen cells separated on day 3 were 72.2 and 8.8 0.6,
respectively. The specific resistance to the infection was lowered in mice transferredspleen cells separated on day 5. Neither protection nor longevity was induced intomice by transferring 5 x 107 or I x 108 immune spleen cells separated on days 7, 14,
and 21 (Table 1).
All of the mice transferred normal mouse sera died 4 to 6 davs after infections
but those transferred immune sera separated on day 3 died 6 to 8 days after infections

and mean survival days of the dead mice were extended to 7.2 0.2. A mighty
resistance to the infection was inducible in mice by transferring immune sera sepa‑
rated on day 7. About one‑third of respective mice transferred immune sera sepa‑
rated on days 5, 14, and 21 conquered the infection and mean survival days of thedead mice were also extended to about 10 days (Table. 1).

1 63

Table 1 Resistance to challenges with Trypanosoma gambiense in mice transferred Immune
spleen cells and immune sera

immune spleen cells

immum‑

zation termof donormice in days

5 x 107 cells/mouse

MSDS of antibody
per cent
MSDS of survival
titer ofin

per cent

survival inrecipientthe deadrecipient

mice

mice

8.8

5

11.8 ( 2117)

6L2 0.680.0 (4/5)

7

O ( O/ 7)
O ( O/ 7)
O ( Oj 7)

4.0

O (0/5)O (0/5)O (0/5)1421

the dead donor

mice micemlce

72.2 (13118)

3

immune seral >< 108 cells/mouse

4.6 0.3
4･2

0.2

mice

MSDS ofthe deadmice

original

O (0/9)

7.2

0.2

5.0 0

l:8

27.3 (3/11)

l O. I

0.8

5.0 0.3

1: 16

90.0 (9/10)

9.0 0

original

30.0 (3jlO)

7.0

I .8

1:8

40.0 (4jlO)

9.5

I .5

0.6

O

per cent

survival inrecipient

4･6

0.4

5.0 0

MSDS : mean survival days (mean S. E.)‑ : not examined
Agglutination antibody titer was measured with pooled serum.
Recipient mice transferred immune spleen cells and immune sera were challenged with 50 and
3 x 103 parasites, respectively.

g

mlce

6.8 0.8(A)20
5 10days after challenge

1015 2:!:O 3' ' 4.5d: O
(O 5

57

. (D) 6

5

5

Fig. 1 Effect of treatment with dextran sulfate 500 on resistanceto challenges with Tr̲ypanosoma gambiense in mice transferredimmune spleen cells (A) : transfer of immune spleen cel]s,(B) : transfer of immune spleen cells and treatment withdextran sulfate 500, (C) : treatment with dextran sulfate

500 alone, (D) : nontreatment, [] : survival, I:
death

To inflict injuries on phagocytes in the immune spleen cells and in the recipients,

mice were injected DS 500 intraperitoneally immediately after transfer of immune
spleen cells separated on day 3 and were chal]enged with the parasites uninter‑
ruptedly. The passive resistance induced in mice by immune spleen cells was cut
off completely by treating the mice with DS 500 (Fig. l).

DISCUSSION
All of the mice immunized with microsomal antigen in adjuvant were able toovercome the challenges given on day 3 or 14 but agglutination antibody titers intheir immune sera were strikingly low (Furuya, 1977; Osaki and Furuya, 1978).

1 64

To analyze the mechanism of defense against infections with the parasites, theresistance to infections was examined in mice transferred immune spleen cells andimmune sera.
Passive transfer of specific resistance was accomplished by the use of immune
spleen cells separated on days 3 and 5 of immunization. Campbell and Phillips(1976) reported that specific resistance to T. rhodesiense was transferable to therecipients by immune sera or B Iymphocytes.

Moreover, Takayanagi and Nakatake (1975) reported that glass‑adherent,
antibody‑forming cell population among immune mouse spleen cells was effective inpreventing experimental infections with T, gambiense in recipient mice. In thepresent study, the spleen cell suspension contains different sorts of cells such as mono‑

nuclear and polymorphonuclear phagocytes, T‑ and B‑lymphocytes, plasma cells
and others. If any one of B Iymphocytes, plasma cells and antibodies produced inrecipients by these two kinds of cells is to be involved in the sweeping away of theparasites, mice transferred immune spleen cel]s taken from different study days, 7
to 21, should have overcome the challenges. But, neither protec.tion nor longevity
was observed in those mice.
Hahn (1974) and Hahn and Bierther (1974) reported the selective damage by

DS 500 to mononuclear phagocytes. In the present experiment, recipient micetreated with DS 500 immediately after transfer of immune spleen cells were not able
to evade the challenges with the parasites. From this, macrophages are likely toplay a key role in the removal of parasites in the recipient mice. The reason why
specific resistance was transferred by spleen cells within only 5 days after immuni‑
zation might be that, in the early stage ofimmunization, the donor's spleen was filledwith macrophages seemingly being partici.pated in the transmission of antigenic

informations to lymphocytes. These macrophages were probably activated by acertain stimulation because normal macrophages do not phagocytize trypanosomesin the absence of specific antibodies. But whether these macrophages have alreadybeen activated by phagocytizing the antigens in the donors and this status has been

maintained in the recipient mice or these macrophages were activated by lym‑
phokines produced by donor's T Iymphocytes in the recipients is not known.

REFERENCESl ) Cambell, G. H. and Phillips, A. M. ( 1976) : Adoptive transfer of variant‑specific resistance toTrypanosoma rhodesiense with B Iymphocytes and serum. Infect. Immun., 14, I 144‑1 150.

2) Furuya, M. ( 1977): Immunogenic activities of subcellular components of Trypanosoma gam‑biense in mice. Jap.J. Parasit., 26, 350‑366.
3) Hahn, H. ( 1974) : Effects of dextran sulfate 500 on cell‑mediated resistance to infection with
Listeria monocytogenes in mice. Infect. Immun., lO, I 105‑1 109.

4) Hahn, H. and Bierther. M. (1974) : Morphological changes induced by dextran sulfate 500 inmononucler phagiocytes of Listeria‑infected mice. Infect. Immun., I I l0‑1 1 19.

5) Lanham, S. M. and Godfrey, D. G. (1970) : Isolation of salivarian trypanosomes from man
and other mammals using DEAE‑cellulose. Exp. Parasit., 28, 52 1‑534.
6) Miller, J. K. ( 1965) : Variation of the soluble antigens of Trypanosoma brucei. Immunology,
9, 521‑528.

1657）OsakiラH．andcomponentsof

Furuya，M．（1978）：Immune
T卵朋o∫o窺α9α励伽58．

responses

in

mice

immunized

withsubcellular

Protozoan Diseases，Jap．一Ger．Ass．Protoz．Dis．，Tokyo，

1，18−20．

8）

Seed，」・R（1963）：The

characterizationofantigens

isolated倉om

T卯αηo∫o御αrho廊」8π58．J．

immunity to

Tり御η050規α伽66∫8α励伽58．

Protozool．ラ10ン380−389．

9）

seedJ

R・（1977）：The

role

of

immunoglobulins

inInt．J．Parasit．，7，55−60．

lo）seed，J・R・and

Gam，A・A・（1966）：Passive

immunitytoexperimental

trypanosomiasis．J．

Enriquez，G．L．（1973）：E価cts of

the

IgG

immunoglobulins

Parasit．，52，1134−1140．

11）Takayanagi，T．and

T卯αη050アηα9α励伽58in飽ctions

12）

in

Takayanagi，T．and Nakatake，Y．（1975）： Tりμη050窺α
glutininandprotection13）weitz，B・（1960）：The

in

subpopulations

properties

and

IgM

in

mice・J・Parasit・ラ59，644−647．

byofsomeimmuneantigens

spleengα撹伽n58：Enhancement

of

ag−

cells．Exp、Parasit．，38，233−239．

of乃蜘ηo∫o灘伽漉J．Gen．Microbiol．，

23，589−600．

聯αη05襯α即励初∬免疫マウスの脾細胞あるいは抗血清を移入した
マウスの同一原虫感染に対する抵抗性
古谷正人・岡 三希生・伊藤義博・岡 好万・尾崎文雄

τ叡ραπ050窺αgα規ゐ∫θη5θ1こ対するマウスの感染防御機構の解明を図る目的で，同原虫由来マイク

ロソーム画分を抗原としてFremd1s

completeadjuvantと共に免疫した。免疫後3〜21日にわたり

経日的に得た免疫マウスの脾細胞あるいは抗血清を正常マウスに伝達し，同原虫感染に対する抵抗性を観察した。

免疫3あるいは5日目の脾細胞を移入したマウスのみ50個の原虫攻撃に対して特異抵抗性を示した。
この抵抗性は脾細胞移入直後の硫酸デキストラン500による処理で消滅した。
抗血清の受身伝達では，免疫7日目の血清に非常に強い抵抗性付与能が認められた。また免疫5，

14，及び21日目の血清を受身伝達したマウスの約1／3は3×103個の原虫攻撃に抵抗したが，免疫3日目の血清にはこれらの効果は認められなかった。

徳島大学寄生虫学教室

日本熱帯医学会誌 第9巻

第3，4号 1981

167

167−173頁

DIC症候群類似の症状を呈したクロロキン
耐性熱帯熱マラリア症の一例
戸田正夫1・石崎達1・高岡

昭和56年7月10日

正敏2

受付

耐性熱帯熱マラリアに罹患し，重症化しDIC819）

ま

え

が

（Disseminatedきintravascularcoagulation）症候群を呈した患者を経験したのでここに報告する。

日本におけるマラリアは，古くは土着のマラリ

アも存在し，地域的に相当数の発生も見たDが大症例（Table1）

正期以降はその数も著しく減少し，第二次世界大

患者は21歳の無職の男性である。中肉中背で生

戦後の一時期は復員兵による外来の輸入マラリア

来健康で特記すべき既往症はなく，家族歴にもアが猛威を振い二次感染も少数ながら発生したが2）レルギー素質その他特記すべきことはない。

現在は土着マラリアは沖縄の一部3）を除いてはほ

昭和55年3月5日より蝶類採集の目的でフィリヒ。ン（PhilipPines）のパラワン（Palawan）島のとんど消滅している。

しかし，近年我国が経済発展をとげ国民の多数
が世界各地に観光，出張滞在をするようになり，

プエルト・プリンセサ（Puerto Princesa）に渡航しガイドをやといバルサハン（Balsahan）の

輸入マラリアが今日大きなトピックスとなりつつジャングル地帯に入り，13日間蝶の採集を行っある。このような情況のもとで，東南アジア・中

た。その行動を要約すると次のとおりである。

南米・アフリカなどの熱帯各地を旅行または滞在した場合はマラリア感染の機会が多く，しかもマ

3月5日マニラ（Manila）に到着し翌日プエ
ルト・プリンセサに渡った。翌日（7日）より，

ラリアに対する啓蒙教育が不完全であったため予

同市を基点に，7日バルサハン，8日イラワン

防および治療に対する関心が乏しいことから，大（lrawan），9日サルコッ1・山（Mt．Salakot），10

友ら4）5）の10年間にわたる連続調査によれぱ我国

日再びバルサハン，11日イラワンにて，それぞれ民で海外で感染し，帰国後発病したマラリア患者

蝶類採集に従事した。さらに，1日おき13日から

は年間わかっただけで平均50名を越え，推定では，

再び同島ジャングル地帯のラングアン（Languun）

100名に及ぶとみられ，そのうち数人の死亡例を及び周辺での蝶採集が開始された。発症日より，みているのが現状である。

通常の平均的潜伏期を逆算すると，同日付近に感東南アジア地域のマラリア症のうち，ベトナム染があったことを推測させる。20日にプエルト・

3国，フィリピンなどの熱帯マラリアはクロロキ

プリンセサに戻り，この翌日，既に過度の疲労感

ン耐性株であることが知られており6），この傾向を自覚しているが，他の所見に欠け明らかな発症は東南アジア全域に拡大されっつあり，更にこのは認めていない。22日さらにマニラに戻り，2泊

