ISSN

Japanese

Joumalof

Tropical Medicine and Hygiene

第17巻第4号

平成元年12月15日

内原O304−2146

容著

大分におけるキアシツメトゲブユの0郷hoo召繊幼虫自然感染
一特に動物寄生性0窺hoo6瓢8による人への伝播に関して一（英文）

・高岡

宏行，馬場

稔，OdileBain279−284

ビルハルツ住血吸虫症の感染の強さ，罹患率と陽転・陰転比（英文）

・嶋田

雅暁，平田

佐藤

瑞城，John克之，野田H．Ouma，伸一，青木 克己

285−290

ケニア，沿岸州におけるビルハルツ住血吸虫症の疫学的研究

一ビルハルツ住血吸虫感染における水との接触行動の重要性（英文）

・嶋田

雅暁，平田

佐藤

瑞城，John克之，野田H．Ouma，伸一，青木 克己

291−301

インドネシア国北スマトラにおけるマラリア高度浸淫地と年齢別生殖母体

血中濃度分布について（英文）・・糸川英樹，高井錬二，石井

ブタ回虫感染マウスにおけるサイトカインとNK活性の動態

明，W．Panjaitan………………陶303−310

道311−321照明323−329

大平肺吸虫の脱嚢メタセルカリア主要外皮抗原と成虫主要外皮抗原を認識する
モノクローナル抗体（英文）……一…………・・………一……・及川陽三郎，池田

南米エクアドル共和国リーシュマニア症の皮内反応とELISA法による疫学調査（英文）………………・………・・…古谷正人，三森龍之，E．A．L．Gomez，

V．V．Corone1，川端 真人，橋口義久

331−338

アマゾン流域日系移住地，トメアスー地区における回虫およびトキソプラズマ
感染状況についての考察

一………………・佐藤久美子，狩野 繁之，Milton

柳澤

症例報告

M．Hida，紘，小河原はつ江，鈴木守

339−346

弘347−350

ヒトクイバエCo吻Jo伽α窺加oρoρ加gπによるハエ症の輸入例

一影井会

昇，村田以和夫，倉橋

報

投稿規定

第17巻総目次

日熱医会誌Japan．J．Trop．

Med．Hyg．

日本熱帯医学会

Japan. J. Trop. Med. Hyg., Vol. 17, No. 4, 1989, pp. 279‑284

279

NATURAL INFECTIONS OF SIMULIUM BIDENTATUM(DIPTERA= SIMULIIDAE) WITH LARVAE OF
ONCHOCERCA SPP., IN RELATION TO A HUMANZOONOTIC ONCHOCERCIASIS IN OITA, JAPANHIROYUKI TAKAOKA*, MlNORU BABAAND ODILE BAIN2

Received June 7 1989/Accepted August 30 1989

Abstract: A total of 579 wild female flies belonging to eight species of Simulium werecollected on human baits, in relation to the transmission of a human zoonotic onchocer‑
ciasis found in Oita, Japan. Simulium bidentatum was the most dominant species. Naturalinfections with filarial larvae were found in ll of 449 S. bidentatum (or 6.5% of the parousfiies) and in I of 23 S. aokii (or 10% of the parousflies). All the infected flies had 1‑4 either

first‑, second‑ or early third‑stage larvae in the thorax. Examination of third‑stage
larvae shows that S. bidentatum is infected with at least two species of Onchocerca. Our
result represents the first record of natural Onchocerca infections in Japanese Simuliidae,

and also suggests the possibility of S. bidentatum as the potential vector of zoonotic
Onchocerca spp. to humans.

INTRODUCTION
Four human cases of zoonotic onchocerciasis have been reported from various areas of
the world (Azarova et al., 1965; Siegenthaler and Gubler, 1965; Beaver et al.. 1974; Ali‑Khan,

1977). An additional case was recently found in Oita, southern Japan (Hashimoto et al.,1990). Examination of histological sections of a female nematode found suggested that, as
in all the previous cases, its morphological features resemble those of Onchocerca gutturosa
Neumann, 1910 and O. cervicalis Railliet et Henry, 1910 infecting cattle and horses respectively(Beaver et al., 1989).

Several Onchocerca spp. including O. gutturosa and O. cervicalis have been reported fromJapan (Sato et al., 1954; Suzuki et al.. 1982). However, no transmission studies of thesefilarial nematodes have been carried out, although biting midges (Culicoides spp.) or
blackflies (Simulium spp.) have been known as the vectors of certain Onchocerca spp.
elsewhere (Bain and Chabaud, 1986).

1 Division of Medical Zoology, Medical College of Oita, Hazama, Oita 879‑56, Japan
2 Laboratoire Des Zoologie Vers. Museum National d'Histoire Naturelle, 61, Rue de Buffon,
75231 Paris Cedex 05, FranceThis study was supported by a Grant‑in‑Aid for Scientific Research from the Ministry of Educa‑
tion, Science and Culture of Japan (No. 01570216)

280

The objective of this study is to investigate the transmission of zoonotic onchocerciasis
which may be acquired by humans. In this paper, we report natural Onchocerca infections of
S. bidentatum Shiraki collected in one of the suburban residential areas of the city of Oita,

where a human zoonotic onchocerciasis occurred.

MATERIALS AND METHODS
The patient infected with a zoonotic Onchocerca, a two‑year‑old Japanese girl, was an
inhabitant of Tabaru, one of the suburban residential areas (altitude ca. 30 m above sea level;

coordinates 131'35'E; 33'10'N), situated in the western part of the city of Oita. It waspresumed that the zoonotic Onchocerca infection might have been acquired by this child at ornear her residential area, because, Iiving in one of the housing complexes (ca. 0.01 km2) , sheused to spend her time with her mother during the daytime at the recreation ground (ca.
1,200 m2) Iocated on the west within their premises, and had never left the city, according to

her mother. It was therefore attempted to collect biting midges or blackflies at the corner ofthis recreation ground. Adjacent to this place on the west lies another huge housing area
recently developed (with an extension of about 0.4 km') . There is a low hill with a shrubbery
just behind these living areas on the south. On the north, a flat farmland mainly used for ricecultivation widely opens and in its center the Oita River runs eastwards which is, at thenearest, about 400 m away from the collection site. There are several cattle sheds but nohorses are raised within a 4‑kilometer radius from the collection site.
Collections were made between 16.00 and 18.00 hours during September‑December 1988.
Flies landing on or flying around the body surface of the human baits were captured by anaspiration tube or by an insect net, and held overnight in small plastic containers. The
following day, fiies were dissected in saline on a glass slide, and parity was determined by thepresence of follicular relics (Detinova, 1962) . Parous females were microscopically searched
for filarial larvae. The larvae found were preserved in formalin‑glycerol (Wharton, 19 9) for

morphometric observations.
About 170 blackfiies collected in December were individually maintained with sucrose
solution at 25'C for seven days, and dissected for the presence of third‑stage filarial larvae.

Generic diagnosis of third‑stage larvae followed that of Bain and Chabaud (1986).

RESULTS
Four collections carried out from September to November 1988 yielded a total of eight
blackfly species, but no Culicoides species was captured. The majority of blackfly species
were Simulium bidentatum, followed by S. arahawae Matsumura, S. quinquestriatum (Shira‑ki) and S. aokii (Takahasi) (Table 1).
Natural infections with filarial larvae were found in 2.5% of 449 S. bidentatum collected(or 6.5% of the parous flies) , and in 4.3% of 23 S. aokii captured (or 10% of the parous fiies) .

Eight of 11 infected S. bidentatum had I to 4 first‑stage larvae, two flies each harbored one

second‑stage larva, and one fly had two third‑stage larvae probably just after the second
moulting (Fig. 1) . One infected S. aokii had one first‑stage larva (Fig. 2) . All the larvae were

found in the thorax of the fiies.

One of the two third‑stage larvae (Fig. 1) recovered from September collection showed281
Table I Species composition, parity and natural infections of blackflies collected in

Tabaru, one of the suburban residential areas, where a human case of
zoonotic onchocerciasis occurred, in Oita, Japan
Date of collection
Sept. 23

Total

Nov. 17Oct, 13

Oct. 6

Simulium

No. No, No. No, No. No, No. No. No, No, No. No. No. No, No,

spp.

col . par , inf , col . pa r . inf . col . par , inf , col , par . inf . col , par. in f ,

aokii

o

a raha wae

33

11

o

o

bidentatum

88

30

4

43

ja ponicumoonikkoenseooquinquestriatumooru fi basistahahasii

4

o

o

3

1

7

4

o

O

9

O

174

69
o

o

O
O

o

2

o

o

3

2

o

7

2

o

2

1

o

O

12

3

o

23

10

3

l

o

36

12

O

144

62

3

449

170

11

1

O

o

2

O

O

1

O

O

O

O

30

10

21

o

33

10

O

o

12

5

O

o

23

2

O

the following measurements: total body length (BL) 743 pm, body width (BW) 26 pm, Iengthof oesophagus (LE) 464 pm, Iength of tail (LT) 35 pm, width of tail (WT) 21 pm.
In dissections of flies maintained for seven days after collection, one of 144 S. bidentatum

examined had one third‑stage larva (Fig. 3) in the thorax, but all of the 22 S. aokii were

negative. The third‑stage larva recovered (Fig. 3) showed following measurements: BL
1,280 pm, BW 25 pm, LE 620 pm. LT 45 I/m, WT 20 pm. This larva, as well as two early
third‑stage larvae mentioned above, seemed to belong to the genus Onchocerca by possessing
the very small, almost indiscernible caudal lappets.
It is generally impossible to identify the young third‑stage larvae. However, the early
third‑stage larva fdund in September seemed to be distinct from the longer third‑stage larva
found in December because the ratio of LE/BL, as well as body length, was clearly different(i. e., 0.62 vs. 0.48) . This conclusion is supported by our observations (unpublished data) thatearly third‑stage larvae of the latter (long type Onchocerca) bear the short oesophagus (lessthan 1/2 of body length) Iike mature third‑stage larva, and measure over 850 pm in bodylength.

It remained to be determined whether the filaria infecting S. aokii belongs to Onchocerca.

DISCUSSION
Beaver et al. (1989) suggested that the human zoonotic onchocerciasis found in Oita wasprobably caused by O. gutturosa or O. cervicalis which have been commonly known in cattle
and horses respectively around the world. Our result shows the least possibility that thecausative worm was O. cervicalis, because its transmitter, Culicoides, was not captured during

the daytime in the study area. This is also supported by the fact that horses, the definitive
host of this parasite, were not raised around this area.

On the other hand, it is demonstrated that there were several anthropophilic blackfly
species in the study area, and that S. bidentatum, the most dominant species, was naturally
infected with larvae of Onchocerca. Examination of the third‑stage larvae recovered shows282'* '* '

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̲= >̲ +'
̲*<{ =****'i:i:;
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'=: #

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Figure I Early third‑stage larva of Onchocerca sp, found in S. bidentatum dissected
soori after collection.
Figure 2 First‑stage larva of unidentified filaria found in S. aokii dissected soon aftercollection.

Figure 3 Third‑stage larva of Onchocerca sp, found in S. bideletatum maintained forseven days after collection.
All scale bars indicate 100 ftm

283that S. bidentatum was infected with at least two Onchocerca species. One of these two fits
in with the body length of third‑stage larva of O. gutturosa given by Bain (1979) , but more

specimens of mature stage are needed to compare with the bovine parasite. The other one,though only one specimen is available, may belong to a new species because it has a very long

body, as compared to those of other known Onchocerca species (Bain and Chabaud, 1986) . At
the present, it is thus difflcult to conclude that the zoonotic Onchocerca acquired by aJapanese child was O. gutturosa, although it might be one of the two Onchocerca species foundin S. bidentatum.

There were many cattle (both Holstein and Japanese Black) at several sheds set up in
the farmland near the study area, and microfilariae of Onchocerca spp. were already found inblood‑fed females of S, bidentatum. S. aokii and S. arakawae collected at one of five cowsheds

examined. Further, three types of third‑stage larvae of Onchocerca spp. were commonly
discovered in the same blackfiy species collected at the same cowshed (Takaoka and Bain,unpublished data) . One of these three types of Onchocerca appears to be the same species as
the longer third‑stage larva found in this survey, and the remaining two types resemble third‑

stage larvae of O. gutturosa and O. Iienalls Stiles, 1908 respectively, although both of thelatter two bovine filariae have been not yet recorded in Oita. Whether all these types of
Onchocerca spp. recovered from Simulium spp. are parasites of cattle or of other animals
should await future studies.
In summary, our findings indicate that there exists a situation, under which the zoonotic
Onchocerca infection may be acquired by humans through biting of the local blackfiy species,in the western suburbs of Oita.

ACKNOWLEDGEMENTS
We are grateful to Dr. I. Tada, Kumamoto University Medical School, for his valuablecomments. Thanks are also due to Miss C. Aoki, and K. Ogata, Medical College of Oita for
their assistance during this survey.

REFEREN CES1 ) Ali‑Khan, Z. (1977): Tissue pathology and comparative microanatomy of Onchocerca from aresident of Ontario and other enzootic Onchocerca species from Canada and U.S.A., Ann. Trop.
Med. Parasitol., 71, 469‑482
2 ) Azarova, N.S., Miretskij, O.Y. and Sonin, M.D. (1965): The first discovered case of nematodeparasitism in the USSR in a human being (genus Onchocerca Diesing, 1841), Med. Parazytol.
(Moscow), 34, 156‑158 (in Russian)3 ) Bain, O. (1979): Transmission de l' Onchocerque bovine, Onchocerca gutturosa, par Culicoides,Ann. Parasitol., 54, 483‑488
4 ) Bain, O. and Chabaud, A.G. (1986) : Atlas des larves infestantes de filaires, Trop. Med. Parasit.,

37, 301‑340
5 ) Beaver, P.C., Horner, G.S. and Bilos, J.Z. (1974): Zoonotic onchocerciasis in a resident of
Illinois and observations on the identification of Onchocerca species, Am. J. Trop. Med. Hyg.,23, 595‑607
6 ) Beaver, P.C., Yoshimura, H., Takayasu, S., Hashimoto, H. and Little. M.D. (1989): Zoonotic

Onchocerca in a Japanese child, Am. J. Trop. Med. Hyg., 40, 298‑300

2847）Detinova，T．S．（1962）：Age−grouping

methodsin

Diptera of

medicalimportance，Monograph

Ser．W．H，0．no．47，pp．217
8）Hashimoto，H．，Murakami，1．，Fuliwara，S．，Takayasu，S．，Takaoka，H．，Uga，S．，Akao，N．，
Kondo，K．and Yoshimura，H．（1990）：A
Dermatol．，17（in9）Sato，K．，Hayashi，S．and
micro且1ariasis（Kasen

human

case

of

zoonotic

onchocerciasis

Tanaka，H．（1954）：Studies

on thecausative

disease），0館6海oo6名oα

of

skin

of equines

召7毎oα傭Railliet et Henry，1910，JPn．J．Parasitol．，2，199−206（inGubler，R．（1965）：ParaatricularesOηohooθ膨），95，1102−110411）Suzuki，Y．，Sugimura，M．，Yagi，K．，Ohbayashi，M．and
Japanese serows，C砂痂o■痂s

Nematodengranulom（einheimische

Shoho，C．（1982）：Onchocerciasis

ino万s餌s，Jpn．」．Vet．Sci、，44，823−82512）Wharton，R．H．（1959）：A simple method
HlthJapan，J，

parasitesof cattle（Wahi Disease），0％oho 6綴gπ枷名osαNeumann，1910，andJapanese）
10）Siegenthaler，R．and

wild

in

press）

ofmounting

andpreserving且1arial

larvae，Bu1L

Wld．

Org．，20，729−730

大分におけるキアシツメトゲブユのOn6hoo6z伽幼虫自然感染
一特に動物寄生性Onohoo6z昭による人への伝播に関して一高岡宏行1・馬場

稔1・Odile Bain2

大分市の西部郊外において見いだされた，動物寄生性0解hoo召κ8線虫による人体例の伝播に関して，患者の居住区において人囮法によって吸血ブユの採集を行い，フィラリア幼虫の感染の有無を調べた。その結果，8種のブユが採集され，そのうちキアシツメトゲブユが優先種である
ことが分かった。また本種ブユでは，調べた449個体のうち11個体の雌に，フィラリア幼虫の感染が見つかった。第3期幼虫の形態的観察により，On6hooθzo4属に属する2種が含まれていることが分かった。これらの結果から，動物寄生性0甥hooθzoαの人体への感染について考察を行った。

大分医科大学医動物学教室
Laboratoire Des Zoologie

Vers．，Museum National d Histoire Naturelle，FranceJapan. J. Trop. Med. Hyg., Vol. 17, No. 4, 1989, pp. 285‑290

285

INTENSITY, INCIDENCE AND CONVERSION/
REVERSION RATIO OF SCHISTOSOMAHAEMATOBIUM INFECTIONMASAAKI SHIMADA', MIZUKI HIRATA,, JOHN H, OUMA*,
KATSUYUKI SATO,, SHINICHI NODA' AND YOSHIKI AoKr
Received May 6 1989/Accepted August 25 1989

Abstract: The changes in the intensity of Schistosoma haematobium infection, incidence
and conversion/reversion ratio in a community were observed over a period of 1.5 years. The
intensity of infection was judged by two indices: egg counts per 10 ml of urine and egg counts
per hour. During the study period, 4 urine examinations were conducted at 6‑month intervals.
Subjects were included in the analysis of the changes in the intensity of infection in a commu‑

nity if they took all 4 urine examinations and were positive for eggs. The changes in theintensity of S. haematobium infection differed considerably according to the indices used. Thechanges in the intensity of infection were compared with the changes in the level of transmis‑
sion expressed by incidence and conversion/reversion ratio or with the changes in the urine

volume. The changes in the intensity of infection expressed by egg counts per hour closely
paralleled the changes in both incidence and conversion/reversion ratio in a community,
regardless of urine volume. By contrast, when the intensity of infection was expressed by egg
counts per 10 ml of urine, it did not correlate with the changes in the level of transmission, andwas negatively related to the changes in urine volume. These results suggest that egg counts
per hour is a reliable index that accurately reflects the changes in the intensity of S.
haematobium infection in a community.

INTRODUCTION
Although various indices have been proposed for the measurement of the intensity of S.
haematobium infection in individual patients (Clarke, 1966; Wilkins, 1977; Stephenson et al..

1984), egg counts per 10 ml of urine at midday has been used most frequently thus far.
However, one of the shortcomings of this index is that it is affected by daily changes in urine

volume, so that the intensity of infection of individual patients varies from day to day.
1 Department of Parasitology, Institute of Tropical Medicine, Nagasaki University, Nagasaki
852, Japan
2 Department of Parasitology. Kurume University School of Medicine, Kurume 830, Japan
3 Division of Vector Borne Diseases, Ministry of Health, Nairobi, Kenya
4 Department of Medical Zoology, Faculty of Medicine, Kagoshima University, Kagoshima 892,
J a panThis study was conducted under the Kenya‑Japan Communicable Diseases Research and Control
Project, with support from the Kenya Medical Research Institute and Japan International Coopera‑tion Agency. The data analysis was carried out at the Nagasaki University Information Process‑ing Center and at the Computer Center, Kyushu University.

286

Therefore, we recommended the use of an index, that is, egg counts per hour, that is notaffected by changes in urine volume. This serves as a stable index for the measurement of
the intensity of infection at an individual level (Shimada et al.. 1986) .

For community‑based longitudinal studies, particularly cohort epidemiological studies orcontrol projects, the change in the intensity of infection is a key variable. At a community
level, however, the intensity of S. haematobium infection has also usually been expressed bymean egg counts per 10 ml of urine from infected people. Urine volume varies not only fromday to day at an individual level but also from season to season, owing to environmental
factors such as temperature and humidity. Therefore, it is highly probable that the mean

intensity of S. haematobium infection in a community differs from season to season eventhough there is not a true change in the intensity of infection.
In the present study, we attempted to examine the possible changes in the mean intensityof S. haematobium infection in a community judged by two indices, that is, egg counts per 10

ml of urine and egg counts per hour, over a period of 1.5 years. The results were thencompared with the changes in the level of transmission expressed by incidence and conver‑sion/reversion ratio, and with the changes of the mean urine volume.

MATERIALS AND METHODS
The present study was conducted at Mwachinga village, Kwale district, Kenya. The
intensity of infection of the patients was expressed by two indices: egg counts per 10 ml ofurine and egg counts per hour. The details were described elsewhere (Shimada et al., 1986) .
Briefiy speaking, urine was collected I hour after the previous urination during midday (11:30‑

13:30). The volume of each specimen was measured. The nuclepore filter method of Peters
et al. (1976) was used to filter and count the number of eggs per 10 ml of urine, and then the

total number of eggs in the urine was calculated. Egg counts per hour was obtained by
dividing total egg counts by the time between two urinations.

The intensity of infection in a community was expressed by the mean egg counts ofsubjects in logarithm. Urine examinations were conducted 4 times during a study period of
1.5 years. The Ist examination was done in May/June 1982, the 2nd in November/December

1982, the 3rd in May/June 1983 and the 4th in November/December 1983. The number ofvillagers taking each urine examination was 699, 710, 869 and 682 respectively. Among these,

219 completed all urine examinations and were positive for eggs. These 219 patients were
subjected to the analysis of the changes in the intensity of infection.

Four urine examinations done during a period of 1.5 years allowed us to calculate theincidence and conversion/reversion ratio in our study area at 6‑month intervals. Incidence
was expressed as percentage of persons who had been negative at the first survey but werepositive for eggs at the second of two surveys. Conversions were the egg positives at a urine
examination who had been negative at the preceding examination, while the reversions werethe egg negatives at a urine examination who had been positive at the preceding examination.

