㏄

、

第1号

内
原

士心

目両

雑岬
会㎞
学漁
医剃
帯田

熱畑
本脚

日J
第1巻

昭和48年8月20日

容著
βJo卿p加」θ7」σ魏顔群〃鷹⑳8」観（Dunker）及び且膨4α勉oα（Martens）のマンソン住血吸虫に対する感受性（英文）

・伊藤

洋一，板垣

博，TEFERRA

WoNDE

1−5

エチオピア南西部におけるOnohooθ7昭拶oJ伽」％s自然感染のブユ（英文）…・田中

生男，井上 義郷，多田功，TEFERRA WoNDE 7−11

皮膚切片からの0耀h㏄θ7昭∂oJ捌」郷マイクロフィラリアの遊出に関する定量的研究（英文）

一多田

功，岩本

功，TEFERRAWoNDE13−24

エチオピア西南部における象皮病の一成因としてのオンコセルカ症（英文）…TEFERRA WoNDE，多田功，岩本功 25−29

沖縄における溶連菌の疫学…塩川優一，村中 正中，只野寿太郎，山田 俊彦31−37

ヘモグロビン濃度およびヘマトクリット値の季節的変動（英文）
一錬石昇太廊，福島 和子，LEoNARD会

A SAGAN 39−50

報
発刊のお知らせ……・…………・・………・………甲・…・…・一

会員一名簿…・・……………・………・…・・…………一一・…
投

稿

規 定

日熱医会誌
Jap．J．T．M．H．

日

本熱帯医学会51

52−71

Jap. J. T*'p. M*d. Hyg., vd. l, N*. 1, 1973, pp. 1‑5l

STUDIES ON THE SUSCEPTIBILITY OF BIOMPHALARIAPFEIFFERI RUEPPELLII (DUNKER) AND B. SUDANICA(MARTENS) TO SCHISTOSOMA MANSONI IN ETHIOPIAYOUICHI ITol, HIROSHI ITAGAK12 AND TEFERRA WONDE3
R***ived fo' p bh**tion 30 Ap*n 1973

Abstract : Biomphalaria pfeafferi rueppellii and B. sudanica collected at various sites in
Ethiopia, were exposed to Ethiopian strain ofSchistosoma mansoni. Striking differences wereobserved in susceptibility of the two species of snail vectors to the Ethiopian strain. The
infection rate of B. pfelfferi rueppellii with S. mansoni ranged from 67 to 100 o/o' while thatof B, sudanica was only 9 o/o' It appeared that B. pfelfferi rueppellii would serve as themost important intermediate vector to S. mansoni in Ethiopia.

Two species of vector snails of Schistosoma mansoni. Biomphalaria pfelfferi ruep‑

pellii (Dunker) and B. sudanica (Martens), are found in Ethiopia (Brown, 1964).
B. pfelfferi rueppellii is fbund throughout the plateaux in a wide variety of habitats,and also in the small streams and temporary pools. On the other hand, the distri‑bution of B. sudanica is confined almost exclusively to the lake areas located in thesouthern part of the Rift Valley.

Ayad (1956), Lemma (1969) and Ito et al. (1973) surveyed the foci of schisto‑
somiasis mansoni in Ethiopia and concluded that the endemic foci of schistosomiasis
mansoni had a sporadic distribution.
It is contemplated that the distribution of vector snails and their susceptibility
to Schistosoma mansoni play a significant role in the sporadic nature of the endemicfoci.

In the present study, two species of Biomphalaria snails from Ethiopia were test‑
ed for their susceptibility to Ethiopian strain of S. mansoni.

MATERIALS AND METHODS
The present experiments were made in the Imperial Central Laboratory &Research Institute, Addis Ababa.
Biomphalaria pfelfferi rueppellii and B. sudanica from Ethiopia were utilized forthe experiments. B. pfelfferi rueppellii was collected from several small streams mainly

along Asmara Road, while B. sudanica from the Lakes, Ziwai and Awasa (Fig. I ) .
l Dept. of Parasitology. National Institute of Health, Kamiosaki. Shinagawa, Tokyo, Japan. 2
Dept. of Parasitology, Azabu Veterinary College, Fuchinobe, Sagamihara, Japan. 3 Dept. of
Medical zoology, Imperial Central Laboratory & Research Institute, P. O. Box 1242, Addis Ababa,
Ethiopia.