熱帯マラリアはファンシダール耐性の傾向7）も示滞在の後24日帰国した。

して来ている。これらのことが熱帯マラリア患者

Nivaquine（ニバキン）を週1回200mg1錠を内の治療を困難ならしめている。

我々はフィリピンのパラワン島でクロロキン
1独協医科大学アレルギー内科教室

その間，マラリア予防の目的でクロロキン製剤

服し，帰国後も発症までに2回内服していた。

2独協医科大学医動物学教室

168

現病歴

Table l Case History（Y．1．ma】e，21age）

Sn切ective
ache

and

symptoms：Remittent

fヒver，Head−musclelimbs

Past history：

Family

pain

on

lower

history：

collecting

感，戦傑を発現し約1時間半の後40．3。Cまで発熱を生じた。29日午前9時頃発汗と共に下熱し

none

た。同日午後1時頃再び約1時間の悪感戦懐の後，

history：none

Present

3月28日の朝に不快感を自覚し，同夜半より悪trip

40。Cの発熱をみ，同午後10時凋協医科大学アレル

in Parawan lsland，Philip−

ギー内科へ，某医よりの紹介で緊急入院となった。

Patient

butterflies

has

experiencedapines，during5th through24th ofMarch，1980．

On28th，March，飴tigue
highfbver attack

40．3。C
body

of

and

the

to

入院時体温39。Cで，脈拍数90／分，血圧108／60

temperature，on29th，March，

mmHgであった。頭色は蒼白かっ軽度黄疸様を

down

up

with

again

perspiration

to40。C

with

up

in

the

chilling

sensation、At

night of29th，10pm，patient wasadmitted

the Dokkyo

Present

instatus：Anemic血ce

University

withphnodefingersandnopressuretemperature39．3。c，no lym−swelling，liver

two

Hospitalslightjaundice，

Puls rate841minute，normal blood（115175），body
wide

現症の概要

moming andwith chilling sens飢ion

bodytemperaturemominginwithenlarging

palpablepain，no spleenas

呈した。頭痛を有し，眼瞼結膜・眼球結膜にはそれぞれ貧血・黄疸はなく，表在リンパ節の腫脹もなかった。胸部所見では聴診上で異常所見はなかった。腹部所見では，肝は軽度腫大し，弾性軟，

圧痛あり，脾はこの時は触知しなかった。しかし，後にシンチグラムで腫大が確認された。その他，

神経学的所見などには，異常は認めなかった。

palpable

検査所見neurological symptom。

（Table2）

入院時，貧血はなかったが，白血球数2900／
Table2RBC
RBC

ClinicalWBC

GOT

92K．U．

GPT

53K．U．

42％

Al−P

21．1K，A．U．

OAP

220G．B．U．

2900／mm3

7％

Meta．
Stab．
Seg、

Eo．

Mo．
Lym．
y−Lym．

52
11
1
8
19
2

3．5104／mm3
6％・
十3

618W．U．

7−GTP

38mU／mJ

Bil

Ret，
ESR（1hr．）

LDH

l3U

M．G．

Pl．

CRP

begin of hospitalization

15．49／d1

518104／mm3

HtAt

Findings at the

2mm

2．O

mg／dJ

D．B．

L3mg／dJ

1．B．

O．7mg／dl

Urobilinogen（U．）

耕

Bleeding Timel

Coag．T．

10min．

PT．
（cont．

PTT．
（cont．

min．

10．8sec．
9。9sec．）

46sec．
32sec．）

Fibrinogen

220mg／dJ

FDP＜40μ9／mJ

169

mm3，血小板数3．5×10

第5病日にP，S剤各々50mg，19を経口投与／mm3と共に著名な減少を示した。白血球分画像では桿状球52％，分葉球したところ翌日下熱したが末梢血中の原虫数に変11％と左方偏位を呈し，単球は8％と軽度増加し化をみなかったため，さらに前日の半量を追加投ていた。血沈は1時間値2mm，2時間値8mmと与し，同日晩より塩酸キニーネL5g／日の投与をむしろ遅延していたが，CRPは3＋であった。

開始したが，第7病日再び発熱し難聴をも発現

GOT92K．U．，GPT53K．U．，A1−P2LIしたためP・S合剤を増量し，さらに同夜塩酸キ

K．AU．，

LDH618W．U，M．G13U．，13il，2．Omg／d1（nB

ニーネ300mgを点滴静注した。同時に下熱の目

1．3mg／dl

的でhydrocortisone100mgを静注した。そのたIBO．7mg／dl）と，軽度〜中等度肝機能障害を示し，黄疸がみとめられた。しかし，血液

めか原虫数はその後に減少をみた。

第8病日には，すでに末梢血中よりのring及

凝固系には異常はなかった。

臨床経過並びに治療

ぴtrophozoitはすでに消失していたが，尚も発熱（Fig、1及びTable3）

熱型はFig．1のごとく，周期不定の間歌熱がは持続したのでPredonisolone15mg／日の経口投持続した。第2病日に末梢血液塗抹標本でring 与を開始した。
第9病日には下熱したが，さらにP．S合剤，
fom及びtrophozoiteのマラリア原虫が確認された。原虫数が次第に増加したので，第4病日より

キニーネは継続投与し，各剤の合計がそれぞれ治療を開始した。

275mg，5．5g，10．3gとなったときに投与を中止した。Predonisoloneは漸減中止した。

通常の投与法に従って，2日間クロロキン塩基にして1200mgまで投与したが，原虫数の減少

第13，14病日に，再び発熱及び白血球数増多をはきわめて軽度だったので，ピリメサミン・スル

見たが，末梢血中原虫は認められなかった。経過

フォモノメトキシン（P剤・S剤）の合剤に切り中，白血球数に関しては入院後，段階的改善を示

換えた。

した。

X103／mm3

W．B．C．B．T． Quinine愚

hvdrochbrid

10．39｛TlTotal
P．farci．

275mg｛T》−sulfomonomethoxine
一suI

5、5g

IT，

？跳締、皿皿

count

100mg
40

同塩

39
38
o

37
103

→

〆

ヤ

、梶・、

！

＼

ノ

以

ラしヤラ

胤、

『−一−WBC

〆

︑︑

36

102

判

ヤー
幅︑b

ー︑噸︑

．ノ■

ノ﹂

ロ

■

へ

10

5

一一一＊t．P．

Predonisobne

106

104

B．T、

Hydrocortison︾冒

mm 313105

Pyrimmethamine
Pyr

メ

、ヌ

＊

t．P．＝totaI

101

P．farcipalum
count

1
Date

29／Mar、31

Hospitalday

1／Apr，

7

10

12

15

1718

1234567 9101112131415161718192021
Fig．

ClinicalCourseI

170
Table3

Changes29

Dat●ofP．色rciparum
30

31

count

1

Ho5pi加1dav

1

2

3

4

Paro5ito

r．t

r．t

r、t

r・t

2

3

4

5

6

5

6

7

8

9

r・t

r・t

r・9

9

s
W

37003400

B C

P！W

to Therapeutic Process

Apr．

m8r．

Stag●

in relation

2900

3800

r．tニringform，

g

6800

5800

5700

法。2覧。14乳15豫。2乳。167蓄※4搬0＋器。0城

mtio

S＝＝schizont

Total

5，735

6、902

測oI

3．75

3．83

5．46

5．46

4．23

428

2．15

0

0

50xu81par85ito｛98metocytol

0

0

0

0

0

16

29

14

19

o3●xu81

289，033290，700

17、204

19，488142

0

0

P8r■5it●8！mm3

10918＆ trophoz置醗曲s

Pのr副t●8！mm31⑧⑧
9＝gamotocyte

1．2

109｛8exuol

1．4

1、1

1，3

瓢〆

por筋ite＄／mm31

ThorapγCh｛pororoquho

male

o5，

lmg，

900

300

Pyrimothamino lmgl

50

25

75

75

25

Sulfomonom●tho翼ho lgl

1

0，5

1，5

1．5

0．5

0，5

1，0

1，0

1．5

Quinin■HClfemalelgl｛9Hi、v，

なお，第6病日より，末梢血中にGametocyte＋1※：1胃gamote
count

＋0、3

上記所見を総合すると臨床症状は出ていないが，が出現し，これにより本症例は熱帯熱マラリアでDIC症候群の検査成積に似ている。さらに，アンあることが確定した。

チトロンビンIII（11．5mg／dZ）の減少，SK活性

凝固系異常 （Fig．2およびTable4）

プラスミン（30min）の尤進もみられたので，こ次に我々が興味を持った，経過中に生じた凝固の考え方は妥当のものであろう。

入院時すでに血小板数は減少し，血漿フィブリ

上記所見に対して患者は新鮮血400mZの輸血と1日9000U約2日間のヘパリン療法（このヘ

ノーゲンは当初正常であったが経過と共に漸時減

パリンの投与量は，通常の投与法に比べ，はるか

少した。FDP（飾rindegradationproducts）はに少量であったが）の後，Fig．2のごとく改善を漸時増加し，第6−7病日以降，すなわち末梢血中見た。前記した難聴も，発症後約1週間の経過のよりのring及びtrophozoiteの減少期あるいは後消失した。

異常に関して述べる。

消失の後にその最大値を示した。この時期の血小その他の検査所見では，Fig．2及びTable4の板数，血漿フィブリノーゲン量及ぴFDPの実測

如く肝機能は入院後ほぼ10日目以降に正常化した。

値は各々1．2×104／mm3，80mg／dl及び10μg／ml 一方経過につれて貧血が出現し，低補体価は当初未満であった。

よりあったが，共にやや長期にわたって段階的改あったが，プロトロンビン時間は12．3秒でコン善をみた。尚，貧血に関して，網状赤血球の増加はあったが，直接・間接クームス試験はいずれトロールの9．8秒に比してあきらかに延長がみらも陰性であった。

同時に，この時期の出血，凝固時間は正常で

れた。

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172を追求した例は必ずしも多くはない。本報告では症状の経過と密接に関係して血小板の著減，血漿察考

フィブリノーゲンの減少，FDPの増加をみとめ，

熱帯性マラリアは別名悪性マラリアと呼ぱれ，

肝障害（これはキニーネ内服が関係する可能性あ

24時間の周期で発熱し，血中の環状体が急激に増り）があり，低補体価およびプロトロンビン時間

加し易いこと，脳症状をおこし易いことでよく知の延長，その他の現象を観察しえた。DIC症候群

られている。この症状は脳症状は起さなかったが，によるショック発現にいたらなかったとはいえ，短時日に体力の消耗が著しく，発病直後に入院し上記所見はDIC所見と関連を有するものである。

たにもかかわらずクロロキン耐性株であった為に熱帯熱マラリア死亡例とDIC症候群との関係

初期の治療が充分に効かず，キニーネの内服でもはなお討議の余地があるが，本症例は討議の材料不充分で，キニーネの点滴およびP剤とS剤のとして充分に価値のあるものである。

内服でやっと危険期を脱出したものである。

疫学的にはパラワン島の熱帯マラリアはクロロ

結キン耐性株が多いことは寄生虫学者間ではよく知語られているが，海外渡航者の殆ど全員が熱帯病のクロロキン耐性の熱帯熱マラリア患者（男21

知識なしに出かけている現在，クロロキン剤の内歳）1名を発病の初期から観察して，各種の臨床服で安心して流行地に滞在することは当然のこと

検査の結果とくに血漿の線溶系を中心にDIC症

で，この点からみてこの症例は貴重な症例である。

候群にいたる段階の成績をみとめ治療により全治

熱帯熱マラリア原虫の診断については，標本虫がクロロキンの影響をうけて変化しているが，特したので，その場合の諸検査の回復も含めてここに症例報告した。

有の熱型や環状体が一個の赤血球中に2個以上出現すること，それが短時日に増加すること，被変稿を終るに臨み御指導と薬剤の供与をいただい性赤血球の形が増大しないこと，schizontの形，