287
log3

A

i i i‑̲i

:,

o

!: 2

RESULTS
Intensity of Infection

When egg counts per hour was used as anindex, the intensity of infection in a community
fluctuated slightly (Fig. IA). The mean log egg

C:

:

Ou 1
el
a,
a,

log3

B
a,

i‑

C
L
5

'

'1¥̲l

O 2

counts per hour increased from 2.732 to 2.804
during the first 6‑month period between Juneand December 1982. During the second 6‑month
period, December 1982‑June 1983, the intensityof infection again increased slightly to 2.839.
Egg counts then decreased to 2.696 during thethird 6‑month period. The ch nges in the inten‑
sity of infection over a period of 1.5 years were
not statistically significant (p=0.3674) .

S

When egg counts per 10 ml of urine wereeused, the pattern of the changes in the intensityu, 1of infection was different from that expressedby egg counts per hour (Fig. IB). Egg counts

C:

5
O
U

C1

increased from 2.394 to 2.585 during the first 6‑

C1,D

month period, then decreased to 2.474 in the
ml
40
a,

E:: 30

e 5'
e ̲

/

e

o>

Q, 20

c
L

second and to 2.289 in the third 6‑month period.

The difference in the intensity of infection

during the study period was statisticallysignificant (p=0.0138). The mean egg counts
per 10 ml of urine were significantly different:, 10

between the second and the fourth urine
examinations by Scheffe's test (p< 0.05) .

June Dec June Dec

1 982 1 983

Figure I Changes in the intensity of S.
haematobium infection in a com‑munity (A, B) and fluctuation ofurine volume (O over a periodof 1.5 years. Intensity of infec‑
tion was expressed by egg counts
per hour (A) and egg counts perlO ml of urine (B) .

represents mean standard
error.

Incidence and Conversion/Reversion Ratio

The results are shown in Table 1.
Although the incidence was not significantly
different from season to season, the incidence
among children under 14 years of age during thefirst and second 6‑month periods was approxi‑
mately 2 times greater than that during thethird period.

Conversion/reversion ratios during the firstand second 6‑month periods exceeded 1, andratios during the third 6‑month period were
smaller than 1, but the difference was not statis‑

tically significant. The changes of the intensity of infection expressed by egg counts per hourclosely paralleled the changes in incidence or conversion/reversion ratio.

Urine Volume
The results are shown in Fig. IC. The mean volume of urine obtained at each examina‑tion showed significant changes; 37.51 ml for May/June 1982, 28.34 for November/December

288

Table l Incidence and conversion/reversion ratio in the study area

periodConversion/Reversion

IncidenceStudy
0‑14 yrs

ratio

15 yrs or more

0‑14 yrs

15 y rs or more

16/73* (21. 9) t

21/81

(25 . 9)16/8t (2 . O) S

21/14 (1.5)

1982‑
1983

13/68

(19 . 1)

23/75

(30 . 7)

13/9 (1.4)

23/23 (1.0)

June 1983‑

7/80(8 . 8)15/70(21 . 4)

7/10

15/19 (0.8)

June 1982‑
Dec.

1982

Dec.

June
Dec.

(O . 7)1983* : No. of egg positives at the second of two surveys who had been negative at the first survey/No.
of egg negatives at the first surveyt : (%)$ : conversion/reversion: conversion=new infection ( ‑ to +) , reversion=spontaneously lost infec‑
tion(+ to ‑)S : ratio

1982, 38.11 for May/June 1983, and 41.23 for November/December for 1983 (p=0.0097) . The
mean volume of urine at the second urine examination was significantly lower than that at
the fourth examination (Scheffe's test, p < 0.05). The fluctuation pattern of the intensity of

infection expressed by egg counts per 10 ml of urine was mirrored by that of the urine
volume.

DISCUSSION
The data presented in our study show that it is important to reconsider the precision of

the indices by which the mean intensity of S. haematobium infection in a community has been

judged in the past. The changes in the mean intensity of infection in a community during acertain period of time differed considerably according to the index used, that is, egg counts
per 10 m I of urine or egg counts per hour. When the changes in the intensity of infection werecompared with the changes in the level of transmission in our study area, the advantages anddisadvantages of the two indices were disclosed.
Incidence and conversion/reversion ratio are considered to indicate the level of transmis‑

sion in, an endemic area of schistosomiasis. When these values are greater during a certain
period of time than those during a previous period, the mean intensity of infection in acommunity is expected to increase during the correnponding period of time, and vice versa.
In our study, when the mean intensity of infection was expressed by egg counts per hour, thechanges in the intensity of infection in a community during a study period of 1.5 years,
although not statistically significant, closely paralleled the changes in incidence and conver‑sion/reversion ratio in that community. By contrast, when it was expressed by egg counts
per 10 ml of urine, the intensity of infection did not correlate with the changes of the levelof transmission.

Variation and stability in S. haematobium egg output have long been cosidered (Wilkins
and Scott, 1978). In discussions on the changes in egg output, urine volume has usually been
indentified as a factor which may affect egg output (Clarke, 1966; Wilkins, 1977; Stephenson
et al.. 1984) . However, there has been no report describing the actual changes in mean urine

289

volume and their effect on the measurement of the mean intensity of infection in a longitudi‑

nal study of a community. Recently, we reported that at an individual level, egg counts per

10 ml of urine are affected by the changes of urine volume (Shimada et al., 1984). The
present study revealed once more that, at a community level, the changes in the intensity of

infection expressed by mean egg count per infection expressed by mean egg count per
infection expressed by mean egg count per 10 ml of urine were negatively correlated to thechanges of mean urine volume (R= ‑0.908, 0.1 >p>0.05, n=4) . By contrast, the mean egg

count per hour was not affected by the changes of mean urine volume.
Although the results of the present and previous studies do not conclusively prove thategg count per hour is the best index for the measurement of the intensity of S. haematobium
infection, it is undoubtedly a reliable index which can be used as a field technique at both theindividual and community levels. Egg count per 10 ml of urine measures the concentrationof eggs in urine, but not the real intensity of infection.

ACKNOWLEDGMENTS
We express appreciation to Prof. M. Mugambi. Director of Kenya Medical ResearchInstitute; Dr. T.K. Arap Siongok, Director, Division of Communicable Disease Control,
Kenya; Dr. J. N. Kaviti, Director, National Public Health Laboratory Services, Kenya; Dr. D.

K. Koech, Director, Division of Vector Borne Diseases, Kenya; and Mr. K. Onoda JICA
Officer, Nairobi, Kenya, for their encouragement in conducting the present study. We are also
grateful to the late Mr. P. Bebora and his staff, and chief of Mwachinga village, for theircooperation during the survey. We wish to express special appreciation to Prof. H. Tanaka,
Tokyo University, for his helpful criticism and suggestions in the preparation of the manu‑
script.

REFERENCES
1 ) Clarke, V. de V. (1966) : The influence of acquired resistance in the epidemiology of bilhar‑ziasis, Cent. Afr. J. Med., 12, 1‑30

2 ) Peters, P.A., Mahmoud, A.A.F., Warren, K.S., Ouma, J.H. and Arap Siongok, T.K. (1976):
Field studies of a rapid, accurate means of quantifying Schistosoma haematobium eggs in urine

samples, Bull. W.H.O., 54, 159‑162
3 ) Shimada, M., Hirata, M., Sato, K., Wambayi, E., Ouma, J.H. and Aoki, Y. (1986): Egg countin urine to determine the intensity of Schl tosoma haematobium infection, Japan. J. Trop. Med.

Hyg., 14, 267‑272 .
4 ) Stephenson, L.S., Latham, M.C., Kinoti, S.N. and Oduori, M.L. (1984): Sensitivity andspecificity of reagent strips in screening of Kenyan children for Schistosoma haematobium
infection, Am. J. Trop. Med. Hyg., 33, 862‑871

5 ) Wilkins, H.A. (1977): Variation in urinary creatinine concentration and Schistosoma
haematobium egg count, Trans. Roy. Soc. Trop. Med. Hyg., 71, 411‑415
6 ) Wilkins, H.A. and Scott, A. (1978): Variation and stability in Schistosoma haematobium egg
counts: a four‑year study of Gambian children, Trans. Roy. Soc. Trop. Med. Hyg., 72, 397‑404

290

ビルハルツ住血吸虫症の感染の強さ，罹患率と陽転・陰転比嶋田

雅暁1・平田 瑞城2・John H．Ouma3・佐藤
野田 伸一4・青木 克己

克之1・

ビルハルツ住血吸虫症の感染の強さ，罹患率，陽転・陰転比の変化を1年半にわたって観察した。感染の強さは2つの指標，尿10ml中の虫卵数と1時間当たりの排泄虫卵数で表した。調査
期間中，6カ月毎に4回尿検査を調査地で行った。すべての調査を受け，かつすべて虫卵陽性であった者を，部落における感染の強さの変化の解析の対象者とした。
感染の強さの変化は，用いる指標によって全く異なった。この変化を罹患率，陽転・陰転比で示した感染の圧力の変化，あるいは尿量の変化と堵較した。感染の強さを時間当たりの排泄虫卵数で表すと，その変化は罹患率，陽転・陰転比と同様に変化した。この変化は，尿量の変化とは無関係である。一方，尿10mJ中の虫卵数で表すと，感染の強さの変化は，感染の圧力とは全く関係せず，尿量とは逆相関した。

これらの結果は，調査地でビルハルツ住血吸虫症の感染の強さを表す指標としては，1時間当たりの排泄虫卵数の方が信頼できることを示している。

長崎大学熱帯医学研究所寄生虫学部門
久留米大学医学部寄生虫学教室
Division of

VectorBome Diseases，Ministry

鹿児島大学医学部医動物学教室

ofHealth，Nairobi，Kenya

Japan. J. Trop. Med. Hyg., Vol. 17 No 4 1989 pp 291 301

291

EPIDEMIOLOGICAL STUDY OF SCHISTOSOMAHAEMATOBIUM INFECTION IN A COASTAL AREA
OF KENYA ‑ THE IMPORTANCE OF WATERCONTACT PATTER'NS IN RELATION TO
S. HAEMA TOBIUM INFECTIONMASAAKI SHIMADA*, MIZUKI HIRATA2, JOHN H. OUMA3KATSUYUKI SATOl, SmNlcm NODA4 AND YOSHIKI AOKI*
Received May 8 1989/Accepted September 5 1989
Abstracts Water contact observation and urine examination were carried out on a
population in an endemic area of Schistosoma haematobium infection, Mwachinga, Kwale
district, Kenya. The change in the mean level of water contact with age and the age‑prevalence or age‑intensity distribution of S. haematobium infection showed similar
trends, but not a direct quantitative relationship. Therefore, a mathematical model wasapplied in the attempt to analyze the relationship between the intensity of S. haematobium

infection and the level of water contact. The worm burden, number of paired worms andproportion of hosts with paired worms were estimated from the level of water contact,
annual incidence, and the proposed life‑span of S. haematobium by using an immigration‑

death model on the basis of simple assumptions. These estimated values were comparedwith the observed prevalence and intensity of infection. The results showed a linear
correlation between observed data and estimated values. We conclude that the character‑istic shape of the age‑egg output curve for S. haematobium in the community is satisfacto‑rily explained solely in terms of 'the change in water contact with age.

INTRODUCTION
The relationship between the age and the prevalence and intensity of S. haematobium
infection is similar in most endemic areas. The characteristic shape of the age‑prevalence
and age‑intensity curve for S. haematobium is a peak between the age of 10 and 14 years anda relatively rapid decline in older people (Warren, 1973; Mott, 1982). Intestinal schis‑tosomiasis also shows a similar age‑related pattern. With regard to influences upon immigra‑
1 Department of Parasitology, Institute of Tropical Medicine, Nagasaki University, Nagasaki
852, Japan
2 Department of Parasitology, Kurume University School of Medicine, Kurume 830, Japan
3 Division of Vector Borne Diseases, Ministry of Health, Nairobi, Kenya
4 Department of Medical Zoology, Faculty Qf Medicine. Kagoshima University, Kagoshima 892, Japan

This study was conducted under the Kenya‑Japan Communicable Diseases Research and Control
Project, with support from the Kenya Medical Research Institute and Japan International Coopera‑tion Agency. The data analysis was carried out at the Nagasaki University Information Process‑ing Center and at the Computer Center, Kyushu University.

292tion and emigration of worms, two major factors have been considered in explaining of thecharacteristic relationship between age and infection (Warren, 1973) . These are 1) the de‑velopment of protective immunity by the age of 10 years (Bradley and McCullough, 1973) and2) the variation in water contact with age (Dalton and Pole, 1978; Kvalsvig and Schutte,1986) .

However, no convincing evidence has been presented on the role of protective immunity
in the decline of the intensity or prevalence of infection after adolescence. Most of theevidence of protective immunity recently reported vyas observed after chemotherapy (Stur‑rock et al.. 1983; Butterworth et al., 1985; Wilkins et al.. 1987) . Meanwhile, epidemiologists
concluded on the basis of human water cOntact studies that the characteristic shape of the age‑egg output curve for S. haematobium is satisfactorily explained solely by the variation inwater contact with age (Dalton and Pole, 1978; Kvalsvig and Schutte, 1986) . Again, however,shortcomings exist in their analysis, one of which is that the death of worms was not taken
into account.
To further discuss the possible role of the level of water contact in the characteristic
shape of the age‑prevalence and/or intensity curve, not only a quantitative analysis of humanwater contact but also an estimation of worm burden is necessary. This is because the egg
output reflects accumulated worm burden resulting from contact with infested water, not thelevel of water contact itself. On the basis of the level of water contact and the proposed life‑

span of S. haematobium (Wilkins et al.. 1984), we attempted to estimate the worm burden in

the community by using an immigration‑death model. Since schistosome needs to mate to lay
eggs, the probability of pairing was also calculated. The comparison between the observed
data and estimated worm pair burden allows us to determine whether or not the patterns of
S. haematobium infection in a community can be adequately explained solely in terms of thepatterns of water contact.

MATERIALS AND METHODSUrine examination
The present study was conducted at Mwachinga village, Kwale district, Kenya. Informa‑tion concerning the geography, population, and prevalence and intensity of S. haematobium
infection in the study area was compiled during an initial survey done in June 1982 and has
been published elsewhere (Shimada et al.. 1987) . The method of urine examination was also
published previously (Shimada et al.. 1986). Briefly speaking, urine was collected I hour
after the previous urination during midday. The intensity of infection was expressed as thenumber of eggs excreted per hour assessed by the filtration method of Peters et al. (1976) .

The geometric mean was obtained by using the n+ I transformation for a series of egg output
including zeros.
Water colctact study

Water contact observation was carried out on the population over a period of one year
(June 1982 to May 1983) by the direct observation method of Dalton and Pole (1978) . The
study sites were 16 major points which had been identified as busy sites by a questionnaire.
The observers were two villagers who have lived there for many years. In order to reduce
the effect of site and seasonal variation in our study area, a 4 x 4 Latin square with three
replicates was used. The study sites were divided at random into two groups of 8 sites. Each293

Table 1 Water contact observation schedule

Week 3

Week 1

Tue
June

Fri

Sat

S un
4

2

3

2

3

4

August34l2

Se ptember

4

1

2

3

July

Sun

Tue

Fri

5

6

7

8

6

7

8

5

Sat

6

8
8

5

6

7

Numbers indicate the study sites.

Table 2 Percentage of parts of body to total body surface
Children (0‑14 yrs)

Adults (15 yrs or more)

One hand2

2

Two hands

4

4

2

2

4. Two feet

44

5. One arm

10

9

20

18

7. One leg

1518

8. Two legs
9. Hands and feet

30

36

8

8

34

40

24

22

12.

Hands and legs
Arms and feetArms and legs

50

54

13.

Whole body

100

lOO

2.

3. One foot

6.

10.
ll.

Two arms

Cited from Behrman and Vaughan (ed.) . Nelson Textbook of Pediatrics, 1986.

group of 8 sites was observed by one observer. Water contact was observed at each site for4 days in each of the 3 "seasons" of 4 months' duration. Table I shows the date and sites ofwater contact observation made by one observer during one season of 4 months. The
observation was carried out from 06 h OO to 18 h 30. The collected information included
names of inhabitants, time of day, duration of contact (in minutes) , and submerged parts ofthe body. The level of water contact of an individual during a single submersion was
expressed by the duration of contact (minutes) multiplied by the percentage of submerged
parts to total body surface, which was calculated from the burn chart shown in Table 2. Thenthe level of water contact of a person for one year was calculated as the sum of each contact.

The level of water contact of a certain age group was expressed by the geometric mean. A
population census was conducted in June 1983.

RESULTS
Water contact observation
Out of 1,195 villagers, 617 came in contact with water during the one year study period.

The overall geometric mean of the level of water contact among observed persons was 1,510.

294

Table 3 shows the relationship between the level of water contact and sex or age. The
geometric mean of the levels of water contact of male and female was 1,580 and 1,450,
respectively. The difference between sexes was not significant ･(t=0.48, p = 0.63) .
' To express the level of water contact of each age group, the mean level of water contact

for the observed person was multiplied by a ratio 'of the number of observed people to thetotal number of people of the group registere,d. The reason for this modification of the levelof water contact is that the proportion of observed people differed much by sex and age, andnon‑observed persons probably came scarcely or less frequently in contact with water.
The modified level of water contact showed that, for males, the level of water contact
reached a peak among those aged l0‑14 years and then declined rapidly to lower levels, withthe exception of a relatively high level of water contact at the age of 40‑49 years. For
females, the level of water contact reached a peak at the age of 5‑9 years, decreased rapidly
to a low level at the age of 15‑19 years, and remained low in older people.

Estimation of worm burden, number of paired female worms and proportion of hosts withpaired worms
The estimation of worm burden, number of paired female worms and proportion of hosts
with paired worms was based on simple assumptions for immigration and emigration of
Table 3 Observed level of water contact and modified level of water contact

Level of water No. of
Age
Males

0‑4
5‑9

contact of persons

Po pulation

person observed observed‑geometric mean‑

Modified levelofwater contact(A) (B)(O(A) x (B) /(O1 , 230

18

82

270

3 , 72056

l02

2 , 04010‑14

3 , 98064

81

3 , 15015‑19

1 , 700

41

56

1 , 240

20‑29
30‑39
40‑49
50‑59
60‑

1 , 070

44

75

630

500

24

52

230

1 , 41027

32

1 , 190

230

17

32

120

370

16

38

160

total

1 , 580

307

550

880

Females

0‑4
5‑9
10‑14

1 , 550

28

121

360

1 , 900

52

llO

2 320

l , 91052

78

1 , 270

15‑19

980

29

55

520

20‑29
30‑39
40‑49
50‑59
60‑

1 , 350

55

94

790

490

total

780

38

60

710

20

43

330

1 , 450

21

46

660

240

15

38

90

1 , 450

310

645

690

295

schistosomes. To estimate the worm burden, we used a immigration‑death model (Anderson,1976) . The number of worms in a host (N) at time t can be expressed as
N ={(A X I)/D} x {1‑ exp (‑D)} + {Nt̲1 x exp ( ‑D)}
where Nt̲1 is the number of ivorms at t ‑ 1. A is a constant, and I is the relative level of watercontact. Thus, A X I is immigration rate and D is death rate of a worm per year. The
immigration rate (A X I) can be calculated from the following equation proposed by Hairston(1965), m= ‑ Iog (1‑ J ), where m is the mean number of female worms acquired by aperson per year and B is the annual incidence. The annual incidence in our study area was
about 0.28 (Shimada et al.. 1989). Solving the equation, therefore, 0.75 female worms wereacquired per person per year and there are 2m or 1.5 worms acquired per person per year. We
assumed that the number of cercariae invading a host refiects the level of water contact.
Therefore, a person with a level as high as the mean annual level of water contact of thevillagers in our study area is expected to acquire 1.5 worms per year. To obtain theimmigration rate of each age group, we gave the constant A the value 1.5 and I the ratio of
the level of water contact of each age group to the mean level of water contact of all thevillagers. The death rate (D) was assumed to be 0.3 per worm per year. This was calculated
from the estimated life‑span of S. haematobium (3.4 years) proposed by Wilkins et al. (1984) .

In addition to the estimate of worm burden in a host, ah attempt was made to estimate
the number Qf paired female worms which reflects the intensity of infection or egg output, andalso the proportion of hosts with paired worms which represents the prevalence of infection.