2
DE SIE
sltlnta R.

Aiowa R.

DEBRE MARCOS Keralu R.
wolke R.
Damoto R.

Agere R.

GEDO

ADDIS ABABA

NAZARETH

Lake ziwai.:l Lake Awasa

Fig. I The map showmg the places of snail collectron m Ethiopra

After breeding the snails in the laboratory for two months or more, snails were re‑moved to a beaker containing a small amount of water, and the beaker was placed
under an electric light to check the natural infection with the trematode cercariae

before the experiments could be undertaken. Only non‑infected snails were used
for the present study.

A strain of Schistosoma mansoni from Ethiopia which was obtained from the stoolof an Ethiopian patient was used for the experimental infection. The Egyptianstrain of B. pfeefferi was exposed to the miracidia and then the mature cercariae were
inoculated intraperitoneally into white mice.
The mice infected with S. mansoni were killed 8 to 12 weeks after the infection.

Then small portions of the liver were examined under microscope for the presenceof ova and two or three infected liver fragments were used for the collection of eggs.

Liver was homogenized in a motor with cold water, and the suspension transferred
to a flask fllled with cold water for the sedimentation. After sedimentation for 20
minutes the sediment was brou ght into a 500 ml conical flask with water warmed
at 28‑30 C for the hatching of miracidia. The flask was covered with strips ofblack vinyl‑tape except its top portion. The flasks were placed under a 100‑wattelectric light bulb and within one hour the miracidia migrated to the surface so thatthey could be used to infect snails. The miracidia were counted under dissecting

3

microscope, 300 miracidia placed into each of the beakers filled with 200 ml tap
water kept at room temperature for 2 or 3 days, to which'30 snails were placed ineach one of them. The beakers were maintained at room temperature overnight.
In the next morning, all snails were removed to an aerated aquarium operated bysmall pumps. The snails were maintained in the aquarium for three months andfed on cabbage leaves twice a week. At the end of a three month period, the snails
were dissected and examined for sporocysts and cercariae of Schistosoma mansoni.

RESULTS AND DISCUSSIONS
A total of 410 Biomphalaria pfeifferi rueppellii and 490 B. sudanica were exposed

to Ethiopian strain of S. mansoni. The results which are summarized in Table 1,
TABLE I Susceptibility of Biomphalaria pfelfferi rueppellii and B. sudanica from Ethiopia
to Ethiopian strain of Schistosoma mansoni

Collecting sites
No. of snails No. of snails No. of snails Mortality
Species of snails

of snails exposed

Silinga River

B. p‑ rue p pellii

examined infected (o/o) (O/o)

60

99(100)

85.0

60

4

4(100)

93.3

6

4(66.7)90.0

109(90.0)66.7

Wolke River
Keralu River
Ajowa River

ll

ll

ll

ll

ll

ll

Aegere River

ll

ll

60
30
70

4

4( I OO)

94.3

Damoto River

ll

ll

l 30

34

32 (94. I )

73.8

410

67

62(92.5)

83.7

430
60

175

19(10.9)

59.3

40

O

33.3

490

215

19(8.8)

56. l

Total

Lake Awasa

B. sudanica

Lake Ziwai

B. sudanica

Total

reveal that almost all B. pfeifferi rueppellii collected from several areas encountered
were infected with S. mansoni; the infection rate ofthe snails collected from the Silinga

River, Wolke River and Aegere River was 100 , 90 of those from Ajowa River,

94 of those from Damoto River, and 67

of those from Keralu River were

found infected with S. mansoni. A Iarge number of B. sudanica collected from theLakes, Awasa and Ziwai, however, remained non‑infected with the Ethiopian
strain of S. mansoni, namely, 1 9 out of 2 1 5 (9 ) of the examined snails were infectedwith S. mansoni.