た，岐阜大学寄生虫学教授大友弘士博士，独協医

gametocyteの出現などから容易である。一般に

科大学医動物学教授山本久博士，独協医科大学ア

マラリアでは白血球の減少はよく知られているが，レルギー内科教授牧野荘平ならぴに日本熱帯医学

治療経過を追って虫体の出現消失および血液所見協会に深い感謝の意を表します。

文

1）

森下

献

薫（1963）：日本に於けるマラリアの疫学的研究，日本における寄生虫学の研究3，70−

103，目黒寄生虫館
2） 沢田藤一郎（1949）：宿題報告，戦後マラリア，日本内科学会雑誌，38，144．

3） 照尾寛善（1976）：沖縄における主要感染性疾患の戦後における消長（沖縄の医療年表），沖縄
県公害衛生研究所報，9，175−217，4）

大友弘士，山口剛，石崎達，加納六郎（1978）：マラリア患者29人の罹患状況に関する疫学的考察，感染症学雑誌，52，41−49．

5）

Nakabayashi，T，Ebisawa，L，Ohtomo，H．＆Ishizaki，T．：Investigation
cases in Japan

6） Bruce−chwatt，LonimportedJ．（1970）：Imported

malaria．A

growing

world

problem．Trans．Roy．Soc．

Trop．Med．＆Hyg．，64：201−209．

7） 大友弘士（1979）：マラリアの最近の動向，公衆衛生，43，865−869．
8）malaria

in1972−1974，エTrop．Med．＆Hyg．（London），Nov．，247−251．

大友弘士（1979）：脳性マラリアの病態について，27，91−98．

g）DaroHンR．B．，Deller，」．J．，Kastle診A．」．＆Blocker，W．W、（1967）：JAMA，202，67⑨

173

A CASE STUDY ON CHLOROQjUINE RESISTEDFALCIPARUM MALARIA COMBINED WITHDIC RESEMBLING SYMPTOMSMASAO TODA*, TATSUSHI ISHIZAKI* AND MASATOshl TAKAOKA**
Received for publication 10 July 1981

A male patient of 2 1 of age was infected with Plasmodium falciparum resisted to chloroquine
during his stay in Parawan Island, Philippines.

Typical fever attacks occurred just after coming back to Japan and he was hospitalized. The
patient turned to worse within a few days due to high fever attacks and acute increase of parasitemia

even by oral administration of chloroquine, and then he was treated with quinine intravenously aswell as orally, in combination with pyrimethamine and sulfamono‑methoxine, which resulted toestablish complete cure of this patient.

Among his clinical course, such findings were positive in high grades as leucopenia, thrombocy‑

topenia, prolongation of prothrombin time, decrease of fibrincgen, antithrombin 111 and fibrin
degradation products (FDP) in blood. Highly activation of SK plasmin was also recognized.
Thcse findir]gs were reserr.b]ed with DIC syndrcm even thcugh wilhcut actualization in thispatient. B:ood tran fusicn, rreditatitn Gf corticcsteroid ard hcparin wcre very uscful fbr recovery
from those situation.

* Department of Clinical Immunology & Medicine and ** Department of Medical Zoology, Dokkyo
University School of Medicine,Japan. J. Trop. Med. Hyg., Vol. 9. I¥ To. 3, 4, 1981, pp. 175 185

1 75

FUNDAMENTAL STUDIES ON THE LEUKOCYTECHEMOTACTIC FACTOR IN LYMPHOKlNES OF
DOGS INFECTED WITH BABESIA GIBSONI
MAMORU ITOH, SUSUMU MAKIMURA AND NAOYOSHI SUZUKI
Received for publication 28 May 1981

Abstract: The agarose plate method using dog neutrophils and human monocytes
as migrating indicator cells were used to demonstrate neutrophil chemotactic factor andmonocyte chemotactic factor, respectively, in dogs infected with Babesia gibsoni. The
experiments were done in the supernatant from in vitro cultures of spleen cells, peripheral
blood lymphocytes as well as extracts of spleen cells.

In particular, it was suitable to use canine neutrophils as migrating indicator cells
for demonstrating neutrophil chemotactic factor in 2.5 >< 105 cells per well of normal canine

leukocytes incubated for 2 hours in agarose plate with I O ･ inactivatecl normal canine
serum. Supernatants from in vitro cultures ofcanine spleen cells (3 weeks postinfection) andperipheral blood lymphocytes (3 days postinfection) showed an increased chemotactic
activity of neutrophils as compared with that of normal dogs. Similarly, supernatantsfrom in vitro cultures of canine spleen cells (2, 3 and 25 weeks postinfection) and peripheral

blood lymphocytes (7 days postinfection) showed a tendency to contain monocyte
chemotactic activity.

INTRODUCTION
Recently, infection with Babesia (B) gibsoni in dogs has been reported fromvarious regions ofJapan (Akashi et al., 1969; Kusunoki, 1971 ; Noda, 1977, 1978).
In a series of previous studies by the authors on experimental protozoan diseases using

animals based on cel]‑mediated immunity, it was reported that macrophage a,cti‑
vation was associated with increased cell size, spreading on glass, adhensiveness to acharged surface, phagocytic activity and digestive capacity, and with increase incontents of some lysosomal enzymes (Ishimine et al., 1978, 1979; Makimura &Suzuki, 1977; Noda & Suzuki, 1977; Sethi et al., 1975; Shirahata et et., 1975, 1976,
1977; Nakabayashi et al., 1978). It was suggested that the cell‑mediated immunity

by the macrophages and lymphocytes towards B. gibsoni infection may be involvedin the protective immunity (Ishimine et al., 1978, 1979; Suzuki, 1976). On theother hand, it has been suggested that the initial reaction of antigen with a few
specifically sensitized lymphocytes results in the production of soluble mediators,
also called lymphokines (LKS), which through their biologic activity, are capableof recruiting host inflammatory cells, activating them, and keeping them at the site

(Boyden, 1962 ; Fudenberg at al., 1976; Weissmann, 1978). However, there is a
Departrnent of Veterinary Physiology. ObihirQ University, 080 Obihiro, Hokkaido, Japan1 76

surprising paucity of research regarding the LKS and monocyte function in normal
dogs and also concerning the basic and clinical cell‑mediated immunity of canine
diseases. In consequence, the authors tried to examine the effect of LKS on the
peripheral blood monocyte chemotaxis, as well as on the neutrophi]s in B. gibsoni
infected dogs by detecting the chemotactic factor in the LKs.

MATERIALS AND METHODS
Babesia species : The strain of B. gibsoni used in this study was maintained bycanine passages in our department (Ishimine et al., 1978, 1979). This was originallyobtained from a hunting dog that contracted the parasite in Hyogo Prefecture in
Japan.
Experimental animals : Six adult mongrel dogs, weighing approximately I O kg,
were used in the experiments. They were inoculated intravenously (i.v.) withapproximately 8.5 x 109 parasitized erythrocytes which had been harvested from asplenectomized dog wherein 300/0 of the erythrocytes contained B. gibsoni. Peripheralleukocytes were examined at the hours of 3, 6 and 12, and on days l, 3, 7, 10, 14,

21 and above 1 75 postinfection (p.i.). For the 2nd experiment, dogs were also
inoculated with the parasitized erythrocytes, and the materials were taken from 2 ofthe dogs on the 2nd and 3rd weeks representing the acute stage, and above the 25th
week p.i. as the chronic stage of the infection. For the chronic cases in the canine
experimental babesiosis the dogs were challenged with the same parasitized erythro‑cytes i.v. 25 weeks after the Ist inoculation to obtain hyperimmunized dogs as donorsof the immune lymphocytes. The hyperimmunized spleen cells were taken on the
4th week after the challenge.
Preparation of Babesia lysate antigen : Three volumes of PBS were added to one

packed volume of the parasitized erythrocytes. The suspension was homogenizedwith a glass homogenizer and ultrasonic vibrator (100 W, Kubota Insonator, Model
200, Tokyo) for 5 minutes, and stored for at least 48 hours at ‑80 C. The antigen
was thawed, rehomogenized, washed 5 times with PBS and recentrifuged at 1 7,300 g
for 20 minutes. The final sediment was homogenized with I .5 vo].ume of PBS andlyophilized in 0.5 ml quantities. When needed, 50 mg of lyophilized antigen was
mixed with 2 ml of PBS in a 10 ml conical centrifuge tube and centrifuged at 365 g
for 30 minutes. The supernatant fluid was collected and stored at ‑80 C to serveas the "Babesia lysate antigen, or BLA".
Preparation of lymphokines (LKs) : Spleen cells and lymphocytes from peripheral

blood were collected from normal and Babesia immune dogs by means of a slight
modification of the Conray‑Ficoll method (Tsuji, 1971). The cells were washedtwice with HBSS. Normal and Babesia immune spleen lymphoid cell suspensions
were incubated with 50 pg BLA in a humidified 50/0 C02 mcubator at 37 C for
24 hours. These lymphoid cell cultures were centrifuged at 1000 g for 30 minutes.
The supernatants thus obtained were filtered through a millipore membrane filter(0.3 p, type PH, Millipore Co., USA) and were used as the normal cultured super‑
natant and Babesia immune LKs. They were diluted I : 2 with medium TC‑199.
Preparation of spleen cell extract: Suspension of I >< 108 spleen cells per ml of1 77

medium TC‑199 was repeated by dry‑melting (‑80 C and 37 C) and sonicated by
the ultrasonic vibrator for 5 minutes. The suspension was flltered through a millipore

membrane filter.
Preparation of agarose plate : 0.12 g of agarose was dissolved in 45 ml distilledwater and 4.5ml double concentrated medium TC‑199 containing I o/heat‑

inactivated canine or human serum was added and kept at 48 C. After suspension,
5 ml of this mixture was poured into a petri dish for tissue culture. Three ml of thismixture was placed on a slide glass at 4 C and allowed to solidify. The agarose
plate used for normal canine neutrophils was prepared to contain 100/0 heat‑inac‑
tivated calf serum (CS) or canine serum. As shown in Fig. 1, these wells, 3.0 mm
in diameter, were punched into the agarose medium with a steel punch. Ten pl ofindicator cells as previously described were poured into the outside well and 10 pl of
medium TC‑199 into the inside well. The plate was incubated for 2 h.ours to evalu‑

ate ch,emotaxis of neutrophils. Incubation was for 18 hours at 37 C in C02 incu‑
bator. After that, the petri dish or slide glass was fixed in methanol overnight at
4 C. The agarose was discarded and the cells were stained with Giemsa or Wrightstain.

Measurement of chemotaxis : Measurement of chemotaxis was made according to
the method of Nelson (1975). The measurement of chemotaxis was calculated asth.e ratio A: B; that is, A, the chemotactic distance between a central well containing
indicator cells and outside well containing the sample for measurement of chemotaxis
or positive chemotaxis, and B, the natural chemotactic distance from central well tothe inside well containing medium TC‑199 as control or spontaneous migration.
This was determined by the use of microscope.

RESULTS
Spontaneous migration of normal canine neutrophils : Patterns and measurements of

spontaneous migration in normal canine neutrophils of the peripheral blood areshown in Chart I . The migration distance of normal canine neutrophils increased
parallel with the incubation period and also with the increase in the number of thecells. Addition of serum into the agarose plate is necessary for the migration of thecells. Migration increased by adding small amounts of serum to the agarose plate.
The migration distance and cell number were observed to decrease remarkably whenheterologous serum was used instead of autologous serum. The difference in the
migration distance between the slide glass and petri dish used as glass plates wasinvestigated to simplify this method. No remarkable difference was demonst,rated
in both materials as shown in Chart I . To incubate, it was necessary to keep theslide glass on a flat surface. In this experiment, the authors found it suitable to use
canine neutrophils as migration indicator cells for demonstrating neutrophil chemo‑

tactic factor (NCF) in 2.5 x 105 cells per well of normal neutrophils. They should
be incubated for 2 hours in agarose plate with I Oo/o inactivated normal canine serum.
Neutrophil chemotactic assay : Neutrophil chemotactic assay was carried out using

supernatant from spleen cell cultures of three normal canine samples. The chemo‑
tactic index ranged from 0,6 to I ,3 and the average was I .O:1:0.2, as shown in Chart 2,

1 78

a. Arrangement of wells

b. Pattern of differential migrationin agarose platein response to chemotactic factor( sam ples )

3.0mm
O

l 3. 5mm

O

OOOO
O

O

Positive chemotaxis

A

l
O

OOO

O O

OO O

i

B
Spontaneous migration( TC‑ 1 99)

Fig. I Differential migration of canine neutrophils in the leukocytes from a well in the supernatantfrom the spleen cells of dogs in the acute infection of Babesia gibsoni.