The probability of male and female worm mating was given as (0.88 x N)/(N+ 1.73) as
proposed by Goddard (1978) . Thus, the number of paired female worms (F) is expressedmathematically as

0.88xN x 1

F=NX N+ I .73

The proportion of hosts with paired worms (P) is expressed as
P= {1 ‑ exp ( ‑ N/2)}2

This is based upon the random distribution of worms among people (Macdonald, 1965). A
calculation program for these formulae in the NEC computer is written by BASIC Ianguage.
Table 4 Estimated number of paired worms and proportion of hosts with paired worms

FemalesMales

Age No,ofpaired worms(mean)

Proportion ofhosts withpaired worms(%)

No. ofpaired worms(mean)

Proportion ofhosts withpaired worms
(%)

0‑4
5‑9

O . 098

O . 2018

2 . 4390

3 . 79

97

10‑14
15‑19

5 . 47100

4 . 1999

4 . 1699

2 . 2891

1 . 6582

l . 85

88

O . 4839

1 . 28

76
55

20‑29
30‑39
40‑49
50‑59
60‑

l . 76

84

O . 73O . 5942

1 . 2876

O . 14

13

O . 4133

296

The estirnated number of paired worms and the proportion of hosts with. paired worm bysex and age is shown in Table 4. The number of paired worms reached a peak at the age of
l0‑14 years and declined to a lower number in older people. In males, however, another peak
was observed at the age of 40‑49. The proportion of hosts with paired worms showed asimilar pattern.
Parasitological study

Of the inhabitants registered, 694 provided urine specimens in December 1982. Table 5shows the prevalence of infection. The difference by sex was not statistically significant as

a whole (Mantel‑Haenszel x2=3.374, p=0.066) . The prevalence of infection reached a peakat the age of 10‑14 years in both sexes. However, the shape of the age‑prevalence distribu‑
tion after the peak differed by sex. Males showed a relatively sharp decline in prevalencefrom the peak to a lower level in older people with exceptionally lower prevalence at the ageof 30‑39 years. In contrast, the prevalence in females decreased from the peak gradually toTable 5 Prevalence of infection by age and sex

Males

Age No.
examined

FemalesNo, ofegg

positive

Prevalence No.
( % ) examined

No. ofe gg

Prevalence(%)

positive

0‑4
5‑9

36

5

14

36

ll

31

74

56

76

71

59

83

l0‑14

54

52

96

47

44

94

15‑ 19

30

27

90

37

33

89

20‑29

19

15

79

64

54

84

30‑39

17

9

53*

42

35

83*

40‑49
50‑59
60‑

22

15

68

41

26

63

22

14

64

32

21

66

24

15

63

36

17

65

Total

298

208

70

396

300

76

* p= O.015, Chi‑square value=5.896

Table 6 Intensity of infection by age and sex

Males

Age No.
examined

Egg counts/hour(geometric mean)

No.

Females
Egg counts/hour

examined(geometric mean)

0‑4
5‑9

36

1.0

36

2.6

74

130 . 871

228 . 1l0‑14

54

1 , 777 . 3

15‑19

30

435 . 54737425 . 6

20‑29
30‑39

19

56 . 564

122 . O

17

4.1*

42

52 . 7*999 . O

40‑49
50‑59
60‑

22

36 . 241

16 . 422

14 . 832

23 . 524

19 . O

36

4.4

Total

298

74 . 8396

76 . 6* p=0.0007, t test

297
(In)

u,2
O
;

e

ID

e Males,U

O Females

o

e o

C:

O

o o

J:l

E
:
C

o
e

iO

1,
,D

E

o

:

V,

LU

o

ee o

LCD1

o

e

1 2 3 ( Iog )Egg counts / hour

Figure l The correlation between estimated number of paired
worms and observed egg counts. The estimated number ofpaired worms and the observed mean egg counts wereplotted by age and sex. The number of paired worms wastransformed to logaritnm. Egg output was expressed by
egg counts per hour and transformed to logarithm. Pear‑son correlation coefficient; for males 0.945 (n= 9, p<0.001) ,for females 0.961 (n=9, p<0.001) .
(%]

o o(‑

1 OO

IJ

o
!::
' u]

os
:o
o

d.

e oo

e Malesu'

O Females

oooo
o

: 50

C
O
o .L‑
L,U

oeo

1'=:

'D1'

d,:,!;
.‑

o

E

1

o

e

50 1 OO( '‑･.)

Observed prevalence of infection

Figure 2 The correlation between estimated proportion of hosts
with paired worms and observed prevalence of infection.
The estimated proportion of hosts with paired worms andthe observed prevalence of infection were plotted by age
and sex. Spearman rank‑order correlation coefficient; formales 0.93 (n=9, p<0.001), for females 0.90 (n=9, p<
0.005) .

298

a lower level in older people. The prevalence of males aged 30‑39 years was signiflcantly
lower than that of females (x2=5.896, p=0.015) .
The overall intensity of infection expressed by a geometric mean egg count was 76 eggs
per hour, that of males being 75 eggs per hour and that of females 77 eggs per hour. No
statistically significant difference was observed. The age‑intensity distribution was similar

to that of the age‑prevalence curve in both males and females (Table 6). The egg output
increased with age and reached a peak at the age of 10‑14 years in both sexes. In males, egg
counts declined rapidly from the peak to a lower level in older pe.ople with lowest counts at

the age of 30‑39 years. In females, by contrast, the intensity decreased gradually from thepeak to a lower level in older people. The mean egg count of males aged 30‑39 years was
significantly lower than that of females (t=3.1923, p=0.0023)
Correlations between observed and estimated data

The estirnated number of paired worms was compared with the observed egg output.
The result is shown in Fig. l. The estimated values and observed data were plotted by sex

and age. A Iinear correlation between observed and estimated data was observed. The
coefficient was statistically significant. The estimated proportion of hosts with paired worm

was compared with the observed prevalence in our study area (Fig. 2). Again a linear
correlation ¥A'as shown between the observed and estimated data. The coefficient was
statistically significant.

When the level of water contact was compared directly with observed egg output, thecorrelation was not as prominent as the above.

DISCUSSION
The main aim of the present study is to explain quantitatively the characteristic shape of

the age‑egg output distribution for S. haematobium by the change in the level of watercontact with age. Dalton and Pole (1978) and Kvalsvig and Schutte (1986) observed theduration and frequency of water contact arnong the villagers and considered the possibilityof a relationship between water contact and infection of S. haematobium. They concluded
that there was a close relationship between water contact activity and the intensity or
prevalence of infection. In their studies, however, important factors infiuencing the intensity
or prevalence of infection‑ that is, the accumulation and death of worms‑ were not taken into

account. Changes in prevalence and intensity of infection result from an imbalance in the
opposing processes of immigration and emigration of worms.
To overcome the shortcomings in the previous analysis of water contact observations
and to examine carefully the relationship between the level of water contact and the intensityof S. haematobium infection, we estimated the worm burden, number of paired worms andproportion of hosts with paired worms from the water contact observation and annual
incidence in our study area and the proposed life span of S. haematobium by using a simple

immigration‑death model and other equations. The estimated values were compared withthe observed data. There was a quantitatively close relationship between both the estimatednumber of paired worms and the observed intensity of infection, and between the estimatedproportion of hosts with paired worms and the observed prevalence. The age‑prevalence andage‑intensity distribution of males in our study area showed an unexpectedly low prevalence
and intensity in the 30‑39 year age group. The present study revealed that this unexpectedly

299

low prevalence and intensity of males 30‑39 years of age could also be explained by theestimated proportion of hosts with paired worms and the number of paired worms. All theseresults imply that in this area the decline in intensity and prevalence of infection is satisfacto‑rily explained solely by the decline in water contact after adolescence.

When we directly compared the level of water contact with the observed prevalence orwith the intensity of infection, the variation in level of water contact by age did not correlate

well with the change in the prevalence or intensity of S. haematobium infection. For example,

the highest level of water contact in females was observed at the age of 5‑9 years although
the intensity of infection showed a peak at the age of 10‑14. This is not surprising, though,because the level of water contact only reflects the degree of immigration of worms.

Our analysis is based on a one year‑long observation of water contact and a cross‑sectional examination of villagers for S. haematobium infection. We presumed that the watercontact behavior of villagers had not changed for decades, and therefore that the infection of
inhabitants was the result of behavior patterns and stayed more or less the same. Indeed, nomajor changes such as the installation of piped water had occurred in this area for several

decades. The direct method of observation for water contact might influence the watercontact behavior of villagers, especially of women. Thus, the level of water contact ofwomen might be underestimated.
The assumptions made for the estimation of worm burden from the data of water contact
may also be too simple. We assumed that the degree of water contact defines the degree of
exposure, that the number of worms infected is proportional to the degree of exposure, thatthe death rate and fecundity of worms are constant throughout the life of the people, and soon. Although some studies have been carried out on these issues, especially with regard o

S. mansoni (Upatham and Sturrock, 1973; Cheever, 1968; Medley and Anderson, 1985;
Crombie and Anderson, 1985; Wertheimer et al.. 1987) , still little is known. It is undoubtedly

necessary to further clarify whether or not these assumptions are applicable to our analysis.

In the present study, no account was taken of other factors which may influence theacquisition of worms. These include the distribution of snails and their infection rates, theconcentration of cercarial density in water and its diurnal fluctuation, and the time of day

when villagers come in contact with water, etc. In our study area, children tended to comein contact with water at midday when the concentration of cercariae in the water was high,while adults were likely to come in contact with water in the morning and evening when theconcentration of cercariae was low (unpublished data) . When these factors are incorporated
in the model, the difference in the worm burden gained by children and adults may be muchwider.

ACKNOWLEDGMENTS
The authors are indebted to the late Dr. P.R. Dalton for his encouragement in carryingout this work. We also express appreciation to Prof. M. Mugambi, Director, Kenya Medical
Research Institute; Dr. T.K. Arap Siongok, Director, Division of Communicable Disease
Control, Kenya; Dr. J.N. Kaviti, Director, National Public Health Laboratory Services,
Kenya; Dr. D.K. Koech, Director, Division of Vector Borne Diseases, Kenya; and Mr. K.
Onoda, JICA Ofiicer, Nairobi, Kenya, for their support and encouragement. We are also
grateful to the late Mr. P. Bebora and his staff, chief of Mwachinga village, for their

300

cooperation during the survey. We wish to express special appreciation to Prof. Y. Wada,
Nagasaki University and Prof. H. Tanaka, Tokyo University, for their helpful criticism andsuggestions in the preparation of the manuscript.

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10) Macdonald, G. (1965): The dynamics of helminth infections, with special reference to schis‑tosome, Trans. Roy. Soc. Trop. Med. Hyg., 59, 489‑506
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samples, Bull. W.H.O., 54, 159‑162
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Trop. Med. Hyg., 17, 285‑290 ‑17) Sturrock, R. F., Kimani, R., Cottrell, B.J., Butterworth, A.E.. Seitz, H.M., Arap Siongok, T.K.

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Houba，V．（1983）：Observations

schoolchildren

after

treatment for

on

Sohガs

possible

immunitytoreinfection

among

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366−37118）Upatham，E．S，and Sturrock，R．F．（1973）：Studies

lengthofexposureon theinfection of19）Warren，K，S．（1973）：Regulation
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mice byprevalencehumaneffectsofcercarialconcentration

andSoh魏osoアnα吻齪so砿ParasitoL，67，219−228

andintensityofschistosomiasis

in

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thefecundityofschistosomiasis

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29−35

ケニア，沿岸州におけるビルハルツ住血吸虫症の疫学的研究
一ビルハルツ住血吸虫感染における水との接触行動の重要性
嶋田 雅暁1・平田 瑞城2・John H．Ouma3・佐藤
野田 伸一4・青木 克己1

克之1・

ヒトと水との接触行動の観察と尿検査（虫卵検査）をケニア，クワレ地区，ムワチンガ村というビルハルツ住血吸虫症流行地で行った。年齢による住民と水との接触量の変化は，住血吸虫症
患者の年齢による感染の強さの変化と，全く同じではないがよく似る。そこで感染の強さと水どの接触量を，数学モデルを介して，定量的に比較してみた。寄生虫体数，雌雄抱合虫体数，雌雄，抱合虫体を有す住民の割合を，水との接触行動観察データ，年罹患率，ビルハルツ住血吸虫成虫の推定生存期間より，虫体の侵入死亡に関する数字モデル，immigration−death mode1，その他の式を用いて算出した。これらの推定値を，調査地住民の感染率と感染の強さと比較した。その結果，推定値は観察値と直線関係を示した。これらの結果より，我々はビルハルツ住血吸虫症流
行地で普遍的にみられる，年齢一虫卵排泄曲線の特長的型は，単に年齢による住民と水との接触量の変化によって，うまく説明出来ると結論する。

長崎大学熱帯医学研究所寄生虫学部門
久留米大学医学部寄生虫学教室
DivisionofVectorBome

Diseases，Ministry

鹿児島大学医学部医動物学教室

ofHealth，Nairobi，Kenya

Japan. J. Trop. Med. Hyg., Vol. 17, No. 4, 1989, pp. 303‑310

303

AGE SPECIFIC ASEXUAL PARASITE AND
GAMETOCYTE DENSITY IN HIGHLY ENDEMIC
MALARIA IN NORTH SUMATRA, INDONESIAHIDEKI ITOKAWAl' 2, RYOJI TAKA11, AKIRA ISHI11' 3

AND W. PANJAITANi' 4
Received June 8 1989/Accepted September 5 1989

Abstract: A mass blood examination was carried out in a coastal village of Perupuk,
Asahan Regency, North Sumatra, Indonesia in August 1982. The parasite rate in all ages

revealed remarkably high (44%). This was caused by an explosive outbreak of Plas‑
modium falciparum malaria. The parasite rate in young children (0‑9 years) was very
high around 60% wheteas low less than 20% in adult (over 34 years) . This indicates apossibility of outbreak of malaria mostly in children posing public health subject in avillage of tropical rural area. Gametocyte carriers of P. falciparum were detected only inthe age group less than 25 years. The high parasite density of P. falciparum with both

asexual form (more than 1,000 ring forms/mm3) and gametocyte (more than 100
gametocytes/mm3) was detected only in the age group less than 12 years. Falciparum
malaria of high intensity repeatedly occurs mostly in younger age group in Anopheles
sundaicus breeding coastal area in north Sumatra. A possible control method aiming at
gametocyte carrier of young age group is discussed.

INTRODUCTION
Although malaria is still a very important disease especially in rural area of most
tropical coutries, detailed epidemiological information is not always provided for malaria
control activities. This was also true in Sumatra Island. It has been told that in North
Sumatra malaria is endemic less than 2% in average. Administration officials considered thisis lower than the level which needs any anti‑malarial action. Previous reports indicated also

low malaria endemicity in Sumatra (Carney et al., 1974, 1975; Stafford et al.. 1976).

Kanbara and Panjaitan (1983) reported the patchy distribution of malarious foci in the

coastal area of Asahan Regency, North Sumatra. Entomological studies revealed Anopheles
sundaicus is a responsible vector in brackish water of the coastal area (Ikemoto, 1982). A.
sundaicus is well known as a common vector of malaria in the coastal areas in southeast Asia.

In Jawa Island, hyperendemic malaria was caused by A. sundaicus once ago in Tanjung Priok
district of Jakarta and Tingal town. Recently in 1984, A. sundaicus transmitted malaria

1 North Sumatra Health Promotion Project, Medan, North Sumatra
2 Department of Medical Zoology, Tokyo Medical and Dental University School of Medicine,Tokyo ll3, Japan
3 Department of Parasitology, Okayama University Medical School, Okayama 700, Japan
4 Provincial Health Office, North Sumatra

304

attacked Kampung Laut in Cilacap, Jawa and caused not less than hundred death as a resultof increase of vectors due to construction of artificial fishing ponds (Kurihara, 1970) .

The endernicity of A. sundaicus transmitted malaria in Sumatra has not been wellinvestigated so far. We report here a result of intensive malaria surveys including mass blood
examinations in the coastal malarious foci. This was conducted as an activities of the projectfor "The Promotion of Health in North Surnat'ra" which is an international cooperation
between the Republic of Indonesia and the Government of Japan. The results of age‑specificparasite rates and parasite density of both asexual and sexual form of P. falciparum showed

patchy highly endemic areas in coastal villages presenting some public health problems anda possibility of strategic malaria control approach to younger age group.

MATERIALS AND METHODS
A community village, Perupuk, kecamatan Lima Puluh, kabupaten Asahan of North
Sumatra was selected as the pilot study village, for it was found as a highly endemic focus
in the project area (Kanbara and Panjaitan, 1983). It is located on the sea side facing to

Malacca Strait and aproximately 150 km southeast of Medan, the capital of North Surnatra.
The mass blood examination was carried out in high prevalence season (August, 1982)
when malaria endemicity was reported the highest in a year in the village frorn the results offormer malariometric surveys (Kanbara and Panjaitan, 1983). In the village, most houses
were built gathered along the road near the harbour as drawn in geographical reconaissance(Fig. i) . Most inhabitants engage in flshery. The population size was about 6,000 in Perupukand that of Lorong (block) 2, where the mass blood examination was conducted, was 372.
House to house visits to collect blood samples were conducted during night time when adults
returned home from their work. Blood specimens were obtained from finger tips, then thickand thin smears were made on microscope slides. Thick smears were air dried for 12 hours

and hemolysed in distilled water. The smears were stained for 30 min with 5% Giemsa
solution diluted with phosphate buffer (pH 7.2). The thick films were examined counting
more than 1,500 white blood cells to secure positivity. Parasite density was calculated inrelation to the number of white blood cells assuming 8,500 white blood cells in I mm3 of blood(Bruce‑Chwatt, 1980) .

RESULTS
The age specific spleen rates were as follows: 21.2% (7/33) for 1‑

years, 20.5% (8/39)

for 5‑9 years, 29.4% (10134) for 10‑14 years, and 14.1% (18/128) for over 14 years old age
groups.
The results of mass blood examination are shown in Table l. In total, 239 persons were
examined in the survey with coverage rate of 64.2% (239/372) . The parasite rate was 44.4%
in all and 60.2% in the age group of 0‑14 years. Two species of malaria parasite, Plasmodium
falciparum (P. f.) and P. vivax (P. v.) were found to exist and the ratio of P. f. and P. v. wasroughly 3 : 1. Gametocyte rate of P. falciparum was 12.6% in all age groups and 20.0% in theage of 0‑24 years. Age specific prevalence curve is shown in Fig. 2. The curve of P.
falciparum infection draws high plateaux in the age group of 0‑9 years and begin to slope
down after that. It reaches to the bottom in 35‑44 years. The curve of gametocyte rate moves

305

N

STRAIT OF MALACCA‑‑ ¥LOron9 2i, '‑ ‑' ‑ """"ee‑ ‑ t‑‑‑,'‑ ̲̲ ̲̲ /̲Pl̲ ¥: ' b J " """" ,1,,,..

,t 'l't"Th""o//o':" "" """I'le"e "‑'' E'e "'It"'t:'i'‑ Irlllllt t .' u I'Ill tIII

.

e
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tt

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It

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II] Bench I km

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Figure I A malaria survey sketch map of village Perupuk, north Sumatra, Indonesia.
Spleen rate (%) is shown in every school.

Table I Age specific distribution of malaria in Perupuk village, North Sumatra, Indonesia in

August 1982

Number Number

Age

Number(years)population

examined positive cases(registered)P. falciparuml7

4 (57.1%)

2 (50.0%)

1‑4

44

33 (75.0%)

17 (51.5%)#g(8)P. vivax

Mix

O

O

2

2 (6.1%)2 (6.1%)21 (63.6%)

Total(50.0%)

5‑956

39 (69.6%)

19 (48.7%)#g (9)

5 (12.8%)

O

24 (61 . 5%)

l0‑14

58

35 (60.3%)

14 (40.0%)#g(9)21 (60.0%)

15‑24

73

38 (52.1%)

lO (26,3%)#g(4)

7 (20.0%)
5 (13.2%)

OO

15 (39 . 5%)

25‑34

45

29 (64.3%)

9 (31.3%)

3 (10.3%)

O

12 (41.4%)

35‑44

28

18 (64.3%)

1 (5.6%)1 (5.6%)

O

2

45‑54

33

24 (72.7%)

O

O

55

28

19 (67.9%)

4 (16,7%)
3 (15.7%)

4 ( 16 , 7%)

2 (10.5%)

O

5

372

239 (64 2%) 79 (33.1%)

25 (10.5%)

2 (o.,8%)All ages(ll.1%)(26.3%)

l06 (44.4%)#g number of gametocyternia cases in falciparum infections

in parallel with the curve for P. falciparum infections and reaches to "zero" Ievel in over 24

years. The curve of age specific prevalence rates and parasite density of P. vivax did notshow so typical pattern like those of P. falciparum.
The parasite density of asexual form in P. falciparum infections is shown in Fig. 3. Highparasite density more than 1,000 ring formslmm3 blood was found only in young children
under IZ years. All cases in the age of 25‑45 years were less than 100 ring forms/mm3.
Gametocyte density in P, falciparum infections is shown in Fig. 4. Six out of 8 cases in the

306

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･:1 1‑4 I0‑14 15‑24 25‑34 35‑44 45‑54

5 9 Age‑groups (years)

55

Figure 2 Age specific distribution of malaria parasite positive cases in village Per‑
upuk. e o : total malaria parasite rate, A‑'‑'‑ : P. falciparum asexual
form, []‑‑‑‑‑‑[] : P. falciparum sexual form (gametocyte) , ･r‑‑‑‑' : P. vivax

asexual form. O mixed infection.

E

l 05

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Age (years)

Figure 3 Age specific distribution of asexual form parasite density in P. falciparum
infections in village Perupuk.

307

l 05
(L,

;

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5 10 15 20 25 30
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Figure 4 Age specific distribution of sexual form gametocyte density in P. falciparum
infections in village Perupuk.

l 05

)<

o
.2

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th

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r"

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5 10 15 20 25 30 35 40 45 50 55 60 65
Age (years)

Figure 5 Age specific distribution of parasite density in P. vivax infections in village

Perupuk.

age of less than 5 years old were found with more than 100 gametocyteslmm3 and two of themshowed more than 1,000 gametocytes/mm3. On the other hand, most cases over 14 years ofage showed low density gametocytemia less than 100/mm3, the parasite density of P, vivax
infections is shown in Fig. 5. Only one case showed parasitemia higher than 1,000/mm3.