Meanwhile, a lot of snails exposed to the miracidia died, and the mortality ofB. pfelfferi rueppellii ranged from 68 to 97 at the end of a three month maintenanceperiod. On the other hand, the mortality of B. sudanica ranged from 20 to 93
and seemed to be slightly lower than that of B. pfelfferi rueppellii. The high mortality

might be caused by unfavorable conditions due to maintenance and infection of
the snails with S. mansoni.

4

Files and Cram (1949) reported that B. pfelfferi from Liberia was readily in‑fected with four strains of S. mansoni from Puerto Rico and Venezuela and the infec‑

tion rate ranged from 38 to 65 ･ Malek (1962) described in his guide book thatboth B. pfetfferi and B. sudanica served as the vector snails of S. mansoni in Africa. It

is apparent from this experiment that B. pfeifferi rueppellii and B. sudanica from
Ethiopia differ in their susceptibility to Ethiopian strain of S. mansoni; B: pfelfferi

rueppellii could acquire infection, while only less than I O of B sudamca were
infected with that of S. mansoni from Ethiopia.

Wright and Brown (1962), Brown (1964) and Suzuki et al. (personal communi‑
cation) assumed that B. pfeafferi rueppellii had ubiquitous distribution on the Ethiopian

plateaux, while B. sudanica seemed to be conflned to the lakes near and around the
Rift Valley. The authors also found that the distribution of B. sudanica was almost
limited to the Lakes, Ziwai and Awasa situated in the southern part of the Rift Valley,

Ethiopia.
These data indicate that B. pfezfferi rueppellii serves as the most important vector
snail to the Ethiopian strain of S. mansoni, while B. sudanica seems to be fairly resis‑
tant to the latter.

It is hoped that comparative studies on the susceptibility of Biomphalaria snails

from the neighbouring countries to the Ethiopian strain of S. mansoni would shedsome light on this problem in the near future.

ACKNOWLEDGEMENT
The authors wish to acknowledge the advices and encouragements received
from lhe Drs. T. Ishizaki and S. Asahina, National Institute of Health, Tokyoand Dr. A. Tekle, former director of Imperial Central Laboratory & ResearchInstitute, Addis Ababa.
The stimulating discussions with the Drs. C.T. Lo, a visiting research worker
at the Institute of Pathobiology, Faculty of Science, Haile Sellassie I University,

Addis Ababa and N. Suzuki, National Institute of Health, Tokyo are highly ap‑
preciated by the authors.

REFERENCESl)

Ayad, N. ( 1956) : Bilharziasis survey in British Somaliland, Eritrea, Ethiopia, Somalia, theSudan and Yemen. Bull. Wld Hlth Org., 14, 1‑ll7
2)Brown, D. S. ( 1964) : The distribution of intermediate hosts of Schistosoma in Ethiopia, Ethi‑

opian Med. J., 2, 250‑259
3)Files, V. S. and Cram, E. B. ( 1949) : Study on the comparative susceptibility of snail vectors
to strains of Schistosoma mansoni, J. Parasit., 35, 555‑560

4)5)Ito, Y., Tada, I.. Itagaki, H., Iwamoto, I. and Wonde, T. (1973) : In press
Lemma, A. (1969): Bilharziasis in the Awash Valley I. An epidemiologica]. study with special
emphasis on its possible future economic and public health importance, Ethiopian Med. J.,7,l 47‑1 76

6) Malek E. A. ( 1962): Laboratory guide and notes for medical malacolo y, Ist ed., Burgess
Publishing Company, Minneapolis

57）Wright，C．A．andBr・wn，D．S．（1962）：Onac・llecti・n・f倉eshwatergastr・P・dm・lluscs黛・m
thc

Ethiopian Highlands，BulL

Brit。Mus．（Nat．Hist．）ZooLコ8，287−311．B∫oηψh痂吻伽塀7∫7卿ρ61痂（Dunker）及び」8・5痂π加（Martens）のマンソン住血吸虫に対する感受性