The chemotactic index of' the supernatant from spleen cell cultures from acute casesof canine babesiosis showed I . I 0.2. A slight increased chemotactic activity ofneutrophils was also detected in some cases. The neutrophil chemotactic index ofchronically infected babesiosis was 1.0 0.2. As shown in Chart 3, supernatantfrom peripheral blood lymphocyte cultures showed a slight increase in chemotactic
activity at 3 and 7 days postinfection (p.i.) ofB. gibsoni compared with that ofnormal

dogs. There was a tendency for this activity to gradually decrease up to the 2lst
day p.i.

Monocyte chemotactic assay : When canine peripheral blood monocytes were usedas migration indicator cells for demonstrating NCF, migration of neutrophils present

in small numbers was also observed at the same time. Thus, it was impossible tocalculate the chemotactic index accurately. For this reason, human peripheral
blood monocytes were used as migration indicator cells for demonstrating NCF inthese experiments. The monocyte chemotactic index of spleen cell extracts fromnormal dogs was shown to be I .0 0. I (0.8‑1 .O). The monocyte chemotactic indicesin the acute stage of 3 weeks p.i. and chronic stage of 29 weeks p.i. were 1.1 0.l(1.0‑1.1) and 1.1 0.2 (0.9‑1.2), respectively. As shown in Chart 4, the differencein chemotactic response to monocytes was demonstrated in some cases of B. gibsoni

infection compared with that of the control. The monocyte chemotactic index ofsupernatant from normal canine spleen cells was 0.9 0.2 (0.9‑1.2) and that of thesupernatant from acute cases of B. gibsoni infected dogs, 2 and 3 weeks p.i., was
1.2 0.1 (1.0‑1.3) and 1. I

0.2 (0.9‑1.5), respectively. Increased monocyte activity

was demonstrated compared with that of normal dogs. The monocyte chemotactic
index, shown in Chart 5, of supernatant from Babesia chronically infected monocyte

chemotactic activity was also observed. Monocyte chemotactic activity was ob‑
served to be present in the supernatant from peripheral lymphocyte cultures 7 days
p,i. as shown in Chart 6,

1 79

Chart la.

Measurement ofspontaneous migration in normal dog peripheral blood neutrophils.
Effects of the number of leukocytes added per well and incubation time

cm ̲̲/
1.0
̲//1 '/5.
̲ 1. OXI05
OXI05
'･ ･
./'
̲̲
O 5 l //
2. 5X I 05

. .

O

3

2

1

4

hours incubation

spontaneous migration (cm)celis/well(hours incubation) 2

41

5.0 X I05

0.8

0.1

1.1

0.2

1 .3 d: 0.6

2.5 X I05

0.6 i 0.1

0.9

0.1

1.1

1 .O X 105

0.5 d: 0.03

0.6 :!: 0.4

0.4

0.9 d:0.3

b. Effect of varying dog serum concentration in agarose medium

Dog‑ ser um

Spontaneous migration (cm)

10 %1 .2 :!: 0.2

2.5 %0.9 d: o. 1O

0.3 :!: 0.2

oc. Differential migration on petri dish and slide glass

Notes :

Materials

Spont neous migration (cm)Petri dish0.8 :!: O. 1

Slide glass

0.7 d: O. 1

The values shown are mean standard error within representative experiments done
independently at least five times

1 80

Chart 2
Chemotactic response of dog neutrophils to supernatants from spleen cells of normal
dogs with acute infection and chronic infection.
infection

ee

1.4

oe

e

e

e

e

ee

eee

e

e

ee
oe

eee･o

ee

1.2

1.0

eee
e

o. 8

e
o. 6

e

ACUTE
(3wKs)NORMALOHRONIC(above 25wKS)

postinfection

No, of examined

M igrat i on

ratio

11

10

11

M ean:!: S. D.

1 . od:o .

1. 1:!:0.2

1. od:o .2

Range

o. 6‑1 .3

O. 9‑1 .4

o. 8‑1 .4

Notes :

Migration rati0=

Distance of positive chemotaxis
Distance of spontaneous migration

DISCUSSION

The major defense mechanism towards Babesia infection in dogs is generally
thought to be the cell‑mediated immunity (Ishimine et al., 1978, 1979; Makimura &Suzuki, 1977; Noda, 1978; Suzuki, 1976). Based on various findings by the authors
in the past experiments, it has been suggested that the cell‑mediated immunity by

the macrophages, Iymphocytes and the LKS may be involved in the protection
immunity (Ishimine et al., 1979; Suzuki, 1976). These flndings included theincrease of the monocytes in the peripheral blood, the phagocytosis of the parasitizederythrocytes by the reticuloendothelial cells, especially the splenic macrophages, theincreased parasitemia caused by splenectomy in the chronic cases, and the acceler‑ated phagocytosis of the parasitized erythrocytes by the immune monocytes fromthe chronic cases in comparison with those from the normal dogs in in vitro cultures.

On the other hand, it is known that polynuclear leukocytes and monocytes in
infectious diseases migrate to the site of infection (Fudenberg et al., 1976 ; Weissmann,

181

Chart 3Chemotactic response of dog neutrophils to supernatants from peripheral blood
lymphocytes in dogs intravenously infected with B. gibsoni.
O. 6

P ost ‑

0.8 1.0 1.2 1.4

Range

Meand: S D

o 7‑1 ,oO. 9d:O .1

O 7‑1 .1

1. 0 0 .2

ee e eo. 9‑1 .o

1. od:o .1

12 hr

ee e eo. 9‑1 .o

1. O:!:O .1

24 hr

eo e e eO. 9‑ I .O

1. O:!:O . 1

3 day

e e

1. 0‑1 .2

1. I :!:0.2

7 day

eee eeo. 8‑1 .3

1. Id:0.2

O. 9‑1 . 1

1. Od:O . 1

o. 8‑1 .2

1. O:!:O .1

1. 0‑1 .1

1. O:!:O .1

infeotion

Migration ratio

O hr

e ee

3 hr

o ee
e

6 hr

e

o

10 day14 day

eee e

e e e
e oe e21 dayNotes :

See Chart 21978) . The movement of leukocytes has been divided roughly in two ways, theoriginal or spontaneous mobility or negative chemotaxis, and movement towards
a factor or positive chemotaxis. Several methods have been used for measuringchemotaxis such as the membrane fllter method (Boyden, 1 962), and the recently
devised agarose plate method (Tautz, 1974; Tsuji & Que, 1976).
In this experiment, the authors thought of using canine monocyte for the mono‑cyte chemotactic measurement, as indicator cells. The purification method of
canine peripheral blood monocyte is not yet well established up to now. For thisreason, canine monocyte chemotaxis was measured by using human peripheral
blood monocytes as migration indicator, instead. This is because there are someproblems encountered in the use of human monocytes. One is the property orfunction of monocytes which may differ in different animal species, and the other isthe nonspecific effect of the materials produced by Babesia sensitized‑1ymphocytes onthese monocytes. The former statement is yet to be considered, but fbr the latter

there have been some reports on positive chemotaxis nonspecifically induced byhuman monocytes in mouse spleen cell extracts in cases of malaria infection (Wyler& Gollin, 1977).

1 82

Chart 4
Chemotactic response of human monocytes to extracts of spleen cells from normal
dogs with acute infection and chronic infection.
ratio

1.4

1.2

,

1.0

e e e

e e
e e

･ e

e
o. 8

,

o. 6

ACUTE
(3 wKs)NORMALCHRONIC( above 25 wKS)

postinfeotion

Migration No, of examlned
ratio
‑4
4
Range

O. 8‑1 .O

1. 0‑1 .1

M ean d: S.D.

1. od:o .1

1. 1:!:0.1

4
o. 9‑ 1. 2
1. 1:!:0.1

Notes : See Chart 2

In the early stage of Babesia infection in animals, an increase in the number ofneutrophils and monocytes is observed in the peripheral blood (Holbrook et al.,

1968; Simpson, 1974; Trubowitz, 1968). Recently, the presence of neutrophil
chemotactic factor in the LKs, produced by sensitized lymphocytes with specificantigen, has been reported (Ishimine et al., 1979; Suzuki, 1976; Fudenberg et al.,1976). However, no report has been made yet concerning the presence ofneutrophil
chemotactic factor in canine babesiosis. In the present study the authors believethat there is small quantity of chemotactic factor present in the spleen cell culture,especially in the peripheral blood lymphocyte supernatant. This suggests that there
is a response of the sensitized lymphocytes to Babesia infection in dogs as a defense
mechanisms against the parasitic infection. In the present study it was observedthat a small quantity of monocyte chemotactic factor is present in the spleen cell
extracts and the culture supernatant from chronically infected dogs.
It has been reported that immunity against Babesia infection is brought about
by the state of nonsterile immunity or premunition, and that the protozoa is hiddenin the host for a fixed period after recuperation from the infection (Noda, 1977). It
is also supposed that Babesia latent infection enables the lymphocytes to keep it

183Chart 5 Chemotactic response of human monocytes to supernatants f ro m spleencells of normal dogs with acute infection and chronic infection.
Ion

e

ratio

e

1.4

e

ee

1.2

e

ee ee
ee

e

1.0

e e

,

e e

e

e e‑

ee

e
o. 8

e e

o. 6

e

e

, ,

eee

e

ACUTE ACUTENORMAL(2wKS) (3wKS)

CHRONIC(above 25wKS)

postinfection

No, of examined

M igration ratio

11

4

11

9

M ean:!: S . D.

O. 9:!!O .2

1. 2d:o .1

1. Id:O .2

1. 1 0 .2

Range

o. 6‑1 .2

1. 0‑1 .3

o. 9‑1 .5

o. 9‑1 .5

See Chart 2

Note :

Chart 6 Chemotactic response of human monocytes to supernatants from peripheral
blood lymphocytes in dogs intravenously infected with Babesia gibsoni.
0.8 1.0 1.2 1 4 1 6
Postinfection (Days)

Migration ratio

Range

M ean
S. D.

eee eee ･eO. 9‑1 .1o

1. 0+̲o .1

1

ee ee e
e

3

･ ee

7

ee ee‑

10

eee eeO. 9‑1 .4 1. 1:!:O .2

14eeee eO. 9‑1 .5 1. Id:0.2

Notes :ee,e

See Chart 2

o. 9‑1 .2 1. od:o .1

O. 9‑1 .2 1. I 0 .1

o. 9‑1 .5

1. 2: :o .2

1 84

sensitized from the speciflc antigen in vivo, hence, the long duration of monocyte
chemotactic factor emission.

REFERENCES
Akashi, M.) Noda. R., Tomimura, T., Kotani, T., Shimakoshi, Y., Onishi. T. and Horie. M., 1969.
Serum transaminase activity in dogs infected with B. gibsoni. Jap. J. Parasit., 18: 689‑690 (inJapanese) .

Boyden. S., 1962. The chemotactic effect of mixtures of antibody and antigen on polymorphonuclear
leukocytes. J. Exp. Med., 1 15: 453‑466.
Fudenberg, H. H., Stites, D. P., Caldwell,J. L. and Wells, J. V., 1976. Basic and clinical immuno‑logy. Maruzen Asian Edit., Tokyo, pp. 587‑614.
Groves. M. G., 1972. Babesia gibsoni field and laboratory studies of canine infections. Exp. Parasit.,
31 : 153‑159.