308

DISCUSSION
From the figures of spleen rate in this study, it was considered that the surveyed area wasmesoendemic in malaria endemicity. The par,asite rate in young children was remarkably
high being around 60%. This high parasite rate was a result of epidemic outbreak of P.
falciparum infections. It has been reported in north Sumatra that malaria infection rate is lessthan 2% according to the existing reporting system through local health centers. This isbecause of uneven distribution of malaria cases in whole north Sumatra. However, as
Kanbara and Panjaitan (1983) reported, malaria endemic areas were found in coastal
villages. The result of this study indicated that there could be a malaria outbreak with publichealth problem even when available statistics show low rate of malaria prevalence. Thisposes a public health subject not to be overlooked because especially high infection rate wasobserved in younger age groups without enough immunity to malaria. The cause of this
explosive outbreak of P. falciparum was not clear although increased man‑biting rate ofAnopheles sundaicus was reported before this survey (C. Imai, personal communication) .
The age specific distribution of asexual parasite and sexual gametocyte of P. falciparum
made us consider of the importance of younger age group in malaria transmission. Parasite
density higher than 1,000 ring forms/mm" blood as well as gametocyte density higher than
100lmm3 were found only in the age of less than 12 years. The number of gametocyte carriers

decreased by age and the density of gametocyte decreased also by age. The background of
the phenomena may be explained by the progress of irnmunity (Boyd, 1949) , however thisremained to be elucidated in further studies. From the practical point of view, this is very

important when one considers to interrupt malaria transmission by any method of interven‑tion. Children constitute the most important reservoir of malaria infection for the transmis‑

sion of the agent. Transmission blocking immunity and vaccine have been studied in somelaboratories (Targett and Sinden, 1985) . Another method of transmission blocking is the useof gametocyticidal drugs such as 4‑aminoquinoline, primaquine (Matsuoka et al.. 1987) .

Infectivity of human population with malaria to mosquitoes has been studied by sorne
workers (Jeffery and Eyles, 1955; Graves et al.. 1988) . According to Rieckmann et al. (1968) ,gametocytemia higher than 100lmm3 is usually necessary to make mosquitoes infective.
Their feeding experiment on volunteers suggested this critical point. Carter and Gwadz(1980) also wrote that density of gametocyte at 1,000lmm3 mosquito infection was the highest

and majority of cases are infectious to mosquitoes at density of 100 gametocytes/mm*.
In this study, gametocytemia higher than 100lmm3 was found mostly in children. The
present results showed that the target population of the transmission blocking is younger age

group especially that of under 12 years. For that purposes primary school children andpreschool children are very important. School health activities and MCH (mother and childhealth) activities would constitute the most rational and useful strategic element for theintervention of malaria.

ACKNOWLEDGEMENTS
The authors wish to thank Drs. J. Simajuntak, T. Suzuki, R. Kano, H. Tanaka, H.
Ohtomo for their help in carrying out and reporting the study. This work was done as a partof an international cooperation project entitled Health Promotion in North Sumatra which309

was suported frorn Republic of Indonesia and Japan International Cooperation Agency.

REFERENCES
1 ) Boyd, M.F. (1949): Malariology, p. 612‑614, W. B. Saunders, Philadelphia, U.S.A.
2 ) Bruce‑Chwatt, L.J. (1980): Essential Malariology p. 144‑146, W. Heinemann Med. Books, U.
K.
3 ) Carney, W.P., Admosoedjono. S., Sajidiman, H. and Joesoef, A. (1974) : Intestinal parasites andmalaria in Sukamentati, Pasaman Regency, West Sumatra, Hlth. Studies Indonesia, 2, 614 ) Camey, W.P., Admosoedjono, S., Sajidiman, H. and Joesoef, A. (1975): Intestinal parasites andmalaria in Musi Banyu Asin and Ogan Komering Ulu Regency, South Sumatra, Hlth. Studies
Indonesia, 3, 55 ) Carter, R. and Gwadz, R.W. (1980): Infectiousness and gamete immunization in malaria. in"Malana" vol 3 pp 263 297 ed Krerer J P Acad. Press, New York, U.S.A.
6 ) Graves, P.M., Burkot, T.R., Carter, R., Cattan, J.A., Lagog, M., Parker, J., Brabin, B.J., Gibson,

F.D., Bradley, D.J. and Alpers, M.P. (1988): Measurement of malaria infectivity of humanpopulations to mosquitoes in the Madang area, Papua New Guinea, Parasitology, 96, 251‑263
7 ) Ikemoto, T. (1982): Studies on the bionomics of Anopheles sundaicus (Rodenwalt, 1925), theprincipal malaria vector in a coastal area of North Sumatra, Indonesia, Med. J. Teikyo Un. iv.,
5(1), 1‑158 ) Jeffery, G.M. and Eyles, D.E. (1955): Infectivity to mosquitoes of Plasmodium falciparum as
related to gametocyte density and duration of infection, Am. J. Trop. Med. Hyg., 4, 781‑789

9 ) Kanbara, H. and Panjaitan, W. (1983): The epidemiological survey of malaria in Asahan
district, North Sumatra, Indonesia, Japan. J. Trop. Med. Hyg., 11(1), 17‑24lO) Kurihara. T. (1970): Malaria in Java‑epidemiology and control, Nettai, 5(2), 98‑105 (inJapanese)11) Matsuoka, H., Ishii, A. and Panjaitan, W. (1987): Chemotherapeutic control trial of Plas‑
modium falciparum with a combination of chloroquin and primaquine on selective age groupin a coastal village of north Sumatra, Indonesia, Japan. J. Trop. Med. Hyg., 15(4) , 257‑268
12) Rieckmann, K.H., McNamara, J.V., Frischer, H.. Stockert, T.A., Carson. P.E. and Powell. R.
D. (1968) : Gametocytocidal and sporontocidal effects of primaquine and of sulfadiazine withpyrimethamine in a chloroquine‑resistant strain of Plasmodium falciparum. Bull. Wld. Hlth.
Org., 38, 625‑632

13) Stafford, E.E. and Joesoef, A. (1976): Intestinal and blood parasites of man in Bireuen and
Takengon, Aceh Province, Sumatra, Indonesia, Southeast Asian J. Trop. Med. Publ. Hlth., 7,

518‑522
14) Smal]ey, M.E. and Sinden, R.E. (1977): Plasmodium falciparum gametocytes: their longevity
and infectivity, Parasitology, 193, 673‑675

15) Targett, G.A.T. and Sinden, R.E. (1985): Transmission blocking vacL ines, Parasitology
Today, 1(6), 155‑158

310

インドネシア国北スマトラにおけるマラリア高度浸淫地と年齢別生殖母体血中濃度分布について
糸川英樹1・2・高井鎗二1・石井明1・3・W．Panjaitan1・4

インドネシア国，北スマトラ州のマラッカ海峡沿岸村落において，夏期にマラリア集団検血を実施した。従来，マラリア感染率は全般的に2％以下とされていたにもかかわらず，沿岸村落に44％と高い感染率が検出された。特に0−9歳小児においては60％にも及び，公衆衛生上の問題となる事が指摘された。マラリア原虫の年齢別分布ならびに血中濃度の算定成績から，熱帯熱マラ
リアと三日熱マラリアが約3：1に存在し，熱帯熱マラリアにおいて学齢前幼児，小中学校生徒に高い感染率が認められ，無性原虫血中濃度も高かった。特に生殖母体の血中濃度は25歳以下の年齢の者に高く，102／mm3以上の者は15歳以下の者に検出され，マラリア伝播に中心的役割を果している事が判明した。この事はマラリア伝播を切断して，マラリア対策を行う際に重要な点を示唆している。三日熱マラリアでは感染率は比較的低く，感染は高年齢者層に及んでおり，103／mm3以
上の原虫濃度は稀であった。

北スマトラ地域保健対策計画，国際協力事業団，東京
東京医科歯科大学医学部医動物学教室
岡山大学医学部寄生虫学教室
北スマトラ州衛生部，メダン

311

日熱医会誌．，第17巻 第4号 1989 311−321頁

ブタ回虫感染マウスにおけるサイトカインとNK活性の動態陶道

平成元年7月10日受付／平成元年9月5日受理

イルス（Bukowskiε∫認．，1985；Mcintyre

はじめに蠕虫感染によって，宿主の免疫機構はなんらかの影響をうけ，液性免疫や細胞性免疫に様々な変化が起こるが，特に抗体産生では蠕虫と無関係な抗原に対する免疫応答が抑制されることが知られている（Crandall

andCrandall1971，1976；

Crandallα認．，1978；Faubert
1971；Faubert，1976；Good

and

and

Welsh，1986），細菌および寄生虫などの感染に対

andTannerMiller，1976；

する防御活性を示す（Murphy

1982；Nabavi

andzJ．，1980；Hatcher

andMcDanie1，

Murphy，1985；Hatcher8孟
andKuhn，1982；Orago

andSolomon，1986；Solomon，1986）のみならず，
自己の免疫応答の調節にも関与するものとされている（Callewaert，1985）。

本研究では，これら宿主寄生虫相互関係の一局
面を，免疫学的観点から細胞レベルで理解するた

Komatsu6厩1．，1979）。

ブタ回虫をマウスに感染させると，その感染時めに，ブタ回虫感染マウスにおける各種免疫担当

期や程度に応じて，マウスの抗体産生に各免疫グ細胞によるサイトカインの産生状況や，NK活性ロブリン（Ig）クラスについて特異的な変化をきの経時的変動を検討した。

たすことが明らかにされてきた（Crandall

andCranda11，1971，1976；Cranda11θオ zl．，1978）。し

材料と方法

かしながら，こういった免疫学的変調には，単なるB細胞のみならず，マクロファージ，丁細胞，

1）実験動物またはナチュラルキラー（NK）細胞などの免疫担

以下の純系マウス（日本チャールズ・リバー社

当細胞の，量的あるいは質的変化も関与しているより購入）を用いた。7週齢雌のBDF1（C57BL／と考えられるが，これらについての解析は乏しく，

6NCrjxDBA／2NCrj）マウス6−8匹を1群としいまだ不明な点が多い。

て感染実験に用いた。またIL−1活性の測定には，免疫応答は丁細胞，B細胞，マタロファージなどの細胞間相互作用によって成り立っているが，5−6週齢雌のC3H（C3H／HeNCrj）マウスを用いた。

これらの免疫担当細胞より産生される各種サイト

2）回虫幼虫包蔵卵の感染

カインは，免疫応答の誘導相や反応相において各

大阪食肉センターにおいて屠殺されたブタより細胞相互間のシグナルの伝達因子として，その応採取したブタ回虫の子宮内卵を，2％ホルマリン

答の発現や調節機構に重要な役割を演じている

液中にて29℃，30日間培養して得た幼虫包蔵卵の（Aarden召∫認．，1979；Gorden

103あるいは104個を含む0．1mlの生食浮遊液を，

1965；Kasakura

and

and

McLean，Lowenstein，1965；Mor・

マウス用ゾンデを用いて経口的に胃内に注入した。

gan6∫α1．，1976）。他方，NK細胞は自然に備わっ

3）培養液および細胞の培養た細胞性の生体防御因子として，抗腫瘍活『生やウ

培養液として5×10−5Mの2一メルカプトエタ

兵庫医科大学免疫学医動物学教室

312

ノール，10％の牛胎児血清（FCS）を含むRPMI

6）IL−1活性の測定

l640を用いた。各種細胞の培養は5％の炭酸ガス

IL−1活性の測定はMizel8緬」．（1978）の方法

存在下，37。Cで行った。

4）腹腔滲出細胞の分離および培養上清の調整

幼虫包蔵卵感染後のマウスの腹腔内に5mJの5％FCS−RPMI1640を注射したのち，それを回旨に準じた。すなわちC3Hマウスの胸腺を無菌的に摘出し，10％FgS−RPMI1640を用いて2×107／
mJの単細胞浮遊液を作成して，96穴マイクロプ
レートの各ウェルに50μ」ずつ分注し，これに5μ収することにより，経時的に腹腔滲出細胞を得た。

9／mJのphytohemagglutinin−P（PHA，和光純

得られた細胞をさらに同培養液で2回洗浄し，ト薬）を50μ」加えた。さらに段階希釈した前述の各

リパンブルー染色液で生細胞を数え，1×106／mJ

種培養上清サンプルを10μ」加えて72時間胸腺細の細胞浮遊液を作成した。この浮遊液をプラス胞を培養し，その回収6時間前に3H−TdRO．5

チックシャーレに入れ，3アC，5％の炭酸ガスのμCiを添加してその取り込みを測定した。なお，培存在下に1時間静置して，非付着細胞を除いたの

養上清サンプルの20，60，180および540倍の希釈

ち，付着細胞をセルスクレイバーで回収し，1×濃度では，その希釈と3H−TdRの取り込みの間に106／mJになるように調節した。

上記の方法で得た付着性腹腔滲出細胞（腹腔マ

直線関係が見られたので，IL−1の測定にはすべて180倍希釈培養上清サンプルを用いた。

クロファージ）に，1ipopolysaccharide（LPS，7）IL−2活性の測定

半井化学薬品）を最終濃度10μg／mJになるように

IL−2の活性は，CTLL−2細胞（C57BL／6マウ

加え，T−25型プラスチック培養フラスコで24時問ス由来Friendvirusinducedleukemia）のIL−2培養した後，その培養上清を回収し，一2ぴCにて凍依存性増殖反応を用いて測定した。すなわち，2結保存して，これをIL−1活性およびPGE2測定（後倍連続希釈した培養上清サンプル100μ」を入れた

述）用腹腔マクロファージ培養上清サンプルとし

96穴マイクロプレートの各ウェルに，5×103個のた。

CTLL−2細胞を含む細胞浮遊液0．1mJを分注

腹腔マクロファージは，非特異的エステラーゼ

して，20時間培養後，ウェル当たりo．5μCiの染色法で同定し，好酸球はギムザ染色法で同定し3H−TdRを加え，さらに4時間培養した。培養終了

た。染色後200個の細胞を数え，マクロファージあ後，セルハーベスターで回収し，3H−TdRの細胞

るいは好酸球の比率を算定した。

への取り込みを，液体シンチレーションカウン

5）脾細胞および培養上清の調整

ターで測定した。

マウスの脾臓を無菌的に摘出し，培養液中で注8）IL−2活性に対する抑制の測定

射針にてほぐした後，ステンレスメッシュを通し

IL−Z活性に対する抑制の測定は，Lelchukandて単細胞浮遊液を作成した。培養液で2回洗浄しPlayfair（1985）の方法に準じた。すなわち，回た後，トリパンブルー染色液にて生細胞数を数え，

虫感染マウスの腹腔マクロファージ，あるいは脾

細胞密度を調節した。

臓付着細胞の培養上清50μ」を，1×105／mJの

5×106／mJの脾細胞単細胞浮遊液に，Con・CTLL−2細胞浮遊液100μ」および10mit／mlのcanavalinA（ConA，MileS社）を最終濃度2μg／

リコンビナントIL−2（武田薬品）50μ」を含む96mJになるように加え，24時間培養した後，その培穴のマイクロプレートのウェルに加えて20時間培

養上清を回収し，一2ぴCにて凍結保存し，これを養後，ウェル当たりo．5μCiの3H−TdRを加え，さIL−2活「生測定（後述）用脾細胞培養上清サンプルらに4時問培養して，3H−TdRの取り込みを測定とした。

し，培養上清の添加による取り込みの減少をIL−

付着細胞の単離と培養上清の調整は1×107／

2活性の抑制とした。

mJの脾細胞について，腹腔マクロファージと同様抑制因子の分子量を概算するために，透析用セな方法で行い，回収した上清をIL−1測定（後述）

ルロースチューブ（分画分子量12，000−14，000）を用脾細胞培養上清サンプルとした。

用いて，上記の培養上清をRPMI1640で透析した313

り，モルカット10，000（Millipore）で分子量

lysisは常法により算定した。

10，000以上を除去した後のIL−2活性への抑制を

なお，NK抵抗性を示すP815細胞（DBA／2マウス由来Mastocytoma）についても同様の測定測定した。

9）IL−2産生に対する抑制の測定を行った。

まず，5×106／mJの正常脾細胞浮遊液5mJに
前項で用いた培養上清サンプルを25％あるいは50％になるよう，またConAを最終濃度2μg／ml

I・Polyになるように加えて10mJとし24時間培養した。

Poly

この培養上清を前項と同じ条件で予め透析し，そ

ウスの腹腔内に投与した。

また，polyinocinicpolycytidylicacid（PolyC）による脾臓内NK活性や血清中IFN活性の誘導を見る場合には，その測定の前日に，
I・PolyC（シグマケミカル社）100μgをマの中に含まれる低分子のIL−2活性抑制因子を除11）インターフェロン（IFN）活性の測定

去したのち，前述と同じ方法で培養上清中のIL−2NK活性の測定に用いたマウスの血清を，96穴

活性を測定した。培養上清サンプルのかわりに，

マイクロプレートにて2倍遭続希釈した。この0．1

培養液を加えた対照よりの減少をIL−2産生の抑mJに同量の106／mlのL細胞（マウス由来の制とし，抑制率（％）を以下の式で算出した。

且broblast）を加え，24時間培養した後，0．1ml当抑制率（％）＝（A−B）／AX100

たり100プラークフォーミングユニット（PFU）の

ここではAは対照におけるCTLL−2細胞の，3H−TdRの取り込み値を示し，Bは培養f清サン

vesicularstomatitisVirus（VSV）を感染させプルとConAを加えた正常脾細胞の培養上清によをもって，血清中のIFN力価とした。

た。血清添加によるVSVのL細胞細胞障害の抑制
IFNの型は抗マウスIFN一α，β抗血清を用いてるCTLL−2細胞の3H−TdRの取り込み値を示す。

10）NK活性の測定
脾細胞単細胞浮遊液1×107／mlを3倍連続希判定した。

釈し，96穴マイクロプレートの各ウェルに0．1ml

結を加え，51Crで標識した0．1mlのYAC−1細胞

果（A／Snマウス由来Moloneyvims−induced

1）回虫感染によるマウス腹腔滲出細胞の変動

lymphoma）と共に4時間培養後，標的細胞から51Crの遊離を測定し，NK活性とした。Speci負c1に見られる如く，15日目を峰として一過性に増回虫感染によりマウスの腹腔滲出細胞数は，図

20
ハ

0
の

18

コ

0
E

16

￥

14

00
＿ 12
⑳ 10
一
じ

コ 8

0口

・1

6
、

00

4

ぎ

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2

5

10

Days

FigurelChange

of

15

20

after

infection

peritoneal

cell

counts

30

in

mice

duringinfection

with103（○）or104（△）eggs

of、4s伽7乞s

さがゆ コ
Mean of six

mice with vertical bars as

the S．D．is

shown．

314

100

Macrophage

EosinophiI7

0

Φ切＄⊂8﹂8

ム、、、
、昏一一

を』、

0

5

』、か一一一一会【一

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一一一・ム

0

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、

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、

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、

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！
！

！

10

、

ノ

0

5

10

15

20

Days

30

after

0

infection

5

10

、
20

15

30

Fig皿e2Percentage of macrophages and eosinophils in the peritoneal cav・
ities of mice infected with103（O）or104（△）eggs of／4．s観吻．
Two hundred cells were counted at least，and percentage of macro・
phages and eosinophils were calculated after that cells were stainedwith nonspeci丘c esterase or
obtained from three mice．

加することが観察された。103あるいは104個の虫Giemsasolutions．

Results

were2）回虫感染による腹腔滲出細胞のIL−1産生

卵感染によりマウスの腹腔滲出細胞数は，正常マの変化

ウスのそれに比べ，感染後5日目において，それ

回虫感染マウスから経時的に採取した腹腔マク

ぞれL2と3．4倍，10日目において9．1と8．1倍，15

ロファージを，そのまま培養してもIL−1産生は認められないが，LPSで刺激すると103あるいは104個のいずれの感染群においてもIL−1の産生が起し，30日目には，103の感染群で正常マウスと同程

5焉Lo9︒2届老一隻工↑︹評︺でに減少した。

ころ，図2に示すように，マクロファージの総細

！

！
1

七

、

妄一一一一一一｛ノ5胞数に対する比率は正常マウスのそれと相異なく，

01染色法を用いて同定し，その細胞数を計測したと

m9×E巳0好酸球を，非特異的エステラーゼ染色法とギムザ︻﹂
1

この腹腔滲出細胞に含まれるマクロファージと

／

／γ
！

度となり，104の感染群で正常マウスの2倍以下ま0
つ二日目において13．8と15．3倍と著明に増加した。しかし，20日目に1．5と4倍と増加率は大幅に減少

感染時期による明らかな変動を示さなかったが，図1に示したように総細胞数が顕著に増加したことから，腹腔内のマクロファージの総数は感染後15日目を最高として，一過性に増加することが示された。好酸球の比率は，5日目に増加し始め，

0

5

Days

10

after

15

2q

30

infection

Figure3Time course of LPS−induced IL−1
production of peritoneal adherent
cells ofみ．s吻吻infectedThe

culturemice．

supematants

of

pe卜

mice

in．

10日目に最高に達し，15日目より減少する傾向を

itoneal adherent cells

示し，30日目に対照群と同程度に復元したが，両fected with103（O）or104（△）eggs
of／1。s観吻were assayed on PHA−
stimulated thymocyte cultures at a者の比較から感染マウスの腹腔内で好酸球はマク
ロファージに比して，約5日間先行して一過性に増加することが明らかとなった。

fromdilution of l：180． Each point repre・sents the mean±S．D．of three experi・
ments．Day O stands for the value of

uninfected mice．

315

25
0

︻

5

−

0

mo一養含

ウ一

5

︒︒葛﹄区﹄︒︒︒；ロ一﹁三ξ﹃︺

5

o

l

o

15

h−

20

；50

Daysafterinfection
Figure4Kinetics of suppression of IL−2 activity by culture
supematants of peritoneal adherent cells from mice
infected with104eggs of z4．s襯彫．
Before（●）or after（▲）dialysis，50μl of the culture

supematants

unit／mlwereadded，together

recombinant

with50μJ

oflO

IL−2to1×104CTLL−2cells

contained in100μ1．After24hr−culture，proliferationof CTLL−2cサlls were measured by3H−TdR incorpora・
tion．Each point represents the mean±S．D．of threeexperimentS．
こり，図3に示すように培養上清中のIL−1活」生は感染後5日目より漸増し，15日目に最高に達した