伊藤洋一1・板垣 博2・テフェラ・ウオンデ3
エチオピア産研o吻hαZαr如属の2種の貝にエチオピア人の患者から分離したマンソン住血吸虫ミラ
シジウムを実験的に感染させ，その感受性を比較した。その結果，各地から採取したB．魏漉ガ耀砂一

p8ZZ∫∫では67〜100彩の感染率が得られたのに比し，B膨4朋foαではわずかに9％の感染率しか得られなかった。また両種のエチオピアにむける分布状態を調査したところ，B魏伽擁耀砂ρθZ躍はエチ
オピア全土に亘り分布しているのに比し，B．躍4αη∫oαは南部湖水地区の一部の湖水にしかその棲息が認められなかった。
これらの結果より，エチオピアにおけるマンソン住血吸虫の主要な中間宿主は13．∫塘漉万耀ψ少θ」漉

であると考えられる。

1国立予防衛生研究所
医動物学部門寄生虫部 2麻布獣医科大学 寄生虫学教室 3エチオピア帝国中央研究所

7

Jap. J. Trop. Med. Hyg., Vol. 1, No. 1, 1973, pp. 7‑11

SIMULIUM DAMNOSUM, NATURALLY INFECTED WITH
ONCHOCERCA VOL VUL US IN SOUTH‑WEST ETHIOPIA*IKUO TANAKA2, YOSIIISATO INOUE3, ISAO TADA4, ISAO IWAMOT05

and TEFERRA WONDE6
Received for publication '19 July 1973

Abstract: The existence of onchocerciasis in Ethiopia, especially in its South‑Western
Region was known. The naturally infected Simulium has not been reported in the region,
though there has been reported the existence of S. damnosum. S. woodi etc. which wereidentified as the vectors of this disease in other parts of Af'rica. The authors obtained anumber of Onchocerca volvulus from S. damnosum collected by biting‑catch method in thefield in August and November, 1971. Dissection was made on the 975 Simulium out of
about 1000 caught at the riverside of Gojeb and those caught at Didessa riverside. The
infection rate ranged from 10.2 to 12.9 /0 in the former and 20.0 to 40.6 in the latter. The

sausage type was most frequently found and the late stage type was detected in I I o/o of

Simulium. An evidence was given that S. damnosum is the main vector of onchocerciasis
in Ethiopia, since this species formed an absolute majority of the flies collected by biting
catch and no filaria was detected in the other species.

The existence of onchocerciasis in Ethiopia was initially ascertained in 1939
at Bonga, Kefa Province, and the collection record on Simulium damnosum was madeby the same author (Giaquinto, M. 1939).
Oomen, A. P. (1969) collected S. damnosum and S.

)oodi, and suggested that

S. damnosum might be the vector of this disease in the south‑west Ethiopia surveyed.
Although S. damnosum has been incriminated to be one of the potential vectors
ofonchocerciasis in Ethiopia, naturally infected S. damnosum has not yet been reported,so far as we are aware.
In order to clarify the vector species of this disease, we visited the endemic areaof South‑west Ethiopia in 1971, and made attempts to check infected adult black
flies.

This paper is a brief report on the discovery of naturally infected S. damnosum
with Onchocerca volvulus in the area.
l The contents of this paper was preliminarily presented at the 24th Annual Meeting of the Japan
Society of Sanitary Zoology, Okayama, 3 April 1972. 2 Division of Pest Control, Japan Environ‑mental Sanitation Center, Kawasaki, Japan. 3 Dept. of Medical Zoology, National Institute of

Health. Tokyo. Japan. 4 Dept. of Medical Zoology, Kanazawa Medical University, Uchinada,
Ishikawa, Japan. 5 Dept. of Parasitology, Institute for Tropical Medicine, Nagasaki University,
Nagasaki, Japan. 6 Dept. of Medical Zoology, Imperial Central Laboratory & Research Institute,
Addis Ababa, Ethiopia.