Holbrook, A. A., Johnson, A. T. and Madden. P. A., 1968. Equine piroplasmosis intraerythrocytic
development of B. caballi and B. equi. Am. J. Vet. Res., 24: 247‑303.

Ishimine. T., Makimura, S.. Kitazawa, S.. Tamura. S. and Suzuki, N., 1978. Pathophysiologicalfindings on blood of beagles experimentally infected with B. gibsoni. Jap. Trop. Med. Hyg., 6 :l 1‑22.

Ishimine, T., Nagasawa, H. and Suzuki, N., 1979. An in vitro study of monocyte phagocytosis in the
peripheral blood of healthy and B. gibsoni infected beagles. Jap. J. Vet. Sci., 41, 487‑495.

Kusunoki, Y., Ito, Y., Okugi, M. and Osaki, F., 1971. A case of dog piroplasmosis in Tokushimacity. Jap. J. Parasit., 20: 53 (in Japanese).

Makimura, S. and Suzuki, N., 1977. Studies on phagocytosis of parasitized erythrocytes from miceexperimentally infected with Plasmodium berghei by mouse peritoneal macrophages. Res. Bull.
Obihiro Univ., lO: 401‑406.
Nakabayashi, T., Omata, Y. and Suzuki) N., 1978. Transmission of parasites and serum antibodies
from mother to litters in rats infected with P. berghei. Protozoan Diseases. Kinokunia Book
Co., Tokyo, pp. 161‑168.
Nelson, R. D., Quie. P. G. and Simmons, R. D., 1975. Chemotaxis under agarose, a new and simple

method for measuring chemotaxis and spontaneous migration of human polymorphonuclear
leukocytes and monocytes. J. Immunol., 1 15: 1650‑1656.
Noda, S. and Suzuki, N., 1977. Infiuence of lymphokines and macrophage inducers on the mouse
peritoneal macrophages. Jap. .J. Parasit., 26: 25 (in Japanese).

Noda, R., 1977. Canine babesiosis. J. Jap. Vet. Med. Assoc., 30: 247‑251 (inJapanese).

Noda, R., 1978. Canine babesiosis in Japan. Protozoan Diseases. Kinokunia Book Co.. Tokyo,
pp. 197‑200.
Sethi, K. K., Pelster, B., Suzuki, N., Piekarski, G. and Brandis, H., 1975. Immunity to T. gondii

induced in vitro in nonimmune mouse macrophage with specifically immune lymphocytes. J.
Immunol., I 15: 1 151‑ll58.

Shirahata, T., Shimizu, K. and Suzuki, N., 1975. An invitro study on lymphocyte mediated im‑munity in mice experimentally infected with T. gondii. Jap. J. Vet. Sci., 37: 235‑243.

Shirahata, T., Shimizu, K. and Suzuki, N., 1976. Effects of immune lymphocyte products and serum
antibody on the multiplication of T. gondii in murine peritoneal macrophages. Zeit. Parasitenk.,
49: 1 1‑23.

Shirahata, T., Shimizu, K. and Suzuki, N., 1977. Studies on production of biolobically active sub‑stance which inhibits the intracellular multiplication of T. gondii within mouse macrophages. Zeit.
Parasitenk., 53 : 3 1‑40.

185
simpson，L．F．，1974．Phagocシtosisof

B．6α繍byneutrophils

in

the

peripheral

circulation．Am．J．

Vet．Res．，135：70レ704．
suzuki，N．，1976． Protozoan

Sciencediseasesinbeagles．studies

onbeaglefbr

researchinJapan．

So丘

Inc．，Tokyo．PP．β57−392（in Japanese）．

Ristic，M．，Lykins，J．D．and
corpuscularantigenSmith，A・R・，197L

isolated

Tautz，W．A，，1974．Assay

of

frQm

blood

leukocyte

Bα傭α6αn歪5andβφ85狛9必50η」・Soluble andofdogs．Exp．Parasit．，30：385−392．

migration

inhibition

under

agarose．Behrlng．Inst。Mitt．，

54572−80、

Tsuji，Y．and

Que，P。G．，1976。study

on theassay ofhun：anleukccyte

nligration・clinicalIm−

mmoL，8：895−899（inJapanese）・

Trubowitz，S．and Masek，R，1968．P傭解04珈μ 吻r翻；Phagocytosis
leukocytes．

Weis5エrarn，1．L．す19フ8．

wylcr，D．T．ard

infセction，abypolymorphonuclear

Science，162：273−274、Eεs（Ltial

Collin，J．1．，1977．

possiblemechanismccnc（ptsinSplccn・derivcdi皿n

unc1（gy，Th（renjε∬irnloronuclear

forsplenic∬aαcphage

Pub．Co．コUSA，pp』8−59、cell（上e琵ctacticfゑctorinn二alaria

accun二ulat三cn．J・1∬rrunoL，118：478−484．

実験的バベシア感染犬におけるリンホカイン中の白血球走化性因子に関する基礎的検討
伊藤

守・牧村

進・鈴木

直義

実験的Bα6θ5彪塵醜o痂感染犬における脾臓細胞培養上清，リンホカインの好中球および単球走化性因子について基礎的検討をおこない，以下の所見を得た。

1）

犬好中球走化性の測定にはアガロース・プレート法を用いて犬末梢血液白血球数，約2。5×105／we11，10彩非動化犬血清含有アガロース・プレートにて2時間の培養条件が最適であった。

2）

犬好中球走化性は，バベシア感染後3週のリンホカインおよび感染後3日の末梢血液リンパ球

培養上清中に認められた。

3）

単球走化性はバベシア感染後3週およぴ慢性期のリンホカイン，ならびに感染後7日以降の末梢血液リンパ球培養上清中に認められた。

以上のことから，犬ピロプラズマ感染に伴って，白血球走化性因子は感染犬の脾臓細胞およぴ末梢リンパ球培養上清中に出現することが明らかになった。

帯広畜産大学家畜生理学教室

Errata of the publication by Takaoka. H. et al. appeared in the Vol. 9, No . 314.
l 98 1 .

page

line

error

correct

187

seventh line from the bottom

l 98 l

l 982

l 88

first line from the top

ll

l 89

eighth line from the bottom
second line from the topmoniteredmonitored

tra c tthorax‑ ‑To)/12]*12
1 [(Ti

12[(Ti ‑ To) / 1 2]

n=1
ll

sixth line from the top

l 98 1

l 982

1 94

Mf. column in the Table 3

OO

0.0

ll

L1 column in the Table 3

l OOO

lO0.0

ll

L2 column in the Table 3

6.0

6.3

ll

seventh line from the bottom

l 98 l

l 982

l 95

first line from the top

1 98 1

1 982

ll

third line from the top

l 98 ll 982

lltenth line from the top

l 98 l

1 982

I 96

sixteenth line from the bottom

oncocerciases

oncocerciasis!lfourteenth line from the bottom

oncocerciases

oncocerciasisllthird line from the bottom

l 98 l

l 982

Japan. J. Trop. Med. Hyg., Vol. 9, No. 3, 4, 1981, pp. 187‑197

187

DEVELOPMENT OF ONCHOCERCA VOL VULUS LARVAE
IN SIMULIUM OCHRACEUM AT VARIOUS ALTITUDESIN GUATEMALA WITH SPECIAL REFERENCE TO
THE AMBIENT TEMPERATURE
HIROYUKI TAKAOKA1,2, KIM M. HANSEN2, HIROSHI TAKAHASH12JOSE O. OCHOA2 AND ELFEGO L. JUAREZ2
Received for publication 2 1 July 1981

Abstract: Females of Simulium ochraceum which were fed on a carrier of the G･uate‑
malan strain of Onchocerca volvulus microfilariae were maintained under natural conditions
at five different altitudes (350‑1,500 m) within and outside of the onchocerciasis endemic
zone. The results suggested the probability that these insects' chance ofsurviving increases

with altitude. The infective stage of O. volvulus larvae was reached on the eighth day at

M ria Santisim.a (alt. 650 m). At higher altitudes ( I ,200‑1,500 m), however, no larval
development beyond the late flrst stage occurred, except in one S. ochracp.u,n female (atGuatemala City, 1,500 m) which yielded late second‑stage worms on day 16. The lengthof the gonotrophic cycle was three or four days irrespective of altitude. Heat accumulation,

calculated by means of a self‑registered thermometer, suggested that the predicted periodrequired for larvae to reach the infective stage in the vector varies considerably withaltitude (4.4‑28 days). The relationship between the rate of development of O. volvulus
larvae at varying altitudes, and ambient heat accumulation, is also discussed.

INTRODUCTION
Many factors influence the transmission dynamics of onchocerciasis. Ambient
temperature is one of the most important of them, affecting as it does the parasite's
development in the blackfly vector as well as the latter's survival. From previous
laboratory experiments, we reported the development of Onchocerca volvulus larvae tothe third stage in Simulium ochraceum Walker (the principal vector of onchocerciasis

in Guatemala) under simulated day and night temperatures (Takaoka et al., 1981).
This occurred even when night temperature dropped below 16.9 C (the predicted
development zero point), although larval development‑time was extended. These
results suggested that the speed of O. volvulus larval development in field populationof S. ochraceum might vary with heat accumulation at differing altitudes.
The present study was thus undertaken to determine the probability of survivalofS. ochraceum, and the development of O. volvulus larvae within it, at selected altitudes
1 Druslon of Medical zoology, Medical College of Oita, P. O. Box 37, Oita, Japan (870 91). 2
Laboratorio de Investigaciones Cientificas para Control de la Onchocercosis "Dr, lsao Tada", Serv‑
icio Nacionai de Erradicaci6n de la Malaria, 5a Avenida, 1 1‑40, Zona I I , Crudad de Guatemala

Guatemala.

,

1 88

wherc ambient heat accumulation was being monitered.

MATERIALS AND METHODS
Dupli･̲,ate experiments were conducted in November and December, 1979,(i.e. during the early clry season ‑ the period when workers harvesting coffee aremost frequently exposed to blackfly bites). Fe .nales of S. ochraceum used in the
project were capture"*" betwee'n 09.00 and 12.00 hr a,t a coffee plantation, (Finca
M6nica lvon ) Iocated on the southwestern slopes of Atitl n Volcano (Departmentof S Lrchitepequez) .

Field procedures for the c.apture and maintenance of simuliids were the same asin previous work (Takaoka ei al., 1981). The O. volvulus microfilaria‑carrier used inthese studies was a 40 year‑old man, with density of 45 mf/mg (based upon one
shoulder biopsy). After collection, tubes holding individual blood‑fed female
blackflies were randomly divided into four (in November) or five (in December)groups, each consisting of 80‑100 insects. Transportation from th.e collecting site

to Escuintla (the lowest experimental altitude) took about two hours. Further
one‑and‑a‑half hours were required for the journey to the other locations. During
this period the simuliids were held in an insulated portable cooler in which ice held

the temperature to 15‑20 C. Immediately upon arriving̲ at the selected locations
where the adults of S. ochraceum were to be maintained, all dead or r^1̲oribund flies
were removed. As a result, 13.1 o/o (in November) and 10.7 /o (in December) of thetotal females were omitted. The tubes containing the remaining blackflies were
packed horizontally in coverless plastic boxes, each with a capacity for 60 individuals.