100

あと，20日目に向って次第に減少したが，この動O4は認められなかった。また同上清中には，いずれO日目に1．2倍であったが，感染による有意な増強と6

清中のPGE2を測定したところ，104の感染群では対照群に比し，5日目，10日目においてL1倍，15∩U

以下いずれも図表に示さないが，上述の培養上こΩ8愛＆房も訳調していた。

8

態は腹腔内マクロファージの総数の動態とほぽ同

20の時期においてもTNF活性は認められなかった。

3）回虫感染マウスの腹腔滲出細胞の培養上清㎞1 ㎞al infected i㎡㏄ted

50％，LPs（一）50％，LPs（＋）25％，LPs（†）印％上Ps（＋）

中のIL−2抑制因子

Culture

前述の感染各時期の腹腔滲出細胞のLPS刺激
培養上清について，IL−2活性に対する作用を調べたところ，図4に見られるように正常マウスのLPS刺激培養上清の場合に比し，感染10日目における53％を最高とする顕著な抑制作用を認めた。

次に，この培養上清中の正常マウス脾細胞のConA刺激によるIL−2産生に，どのような影響を与えるかを調べた。図5に見られるように，正常

Figure5Suppressive
tion

of

supernatants

activity

nornlalonspleenIL−2produc−

cells．

The culture supematants of per・itoneal adherent cells from mice10
days after infection with104eggs of

且．s吻郷and fromnormalspleencells（cultured with or without LPS）

were

incubatednormalspleen

with

cells

at

ConA

activatedindicatedcentration．After24hr

con・

culture，theマウス脾細胞のConA刺激で産生されるIL二2活性

remaining activity of IL−2in theは，正常マウスの腹腔滲出細胞のLPS刺激培養上

free supematants were assayed using
CTLL−2cells．Results represents the

清の50％添加により約11％抑制されたのに対し，mean±S．D．of

triplicatedcell−

cultures．

316

感染10日目の腹腔滲出細胞の培養上清は，25％あ14

るいは50％の添加により正常脾細胞のIL−2産生

、

、

、
、
＼

4

の分子量の物質を除去したところ，図4に見られ

￥
、

口U

スチューブを用いて透析し，12，000−14，000以下

、
￥

QU

子サイズを調べるために，上清を透析用セルロー

O︵﹄E＞で⊃︶創−﹂一

このような培養上清中に含まれる抑制因子の分つ乙をそれぞれ15％，64％抑制した。

るように，感染後いずれの時期の上清についても

2

IL−2活性への抑制活性は検出できなくなった。次ク

ト、、

いで，同じ培養上清をモルカット10，000で濾過し，その濾液についてIL−2活性抑制を調べたとこ

05101520

Days

ろ，データは示さないが，濾過前と同じ程度の抑

afterFigure6Time course

制活性が確認された。

The

なお，感染マウスの脾臓付着細胞のLPS刺激培

spleeninfectionof Iレ2production．
cells

from

mice

infected

4）回虫感染によるマウス脾細胞のIL−2産生

with103（○）or104（△）eggs of、4．
s観吻were stimulated with ConA for
24hr and culture supematants wereassayed using CTLL−2cells． Eachpoint represents the mean±S．D．of

次に，マウス脾細胞のConA刺激によるIL−2産

three

養上清については，いずれの感染時期においても上記のような抑制作用が認められなかった。

生能の回虫感染による変化を調べた。その結果は，図6に示すように，103個の感染でexperiments．

40

く抑制されていた。いずれの群においても，その抑制は15日から回復する傾向を示した

カ㍉20日以降まで持続した。

ぐ︑︑と10日目の両時期において，IL−2産生は強

辺
の30
≧
．9
士
820
房

5）脾細胞のNK活性も脾細胞のNK活性の感染による変化を，

訳10

弍

！

＼︑

は主として10日目に，104の感染では，5日

！

！

！

！
ノ

、

、

！

ノ
・1！

YAC−1細胞に対する細胞障害作用を指標として調べたところ，データは示さないが，0

10日目に若干の低下が見られたものの，有

5
Days

after

infection

Figure7Anti−YAC−1cytotoxic

activity

意の変化は認めなかった。そこで，その差を明らかにするために，脾細胞採取前日に100μgのPolyI・PolyCを投与することにより，積極的に誘導したNK活性の増強が，感染によって変化するか否かを検討した。

その結果，図7に見られるように，感染マ
ウス脾細胞のNK活性は，103と104個のい

15

10inducedby Poly I・Poly C in mice infected（○）or104（△）eggs of／1．sz z4 z．

with103

Spleen cells were removed on day5，10
and15after infection from mice whichwere intraperitoneally received100μg of
Poly

I・PolyCytotoxicity

C

one

was

day

beforemeasuredsacrifice．

by releaseof

ずれの感染群においても上述と同じく感染

51Cr

10日目に有意に低下し，15日目には回復し

4hr incubation at100：1E／T ratio．Day O
stands for the value of uninfected andPoly I・Poly C injected mice．Each point

た。なお，データは示さないが，P815細胞

fromに対する細胞障害作用も同時に調べたが，represents

両群いずれも対照群に比べ，明らかな変化

ments，

labeledYAC−1target cells

the mean±S．D．of

three

after

experi−317
はなかった。

！

へ

∠△

︑

6

！

O

り∠

一

1

O

2

﹃

一

︸

1

O

＝込

！

O

0U

照群に比べ増強したが，10日目には逆に低0

いても，感染5日目の血清中IFN活性は対nU

図8に示すように，いずれの感染群におり∠も検討した。

4

その血清中IFN活性の感染による影響を

nU

C刺激マウスについては，︵一ミ．⊃．一︶⊆oおtΦゼ一

I・Poly

つ乙

Poly

8

6）血中IFNの活性

！
！

￥

nU

v

4

下し，15日目には復元した。

ノ

！

！

これらIFN活性は，抗マウスIFN一α，β抗血清の処理で中和されたので，Poly

I・

0

5

Days

Poly Cによって誘導された血清中IFN活
性は，α，β型であることが確認された。

Figure81nduction

of

10

after
IFN

s謝勉infected考察

蠕虫感染時における宿主の免疫系の変化の特徴

by

15

infection
Poly

I・Polymice．The

C

serum

in／1．

samplesoriginated from the same donor mice in・
fected with103（O）or104（△）eggs of・4．
s％z 吻justvalue

as described

represents

thein Fig．7． Eachaverage

of

six

sam・

ples．

として，非特異的IgE産生の増強や好酸球増多があげられるが（JarrettandMiller，1982），同時に液性あるいは細胞性免疫能が非特異的に低下すを見出し，幼虫の体内移行に伴って一過性に全身ることもよく知られている。この免疫低下は，宿状態が悪化することを示して来た（未発表）。今回

主寄生虫相互関係を免疫学的観点、から理解する上は，回虫感染によって腹腔内滲出細胞数が感染15で重要な課題であり，すでに多くの虫種について，

日目を最高として，一一過性に著しく増加することその機構の解析がなされて来たが，その様相は虫を見出すとともに，この滲出細胞のうち好酸球数

種によって一様でなく不明な点が多く残されていは10日目を，マクロファージ数は15日目を最高とる。

する経時的変動を示すことを明らかにした（図1，

この点に鑑み，本研究では，ブタ回虫の経口感

2）。次に，これら腹腔マクロファージを初∂伽o染をうけたマウス（非固有宿主）の腹腔内滲出細

でLPS刺激して産生されてくるIL−1活性の経時胞の量的変動や，モノカインの産生状況を調べる

的変動を調べたところ，マクロファージ数の増加とともに，脾内の丁細胞の機能やNK細胞活性，さにほぼ同調して，感染後15日目を最高として一過らには血中IFN活性に対する感染の影響を検討

性に増加するのを認めたが（図3），このような好し，この感染系における免疫低下の成因をさぐろ

酸球やマクロファージ数の増加や活性化は腹腔内うとした。

を移行する幼虫に対する防御反応の一面を反映す

すでに，著者らはブタ回虫幼虫包蔵卵104個をマるものと思われる。

ウスに経口感染させたのち，小腸で艀化した幼虫

他方，回虫感染後の脾内ヘルパー丁細胞の機能の体内移行状況を調べ，肝臓では5日目，肺臓での経時的変動を知るために，感染後の各時期におは7日目で回収幼虫の数が最大となり，約14日以いて，脾細胞を初θ♂加でConA刺激して産生され後には，いずれの臓器においても検出できなくなてくるIL−2活性の動態を調べたところ，感染後5ることから腸，肝，肺の移行経路を確認するとと日目より脾細胞のIL−2産生は減少し始め，10日目

もに，マウスの体重が感染6日目より減少しはじには最低に達したあと，15日目以後回復に向うこめ，10日目には最低に達し対照群の75％となるとが明らかになった（図5）。これは，ブタ回虫感が，その後漸増し，約16日目に復元することなど

染後11−12日目において，マウス脾細胞のConAや

318

PHA刺激による増殖反応や遅延型過敏反応の低清）をも明らかに抑制した（図4，5，6）。この

下を見出したCrandall6∫認（1978）の報告と一一

培養上清中には上述のようにIL−1活性も含まれ致し，この時期にヘルパー丁細胞の潜在的IL−2産ているので，回虫感染によって腹腔マクロファー生能が障害されていることが示された。次にIL−2ジが活性化されIL−1を産生すると同時に，抑制因によって誘導される，NK活性の感染マウス脾内

子をも産生していることが明らかとなった。このの変動を調べたところ，データは示していないが，抑制因子は，透析および濾過実験の結果から，分無処置の感染マウスの脾細胞では，正常マウスの子量10，000以下であることが推定されたが，更にそれとの差は認めなかった。しかし，NK活性の誘

この因子の経時的変動を調べたところIL−1の産導物質であるPoly

生と同調して，感染5日目より増加しはじめ10日

I・PolyCをマウスに予め投与して，NK活性を積極的に誘導させると，感染と非目に最高に達したあと減衰し，15日目には復元し感染の両マウス間に脾内NK活性の差が見られ，た（図4）。

上記IL−2産生が低下する感染10日目においてNK

マクロファージに由来する低分子の抑制因子と活性の誘導が抑制されていたが，15日目には復元

してはPGE2が挙げられるが（Davis召麺」、，1984；

していた（図7）。PolyI・PolyC投与によって血GoodvinandCeuppens，1983；Kunkel8渉α1．，中に誘導されるIFN活性についても同様な変動

1986），本実験では，データは示していないが，感が見られ，感染5日目では活性が若干上昇するが，染マウス腹腔細胞のLPS刺激によって誘導され10日目には低下し，15日目には復元していた（図るPGE2の量は正常マウスにくらべ，明らかな変8）。一般にNK活性はIFNを介して増強されるの化を示さなかった。また，TNF活性はいずれの群で，感染マウスの両活「生が同調して低下するのはにおいても上清に認められなかった。同様な抑制

当然と考えられるが，感染5日目にIFN活性のみ

因子が，感染マウスの脾臓付着細胞のLPS刺激にが対照群より上昇した原因は不明である。なお・

よっても産生されるかどうかを検討したが，そのデータには示していないが，脾内におけるLymphokineactivatedkiller（LAK）細胞の存

培養上清にはIL−2活『生，あるいはIL−2産生に対す在を，P815細胞を標的細胞とする障害活性で調べ

認（1978）の系で，感染により免疫低下を示すマ

たところ，感染による変化を認めなかった。

ウスの脾細胞中に抑制性細胞の存在を認めなかっる抑制作用を認めなかった。これはCrandall8渉以上のように，回虫感染による脾内ヘルパー丁たことと相通じるかも知れない。幼虫の移行経路

細胞のIL−2産生能の低下は，N照活性やIFN活性に当たる腹腔内のマクロファージが，虫体，あるの誘導にも影響を及ぼしつつ，感染マウスの免疫

いは虫体成分による直接刺激によって活性化され応答性の低下をもたらすのであろうと考えられる抑制因子を産生するのであれば，幼虫が移行しな

が，IL−2産生能の低下を惹起する機序については

い脾臓では付着細胞がその因子を産生しないのは不明のままである。すでに種々の嬬虫感染におけ当然であろうが，もし感染による免疫低下が，主る免疫低下の原因として，虫体自身あるいは虫体として上記の抑制性腹腔マクロファージの産生す成分による丁細胞の直接的な障害や，抑制性丁細る抑制因子によるものとすれば，多分循環を介し胞やマクロファージの誘導などが報告されているて遠隔の脾の免疫系に影響を与えるのであろうが，が（Barriga，1975；Faubert

その機序は今後の課題として残されている。

andTanner，1975；

Lubiniecki andCypess，1975），これまでの実験

今回の研究により，ブタ回虫感染初期における系ではこれらの検討はまだなされていない。そこ

丁細胞の機能低下には，腹腔マクロファージよりで，本研究ではIL−2活性に影響する因子を検索す

産生される1レ2抑制因子が関与している可能性るため，感染マウス腹腔内滲出細胞をLPS刺激しがあり，回虫の宿主体内移行部位にある腹腔内マて得た培養上清の1レ2産生に対する影響を調べ

クロファージは生体防御および免疫抑制という両たところ，既存のIL−2活性を抑制するとともに正面で，感染に対応していることが示唆された。ま常脾細胞によるIL−2産生（感染10日目の培養上た，回虫感染はNK細胞やIFN活性の変動を介し

319
c,
r

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,

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:n

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production by activated T Iymphocytes, J. Immunol., 120(5), 1497‑1503
27) Morgan, D.A., Ruscetti, F.W. and Callo, R.C. ( 1976 ): Selective in vitro growth of T ‑lymphocytes from norrnal human bone marrows, Science, 193, 1007‑1008
28) Murphy, J.W. and McDaniel, D.O. (1982): In vitro reactivity of natural killer (NK) cells
against Cryptococcus neoformans, J. Immunol., 128, 1577‑1583
29) Nabavi, N. and Murphy, J.W. (1985): In vitro binding of natural killer cells to Cryptococcus
neoformans targets, Infect. Immun., 50, 50‑5730) Orago, A.S.S. and Solomon, J.B. (1986): Antibody‑dependent and independent cytotoxic
activity of spleen cells for Plasmodium berghei from susceptible and resistant rats, Im‑munology, 59, 283‑288
3D Solomon, J.B. (1986): Natural cytotoxicity for Plasmodium berghei in vitro by spleen cells
from susceptible and resistant rats, Immunology, 59, 277‑281

321

CHANGE OF CYTOKlNE AND NATURAL KILLER ACTIVITIESIN ASCARIS SUUM INFECTED MICETAO DAO
Received July 10 1989/Accepted September 5 1989

In order to analyze the cellular mechanism of immunosuppression during acute infection

by Ascaris suum, cytokine and natural killer activities in A. suum infected mice were
investigated.

Ten days after infection with 103 or 104 embryonated A. suum eggs, Concanavalin A
induced interleukin 2 (IL‑2) production by splenocytes were reduced. At the same time, NK
activity of splenocytes and the levels of serum interferon induced by Poly I･ Poly C were also

reduced. On the other hand, culture supernatants of peritoneal adherent cells from A. suum
infected mice showed significantly high interleukin I activity which reached peak level oh day

15 following lipopolysaccharide stimulation. Increase in the number of macrophages in theperitoneal cavity were also observed in this period. Furthermore, the culture supernatants ofperitoneal adherent cells from 10‑days A. suum infected mice exhibited a suppressive effecton both IL‑2 production by normal splenocytes and IL‑2 activity assayed using CTLL‑2 cells.

Dialysis and molecular filtration studies indicated that the factor with molecular weight
below 10,000 related to the suppressive effect. But there was no significant increase of
prostaglandin E2 in the culture supernatants.

These results suggest that activated macrophages may play an important part in the
immunosuppression by producing a soluble suppressive factor during acute infection by A.
suum .

Department of Immunology and Medical Zoology, Hyogo College of Medicine, 1‑1 Mukogawa‑cho,
Nishinomiya 663, JapanJa pan. J. Trop. Med. Hyg

Vol 17 No 4 1989, pp. 323‑329

323

MONOCLONAL ANTIBODIES RECOGNIZINGTHE MAIN SURFACE ANTIGENS OF NEWLY
EXCYSTED METACERCARIA OR ADULTFLUKES OF PARAGONIMUS OHIRAIYOSABURO OIKAWA AND TERUAKI IKEDA
Received July 17 1989/Accepted October 5 1989

Abstract: This study was undertaken to prepare monoclonal antibodies which
recognized the tegumental antigens of newly excysted metacercaria (NEM) and adultflukes of Paragonimus ohirai. From splenic cells of mice immunized with either the Triton

X‑100 extract of adult fiuke surface or NEM sonicate, monoclonal antibodies were
produced by ELISA screening followed by trial immunostaining of fiuke sections. We
prepared a monoclonal antibody (AS‑Mab) recognizing the tegumental antigen of theadult fluke and a monoclonal antibody (MS‑Mab) for the NEM tegumental antigen. AS‑
Mab also bound to the tegument of 1‑week‑old juveniles but not to that of NEM. MS‑
Mab bound the tegument from the metacercaria to adult stages, but the degree of im‑munostaining appeared to decrease with maturation of the flukes. By the double
immunodiffusion technique, the two monoclonal antibodies formed crossing precipitinlines against a mixture of the adult surface antigen extract and the NEM antigen extract.

MS‑Mab and AS‑Mab gave precipitin line against the incubation fluid from NEM andadult flukes, respectively.

INTRODUCTION
The surface of a parasite plays a crucial role in the immunological interaction between

host and parasite. This is well‑known in African trypanosomes, which undergo antigenic
variation of their surfaces according to the host immune response (Cross, 1979) . In parasitic

helminths, changes of the surface antigens occur during development (Philipp et al., 1980;
Snary et al., 1980; Maizels et al., 1983) and might be one of the ways in which they evade thehost's immune attack. There have been a number of rep9rts about the stage‑specific surface
antigens of helminths, as reviewed by Philipp and Rumjaneck (1984) , but most of the studies
went no further than identification of the surface antigens using surface‑labelling and sodium

dodecyl sulphate polyacrylamide gel electrophoresis (SDS‑PAGE) . It is important forunderstanding fully the immunobiological phenomenon occurring in helminth infections toinvestigate the various immunochemical and biochemical characteristics of surface antigens.
In the lung fluke, an anti‑surface antibody response has been observed to occur during thecourse of Paragonimus ohirai infection (Ikeda and Tani, 1985; Ohara et al., 1985) , the surface

antigens have not yet been immunochemically and biochemically analyzed. Therefore, weDepartment of Medical Zoology, Kanazawa Medical University, Uchinada, Ishikawa 920‑02, Japan324

planned to use monoclonal antibodies to characterize as well as identify the individual
antigens. The present study was undertaken to produce monoclonal antibodies against thesurface antigens of newly excysted metacercariae (NEM) and adult flukes.

MATERIALS AND METHODS
Crude NEM antigen was prepared as described previously (Ikeda and Tani, 1988). In
brief, NEM obtained by culturing metacercariae were sonicated twice for 30 sec in an ice
bath. After the sonicate was centrifuged at 20,000Xg for 30 min, the supernatant was
dialyzed against 10 mM phosphate buffered saline (PBS, pH 7.2) and used as the NEM
antigen. Adult surface antigen was prepared as follows. Living adult fiukes obtained from
infected rats were incubated in several changes of saline for 5 h at room temperature. The
washed fiukes were incubated for 10 min at 4'C in PBS contaihing 0.5% Triton X‑lOO. The
extract was centrifuged at 20,000 X g for 30 min, dialyzed against PBS, and used as the adultsurface antigen.

BALB/C mice were immunized with either NEM antigen or adult surface antigen.
Splenic cells from the immunized mice were fused with P3Ul myeloma cells by using
polyethylen glycol. Hybridoma cultures were screened by enzyme‑1inked immunosorbent
assay (ELISA) and then positive cultures were assessed by immunoperoxidase staining ofsections of NEM or adult fiukes embedded in paraffin. The hybridomas which secreted
antibodies against the surface antigens of NEM or adult flukes were cloned by limiting
dilution. Monoclonal cells were injected into the peritoneal cavities of pristane‑primed miceand the ascitic fiuid was collected as the source of monoclonal antibodies.

ELISA was performed in microtiter plates using horseradish peroxidase‑conjugatedrabbit anti‑mouse lgG according to the conventional procedure. Microtiter wells weresensitized with 100 pl of NEM or adult fluke antigen (10 pg protein/ml) in 0.1 M NaHCO*
buffer (pH 9.6) . As the substrate for peroxidase, 2, 2'‑azino‑di‑(3‑ethylbenzthazoline
sulfonic acid) diammonium salt (ABTS) was used. Immunoperoxidase staining was perfor‑
med as follows. Flukes were fixed with cold 95% ethanol for from 5 h to I day, dehydrated
in absolute ethanol, and then embedded at 60'C with paraffin. Fluke sections were cut andstained essentially by the indirect immunoperoxidase technique of Nakane and Pierce (1966) .
The sections were deparafiinized with xylene and ethanol. After washing with PBS, sections

were incubated with diluted monoclonal antibody or normal mouse serum for I h, washed in

PBS, and then incubated for I h with peroxidase‑conjugated anti‑mouse lgG antibody(Cappel Laboratories, Westchester, PA) diluted to 1/200. After washing, the sections weredeveloped in substrate of 0.02% 3, 3'‑diaminobenzidine and 0.003% hydrogen peroxide in 0.05

M Tris‑HCI buffer (pH 7.6).