8

MATERIALS AND METHODS(a) Date of the survey and collection sites (Fig. I )March 18‑25, 1971 : riverside of Gojeb, Kefa Province
July 31‑August 15: riverside of Didessa and adjacent plateau I km from theriverside, Ilubabor Province

November 15‑18 : riverside of Gojeb and Didessa(b) Catching method for the adult black flies
Collection was made by two or three volunteers seated on a rock or the groundat the individual sites. A11 the black flies settled on the naked parts of the body
were caught using a sucking tube every 30 minutes from sun rise to sun set.
(c) Treatment and dissection of the flies

Collected flie were anesthetized with ether and, in every 60 minutes, weregathered in batches in one small glass tube. After identification, the flies weredissected in a droplet of physiologic saline solution on a slide glass under a diss‑ecting microscope in order to find the parasite.

,;'

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Map of Ethiopia showing the rivers where S. damnosum were collected.

9

RESULTSAs shown in Table I , the infection rate of S. damnosum with the larvae of O. vol‑vulus ranged from 20.0 to 40.6 at the several collection sites in Didessa area andranged from 10.2 to 12.9

at the Gojeb riverside. The other species of black fly

had not been found to be infected with filarial worms.
TABLE 1

Number ofthe naturally infected Simulium damnosum with the filarial worm, Onchocerca volvulus

DidessaGojebriverside

No. flies collected

No. flies infected
Infection rate o/oplateauriverside

Aug 13 15 Aug 15
Mar. Nov.
24 18Nov.
5

32

237(96)l (O)

1 3 (4)

32.6

20.0

40.6

12

O

2

6

127

726

l I (‑)

13(8)

12.9

l0.2

85

,)cS
'H

m

2

l 16

1

11
>
1"L'

1

7

64

O

3

m+s
m+1

o

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O

2

O
O

O

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5

, E,,

bO

Sl '

0

c ;

'c,

O h'cs

; ::

29

s+1

m+s+1

o

O

O

* Stage type of the larvaem : microfilaria types : sausage type
1 : Iate stage type‑: each stages not checked
Parenthesis : total flies possessing late stage type worm

In adult S. damnosum dissected, all the three developmental stages of O. volvulus
i.e., microfllaria type, sausage type and late stage type were found. Of these three
types, the sausage type was most abundant and this was followed by the late stage
type and then microfilaria type. Finding the black flies possessing the late stage typeis especially important from the epidemiological viewpoint.
In August 1971, at the Didessa riverside, out of 237 infected S. damnosum, 96

harboured the late stage worms. The body length measured ranged from 300p
to 700p.
Regarding the frequency of harbouring worms in one dissected fly, usually only
one type was recognized. In a few case, however, two or three types of fllarial larvae

were simultaneously found.
The highest number of the sausage type larvae recognized was 5 1 , while that of
the late stage type, 1 7.

Some smeared specimens were identified as the larval stage of O. volvulus by
Dr. R. L. Muller, of London School of Tropical Medicine and Hygiene.

10

DrscUssroN
It has been already known that onchocerciasis is endemic in South‑west Ethiopia(Cohen, L. B. 1960, Oomen, A. P. 1969, Iwamoto, I. et al. 1973). There is a generalagreement that S. damnosum and S. neavei complex are the main vector of onchocer‑

ciasis in many tropical regions in Africa (De Meillon, B. 1957). Oomen, A. P.
(1969), in particular, collected S. damnosum and S. woodi, and postulated that S.
damnosum might be the vector of onchocerciasis in Ethiopia. Ogata, K. et al. (1970)
reported that they collected seven anthropophilic Simulium species in Ethiopia and
S. damnosum was recognized as the most cornmon one.
Through our study, naturally infected S. damnosum with Onchocerca volvulus wasabundantly found in natural population, whereas the other black fly species were
found free from filarial larvae.

Thus, the present study brings an evidence that S. damnosum is the main vectorof onchocerciasis in South‑west Ethiopia, yet no possibility is denied if onchocerciasis
could also be transmitted by the other Simul'ium species in this area. It is hoped thatfurther studies will throw more light on this problem.