The boxes wrapped in damp towels were placed in a plastic bag to secure highhumidity. These boxes were then aligned in wooden weather‑instrument shelters(40 cm x 40 cm x 50 cm), standing one metre above the ground. Each shelter also
held a hydrothermograph to record air temperature and humidity during the courseof the experiment. The environmental shelters for housing the simuliids were
stationed near the national highway from Escuintla to Guatemala City ‑ at the
Health Centre in Escuintla (alt. 350 m), at finca M ria Santisima (650 m), at finca

Barret l (1,250 m), at finca Rinc6n (1,200 m) and at the laboratory, Servicio

Nacion l de Erradicaci6n de la Malaria, Guatemala City (1,500 m). Humanonchocerciasis and S. ochraceum adu]ts were known to be present, only at M ria
Santisima, Barret l and Rinc6n. A11 sites were visited each day thereafter. The
number of live blackflies was recorded, and dead ones, if present, were removed
and preserved in the laboratory freezer (at ‑ 19 C) for later dissecti,on.

All dead examples were dissected into head, tract and abdomen, and teased out
in 0.90/0 saline solution using a binocular stereomicroscope. The number of larvae

present was counted and the developmental stages were identified by size andmorphology, following Duke (1968) and Bain (1969). Also, follicular developmentof the ovaries was examined to estimate the duration of the gonotrophic cycle atambient temperatures.
Heat accumulation (Day'C) reveals the sum of daily heat accumulation (sup‑posedly allowing the larvae of O, volvulus tQ develop in the vector). Daily heat

1 89

accumulation was directly calculated from the air temperature grapk̲s according to*
the following formula: 12[(Ti‑To)/12],
always providing that Ti (the mean,m= ltemperature of every two hours) exceeds To (the predicted developmental zero point
for O. volvulus larvae in S. ochraceum). The predicted developmental zero point andthe thermal constant used in this study are 16.9 C and 45 Day'C respectively ac‑
cording to our previous study (Takaoka et al., 1981).

RESULTSHeat accumulation atfive locations of deffering altitude

Table I presents the temperature conditions at the five experimental stations.
The temperature‑fluctuation patterns together with the daily h.eat accumu]ations

during four days in December are shown in Figure I . The December‑thermal
pattern for Barret l was lacking due to a faulty thermograph. As was to be ex‑pected, the mean daily temperature decreased as altitudes rose, and temperaturesfluctuated remarkably by day and night at all altitudes observed.

Actual heat accumulation during the November experiment is illustrated in
Figure 2. At Barret l and Guatemala data were insufficient for prediction of thetime required for larvae to develop into the infective stage. Heat accumulation
increased linearly at Escuintla and M ria Santisima, but irregularly in Guatemala
Escuintla ( 350m )30

27 l'C

20
lO

30

M.Santisima ( 650m )24. 2lc

' 20
uJ
oc

:) lO

H<(:: 30

Rlnc6n ( i200m )

u J

CL

20

‑‑･ 18.6 C

H
lO

30

Guatemala ( 1500m )20
16.8 'ClO

Figure l

The fluctuations in air temperature during four days in December at four
distinct altitudes, and effective temperatures above 16.9 C for O. volvulus

larval development in S. ochraceum. (Areas indicated by oblique lines
represent heat accumulation; solid horizontal line represents 16.9 C (deve‑

lopmental zero point) ; dashed line represents mean air temperature).

190

City, due to the fact that the temperatures did not reach 16.9 C on the seventh
eighth and ninth days.
Table 2 shows the predicted periods (in days) required for larval development

to the infective stage, based on the mean daily heat accumulation. Predicted

Thermal constant( K = 45 Day.C )

so

J

(J

P
l
l
l
l
l
l

) 40

f
l
l
l
l
,
l
l
l

uJ

>
Oco 30
<
Z
o
H 20
<J

l
l

l
,
,

ll

l

d

::)

l
l

:D

o

Escuintla (350m)
M.Santfsima ( 650m)
Barretal (1250m )JL

Guatemala ( ISOOm)

e

c

o
<o lO
X
<H
lc
/
/
I l'
,.
l

l
l
l
l

,

LLl

/

l

o

lO

DAYS POST‑INGESTION
Figure 2 The heat accumulation during the course of the November experiment atfour different altitudes.

Table I Daily temperature (C) at five localities of differing altitude in Guatemala ( 1979)

Place

Minimum
Maximum
Mean (Range) Mean (Range)

Date

Mean

(Range)Nov. 4‑lO
Dec. 8‑20
Nov. 4‑14

27.2(25.7‑29.5)

3 1 .4(30.8‑32.2)

23.0(2 1 .8‑2 7.0)

27. l

(26. 1‑2 7.9)

33. 1

(32.3‑35.0)

22.6(2 1 .0‑24.0)

22.8(2 1 .8‑23.3)

28.9(26.0‑30.8)

18.5(17.0‑19.6)

Dec. 8‑2 l

24.2(22.2‑26.5)

30. 1(29.0‑31.6)

19.6( 1 7.0‑2 1 .6)

Dec. 8‑2 1

18.6( 1 7.0‑20.2)

26.7(24.8‑3 1 .O)

13.l( 8.5‑16.2)

17.9( 1 7.4‑18.5)

22. 1(2 1 .4‑23.0)

13.6( 1 3.0‑ 1 4.0)

GuatemalaNov. 4‑ 7
Nov. 4‑16

17.2( 1 3. 1‑1 9.3)

23.0

( 1 5.8‑27.6)

12.8( I I .2‑1 5.5)

( 1 500 m)

Dec. 8‑2 l

l 6.8( 1 4. 7‑ 1 8. 7)

23.0( 1 9.9‑26.2)

12.7( 8.6‑14.5)(Altitude)

Escuintla( 350 m)M ria Santisima( 650 m)Rinc6n( 1 200 m)

Barret l( 1250 m)

191

Table 2 Daily heat accumulation and predicted period required for development of O.
volvulus larvae ot the third stage in S. ochraceum

Daily heat accumulation

Place and Month

Period for larval

development in days

(Range)Mean

9.8(8.8‑1 1 .7)

4.6( 3.9‑ 5.1)

l0.2

(9.2‑1 1 .O)

4.4( 4.1‑ 4.9)

6. l

(4.9‑ 8.5)

7.4

( 5.2‑ 9.1)

7.5

(5.3‑ 9.6)

6.0

( 4.7‑ 8.5)

2.9

(2.0‑ 4.0)

15.5

( 1 1 .3‑22.5)

l .8(1.5‑ 2.0)

25.0

(22.5‑30.0)

l .8(0.0‑ 3.7)

25.0

(12 8‑ )l.6(0.6‑ 2.6)

28.0

( 1 7.3‑75.0)

Mean(Range)Escuintla

November
December
M ria Santisima

November
DecemberRinc6nDecemberBarretaiNovember
GuatemalaNovember
Decemberdevelopmental periods varied considerably, with a tendency for prolongation as
altitude rose.
Survival of the blackfly hostFigure 3 illustrates the survival curves for batches of female S. ochraceum fed on acarrier of O. volvulus and maintained at five different altitudes. The data suggest
the probability that survival decreases with loss of altitude. In both experiments,
very small number of simuliids survived beyond the predicted day when third‑stage
larvae should have appeared in the head. At Escuintla, I . I o/o (1/90 blackflies in
November) and I .6 /o (1/62 blackflies in December) of the initial host population
survived through the fifth day, while at M ria Santisima, 4.40/0 (4/90 blackflies in

November) and 6.30/0 (4i64 blackflies in December) Iived beyond the eighth andsixth day respectively. At Rinc6n. Barretal and Guatemala City, the only S.
ochraceum to survive to the predicted day was one female (out of 90 experimented withat Barretai in November) which lived for 26 days.
Development of O. volvulus larvae in S. ochracrum

Figures 4 and 5 show the larval development of O. volvulus in S. ochraceum inrelation to ambient heat accumulations at five differing altitudes. The mean dailyheat accumulation obtained from the first four day's observation in November wasused for Barretal instead of the actual one. Among 152 blackflies examined at
Escuintla (350 m) no third‑stage larvae were observed although two blackflies whichdied on the seventh day (in November) and sixth day (in December) yielded latesecond‑stage larvae (Duke's stage K). However, at M ria Santisima (650 m),
where the mean temperature was 22.8 C in November and 24.2 C in December,

1 92

l.OOO

0.869
N

'
k,
t ･,L

l

,

t

0.500

tt "c.
･ N

,,,

t
L

1

tb.

>
H

.
t

x

JCD
<CD

November

x
"‑

jLN

.bO.:DoGz 0.000l(J> 1.000

0,893

D

,,, Escuintla ( 350m )

(1)

‑‑O‑‑

IO‑
k

･･‑XllI' JL ･‑

:,

0.500

M. Santfsima ( 650m )Rinc6n ( 1200m )Barret l ( 1250m )Guatemala ( 1500m )

L
N

'
1

x

December

t

t

‑
"' 't

0.000

5

:IC･‑

tX

20

DAYS POST‑lNGESTION
Figure 3

The survival curves for S. ochraceum females which were fed on a carrier ofO. volvulus microfilariae and maintained at five different altitudes.

larval development to the infective stage occurred on the eighth day (in November)and ninth day (in December) , although it was somewhat asynchronous as evidenced
by several stages within individual blackflies (Table 3). On the other hand, nodevelopment beyond late first stage (H) was observed in simuliids held at Barretal(1,250 m) and Rinc6n (1,200 m). Unfortunately, the single example that survived

26 days at Barret l did not harbour parasites. Similarly, Iarval development
beyond stage F was not seen in Guatemala City (1,500 m), except in the case of one
female which died on the 1 6th day in November, harbouring five late second‑stage
larvae (K and J).
Length of gonotrophic cycle

At all selected locations, except in Rinc6n, fully‑grown eggs were first found in

193

80

皿 L

く＞匡︵﹂
﹂O
﹈Oく﹂■匂o

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1J
1
H
G
F
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工
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A
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！

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！

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！

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1011！

！

芥一一捧7一一x！

1！13／4●Escuintla（350m）oM．Sant1sima（650m）

x

Barrcta1（1250m）▲Guat￠mala（1500rh）

イ

3∠l
ノ

，1

，

●

2o3

書

， 1

0

ノ

1

！

ThermalC。nstant一→

ノ

1

／●

ノ
貞 ・rO

10

20

HEAT

Figure4

71！

﹁ノ●

16
▲

relationshipbetween theshow days a丘er ingestion

development of

at

50

40

60

four

O．∂oJ∂ぬ51arvae

altitudes

in

in

the

the丘gure

ofmicrofilariae）、

90

一

ノ

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56
苓■嘱『

58

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﹂O ﹈Oく﹂■OO

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6

5，／

4
一

＿o
●

Escuintla（350m）oM．Santlsima、（650m）

ロRinc6n（1200m）x

Barrεtal（1250m）

ムGuatcmala（1500m）
C◎nstant→

善淫
10

20

30

HEAT
The

in

13

0

Figure5S．06hr召o卿ηandNovember．（Numbers

皿

く＞配

70

POST−INGESTION （Day・C）

ACCUMULATION

ambient heat accumulation

L
K
HJ
1
H
G
F
E
1D
C
B
A

30

relationshipambientheat

betweentheaccumulation

at

40

50

60

70

ACCUMULATION POST−INGESTION（Day・C）
development offive

O．ooJoぬ51arvae

altitudes inshow days a疵er ingestion ofmicrofilariae）．

in

S．o

hrα68麗配andDecember． （Numbers in thethefigure1 94

Table 3Development of Onchocerca volvulus in Simulium ochraceum which were fed on acarrier of the Guatemalan strain of O. volvulus microfilariae and kept undernatural conditions at M ria Santisima (650 m above sea level)

Days
post‑
infection

No. fiies No. flies Total no.

examined harboring larvae
larvaeMean no. Iarvae/

Stage of larvae ('/ )fiy (Range) Mf.