RESULTS

The monoclonal antibody (MS‑Mab) prepared from NEM antigen‑immunized micegave a high ELISA Ievel on the NEM antigen‑coated plate but a low level on the adultantigen‑coated plate. The monoclonal antibody (AS‑Mab) prepared from adult surface
antigen‑immunized mice gave a high ELISA Ievel on the adult antigen‑coated plate but a low
level on the NEM antigen‑coated plate.

325

Figure 1 Immunostaining of P. ohirai NEM sections. Sections were treated with themonoclonal antibodies MS‑Mab (a) or AS‑Mab (b), or with normal mouseserum (c). The surface syncytium, the tegumental cells, and the ventral

sucker were stained by MS‑Mab, but not by ASMab or normal mouseserum.

Figure 2 Immunostaining of sections of 1week‑old P, ohirai juveniles obtained fromthe peritoneal cavities of infected rats. Sections were treated with themonoclonal antibodies MSMab (a) or AS‑Mab (b), or with normal mouseserum (c). The surface syncytium and the tegumental cells were stained by

MSMab and AS‑Mab, but not by normal mouse serum,
Fig. I show8 the immunostaining of NEM sections. MS‑Mab diluted to l/800 produced
strong immunoperoxidase staining of the surface syncytium and the tegumental cells of
NEM. When MS‑Mab diluted ta l/100 was used, nonspecific staining was observed over thewhole body. AS‑Mab and normai mouse serum did nOt stain the NEM sections even at adilution af l/lOO.

326

Figure 3 Immunostaining of sections of adult P, ohirai fluke8 obtained from the lung8
Of infected rats. Sections were treated with the monoclonai antibodies MS‑

Mab (a) or AS‑Mab (b), or with normal mouse serum (c'.). The surface
syncytium and the tegumental cells were stained by MS‑M' rb and AS‑Mab,
but not by normal mouse serum.
Incubation Fluid

AS‑MabMS‑MabAdult

AS MabAntigens(NEM Adult)

Figure 4 Double immunodiffusion in agar F', ig

MS=Mab

Inctibation Fluid

NEM
ure 5 Double immunodiffusian in agar

between the two monoclonal usin'g^ the two monoclonal anti‑

antibodies and a mixture of the bodies and incubation fluid (2 mg

antigen preparations from NEM protein./ml) from either NEM or

and adult flukes. MS‑Mab and adult fukes. MS‑Mab formed a

AS‑Mab produced independent precipitin line against NEM incu‑

precipitin lines. bation fluid,against
and adult
AS‑Mabdid sofluke fluid.

In sections of l‑week‑old juveniles from the peritoneal cavities of infected rats, thesurface syncytium and the tegumental cells were stained by bath MS‑Mab and AS‑Mab
diluted to l/lOO (Fig. 2). In adult sections, the same sites as in the juveniles were stained by

the two antibodies diluted to 1/lOO (Fig. 3). The immunostaining wit,h AS‑Mab was̲ rela‑
tively stronger than with MS‑Mab. Throughout the development of the fluke, the strongest
staining was observed on the tegumental apical surface,
Double immunodiffusion of MS‑Mab and AS‑Mab against a mixture of the adult surface
antigen extract and the NEM sonicate produced crossing precipitin lines (Fig, 4) . When the327

two monoclonal antibodies were diffused against incubation fiuid of the fiukes, MS‑Mab and

AS‑Mab formed precipitin line against the fluid from NEM and adult fiukes, respectively(Fig. 5) .

DrscUsslON

We prepared the two monoclonal antibodies, MS‑Mab and AS‑Mab. Both of themrecognized the P.o. tegumental antigens present in the tegumental syncytiurn and cells and on

the apical tegumental surface, and both showed the strongest immunostaining of the apicalsurface which may have glycocalyxes. MS‑Mab bound to the tegument throughout develop‑
ment of the flukes from metacercaria to adult, while AS‑Mab bound to the tegument of adultflukes and 1‑week‑old juveniles but not to that of NEM. The intensity of immunostaining
with MS‑Mab decreased with the development of the flukes, whereas it increased with AS‑

Mab. Thus, the two monoclonal antibodies appeared to recognize different tegumental
antigens, and this was verified by the formation of independent precipitin lines on double
immunodiff usion.

Bennett and Threadgold (1975) observed at the electron microscopic level that theglycocalyx of F. hepatica was formed by the discharge of tegumental granules which theymorphologically divided into 3 types; TO, T1, and T2 granules. Hanna (1980) proposed thatthe antigenicity of the glycocalyx changed during the development of F. hepatica and that thechange seemed to be related to the appearance of T2 granules. In Paragonimus species,
Fukuda (1986) reported the presence of tegumental granules resembling those of F. hepatica.

We have observed at 'the ultrastructural level that MS‑Mab bound to some types oftegumental granules and to the glycocalyx (Fujino et al., submitted for publication) , but wehave not yet studied AS‑Mab. From the similarity in immunostaining features and thepresence of antigens in the fluke incubation fluid, AS‑Mab is predicted to recognize theantigen present in the other type of tegumental granules.

Hanna and Trudgett (1983) prepared six monoclonal antibodies recognizing a
tegumental antigen present in T1 granules and the glycocalyx of F. hepatica. However, themonoclonal antibodies also strongly labelled the glycocalyx of gut cells and the excretoryduct in juvenile and adult flukes. Our monoclonal antibodies showed little binding to any
antigens except the tegumental antigen, especially in juvenile and adult flukes. It is interest‑

ing that the epitope of their antibodies appeared to be a polypeptide while ours appeared tobe sugar chains (unpublished data) . Neither MS‑Mab and nor AS‑Mab reacted with crude
F. hepatica adult antigens.

The two monoclonal antibodies prepared in this study should aid investigation of thecharacterization and bioimmunological functioning of these surface antigens.

REFERENCES
1 ) B,ennett, C.E. and Threadgold, L.T. (1975): Fasciola hepatica: Development of tegument during
migration in mouse. Exp. Parasitol., 38, 38‑55
2 ) Cross, G.A.M. (1979) : Immunochemical aspects of antigenic variation in trypanosomes, J. Gen.
Microbiol., 113, 1‑123 ) Fukuda, K. (1986) : Differentiation and degeneration of tegumental cells in adult lung flukes

328

Paragonimus species. (Trematoda: Troglotrematidae) , Int. J. Parasitol., 16b 147‑156
4 ) Hanna, R.E.B. (1980): Fasciola hepatica: An immunofiuorescent study of antigenic changes inthe tegument during development in the rat and the sheep, Exp. Parasitol., 50, 155‑170

5 ) Hanna, R.E.B. and Trudgett, A.G. (1983): Fasciola hepatica: development of monoclonal
antibodies and their use to characterize a glycocalyx antigen in migrating fiukes, Parasite
Immunol., 5, 409‑425
6 ) Ikeda, T. and Tani, S. (1985): IgG antibody response against excysted juvenile detected byindirect immunofluorescent method in Paragonimus ohirai‑infected rats, Japan. J. Parasitol.,
34, 127‑132
7 ) Ikeda, T. and Tani, S. (1988) : Induction of secondary lgE antibody response in rats immunizedwith X‑irradiated metacercariae of Paragonimus ohirai, Int. Arch. Allergy Appl. Immunol., 85,

213‑2188 ) Maizels, R.M., Meghji, M. and Ogilvie, B.M. (1983): Restricted sets of parasite antigens fromthe surface of different stages and sexes of the nematode parasite Nippostrongylus brasiliensis,

Immuno]ogy, 48, 107‑121
9 ) Nakane, P.K. and Pierce, G.G. (1966): Enzyme‑labeled antibodies: preparation and applica‑tion for localization of antigens, J. Histochem. Cytochem., 14, 929‑931
10) Ohara, H., Ikeda, T., Oikawa, Y. and Tani, S. (1985): Studies on antibody response in rats
infected with Paragonimus ohirai by immunofluorescent staining method, Japan. J. Parasitol.,
34, 245‑252ll) Philipp, M. and Rumjaneck, F.D. (1984): Antigenic and dynamic properties of helminthsurface structures, Mol. Biochem. Parasitol., 10, 245‑268
12) Philipp, M., Parkhouse, R.M.E. and Ogilvie, B.M. (1980): Changing proteins on the surface ofa parasitic nematode, Nature, 287, 538‑540
13) Snary, D., Smith, M.A. and Clegg, J.A. (1980): Surface proteins of Schistosoma mansoni andtheir expression during morphogenesis, Eur. J. Immunol., lO, 573‑575

329

大平肺吸虫の脱嚢メタセルカリア主要外皮抗原と成虫主要外皮抗原を認識するモノクローナル抗体
及川陽三郎・池田照明

大平肺吸虫（P．o．）の脱嚢メタセルカリア（NEM）抗原あるいは成虫トリトンX−100抽出表層
抗原でマウスを免疫し，それらマウスより，それぞれP．o．主要外皮抗原を認識するモノクローナル

抗体，MS−MabおよびAS−Mabを作製した。ELISAでは，MS−MabはNEM抗原と極めて高い反応性を示し，成虫抗原との反応性は低かった。一方，AS−Mabは成虫抗原のみに高い反応性を示した。

P．o．のNEM，1週目幼虫，および成虫のパラフィン包埋切片を作製し，2種モノクローナル抗体が認識する抗原局在部位を，酵素抗体法で観察した。NEM切片では，MS−Mabのみが外皮および外皮細胞に免疫染色性を示した（図1）。1週目幼虫および成虫切片では，両種モノクローナル
抗体とも，外皮および外皮細胞に免疫染色性を示した（図2，3）。成虫切片では，両種抗体に染色性の差が見られ，AS−Mabがより強い染色性を示した。いずれの発育期でも，両種抗体とも外皮最外層に最も強い染色性が見られた。NEM抗原と成虫表層抗原の混合液に対してオクタロ
ニー反応を行うと，両種抗体はそれぞれ交差する沈降線を形成した（図4）。また，NEMあるいは成虫の排泄・分泌（E・S）抗原に対するオクタロニー反応では，MS−MabはNEME・S抗原に，
AS−Mabは成虫E・S抗原にそれぞれ沈降線を形成した（図5）。免疫染色像，およびE・S抗原における認識抗原の存在などの結果は，これら2種のモノクローナル抗体がP．o．外皮glycocalyxを抗原として認識していることを示唆し，この外皮抗原の主要抗原が発育期に伴って変化することを示した。

金沢医科大学医動物学教室

Japan. J. Trop. Med. Hyg., Vol. 17, No. 4, 1989, pp. 331 338

331

EPIDEMIOLOGICAL SURVEY OF LEISHMANIASISUSING SKIN TEST AND ELISA IN ECUADOR
MASATO FURUYA1, TATSUYUKI MIMORI', EDUARDO A. GOMEZ L.3
VICENTA VERA DE CORONEL3, MASATO KAWABATA' AND YOSHIHISA HASHIGUCH15
Received June 8 1989/Accepted October 7 1989

Abstract: The present study was designed to evaluate the intradermal skin test (ST)and the ELISA as diagnostic tools in the screening for Ecuadorian cutaneous andmucocutaneous leishmaniasis. The antigen for skin testing was prepared from ruptured
promastigotes of Leishmania braziliensis. The ST and ELISA positive rates among 72
subjects with active dermal lesions were 81.8% (36144) and 81.3% (52/64), respectively,
while parasites were observed in 31 (44.9%) of 69 subjects presenting active lesions. In theparasites positive cases, all subjects proved to be positive for the two tests except for one

in ST and two in ELISA. In 35 healed cases, the ST and ELISA positive rates were 86.2%(25/29) and 72.4% (21/ 9), respectively. On the other hand, the positive rate in subjects
without clinical signs was only 3.8% in ST and 8.2% in ELISA. An epidemiological survey
in Selva Alegre, Esmeraldas, revealed that among 115 inhabitants 38 were positive for theclinical signs, 10 active and 28 healed cases. Of these subjects 33 (86.8%) showed positive

reactions against ST and/or ELISA. Based on the results obtained, therefore, we conclud‑ed that the present skin testing antigen and ELISA were very useful for the screening ofleishmaniasis in the endemic areas of Ecuador.

INTRODUCTION
Since the first human case of leishmaniasis was described in Ecuador in 1920, many,
additional cases of the disease have been reported (Rodriguez, 1974; Hashiguchi et al., 1984) .

The causative agent has been considered L. braziliensis complex based mainly on theirclinical manifestations in humans and epidemiological features (Rodriguez, 1974) . Recently,
we characterized six strains of Leishmania isolates in Ecuador, using isoenzyme electrophor‑esis and monoclonal antibodies. Three strains, isolated from cutaneous lesions of humans,
were identifled as L. b. panamensis, while the remainings from wild animals were identified

as L. mexicana amazonensis (Mimori et al., 1989). Thus, the species of Leishmania mostcornmonly found in our study areas endemic for leishmaniasis in humans was considered to12Institute for Laboratory Animals, Kochi Medical School, Kochi 781‑51, Japan
Department of Parasitic Diseases, School of Medicine, Kumamoto University, Kumamoto 860,

Japan
3 Departamento de Parasitologia, Instituto Nacional de Higiene y Medicina Tropical, Apartado
43961, Guayaquil, Ecuador
Department of Clinical Pathology, School of Medicine, Nihon University, Tokyo 173, Japan

5

Department of Parasitology, Kochi Medical School, Kochi 781‑51, Japan332

be L. braziliensis complex.

Little information, on the other hand, has been available on epidemiological features ofthe disease in this country (Hashiguchi et al., 1984). Such insufficient epidemiologicalinformation has partly been due to the lack of reliable diagnostic tool in field studies.
Recently, it has been reported that a soluble leishmanial extract prepared from L. donovani
chagasi promastigotes was highly sensitive and specific for an intradermal skin test for
American visceral leishmaniasis patients (Reed et al.. 1986). In order to obtain a better
understanding of the epidemiology of leishmaniasis in Ecuador, we therefore designed apreliminary survey to evaluate a similarly prepared skin testing antigen.
The present paper deals with the finding that the soluble antigen prepared from L.
braziliensis promastigotes can be readily employed in skin testing for the screening of
cutaneous and mucocutaneous leishmaniasis in areas of Ecuador endemic for these diseases.

MATERIALS AND METHODS
Study areas and subjects

The present examinations were carried out during the dry season from July to August,1986 in Ecuador. A preliminary study using skin test (ST) and enzyme‑linked immunosor‑
bent assay (ELISA) was made on 63 subjects with active dermal lesions who visited theInstituto Nacional de Higiene y Medicina Tropical (INHMT) , Guayaquil, and rural healthcenters and hospitals in several endemic areas of Ecuador. An epidemiological survey was
conducted on 115 inhabitants in Selva Alegre, Esmeraldas, Ecuador by performing ST and
ELISA. Thirty four school children in Gramalote Chico. Los Rios, were also ex mined by ST
alone. Thus, a total of 212 subjects were tested by leishmanial ST and/or ELISA in thisstudy.

ELISA
L. b. braziliensis (MHOM/BR/75/M 2904) obtained from Dr. P. Desjeux. PDP, WHO(formerly Instituto Boliviano de Biologia de Altura, Bolivia) was cultured with Pan's
medium (Pan, 1984). Soluble extract prepared from the harvested promastigotes was used
as an antigen. The ELISA procedure was performed according to the method described byMimori et al. (1987). All the serum samples tested were diluted 1:10. Absorbance values ofmore than OD 0.25 at 500 nm were employed as criteria for evaluating positive serum, since
the absorbances in tested control sera frorn 66 healthy individuals in Ecuador were less thanOD 0.25.
Intradermal skin test
A soluble antigen used for skin testing in this study was prepared by the method of Reedet al. (1986). The promastigotes of L. braziliensis were harvested and washed five times witha balanced salt solution. After the final washing the parasite pellet was resuspended in 5

volumes of distiled water, and a freeze‑thawing procedure with aceton‑dry ice and tepid
water was repeated 10 tirnes. The disruped parasites were diluted in PBS and centrifuged at
10,000 Xg for 30 min at 4'C. The supernatant was adjusted to 250 pg protein concentration
per ml before filtration with a 0.45 p sterile filter. One‑tenth ml of the antigen solution was

injected intradermally in the flexor surface of the forearm. Induration size of more than 5 mm
at the site 48 hours after the injection was considered to be a positive reaction based on thecriteria employed by Reed et al. (1986).

333

Biopsy smear specimens
Smear samples were taken froin the margin of ulcerated lesions, stained with Giemsa or
Wright solutions, and examined with an oil immersion objective. At least 100 fields were

examined.

RESULTSIn our initial study, an evaluation of ST and ELISA was made on 63 subjects with active
dermal lesions who visited INHMT, rural health centers and hospitals in Ecuador (Table 1) .

In this case 45.5% were positive for leishmanial amastigotes in smear specimens. The ST and
ELISA positive rates were significantly high in the parasite positive group compared with theparasite negative one (P<0.1 in ST, P <0.05 in ELISA, and P <0.05 in the both tests) .
However, a relatively high positive rate for ST and ELISA was observed in the parasite
negatives. This high rate suggests that a considerable number of true leishmanial patients
may be included in those parasite negatives. No significant difference in average induration

size between parasite positives (17.4d:9.0) and negatives (13.6i5.7) was observed. These
data indicate that the ST and ELISA are very suitable tools in the screening of Ecuadorianleishmaniasis.
Using these diagnostic tools, an epidemiological survey was conducted in Selva Alegre,

Esmeraldas (Table 2). Of 115 subjects examined dermatologically for leishmanial ulcers,
nodules and scars, 10 active and 24 of 28 healed cases were discovered from the physicalexamination and interview prior to the skin testing. The remaining 4 of the healed cases weredetected by re‑examination after these had shown ST‑positive results. All the leishmanial

scars in such cases were observed on the usually unexposed areas of the body. We went
through the same experience in another endemic area, Gramalote Chico, Los Rios. In thesurvey, typical leishmanial scars were confirmed in 6 out of 8 ST‑positives among 34
examinees after skin testing.
In Selva Alegre, a parasitological examination was performed on 7 out of 10 subj cts

with active dermal lesions. Only one patient was positive for leishmanial amastigotes, andhe also showed positive responses to ST and ELISA. The ST and ELISA positive rates
among these subjects were 80.0% (8/10) and 66.7% (6/9), respectively. In the subjects withhealed dermal lesions, the positive ST and ELISA rates were 79.2% (19/24) and 71.4%
Table I Results of a preliminary evaluation of intradermal skin test and ELISA in 63 subjects
with active cutaneous (leishmaniaD Iesion in Ecuador

Smearspecimens*

+

Total

No, examinedf emalemale

Positive rate per examineest
skin test

ELISA skin test & ELISA( age)(age)

14(2‑66)

16(1‑66)

93 . 3 ( 14/15) 92 . 3 (24/26)

90 . 9 ( 10/ll)

18(1‑66)

15(7‑61)

73 . 7 (14/19) 75 . 7 (22/29)

56.3 (9/16)

32

31

82 . 4 (28/34) 83 . 6 (46/55)

70 . 4 ( 19/27)*Materials from ulcer lesions were fixed, stained and then examined microscopically.
t Examinees were not the same number in each test, mainly because of follow‑up difficulties.

334

Table 2 Correlation between clinical diagnosis and immunodiagnosis in 115 inhabit‑
ants in Selva Alegre, Esmeraldas, Ecuador, using skin test and ELISA
Reaction toskin test

ELISA

active*healedt

+
+

+

5

15

2

4

+
(sub‑total)

N D

ND

No.
without

No. with signs

Total

signs
20
2

8

1

1

6

8

l

4

64

69

9

24

72

105

1

1

4

4

ND

ND

4

4

l

1

(sub‑totaD

1

4

5

10

Total

lO

28

77

115

*Persons with leishmanial ulcers.
tPersons with leishmanial scars.

*Not done.

Table 3 Summary of the results of clinical, parasitological and immunologicalexaminations in 212 subjects in Ecuador

No. with signs

Reaction toskin ELISA

smear specimenst

+
+

+

11

12

l

4

+
(sub‑total)

1

5

No.
without

15
4

l

39
2

10

6

13

1

4

64

69

3

131

12

21

23

72

4

4

6

2

13

8

6

2

5

(sub‑total)

19

17

Total

31

38

NDS

ND

ND

ND

Total

signs

NDS

+

test

healedt

active*

16

27
30

30
8

3

12

33

81

35

105

212

*Persons with leishmanial ulcers.
tPersons with leishmanial scars.
tMaterials from ulcer lesions were fixed, stained and then examined microscopically.
SNot done.

335

Table 4 Frequency distribution of induration size in leishmanialskin test positive subjects ¥ 'ith active or healed
cutaneous (leishmanial) Iesions

Induration No. Active lesions

size (m,m)
Positive%
sub jects
No.

Healed lesions

No.

%

2

8.0

5‑10

11

9

25 . O

11‑15

10

10

27 . 8

16‑20

18

7

19 . 411

44 , o

21‑25

16

6

16 . 71040 . o

26‑30

2

2

5.6

31

4

2

5.6

2

8.0

(20/28), respectively. Twenty subjects who showed positive reactions for both tests had
either active or healed lesions. It was found that five subjects with those signs were negative

for both the ST and ELISA, while eight without active or healed lesions were positive for oneof the two tests. Coincidence rates of ST and ELISA among 105 subjects, 33 with and 72
without the signs, who received both tests in Selva Alegre, were as follows: both positive,
19.0%; both negative, 65.7%; and positive against one of the two, 7.5%.