ACKNOWLEDGEMENT
Acknowledge is made to the support of Dr. T. Aseffa, the Exdirector, Imperial
Central Laboratory & Research Institute, Addis Ababa and Dr. S. Asahina, Chiefof Medical Entomology, National Institute of Health, Tokyo.
We also wish to thank to Dr. R. L. Muller, of the London School of Tropical
Medicine and Hygiene, for confirming the identification of the filarial larvae in our
black fly specimens.

This work was supported by Overseas Technical Cooperation Agency, Japan.

REFERENCES
1)

Cohen, L. B. ( 1960) : Idiopathic lymphoedema of F.thiopia and Kenya, East Africa Med. J.,

2)

De Meillon, B. ( 1957) : Bionomics of the vectors of onchocerciasis in the Ethiopian geographical3)lwamoto, I., Tada, I. and Wonde, T. ( 1973) : Clinical manifestation of onchocerciasis in endemic

37, 53

region. Bull. Wld Hlth Org., 16, 509‑522

4)5)foci of llubabor Province, Ethiopia, Trop. Med., 15, 36‑45
Muller, R. L. ( 1972): Personal communication to Dr. I. Tada
Ogata, K., Takahashi, M. and Ohse, T. ( 1970) : Distribution and epidemiology of black fliesin Ethiopia (Summary in Japanese), Jap. J. Sanit. Zool., 21, 134

6)

Oomen. A. P. ( 1969) : Studies on onchocerciasis and elephantiasis in Ethiopia, I 15, De Erven

7)

Tanaka. I. and Inoue, Y. ( 1972) : Simulium damnosum and its feasibility of the vector of onchocer‑

F. Bohn, N. V., Haarlem
ciasis in Ethiopia (Summary in Japanese), Jap. J. Sanit. Zool., 22, 250

11エチオピア南西部において0π6ho6676α∂ol∂ぬ5自然感染の見られた
8珈％1勉規磁窺η05μ勉
田中生男1・井上義郷2・多田功3・岩本功4・テフェラ・ウオンデ5

エチオピアにおいては，とくに，その南西部にオンコセルカ病が存在することが知られ，また，他のアフリカ地域で，そのベクターと判明している，S加μZ劾窺4α窺ηo躍窺，S．ωoo4」などのブユが生息することが確認されていたが，自然感染ブユは発見されなかった。筆者らは1971年同国に滞在し，8月と11月に行なったbitingcatchによって得た，野外のS．4α解no3襯から，多数の0窺hoo8質

α∂oZ∂ぬ5を得た。

Gojeb，Didessa両河岸から得た個体約1000のうち，975個体について解剖を行なった結果，前者で10．2〜12．9％，後者で20．0〜40．6％の陽性個体を得た。ブユ体内における0．∂oZ∂％Zμ5の発育段階別

では，sausagetypeのものが最も多かったが，約1％の個体が発育終期のものを保有していた。
bitingcatchによって採集されたブユは，ほとんどがS・4α吻πo鍬窺であったこと，および，他種のブユからは全くフィラリアが発見されなかったことから，エチオピアにおいても，S．4α吻no躍解がオンコセルカ病の主要媒介種であるという確証を得た。

1

日本環境衛生センター

医動物学教室 4
物学部門

環境生物部

2

長崎大学熱帯医学研究所

国立予防衛生研究所
寄生虫学部門

5

衛生昆虫部

3

金沢医科大学

エチオピア帝国中央研究所

医動Jap. J. Trop. Med. Hyg., Vol. 1. No. l, 1973, pp. 13 24

13

QUANTITATIVE STUDIES ON THE EMERGENCE
OF ONCHOCERCA VOL VUL US MICROFILARIAE
FROM SKIN SNIPSISAO TADAl, ISAO IWAMOT02 AND TEFERRA WONDE3
Received for publication 19 July 1973

Abstract : The authors made studies on the emergence of O. volvulus microfilariae fromskin snips in order to assess accurate MFD in onchocercal infections from quantitative view

point. The results obtained are as follows : Skin snips should not be teased into small
pieces, but the intact snips should be incubated for a longer period than the teased ones.
The distribution ofmicrofilariae in a minute skin area is quantitatively even in most cases.