L1

L2

L3

November, 1979
l
24

24

8,291

345.5 (20‑893)

100.0

0.0

0.0

0.0

2

22

22

3,064

139.3 ( 7‑512)l O0.0

0.0

0.0

0.0

3

15

13

l ,24482.9 ( 0‑268)

94.7

5.3

0.0

0.0

4

2

2

4

2.0 ( l‑ 3)

50.0

50.0

0.0

0.0

1.5 ( 1‑

2)

0.0

lO0.0

0.0

0.0

5

2

2

3

6

4

3

16

4.0 ( O‑

7)

0.0

93.7

6.0

0.0

2.6 ( O‑

6)

0.0

30.8

69.2

0.0

4.3 ( O‑ 11)

0.0

0.0

23. l

76.9

l)

0.0

0.0

0.0 1 O0.025

12.5 (10‑ 1 5)

0.0

0.0

0.0

lO0.0

7

5

4

13

8

3

2

13

9

O

lO

2

ll

2

‑(

0.5 ( O‑2)

December, 1979
1

25

25

8,382

335.3 (54‑825)

lO0.0

0.0

0.0

0.0

2

14

14

1 ,44 1

l02.9 ( C 357)

99.9

O. 1

0.0

0.0

3

9

9

l ,639182.1 (l0‑398)

98.8

l .2

0.0

0.0
0.0

4

9

8

40
8

4.4 ( O‑ 22)

52.5

47.5

0.0

8.0 ( 8.0 )0.0 1 O0.0

0.0

0.0

2

1

3

l.5 ( O‑ 3)

OO

lOO O

0.0

0.0

4

4.0 ( 4.0 )

0.0

0.0

lO0.0

0.0

7

3.5 ( O‑ 7)

0.0

0.0

42.9

57. l

0.0

8.3

25.0

66.7

5
6
7

8

O

9

2

lO

O

11

1

‑( ‑ )‑( ‑ )

1

12

12 (12 )experimental hosts three days after ingestion of the blood‑meal. Essentially, thelength of gonotrophic cycle proved to be three days irrespective of altitude. The
single exception was Rinc6n, where four days were required.

DISCUSSION

In the laboratory study (Takaoka et al., 1981), it was shown that O, volvulus

larval development in S. ochraceum occurred at diurnaljnocturnal temperatures
where the latter were below 16.9 C (the predicted developmental zero point). In
the present study, Iarval development in the vector species was investigated inrelation to arnbient heat accumulation at different altitudes (350‑1,500 m).
Unfortunately, the results demonstrated significantly‑reduced probabilities of host‑survival in the field when compared with corresponding laboratory temperatures

195(Takaoka et al., 1981). Increased mortality might well have been caused by highmidday temperatures, since temperatures above 28 C are debilitating for S. ochraceum(Takaoka et al., 1981 and Monroy, 1979). Under these circumstances, only a few
blackflies survived to the day when larvae might have reached the infec̲tive stage.
The completion of O. volvulus larval development in S. ochraceum was observed only

at M ria Santisima (where mean temperatures were moderate, ranging from 21.8‑
26.5 C) with the developmental period being 8 or 9 days, nearly equal to that pre‑dicted by heat accumulation at this medium altitude. It is notable that the mean
temperature range there coincides with that optimal for the vector's ability to trans‑
mit O. volvulus in the laboratory experiment (Takaoka et al., 1981). On the other
hand, at lower altitude (350 m) with high mean temperature (25.7‑29.5 C) Iarvae
reached only the late second stage even in females (which died on day 6 and 7)
surviving beyond day 5 (i.e. the predicted day when larvae might be expected todevelop to the infective stage at this altitude). This somewhat retarded develop‑
ment despite the sufficient heat accumulations might be associated with the initialcooling of females to 1 5‑20 C during the transportation soon after the 'ingestion ofblood meal. At higher altitude (1,200‑1,500 m) no blackflies lived to the predicted

critical day (15th‑28th day) except one female at Barret l, and no development
occL!rred beyond the late first stage in blackflies dying during the seventh through13th day after ingestion of microfilariae. Therefore, it was not ascertained whether
O. volvulus larvae could develop to the infective form in accordance with ambient
heat accumulation in nature ; although as shown in Figures 4 and 5, Iarval develop‑
ment (though limited to the first stage) appeared to require less heat accumulation
at higher altitudes. This suggested that the first‑stage larval development could

occur even at temperatures below 16.9 C. The parti l development to the latesecond stage seen in one female dying on day 16 at Guatemala City (1,500 m) seems
to indicate that the extended larval development to the infective stage would takeplace in low percentages even at higher altitudes where night temperature drops far
below the developmental zero point (about 17 C). In such cases the developmental
period would probably require more than two weeks as suggested in Table 2. Fromthe epidemiological point of view, it appears of great importance that the durationof extrinsic development of O. volvulus larvae in S. ochraceum varies considerably byaltitudes (i.e. with temperature conditions) within endemic areas. As alreadyreported by previous workers (Garms, 1975, Collins, 1979, and Garms and Ochoa,
1 980) natural infection rates of S. ochraceum for third‑stage larvae of O. volvulus in

Guatemalan endemic zones were variable ranging from 0.09‑3.20/0' This would
be probably explained partially by the different speeds of larval development in thevector simuliids and by the different longevities of the host insects within endemicareas. In this connection it should be also pointed out that the increased probabilityof simuliid's survival at higher altitudes may be to some extent reduced by theincreased risk of exposure to natural hazards when the female oviposits and takes thesubsequent blood‑meals. This is because more frequent gonotrophic cycles might
be repeated in the host blackfly before the larvae therein becomes infective at higher
altitudes than at lower or medium ones, since the duration of one gonotrophic cycle
is nearly the same ‑ three days ‑ even at higher altitude.

1 96

ACKNOWLEDGEMENTS
The authors wish to thank Dr. Y. Wada, National Institute of Health, Tokyo,
and Dr. M. Miyashita, National Institute for Environmental Studies, Ibaraki, .Japanfor their supplying the valuable references which were needed for the present work.
Thanks are also due to Miss C. Aoki, Medical College of Oita, Japan for her help

in preparation of the manuscript. We are grateful to Dr. I. Tada, Kumamoto
University for his critical comments on the manuscript. Finally, we are indebted to
Dr. Marshall Laird, Memorial University of Newfoundland, Canada, for his criticalreview of the manuscript.
This study was supported by the Ministry of Public Health, Guatemala and theJapan International Cooperation Agency, Japan (GJCRCPO‑MENSAP series
No. 35).

REFERENCES
1 ) Bain, O. (1969) : Morphologie des stades larvaires d'Onchocerca volvulus chez Simulium damnosum
et redescription de la microfilaire, Ann. Parasitol., 44, 69‑81

2) Collins, R. C. ( 1979) : Onchocerciasis transmission potentials of four species of Guatemalan

Siinuliidae, Am. J. Trop. Med. Hyg., 28, 72‑75
3) Dalmat, H. T. (1955) : The blackflies (Diptera, Simuliidae) of Guatemala and their role asvectors of onchocerciases, Smithson. Misc. Coll., No. 125, 425 pp. (Abstracted in Rev. Appl.
Entomol. B, 44, 47)4) Duke, B. O. L. (1968) : Studies on factors influencing the transmission of onchocerciases. V:
The stages of Onchocerca volvulus in wild 'forest' Simulium damnosum, the fate of the parasites in thefly, and the age‑distribution of the biting population, Ann. Trop. Med. Parasitol., 62, 107‑116
5) Garms, R. ( 1975) : Observations on filarial infections and parous rates of anthropophilic black‑flies in Guatemala, with reference to the transmission of Onohocerca volvulus, Tropenmed. Parasitol.,

26, 169‑182
6) Garms, R. and Ochoa, J. O. ( 1979) : Further studies on the relative importance of Guatemalan
blackfly species as vectors of Onchocerca volvulus, Tropenmed. Parasitol., 30, 120‑128

7) Monroy, M. C. (1979) : Infeccion experimental de Simulium ochraceum con microfilarias deOnchocerca volvulus, Informe de Tesis, Escuela de Biologia, Universidad de San Carlos de Gua‑
temala, 90 pp.

8) Takaoka, H., Ochoa, J. O., Juarez, E. L. and Hansen, K. M. ( 1981) : Effect of temperature on
development of Onchocerca volvulus in Simulium ochraceum, and longevity of simuliid vector, J.
Parasitol. (in press)

197

中米グァテマラの温度条件の異なる種々の標高におけるOn6ho6θ76αool∂％」狐幼虫の媒介ブユS珈初伽勉06h7α66徽体内での発育高岡宏行1・2，KM．HANsEN2，高橋

弘2，」．O．OGHoA2，E．L．JuAREz2

中米グァテマラの標高の異なる5地点（350−1，500m）に設置した百葉箱内で0％choo8roα拶oあぬ5
ミクロフィラリア保有者から吸血させた媒介ブユS吻㍑1彪勉oohr鷹θμ勉を飼育し，その後のo．か．幼虫の発育と同時に設置した自記温度計より算出された有効温量との関係を調べた。標高350mのEscuintlaでは有効温量から4日目に第3期幼虫の出現が予測されたが第6日，7日目に死亡したブ
ユ雌でもまだ第2期後期幼虫しか見られなかった。一方，標高650mのMaria Santisimaではほぼ
有効温量から推測されるとおりに第8日目に第3期幼虫への発育が観察された。ここより標高の高い
3地点（1，200m，1，250m，1，500m）では，各々15日，25日，25−28日目に第3期までの幼虫発育が予測されたが飼育したブユの大半がこれらの期間内に死亡したことにより十分な観察を行いえなかった。

これら3地点では，飼育開始後7−13日の間に死亡したブユ体内で見出された幼虫はまだ第1期後期までしか発育していなかった。しかしながら標高1，500mのGua七emala市において飼育開始後16日目に死亡した1個体のブユから第2期後期まで発育した幼虫が見出されたことから，標高の高い場所
でも非常にゆっくりとではあるがo．∂．幼虫の第3期までの発育は可能であることが示唆された。

1大分医科大学医動物学教室
Onchocercosis

2LaboratoriodeInvestigaciones

Dr，IsaQ Tada アServicio NaciQnalde

Cienti批as

Erradicaci6n depara

Controldela Malaria，Guatemala．

laJapan. J. Trop. Med. Hyg., Vol. 9, No. 3, 4, 1981, pp. 199 205

1 99

THE FIRST RECORD OF HUMAN INFECTION WITHDIPHYLLOBOTHRI UM CAMERONI RAUSCH, 1969*
H KAM02, Y. YAMANE3, AND K. KAWASHIMA4
Received for publication 2 November 1981

Abstract: A diphyllobothriid cestode, which has been spontaneously expelled from a
57‑year‑old seaman at Fukuoka City, on December 1969, was sent to us for identification.

The appearance of yellowish‑brown, thickset, complete strobila (about 600 mm in length)

was evidently different from usual forms of the known diphyllobothriid cestodes fromhumans. It was tentatively reported as "another marine species of the genus Diphyl‑

lobothrium from a man in Japan" (Kamo et al., 1978). After careful morphologicalexaminations it is now identified as Diphyllobothrium cameroni Rausch, 1969, which has
been found in a Hawaiian monk seal (Monachus schauinslandi) from Midway Atoll, Leeward
Island in the Pacific Ocean. In the relative position of the uterine pore, within the
genital atrium, and other morphological details, it agrees closely with Rausch's description.

The discrepancies in dimensions ofthe seminal vesicle and egg size are probably attributable

to the considerably greater size of our specimen, which had grown well in larger space ofhuman intestine. Its eggshell surface observed by SEM exhibited the typical character‑istics (deep, numerous pits, with rough intervening surface) as the eggs of cestodes fromthe marine habitat according to Hilliard ( 1972). It seems to be the first and unusualcase of human infection with Diphyllobothrium cameroni Rausch, 1969 (new Japanese name :cameron retto jochu) . The source of human infection is quite obscure.

INTRODUCTION
In such a country as Japan, where raw or undercooked marine fishes are an
important and traditional part of diet, it seems to be fairly strange that a diplo‑gonadic tapeworm, Diplogonoporus grandis has been the only known species fromhuman cases among various diphyllobothriid cestodes from marine habitat.
In order to explore such paradoxical situation, a review study of the so‑called

broad tapeworm in Japan was initiated by Kamo et al. in 1972, suspecting theoccurrence of human infection with some kind of marine diphyllobothriids mistaken
for the common species, Diphyllobothrium latum.