Table 3 summarizes the results of clinical, parasitological and immunological examina‑
tions of 212 examinees in the current study. Coincidence rates between both tests among 59

subjects who had clinical signs and received the two tests were as follows: positive andnegative for the two tests, 66.1% and 8.5%, respectively; positive against one of the two,
13.5% in ST and 11.9% in ELISA. Furthermore, 64 (88.9%) out of 72 subjects who had no
clinical signs and received the two tests did not react against these tests, completely. Thus,a close agreement between dermal clinical signs and immunodiagnosis was demonstrated
with a few exceptions.
Frequency distribution of the ST induration size in positive subjects with active or healed

dermal lesions is summarized in Table 4. Average induration size was 15.7i7.7 in active, and20.0d:5.4 in healed cases. The size was significantly different between both cases (P <0.05) ,showing a strong ST reaction in the healed one. No siguificant difference was found in theinduration size between the patients with active and healed cutaneous lesions, when consid‑
ered with regard to either sex or age.

DISCUSSION

The present study was carried out to evaluate two immunodiagnostic tools, ST and
ELISA, in the screening of cutaneous leishmaniasis in the endemic areas of Ecuador. In theepidemiological survey in endemic areas, a presumptive diagnosis is often made on the basisof clinical diagnosis and Montenegro skin test (Mayrink et al.. 1979; Werner and Barreto,1981). A definitive diagnosis of leishmaniasis requires demonstration of the etiologic agent
from the lesions. However, the visualization of protozoa in tissue samples and the isolationof parasites from active lesions by culture methods were fraught with difiiculties. Weigle etal. (1987) recommended two diagnostic procedures based on their studies, i.e., dermal
scraping smears for immediate diagnosis and culture of aspirates for a definitive para‑336

sitological diagnosis of cutaneous lesions. In the present survey, parasites were observed in

31 (44.4%) of 69 subjects presenting active lesions (Table 3).

As shown in Table 3, ST‑positive rate was 81.8% (36144) of the total skin tested
examinees with active lesions. In the parasite positive cases, furthermore, all subjects but one

proved to be positive for the ST. On the other hand, the positive rate in subjects withoutclinial sigus was only 3.8% (4/104). It is well recognized that the immunological methods,

especially ST and ELISA, are useful tools for diagnosis of New World cutaneous leish‑
maniasis (Bray, 1980). The reliability of these tests, however, has been hampered by the
problem of cross reactivity with various species within the family, Trypanosomatidae, as wellas with other microorganisms such as mycobacteria (Mauel and Behin, 1982). We have nodata with regard to cross reactivity of the present L. braziliensis antigen preparation. Reed･et al. (1986) , however, reported that their similarly prepared L. d. chagasi‑soluble extract

produced no positive responses in either normal controls, tuberculosis patients, or schis‑tosomiasis patients, and less than 5% positive responses in persons with Chagas' disease.
From this information and our results, it is zissumed that the present preparation also showsa lesser degree of cross rezictivity.

Table 3 suggests that there may be close agreement between clinical signs and results ofimmunodiagriosis, except for 12 subjects in Selva Alegre and 2 in Glamalote Chico. Foursubjects in Selva Alegre, showing negative reactions against both tests, may have had otherderrnatological lesions mistaken for leishmanial scars. The remaining cases suggest thepossibility of either cross‑reactions with other infectious agents or oversight of leishmaniallesions.

Diagnosis using ST and ELISA is commonly used against New World cutaneous leish‑
maniasis. However, the standardization of skin test antigen has not yet been done sufficiently
to hav6 a satisfactory results. In this paper, we evaluated the soluble extract obtained by themethod of Reed et al. (1986) from ruptured L. braziliensis promastigotes, as a ST antigen.
Our results obtained showed that the extract was found to be highly sensitive and specificagainst Ecuadorian cutaneous and mucocutaneous leishmaniasis patients. It was concluded
that the use of the present ST, together with ELISA, would be useful in diagnosis for patientswith active and healed leishmanial lesions in the endemic areas of the disease.

ACKNOWLEDGEMENTS
We express our heartfelt thanks to Dr. F. Parra Gil. Director of the INHMT, Guayaquil,
Ecuador, and also express our appreciation to Dr. S.G. Reed of Seattld Biomedical ResearchInstitute, U.S.A. for critical reading of the manuscript. We are much indebted to all themembers of the Departamento de Parasitologia, INHMT, for their support. We are extreme‑ly grateful to Dr. P. Desjeux of PDP. WHO (formerly Instituto Boliviano de Biologia de
Altura, Embajada de Francia, Bolivia) for his generous supply of Leishmania reference
strains.

This study was supported by the Grant‑in‑Aid for Overseas Scientific Research of the
Ministry of Education, Science and Culture, Japan (Nos. 61041059 and 62043055)

337

REFERENCES
1 ) Bray, R.S. (1980): Leishmaniasis, In: Immunological investigation of tropical diseases, ed. by

Houba, S.V., 65‑74, Churchill‑Livingstone, Edinburgh2 ) Hashiguchi, Y., Coronel, V.V. and Gomez, E.A.L. (1984): An epidemiological study of leish‑
maniasis in a plantation "Cooperativa 23 de Febrero" newly established in Ecuador, Jpn. J.
Parasitol., 33(5) , 393‑4013 ) Mauel, J. and Behin, R. (1982): Leishmaniasis: Immunity, immunopathology and im‑
munodiagnosis, In: Immunology of parasitic infections, ed. by Cohen, S. & Warren, K., 2nd ed.,299‑355, Blackwell Scientific, Oxford
4 ) Mayrink, W., Williams, P., Coelho, M.V., Dias, M., Martins, A.V., Magalhaes, P.A., DaCosta,

C.A., Folcoa, A.R., Melo, M.N. and Falcao, A.L. (1979) : Epidemiology of derma] Ieishmaniasis
in the Rio Doce Valley, State of Minas Gerais, Brazil, Ann. Trop. Med. Parasit., 73 (2) , 123‑137
5 ) Mimori, T., Grimaldi, G.Jr., Kreutzer, R.D., Gomez, E.A.L., McMahon‑Pratt, D., Tesh, R.B. andHashiguchi, Y. (1989) : Identification of Leishmania isolated from humans and wild animals in
Ecuador, using isoenzyme electrophoresis and monoclonal antibodies, Am. J. Trop. Med. Hyg.,
40(1), 156‑160

6 ) Mimori, T., Hashiguchi, Y., Kawabata, M., Gomez, E.A.L. and Coronel, V.V. (1987): The
relationship between severity of ulcerated lesions and immune responses in the early stage of
cutaneous leishmaniasis in Ecuador, Ann. Trop. Med. Parasit., 81 (6) , 681‑685
7 ) Pan, A.A. (1984): Leishmania mexicana: Serial cultivation of intracellular stages in a cell‑freemedium, Exp. Parasitol., 58(1) , 72‑80

8 ) Reed, S.G., Badaro, R., Masur, H., Carvalho, E.M., Lorenco, R., Lisboa, A.; Teixeira, R.,
Johnson, W.D.Jr. and Jones, T.C. (1986) : Selection of a skin test antigen for American visderal

leishmaniasis, Am. J. Trop. Med. Hyg., 35(1), 79‑85
9 ) Rodriguez, J.D.M. (1974): Genero Leishmania, In: Lecciones de Parasitologia Humana, 5thed., 170‑185, Univefsidad de Guayaquil, Guayaquil, Ecuador
10) Weigle, K.A., Davalos, M., Heredia, P., Molineros, R., Saravia, N.G. and D'Alessandro, A.
(1987): Diagnosis of cutaneous and mucocutaneous leishmaniasis in Colombia: A comparisonof seven methods, Am. J. Trop. Med. Hyg., 36(3), 489‑496
11) Werner, J.K. and Barreto, P. (1981): Leishmaniasis in Colombia: A review, Am. J. Trop. Med.
Hyg., 30(4) , 751‑761

338

南米エクアドル共和国リーシュマニア症の皮内反応と
ELISA法による疫学調査
古谷 正人・三森 龍之・E．A．L．Gomez
V．V．Coronel・川端 真人・橋口 義久

南米エクアドル共和国における，皮膚型および皮膚粘膜型リーシュマニア症の疫学調査での皮内反応（ST），およびELISA法の有用性について検討した結果を報告する。
皮内反応抗原は，L鵡h規α％∫αわ観g漉εηsゑs promastigote型原虫から作成した。潰瘍病変（1eish・

manial ulcers）保有者72名におけるSTとELISAの陽性率は，それぞれ8L8％（36／44）および
81．3％（52／64）であった。これらの患者の内，原虫が認められた者は検査が実施できた69名中31名（44．9％）であった。原虫が証明された患者において，STで1名，ELISAで2名が陰性であった以外は，全ての者が両検査に陽性であった。治癒病変（leishmanial scars）保有者35名におけるSTとELISAの陽性率は，それぞれ86．2％（25／29）と72．4％（21／29）であった。一方，病変が認められない者におけるSTとELISAの陽性率は，3．8％（ST）および8．2％（ELISA）と非常に低率であった。

同国Esmeraldas地方のSelva

Alegre村で，リーシュマニア症の罹患状況を調べた。その結果，115名中38名に何らかの病変（潰瘍病変10名・治癒病変28名）が認められ，その内の33名（86．8％）

がSTとELISA両検査法に陽性であった。
以上の結果から，同国リーシュマニア症流行地域での疫学調査に，今回の両検査法が有効であることが示された。

日熱医会誌．，第17巻

第4号

339

1989 339−346頁

アマゾン流域日系移住地，トメアスー地区における回虫およびトキソプラズマ感染状況についての考察
佐藤久美子1・狩野 繁之2・MILTON M．HIDA3・柳澤
紘4・小河原はつ江1・鈴木
守2
1989年6月10日受付／1989年10月10日受理

はじめにで，それらの結果を併せて報告する。調査は何れも，免疫学的手法による抗体検査によって行った。

日本国内における寄生虫感染症は，戦後の混乱期にあたる1945年から1950年にかけて急激に増方加したが，1950年を境に急速に減少の傾向を示し
た（Kobayashi，1983）。厚生省および日本寄生虫

法

調査地域

予防会の報告による都道府県別寄生虫感染状況に

調査地トメアスーは258km2の開拓地であり，よれば，最近の蠕虫類感染者は嶢虫を除くと0．5％

州都ベレン市の南，直線距離にして125kmに位にも満たない（予防医学ジャーナル，1980−

置する（図1）。1962年より入植が開始され，熱帯

1989）。しかG国外，特に衛生状態などに問題の雨林地帯の気候の中，現在70家族350人余りの日系多い発展途上国では，蠕虫感染者はかなりの数に人たちが，胡椒，カカオ，コーヒーの栽培や，畜昇っている。一方原虫類感染については，我が国産により生活を営んでいる。今般の調査対象とでは全国的な調査は行われておらず，したがってなったトメアスーの住民のうち，日系人は102名，

その感染の実態については必ずしも明らかではな

現地労働者は119名であった。．

いが，トキソプラズマについては日本においても被検血清

感染者が多数報告されている（中山，1968；伊勢被検血清は1988年8月にブラジル，アマゾンのら，1979；白坂ら，1972）。我々はこのたびブラジ

トメアスー地区において5歳から85歳までの男女

ル，アマゾンのトメアスー地区（図1）で，マラから採取した。分離した血清にはNaN3を添加しリアに関する疫学調査を行う機会を得たが，そのて低温で空輸し，一2ぴC下に保存した。また比較対・際に，住民における抗回虫抗体の高い保有率が見象とした日本人の血清としては，群馬大学医学部いだされた。さらに，その地域における比較的濃付属病院の供血者，17歳から59歳までの健康人男

厚なネコの棲息密度から，同地域には高率にトキ女から交差試験用に採血し，低温に保存した血清

ソプラズマ感染者が存在することも予想された。

を使用した。

そこでこれら住民におけるトキソプラズマの特異

回虫抗原特異IgE抗体の測定

抗体保有状況について調査するとともに，比較の回虫抗原としてヒト回虫と抗原性がほとんど同

ため日本国内における一般人の抗回虫抗体およびじといわれている豚回虫。4s昭漉s鰍吻を前橋屠

抗トキソプラズマ抗体保有の実状をも調査したの場より入手し，虫体をリン酸緩衝液（pH7．2）中群馬大学医療技術短期大学部
群馬大学医学部寄生虫学教室
Faculdade de Medicina de Botucatu−UNESP，Brazil
杏林大学医学部公衆衛生学教室

340

B〔azilズ
T㎝e−Agu

Bel6m

口
■

−

The細旧zon

Figurel Tom6−Agu is a Japanese settlement covering10by25．8km space，
located l25km to the south of Bel6m（latitude2。31 S，longitude48。
22 W）．The temperature of Tom6−Agu is21−33℃and the rain fallina yearamounts2，635mm

on

the

average．The

place makes

highlyvegetatedflatareaat11−30mabovesealevelandwith
naturalwater

networks

byAmazon branches．

でヒスコトロンを用いて磨砕し，その遠心上清を蛋白量25μg／mJに希釈して使用した。特異IgE抗体の測定は，佐藤ら（1986）がすでに報告した

結果

回虫抗原特異IgE抗体保有状況

nUOreSCenCeenzymeimmUnOaSSay（nuOreS・

図2にトメアスー地区の人を対象として調査しcence−EIA）法に準じて行った。特異IgE抗体量た，回虫抗原特異IgE抗体保有状況のヒストグラ

は，Micro FLUOR readerの示すrelative
fluorescencemit（RFU）をそのまま使用した。

値は，10−924まで幅広く分布していた。ここで，抗原蛋白量の測定は，フェノール試薬を用いた

ヒストグラムより，通常我々が本法に適用していFolin法のLowryらの改良法によって行い，標る陽性判定算定基準（林，玄番，1986）に従っ

準蛋白として牛血清アルブミンを使用した（Low・て，RFU値≧73．5を示す例を陽性者と決定した。

ムを示した。被検者221名の，特異lgE抗体RFU

ry6麺乙，1951）。

その結果，抗回虫IgE抗体陽性者は221名中99名

トキソプラズマ特異lgG抗体の測定・で，抗体陽性率は44．8％であった。

トキソプラズマ特異抗体の測定は，平井ら

次に，群馬大学医学部付属病院供血者血清の，（1977），坪田ら（1977）の報告に従ってトキソテ

抗回虫抗体保有状況について図3にまとめた。被ストーMT（栄研化学KK）を用いたラテックス凝検者182名の特異IgE抗体RFU値は，1H23ま集反応のマイクロタイター法によって行った。判でに分布しており，上記の陽性判定基準に従えば，

定はトキソテストーMTの判定基準に従って抗体

抗体陽性者は182名中4名であり，抗体陽性率は価で表し，32倍陽性以上を陽性者と判定した。

2．2％であった。

トキソプラズマ特異IgG抗体の保有状況
トメアスー地区の人を対象として，ラテックス

凝集反応により検査した抗トキソプラズマ抗体の抗体価と，その出現頻度を図4に示した。また，

341

RFU
RFU

918〉
864〉
810〉
756〉
702〉
648）

108．
99．

90．
81．
72．
63．
54．

59角〉

45．

540〉
486〉
432〉
378＞
324〉
270〉
216〉
162〉
108）

36．
27．
18．

9．
0．
30

二〇

・午

10

5η〉

0〉

0

200

100
髄o．of

Figure2Histogram of

the frequencies

bodies（RFU values）in

Cases

of／4soσ漉一antigen−speci且c−IgE

Tom6−AGu

anti・inhabitants，

RFU

117〉

81〉
72〉
63＞
54〉
η5〉
36〉
27〉
18〉
9〉

10

20

Figure 3Frequencies ofvalues）of

30

η0

50

髄0，0f

Cases

60

70

the∠Ls砒7蕗一antigen−specific−IgEJapanesedonorsof

Gunma

University

80

antibodies（RFU

Hospita1．

群馬大学医学部付属病院供血者400名についての400名中の抗体陽性者は65名で，陽性率は16．3％

結果もあわせて示した。トメアスー地区住民の被であった。次にトメアスー地区住民の年齢別の抗検者218名中抗体陽性者は164名で，抗体陽性率は体価を検討した結果を表1に，年齢別の抗体陽性

75，2％であった。男女別にみると，男は118名中90率を表3に示した。また，群馬大学供血者の年齢

名陽性であり陽性率は76．3％，女は100名中74名

別抗体価の分布は表2に，年齢別の抗体陽性率を陽性であり陽性率は74％とほぼ等しい値を示した。

表3にまとめた。この結果から，トメアスー地区

これに比較して，群馬大学医学部付属病院供血者のトキソプラズマ感染者はどの年齢層にも広がっ

342
n

32788
163q8
81 92

I

4096

Tom6‑A u inhabltants

C:l Gunma Unlv. blood donors
20q8,: 102q

L

512

Q,

*‑ 2SSl 28

8

Pas i t lve

32
Ne9at I ve

l 6(

lO 20

50 60 70 80

50 qC

90 1 OO

Frequency of anti‑Toxoplasma antlbodies (X)Figure 4 Comparative frequencies of anti ‑ Toxoplasma antibodies

between Tom6‑Acu inhabitants and blood donors inGunma University Hospital.

Table l High anti‑ Toxoplasma antibody titers in age groups of Tom6‑Acu inhabitants

Age group

Titer (No, of cases)
< 16

32

64 128 256
1*

It

2

5

6

3

4

9

7

512

l,024 2,048 4, 096 8,192 16 384 32 , 768
1$

< 10

1*

10‑19

22

20‑29
30‑39

14

8

1

4

3

7

2

3

3

40‑49

4

1

3

2

8

6

6

3

50‑59

3

2

2

l

60‑69

1

2

4

3

3

1

1

70

1

2

'2 years old3t8 years old

1

5

2

4

4

4

2

2
2

l
1
l

l

9 years old

Table 2 Distribution of anti‑Toxoplasma antibody titers in age groups of blood
donors in Gunma University HospitalAge group

Titer (No. of cases)
< 16

32

64

1 28

256 512l , 024

2 , 048

2

1

10‑19

32

20‑29

133

1

2

1

4

30‑39
40‑49

110

4

4

4

8

5

4

55

2

3

5

4

7

l

50‑59

5

1

343

Table3Comparison
body

of

titerspositive ratesin

Positive

Ageg「oupofanti−To劣oμ召s彫8anti・each grouprate，％（No．of

examined）

T。m←AguinhabitantsGunmaUni玩bl。。dd。n。rs

ND
（32）

＜10

75．0 （4）10−19

63．3（60）

20−29

73．1（52）30−3976．5（34）

20．9

40−49

88．2（34）28．6（77）50−59

76．9（13）

28．6

60−69

94．1

70≦

75．0 （4）

ND：Not（17）O

8．3（145）
（139）

（7）

ND
ND

done

ており，5歳一10歳に75％の陽性率，10歳代でもう場合でも，検便というオーソドックスな方法は63．3％の抗体陽性率を示した。また，抗体価は32−

しばしば困難である。従って，マラリア調査など

32，764倍陽性まで分布していたが，高い抗体価をに伴い採取される血清を使って，免疫学的方法に示す人が若年から老年にわたってほぼ同じ比率みより感染率を算出することが時として現実的となられた。一方，群馬大学供血者では，10歳代の被る。免疫学的方法の中でも，本報告において用い検者32名中に抗体陽性者はみられず，20歳代以上た特異的IgE抗体を測定する手法は，感染との相ではどの年齢層でも32−1，024倍の抗体価を示し，関がかなり高いものと想定される。これまでに述

抗体陽性率は年齢が高くなるにつれて上昇した。

べた日本国内の感染状況から考えると，群馬大学

ネコの飼育状況

病院の検体での陽性者が182名中4名であること

ペットとしてネコを飼っているかどうかを，個は，周辺環境が農村であることから十分にうなず

人別に採血時にアンケート調査した。解答のあっける数字であり，このことから考えてトメアスーた278名中，211名（77．6％）がネコを飼っている地区での抗体陽性率が43．4％という事実は，この家庭に居住していた。

地区の回虫感染者が相当数に上ることを意味する
ものと考えられる。いうまでもなく，本法によっ

食肉嗜好

ブタ肉と牛肉とどちらを食べるか，さらに，生た場合，他の線虫類内の交差反応による偽陽性反焼けを好むかどうかの質問を採血時に行った。牛応を含むことを，考慮しなければならない。また，

肉の生焼けを好む人は10％にすぎず，ブタ肉を生特異的IgE抗体は，虫体排出後も長期間陽性を維焼けで摂食する人はいなかった。

持することをも計算にいれなければならない。

対象となったトメアスー地域の日系人は，幼児

考

察を除いてそのほとんどが戦後移住した一世農民であり，食習慣等をはじめとする一般生活形態は，日本国内における回虫感染率は，多くの府県で本邦の日本人に類似している。しかし，上下水道

今や0．5％にも満たない感染状況であるが，アジ

設備や汚水，し尿処理等，生活に基本的な衛生条

ア，アフリカ諸国における寄生虫検査成績を見る

件が現在の日本とは依然として大きくかけ離れてと，回虫感染者は多数である（建野ら，1984；横おり，回虫の陽性者が高いことはその反映と思わ

川ら，1983）。

れる。戦後の日本においては，駆虫薬を集団投与

熱帯地調査においては，採取材料の種目を限らすることにより，環境衛生状態の改善がr般化すざるを得ない制約があるため，回虫感染調査を行る以前に，回虫，鈎虫等の駆逐に成功している