Recently the evidence of human infection with a certain marine species of thegenus Diphyllobothrium was demonstrated by Kamo et al.. 1977, and subsequently a
new marine species from a man was described as Diphyllobothrium yonagoensis n. sp.
l This work was supported in part by Grant‑in‑Aid for Scientific Research from the Ministry of
2 Department of Medical Zoology, Tottori University School
Education, Science and Culture.
3 Department of Environmental Medicine, Shimane Medicalof Medicine, Yonago City, Japan.
4 Department of Parasitology, School of Medical Technology,
Ufiiversity, Izumo City, Japan.
Kyushu University, Fukuoka; Japan,

200

by Yamane et al., 1981.
In addition the auth.ors, in 19/ 8, briefly reported another Diph)llobothrium
species of marine origin which had been spontaneously expelled from a 57‑year‑old

seaman at Fukuoka City in 1969. The species was identifled as Diphyllobothrium
cameroni Rausch, 1969, and its description is given here.

MATERIALS AI IID METHODS
The worm was preserved in 100/0 fbrmalin solution, and then it was in a con‑tracted condition which had been spontaneously expelled from a 57‑year‑old seaman,

H. U.) who lives in Fukuoka City. Kyushu, Japan, on a day in December) 1969.
A whole mount preparation of some mature proglottids was prepared, staining in
Semichon's acetic carmine. Serial sections of mature proglottids were also preparedin horizontal, sagittal and transversal planes, staining in modified tric.hrome stain
solution. The eggshell surf'ace was observed by the scanning electron microscopy.

OBSERVATION
Strobila muscularyellowish‑brown in colour; margins slightiy serrate. Strobila

650 mm long with maximum width 9.5 mm, attained about 200 mm behind fromthe scolex. Segments wider than kmg, with length increaaing posteriad. Length!
width ratio of premature segments about:, I : 12j af. mature segments with maximum
width about I : 8, and of terminal segments as much as I : 2.5.
Maximum thickness ofstrobila 2.a mm (Figs, l, 2) . Excretory syst,em including

10ngitudinai and transverse ducts in parenchyma and numerous interconnected

Fig, I ¥ rhole bady,20 1

Fig. 2 Proglottids (no staining).

A: 100 mm behind frOm the sc.olex> B: 200 mm behid, C: 300 mm behind,
D : 400 mm bchind, E : terminal

ducts in cortical region. Innermost layer of longitudinal muscle fibres strongly
developed, as much as O.5 mm thick. Scolex lanceolate, p, ointed at apex, 2.2 mm
10ng x l.3 mm wide (lat,eral view) x O.96 mm thick. Bothria deep, extending fulllength of scolex and diversing at apex ; Iips of bothria slightly rolled. Neck absent(Fig. 3). Genital pore situated ventrally on midline at anterior margin of segment,

covered by velurn of preceeding segment. Cirrus sac elongate, O.515 mm long x
0.288 mm in diameter. Apex of cirrus sac attached dorsally by fibres confluent withthose of longitudinal muscle layer. Cirrus sac opening anteriorly into genitalatrium. Seminal vesicle elongate, 0.340 mm dorsoventral dimension by 0.247 mm
in diameter, situated posteriorly near dorsal end of cirrus sac, and connected withlatter by short duct. Subspherical testes numerous, arranged in one layer in lateralfields (in irregular layer two to three deep in near central field) . Vagina running
anteriad, then turning ventrad along posterior surf)ac.e of cirrus sac, opening in
genital atrium immediately posterior to opening of cirrus sac. Bilobed ovary situatedtransversely at posterior margin of segment dorsal to uterus ; ovarian lobes extending
laterad beyond uterine loops. Vitellaria abundant, forming two lateral flelds ;medial margins of vitelline (and testine) fields diversing Posteriad, only slightly
overlapping ends of uterine loops and ovarian lobes. Jterine loops extending laterad
and anterolaterad, reaching level of anterior margin of genital atrium (Fig, 4) .

When much distended with eggs, gravid uterus may extend posteriad into
adjacent segment. Uterus opening into posterior wall of genital atrium (Fig, 5).
T. erminal portion o. f uterus thick walled, with valvelike structure near Orfice. Eggs

202驚．︑鐵灘難藩︒

Fig、3

F鳩4・

20

'

*)

Fi * , r)̲ Sec.tions of mature proglotti,ds.

A, B: sagittal sections, showing arran, gement of genit.a,1 organs, C

D cross sectlons E hon:ontal sectionccirrus sac, ga: genital atril rn* Im : longitudinal n.luscle layer, s:u : uterus

eminal vesicle* t : testis,uo; u'terinc Opcuing, vo : vaginal opening, vt: vitelline follicle

ellipsoidal) without. apical knob) 45.956.7/
39.5i 1.5). Larg, e (irregular sized)(avg 50.9i:2.5) X 35.1.42.4 /･ ( avg

deep pits distributed densely on the surf'ace ofFig, 3

Scolex; dorsovent‑ral (left) and lateral (right) view.

Fig, 4

1/Vhole mO, unt preparation.

right: uterine field (larger magnifica,tion)

20 4

Fi ̲, 6 Eggs nd egg shell su face by StiM( x 5000)

eggshell (O.81 .4 /1 thick in lateral) (Fig. 6).

DrscUssroN
As described above our specimen from a, seaman seems clearly to be identical inalmost all morphola. gical details with the description of Diphyllobothrium cam.erani

Rausch, 1969, which had becn f'ound f'rom the Hawaiian monk sea,1 (Monachus
schauinslandi) collected at Midway Atoll, Leeward rslands. Ir'he discrepancies insome values of measurements such as diameters of the seminal vesicie and egg sizes
are probably attributable to the conaiderably greater aize of aur specimen, which had
grown in larger space of human intestine.
The Hav 'aiia,n monck seal is distributed in lat. 2030 N around the Hawaii
Islands, while its faod habits still obscure according to Nislliwaki (1965). The
source of human infection with this vvorm is quite unknown, although it should befound from s(,)me marine fishes as tlle common food between sea] and human.

AcKNo'wu"'.DC'.EMENTs

The authors wish to express our heartf'clt thanks to Dr. R. L, Rausch, Divisionof Animal Medicine, School of Medicine5 University of Washington

Seattle;Washington, U. S. A., for his kindness and encouragements through providing withmany specimens or papers of various diphyllobothriid cestodes. To Mrs. S. Waka‑
hara Miss M. Hasegawa, and Mr. Y. Sugihara, Dept. of Medical Zoology Tottori
Univ. School of Med., apprec,.iation is expressed for their technical assistances.

205

REFERENCES
1） Hilliard，D，K（1972）： Studies
lbrmation

2）

in

some

on thehelminth

fauna

of

Alaska

LL

from

man

inJapan，Jap・」・Parasit．，21（supPL），71（in3）Kamo，H・，Yamane，Y・，Maφma，J・and Yazaki，S・（1977）：
lobothriidoneggshelldiphyllobothriid cestodes，can．J．zool．，50，585−592．

Kamo，H、，Yamane，Y．and Hatsushika，R．（1972）：Reexamination
tapeworm

Observations

cestode other than D・」伽m

fbund

from

human

of

the

so・called

Koga−Okamura

cases

in

broad

Japanese）

type

diphyl−Japan，NipPon一工ji−shinpo，

2795，43−45．

4）KamoコHりYamaneンY、andJoゐo∫h吻解fromaman

Kawashima，K．（1978）：Another5）Nishiwaki，M．（1965）：Cetaceans andPinnipeds，Tokyo

6）Rausch，R．L．（1969）：Diphyllobothriid
5σhα痂5」α漉Matschie，from Midway

Cestodesofthe

University

from themarinePress，Tokyo．

HawaiianMonk

Seal，ルfoηα h鋸Ma句ima，J．（1981）：On

Genus Dψ妙〃oわo孟hr伽（cestoda：Pseudophyllidea）Found

Jap．J．Parasit、，30，101＿I

species of1）ψ塀一

Japanese）

Atoll，J．Fish．Res．Bd．canada，26，947−956．

7）Yamane，Y・，Kamo，H・，Yazaki，s・，Fukumoto，S。andspeciesform ofinJapan・JapJ．Parasit．コ27（supPL），41（in

from

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Man

Marinei血Japan，

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1）ψ々アJlo60渉h7彪ηz6

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Rausch，1969

一カメロン裂頭条虫（新称）一の人体寄生第一例
加茂甫・山根 洋右・川島健治郎

福岡市在住の男子船員（当時57歳）から，1969年12月に自然排出された，裂頭条虫と思われる虫体が同定のため送られてきた。虫体は頭節を有する完全な標本であったが，小柄（約600mm）で，肉厚の，ずんぐりした黄褐色の外観を呈し，一見してこれまで人体寄生種として知られている裂頭条虫とは異なるもののように思われた。著者らはこれを，人体寄生裂頭条虫のさらに新しい海洋種として，特徴の概要をとりあえず学会に報告しておいたが（加茂ら，1978），このたびP塑hッ伽みo∫hrル〃z

6α耀ro痂Rausch，1969として同定を確定し，和名としてカメロン裂頭条虫を提唱した。本種はミッドウェー諸島近海で捕獲されたタイヘイヨウモンクアザラシ（Moηαohμ550hαμ初51αη4げ）から得られた標本に基づいて、Rausch（1969）が命名記載した種類である。人体から得られた標本は，形態的特徴が極めてよくRausch（1969）の記述に一致し，とくに子宮孔が生殖孔の後壁に開口する点が他種に見られない特徴である。貯精嚢や虫卵などの計測値が人体排出標本で全般的に大きいのは，アザラシの腸管にくらべて広大な人の腸管内で，大きく成長できたことによる差異であろうと思われる。走査電顕による卵穀表面像は，深い点刻が密に分布し，点刻間の面がきめ粗い性状を示し，Hilliard（1972）のいう海洋性の特徴を認めた。これはカメロン

裂頭条虫の人体から見出された最初の例であるが，恐らく偶発的なものであろう。感染源は人問とタイヘイヨウモンクアザラシとの共通の食物となっている海産の魚類であろうと思われるが，まったく不明である。

1鳥取大学医学部医動物学教室
3九州大学医療技術短期大学部

2島根医科大学環境保健医学教室

JAPANESE JOURNALOF
TROPICAL MEDICINE AND HYGIENE
December, 1981

Vol. 9 No. 3, 4

CONTENTS
Original article
HoRI, S., TSUJITA, J., MAYUZUMI, M. AND TANAKA, N.

Comparison of body shape, body composition and sweating reaction between
young male highlanders of Papua New Guinea and young male Japanese. 151‑159

FuRuvA, M., OKA, M., ITO, Y., OKA, Y. AND OsAKI, H.
Resistance to challenges with homologous Trypanosoma gambiense in mice transferredmouse immune spleen cells or immune sera . . . . . . . .

ToDA, M., ISHIZAKI, T. AND TAKAOKA, M.
A case study on chloroquine resisted falciparum malaria combined with DIC
resembling symptoms. 161‑165. 167‑173

ITo, M., MAKIMURA, S. AND SuzuKI, N.
Fundamental studies on the leukocyte chemotactic factor in lymphokines of dogs
infected with Babesia Gibsoni. 175‑185

TAKAOKA. H., HANSEN, K. M., TAKAHASHI, H., OcHoA, J. O. AND JUAREZ, E. L.
Development of Onchocerca volvulus larvae in Simulium ochraceum at various altitudes
in Guatemala with special reference to the a,mbient temperature . .

, 187‑197

KAMo, H.. YAMANE, Y. AND KAWASHIMA, K.
The first record of human infection with Diphyllobothrium cameroni Rausch, 19b 9 . . . . . .

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

12

Sakamoto‑machi, Nagasaki, 852, Japan. 1 99‑205