344（Kobayashi，1983）。トメアスーという比較的閉特別の要因を考えなければならない。本調査での鎖した地区では，戦後日本の回虫・鈎虫駆除計画

問診により，当地の77．6％の人がネコを飼っていを応用して，一定の方針のもとに集団駆虫を繰返る事実が判明している。しかし，ほとんどの人はすことが効果的と考えられる。

生肉を食べる習慣は無い。ノ」・林ら（1976）による

日本におけるトキソプラズマ感染状況について

養豚地帯における調査によれば，ブタのトキソプ

は，これまでにも多数の報告がなされ，感染者数

ラズマ抗体陽性率の高い地域においては，ヒトもは成人のおよそ1／3に見られると成書に記されてまた高い陽性率を示し，両者がよく相関する事実いる（吉田，1987）。また，年齢の値％，即ち20歳が見出されている。これは恐らく，ブタもヒトも代ならば20％の感染があるともいわれてきた。最ネコから排出されるオーシストによる感染を受け

近，平井ら（1989）は東京における感染状況を調る結果であろうと推測される。即ち，ヒトもブタ査し，一般集団のトキソプラズマ抗体保有率はも高い陽性率を示す地域においては，ネコが高率

25．7％であり，．年齢の増加に従って保有率も上昇にトキソプラズマに感染し，オーシストを排出すしたと報告した。また，妊娠中の女性の抗体保有る例が多いことが想定される。トメアスーにおい

率は1982年一1986年までほとんど変らず，平均ても，ネコのトキソプラズマ感染率，オーシスト

15．9％の保有率であったという。我々も1987年に排出率などについて調査を進め，対策をはかる必

群馬大学医療短大学生と群馬大学病院供血者につ要がある。

いて調査したところ，学生の抗体保有率はおよそほぼ同一の生活習慣を営む同一民族が，移民な4．0％であり，20歳代から50歳代にわたる病院供血どによる異なった環境におかれた時，ある特定の者では20．8％であることを見出している（佐藤，

寄生虫病に対して著しく異なった感染率を示すこ

小河原，1987）。今回の調査結果でも，若年層におとがあり得る。このような場合には，寄生虫感染

けるトキソプラズマ感染率は低く，高齢者になるの背後にある生態系とのかねあいを研究する最もに従って感染率が上昇する傾向が確認された。一

よい機会となるだろう。トメアスーにおけるトキ方，国外でのトキソプラズマ感染状況についても

ソプラズマ感染例は，本邦の日本人に比較して異多数の報告が見られ（Arene，1986；Shenone窃

常に多い事実が証明された。トメアスーは上記の認，1982−1986），地域を問わず全世界的規模で多事例の1つとして，トキソプラズマ感染の生態を数の感染者が存在しているようであり，日本国内

さらに解明する上に，有益な野外研究の場を提供

より高感染率を示す地方も多い。ブラジルにおけすることと考える。

るLamb，Feldman（1968）の報告によると，抗トキソプラズマ抗体陽性率は，全土で52％，アマ
ゾン地域で56％である。今回の我々の調査では，

まとめ

トメアスー地区住民の抗体陽性率はおよそ70％にブラジル，アマゾン流域の日系移住地トメア

達し，また若年者から老年者にかけてどの年齢層

スー地区住民について，回虫特異IgE抗体およびの感染率も等しく高く，定量的にみた場合，抗体

トキソプラズマ特異IgG抗体保有状況を，免疫学価が日本国の感染者の平均を大きく上回っている的手法により調査した。比較対象として，群馬大

事実が証明された。特に，10歳以下の小児4人中

学付属病院健康人供血者血清について，同時に検3人までが陽性例であった点が注目される。陽性

査した。トメアスー地区の抗回虫抗原特異IgE抗例は8歳児2名，9歳児1名の内訳となるので，体陽性者は，被検者221名中99名で，陽性率は母体から胎盤を経て移入された抗体が残存した結44．8％であった。これに比較して行った群馬大学

果とは考えられず，いずれも後天性感染の結果と供血者の結果は，被検者182名中抗体陽性者4名判断される。トメアスーの小児の間での感染率にで，陽性率は2．2％であった。トメアスー居住者のついては，今後改めて調査を行う必要がある。

抗体陽性率が高いのは，その地域の上下水道設備

トキソプラズマ感染が高率に見られることは，や汚水，し尿処理等の衛生条件が一因となってい345

ると考えられた。一方トキソプラズマ感染者につれ』た。

いての調査は，トメアスー地区住民218名中，抗体陽性者は164名で，陽『生率は75．2％，群馬大学供血

謝者400名中抗体陽性者は65名，陽性率は16．3％で辞あった。トメアスー地区住民陽性者の抗体価は，稿を終るにあたり，技術協力を頂きました石倉

若年者から高齢者まで異常に高い値を示した。問知枝子氏（群馬大学医療技術短期大学部）に深く診の結果，77．6％の人が家庭においてネコを飼っ

感謝致します。本研究は，1988年度日本学術振興ていることが判明した。しかし肉を生，または生会海外共同研究（研究代表者・鈴木に近い状態で摂食する習慣は認められなかったたとして行われた。

守）の一環

め，ネコが高い感染率の原因であることが推定さ

献

文1〉Arene，F．0．1．（1986）：The prevalence of toxoplasmosis among inhabitants of the Niger
Filia

Delta，

Parasitologica，33（4），311−314

2）林泰之，玄番昭夫（1986）：正常値ガイドブック，その臨床応用，55−57，宇宙堂八木書店，東京
3）平井徳幸，永井佳子，小林昭夫（1977）：トキソプラズマラテックス凝集反応（栄研マイクロタイター法）の検討，寄生虫誌．，26（増〉，31
4）Hirai，N．，Kobayashi，A．，Watanabe，N．，Makioka，A．and Kamio，M．（1989）：The prevalenceofToκoρ如s吻αantibodies

amonggeneraloutpatients

andpregnant

women

in

Tokyo

area，Jpn．

J．ParasitoL，37（4），261−262

5）伊勢やよい，有滝千恵子，竹谷扶美子，佐藤功栄，飯田 孝，島田孝吉（1979）：東京都における妊婦の抗Toxoplasma抗体について，寄生虫誌．，28（増），64
6）Kobayashi，A．（1983）：Changing
andinfectious

pattems

diseases，ed．by

Neil

of

parasitic

A．Crollandinfections

John

In

Japan，∫n

Human

H．Cross，137−168，Academic

ecologyPress，New

York，London
7）小林昭夫，西川洋昭，平井徳幸，飯田辰夫，佐々木栄英，土屋好文，鈴木 守，鈴木 守，辻岡孝，桜井勝男（1976）：一養豚地帯における人獣間の7b％o餌αs耀感染についての疫学的考察，寄生虫誌．，25（5），350−3558）Lamb，G．A．and

Feldman，H．A．（1968）：A

recmits，1964，III．Toxoplasma

dye test

nation9）Lowry，0．H．，Losebrough，N．」．，Farr，A．L．and
the

Folin phenol reagent，J．BioL

wide

serum

surveyofBrazilian

military

antibodies，Am．J．Epidemiol．，87（2），323−328
Randal1，R．」．（1951）：Protein

measurement

with

Chem．，193（2），265−275

10）中山一郎（1968）：トキソプラズマ，モダンメディア，14（6），312−322
11）佐藤久美子，中沢次夫，原 文子，小河原はつ江，土屋 純，巨智部直久（1986）：Release Fluoroim・

mmoassay（RFIA）法を用いた特異IgE抗体の検出法，アレルギー，35（6），313−319
12）佐藤久美子，小河原はつ江（1987）：若年層における最近のトキソプラズマ感染状況，群馬大学医療技術短期大学部紀要，8，187−190
13）Shenone，H．，Contreras，M．C．and
II．Prevalenceinregions

of

IV，V

the

human

Salinas，P．（1986）：Epidemiology

infection，studied

and VI，1982−1986，Boletin

by

means

of

an

of

toxoplasmosis

indirectinhaemagglutination

Chile．
test，

Chileno de Parasitologia，41（314），82−86

14）白坂龍暖，大須賀道代，和田芳武（1972）：伊豆七島におけるトキソプラズマ感染調査，寄生虫誌．，21（増〉，45

15）建野正毅，竹内勤，小林正規，田辺正信，三浦左千夫，浅見敬三，山口直人（1984）：インドシナ

難民の寄生虫感染状況，寄生虫誌．，33（増），110

16）坪田宣之，平岡謙一，大島寄生虫誌．，26（増），31

慧，渡辺俊子（1977）：トキソプラズマラテックス凝集反応について，

34617）予防医学ジャーナル（1986−1989）：（資料号）

18）横川宗雄，小林仁，畑 英一，稲臣成一，辻 守康，小島荘明，H．Miranda，N．Ibanez（1983）：
ペルー国，サン ホワン地区住民の寄生虫調査成績，寄生虫誌．，32（増），43
19）吉田幸雄（1987）：図説人体寄生虫学，2版，36−39，南山堂，東京

HIGH ANTIBODYPOSITIVETOXOPLASMOSIS IN AN

RATES

OF

AMAZONIAN

ASCARIASISJAPANESEAND

SETTLEMENTKuMIKo SATo1，SHIGEYuKI KANo2，MILToN M．HIDA3
HIRosHI YANAGIsAwA4，HATsuE OGAwARAI AND MAMoRu SuzuKI2
Received Jme101989／Accepted

Seroepidemiological
Brazilianstudiesonascariasis

October

and

lO

l989

toxoplasmosis

wereconducted

inaJapanese settlement，Tom6−Agu，in August，1988．The specinc IgE antibodies
antigen were assayed and99individuals out of221inhabitants in the area

against。4s侃冠s

were

found

positive（44．8％），while

Hospital，Japan，weresupplyor sewerageascarispositive（2．2％）．The

systemantibody。The

218were positive

only40ut

in

Tom6−Agu

specific

in

IgG

may

poor
be

antibodies

University

Hospital（16．3％）．The

results

in

in

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against

kept

cats

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of

their
of

cats

the

in

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conditions

thepositivehighToκ砂勉s耀wereof400werequestionnairehouses．Very
wasdonorssanitaryofTom6−ACu（75．2％），while650ut

inhabitants
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as

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that77．6％of

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answeredtobeattributable

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College of Medical Care and Technology，Gunma University，Maebashi371，Japan
Department of Parasitology，Gunma University School of Medicine，Maebashi371，Japan
Faculdade de Medicina de Botucatu−UNESP，Brazil
Department of Public Health，Kyorin University School of Medicine，Tokyo181，Japan

347

日熱医会誌．，第17巻 第4号 1989 347−350頁

症例報告

ヒトクイバエCoz4yZo扉αα窺hzoφoφh卿1こよる
ハエ症の輸入例

影井

昇1・村田以和夫2・倉橋

弘3

平成元年6月7日受付／平成元年8月26日受理
人体ハエ症は我が国でも数多くの症例が報告されているが，その殆どは偶然の寄生によるハエ症

タンザニア，ザンビアを訪問，同年2月20日帰国した。

である。ところが熱帯地域では，ある種のハエ幼旅行の途中，何回か睡眠中に，チクッとした虫

虫がその発育の為に宿主を必要とするものがあり，刺され感を経験したという。その後右腹側に5×

まれに人もその宿主にされるため，その際に病状5cm大の発赤を伴った腫張が見られ，中央部は潰の発現を見ることがある。その様なハエ幼虫感染

瘍を形成した。

は，その土地での生活環境に無知な旅行者に対し帰国翌日の2月21日午前9時頃，本人自身の指て起こっても決して不思議ではなく，Kagei6渉

圧により膿汁排出を試みたところ，無色の液と共磁（1974）が，ブラジルで感染し，日本で発見さに，体長1．5cmの虫体が飛び出してきたので，本人れたヒトヒフバエ（Z）飾㎜云06彪ho郷初瑚の患者

自ら東京都立衛生研究所に持参した。

を報告してから後，同種のハエによる感染例が藤原ら（1977），大滝ら（1978）によって中南米から寄生虫学的検索の帰国者から報告され，更に堀ら（1984）により3人のアフリカからの帰国者からCoz吻Jo伽属

持参時の摘出虫体はまだ生きており，写真1にハエ幼虫寄生例が発見され報告されている（1人示す様に，体長1．5cm，円柱状で体節は12節に分かはロダインコブバエC名04hαづ雇の寄生例，他のれ，2本の鉤型の口部硬皮（1abialsclerites）が2名はヒトクイバエσα窺h名砂oρh㎎1πの寄生例

突出し，それぞれの側に6本の小さな歯を持ったである）。

Sclerotized

本報告は，同じくアフリカから帰国した人でのintegmentがある。各体節には後方に向かって曲がる棘が密生し，後端には気門が見ヒトクイバエによる，ハエ症の患者の追加症例報られる。以上の形態から，本虫はヒトクイバエ

告である。

Co7めJoわ毎4η孟h名oρoρh㍑gπ（Blanchard，1893）の3令幼虫と同定された。

症
25歳

男子

例

ミュージシャン

1989年1月14日から国際交流基金による「ジャズおよび和太鼓」の公演の為，アフリカのセネガ
ルを皮切りにナイジェリア，ザイール，ケニア，

国立予防衛生研究所寄生虫部
東京都立衛生研究所
国立予防衛生研究所衛生昆虫部

なお，本幼虫のさなぎ化，更に羽化させての成虫での同定を考えて，しばらくその飼育を試みたが，途中で幼虫のまま死亡した。

348

Photo．1

Dorsalviewo∫third玉arva至stage

擁㎎πexpelied歪rom

t熱e

of

Co〆緩y♂oゐ臨虜％孟h鶉oρびlesion of rightabdomenofapat重ent，

考察

かったが，先にK』agei6オ認（1974）が報告した
ヒトヒフバエ寄生例に見られた様に，確実な診断

ヒトタイバエはサハラ砂漠以南の西アフリカーのつかないまま，良性の肉芽腫疑いで大きく組織

帯に広く分布し，その宿主としてチンパンジーやを切除されることもあるので，臨床医は本虫に対サル，ヒョウ等極めて多くの野生動物が報告されする十分な知識を必要とする。

ているが（Zumpt，1965は16種の野生動物と数種

一方，人の感染例ではさして問題はないと考えの家畜を挙げている），主たる保虫宿主はネズミ類られるが，感染動物の輸入例では，3令幼虫が輸といわれる。人体も宿主になりうる事が過去より入動物から脱落，さなぎ化，羽化，産卵して，再知られているが，その土地の者でない旅行者は，び人を始めとする動物へ寄生する可能性がある事

本症の存在を知らない。従ってアフリカで感染しは否定できないし，あるいは我が国に土着するかて，帰国後自国で発見されても不思議ではない。

も知れないという問題点を持っている。

その様な症例は現在迄に，我が国では堀ら（1984）

ヒトクイバエの成虫は，動物の排泄物で汚れたによってアフリカのコートジボワールのオフィス

乾いた砂や，着物の上に産卵し，そこで醇化した

街マンションに居住していて感染し，虫体が自然

幼虫が人を含む動物を襲い，皮下に侵入するので，脱出した1例と，母親の手で押し出して虫体を摘本症例もアフリカ公演中に幼虫との接触があった

出したという症例の2例が報告されている．そのものと考えられる。そのことは日時は定かではな

他，人体症例ではないが，ヒトクイバエの輸入例

いが，旅行中に何回か睡眠中に虫刺されを経験しは加納（1967）により，東アフリカより千葉県血たと本人も述べている事も，これを裏付けている。

清研究所に実験動物として搬入されたミドリザル

摘出時の虫体が，既に3令幼虫まで発育していたからも報告されており，恐らく今後も本虫の我が事は，幼虫侵入後5−6日で3令に達し，8日目

国における人，および動物での輸入例が見出され位で脱落する事を考えると（Zu鵬pt，1965），本公

るであろうことは十分示唆される。

演旅行の日程のかなり後の方の行程で感染した事

本虫輸入に伴っての問題点は，人による輸入時が窺えるが．本虫の分布域と行動の上からは若干の確実な診断と治療についてである。幸いにも，のずれが見られ，もう少し早かったのかも知れな

堀ら（1984）の症例も今回の著者らの例も，幼虫

いo自身が自然に脱出したり，患者自身が虫体を押し出したりして，大きな症状の出現までには至らな

349

ま

脱出するのを見た。その虫体についての寄生虫学とめ的検索を行った結果，ヒトクイバエCozみJo伽

1989年1月から1カ月以上にわたるアフリカ

α鋭h名砂oρ加即（Blanchard，1893）の3令幼虫ででの音楽公演旅行を終えて帰国した患者が，帰国ある事が解り，我が国における本虫寄生輸入症例

直後に，右腹側部に形成された潰瘍部を，患者自の第3例として報告した。

身の手指によって圧迫したところ，1匹の虫体が

参

考

文 献

1）堀栄太郎，山口勝幸，和田芳武，山浦常，加納六郎，篠永哲，藤野信之（1984）：カメルーン
及びコートジュボアールでみられたクロバエ属，Co幼1Jo伽幼虫によるハエ症myiasis3例，衛生動物，35（1），87−90

2）藤原紀男，伊藤洋一，柳沢十四男（1977）：Z）召7耀ホoδ毎ho窺 ％づ こよるハエ蛆症，日熱医会誌．，

5，303）Kagei，N．，Hirama，T．，Ogawa，Y．，Toga，N．，Takaoka，M．and Kurahashi，H．（1974）：A
cutaneous myiasis causing by1）6 n碗o伽ho痂脇（Cuterebridae；Diptera）imported from
Brazi1，Jap．」．Trop．Med．Hyg．，2（3／4），181−185

4）加納六郎（1967）：日本にはいった動物寄生バエ，インセクタリウム，4（3），12
5）大滝倫子，小林美咲，佐藤吉昭，加納六郎，寺田雄之助（1978）：メキシコ産ヒトヒフバエによる皮膚蝿症の1例，皮膚臨床，20（6），461−4646）Zumpt，F．（1965）：Myiasis

70−75

in

man

and

animalsinoldworld，Butterworths＆C．Ltd．，London，

350

AN IMPORTED CASE OF MYIASIS CAUSED BY A LARVA OF
CORD YL OBIA ANTHROPOPHA GA FROM AFRICA
NoBORU KAGEll, IWAO MURATA' AND HIROSHI KURAHASHI'
Received June 7 1989/Accepted August 26 1989

A 25 year‑old Japanese man (musician) had been in Middle‑Africa for the concert‑tour
from 14 January to 20 February 1989. On 21 February after homecoming to Japan, a lesion
with a severe pain was found in the right abdomen. He expelled a white larval worm (1.5 cm
in length) from this lesion by pressure of his fingers. This larval worm taken out from thelesion was identified as the third‑stage larva of Cordylobia anthropophaga (Blanchard, 1893) ,by their pair of toothed, spade‑like oral hooklets and by several pairs of posteriorly directed

fieshy processes arising from the twelve abdominal segments, as well as by the character ofthe posterior spiracles, which lack a distinct chitinous rim.

The disease caused by the larva of C. anthropophaga is a familiar form of myiasis in
Africa. This fly lays its eggs on dry sand polluted with the excrement of animals, or clothing.

The hatched larva invade the skin of human and animals. This patient may be contacted witha larva of this fly in Africa.

3

Department of Parasitology, National Institute of Health, Tokyo 141, JapanThe Tokyo Metropolitan Research Laboratory of Public Health, Tokyo 169, Japan
Department of Medical Entmology, National Institute of Health, Tokyo 141, Japan

JAPANESE JOURNALOF
TROPICAL MEDICINE AND HYGIENE
Vol. 17 No. 4

Decelnber 1989

CONTENTS
Original article

Takaoka, H.. Baba, M. and Bain, O.
Natural Infections of Simulium bidentatum (Diptera: Simuliidae) with Larvae of
Onchocerca spp., in Relation to a Human Zoonotic Onchocerciasis in Oita, Japan

279‑284

Shimada, M., Hirata, M., Ouma. J.H., Sato, K., Noda, S. and Aoki, Y.
Intensity, Incidence and Conversion/Reversion Ratio of Schistosoma haematobium
Inf ection

285‑290

Shimada, M., Hirata, M., Ouma, J.H., Sato, K., Noda, S. and Aoki, Y.
Epidemiological Study of Schistosoma haematobium Infection in a Coastal Area of

Kenya‑The Importance of Water Contact Patterns in Relation to S. haematobium
Inf ection

291‑301

Itokawa. H., Takai, R., Ishii, A. and Panjaitan, W.

Age Specific Asexual Parasite and Gametocyte Density in Highly Endemic Malaria in

North Sumatra, Indonesia

303‑310

Tao, D.
Change of Cytokine and Natural Killer Activities in Ascaris suum Infected Mice(in Japanese)

311‑321

Oikawa, Y. and lkeda. T.
Monoclonal Antibodies Recognizing the Main Surface Antigens of Newly Excysted
Metacercaria or Adult Flukes of Paragonimus ohirai

323‑329

Furuya, M., Mimori, T., Gomez, L. E.A., D. Coronel, V.V., Kawabata, M. and Hashiguchi, Y.
Epidemiological Survey of Leishmaniasis using Skin Test and ELISA in Ecuador
Sato, K., Kano, S., Hida, M.M., Yanagisawa, H., Ogawara, H. and Suzuki, M.
High Antibody Positive Rates of Ascariasis and Toxoplasmosis in an Amazonian
Japanese Settlement (in Japanese)

331‑338

339‑346

Case report
Kagei, N., Murata. I. and Kurahashi, H.

An Imported Case of Myiasis Caused by a Larva of Cordylobia anthropophaga fromAfrica (in Japanese)

347‑350
Published byJAPANESE SOCIETY OF TROPICAL MEDIClNE
c/o Institute of Tropical Medicine, Nagasaki University

12‑4 Sakamoto‑machi, Nagasaki 852, Japan