This finding suggests the usefulness of this method to compare MFD of the adjacent skinregions to each other. However, the comparison of MFD with extremely different‑sized
snips should be avoided. There were no significant changes in MFD by warming skinsurface.

For the diagnosis of human onchocerciasis, the skin snip method has been widelyused as an essential and standard method. During a period of epidemiological sur‑vey of onchocerciasis in llubabor Province, Ethiopia, the present authors attained
the conclusion that the microfilaria density. (MFD) of skin snips obtained was highly
affected by the teasmg process and mcubatton time. For example, when the biopsies
were teased by the technique recommended by several previous workers, there weresome microfilariae which were still migrating out from the newly cut surface of thefragmented tissue 1 5 to 20 minutes after incubation. Small numbers of microfi‑
lariae newly released were observed from time to time by additional incubation.
Furthermore, many microfilariae were found torn into small pieces so that they
were immobile. These findings suggested the possibility that the teasing processcaused mechanical damage to the microfilariae in the biopsies. This process natural‑ly might lead to an inaccurate MFD. It was inconvenient for the authors to assessthe densities of microfilariae under several chemical stimulants, unless a standard

method was established. For this reason, the authors made quantitative studies
on the emergence of Onchocerca volvulus microfilariae from skin snips in order to es‑
tablish a standardized method for the skin snips which could be of epidemiologicall Dept, of Medical Zoology, Faculty of Medicine, Kagoshima University, Kagoshima, Japan (Pre‑sent address : Dept. of Medical Zoology, Kanazawa Medical University, Uchinada, Ishikawa, Japan).
2 Dept. of Parasitology, Institute for Tropical Medicine, Nagasaki University, Nagasaki, Japan(Present address : Dept. of Internal Medicine, in same Institute). 3 Dept. of Medical Zoology,
Imperial Central Laboratory & Research Institute, Addis Ababa, Ethiopia.
The present work was supported by a research grant from the Imperial Central Laboratory & ResearchInstitute of Ethiopia and the Overseas Technical Cooperation Agency ofJapan.

14and experimental importance.

MATERIALS AND METHODSIn Abdella, 1lubabor Province of Ethiopia, the experiments were performed,
the first one in August and the second in November, 1971. Our preliminary sur‑vey with skin snippings revealed that the microfilaria rate was 80.70/0 in male adults

from this village. Volunteers were then picked among these adults and brought
to the Dabana Missionary Station for the experiments.

A11 of the experiments were undertaken at room temperature ; in August, it
ranged from 18.0 to 23.0 C., and in November, from 17.5 to 21.5 C.
Every skin snip was taken from the left buttock of volunteers with a needle anda surgical blade. The detailed technique to obtain the skin snip was described byDuke ( 1962). When multiple snips were needed from one volunteer, every snip
was taken I cm apart from the others. Thereafter, skin snips were placed intodrops of physiological saline on slides.

The slide was then placed in an optical apparatus which magnified and project‑
ed the shape of the skin sn p on a prece of paper put on the top of the apparatus.
The area of the skin snip was obtained by counting the number of smallest sections(1 mm2) encircled by the outline of the projected biopsy. One square millimeterof the actual skin snip area was equivalent to 49 mm2 on the paper. The precisearea of the biopsy was obtained by calculation in mm2. The measured snips inphysiologic saline were incubated at room temperature for a determined time andwere transferred to the saline on the next slide carefully with a small forceps. This

process was repeated continuously. The number of microfilariae which were left
behind was immediately counted under 50 x magnification of a binocular microscope.

The microfilaria density (MFD) was calculated by dividing the total number ofmicrofilariae which were released from one snip by the individual snip area in mm2.
The microfilariae found from these volunteers were identified as those of On‑
chocerca volvulus from the morphological feature of the stained specimens and fromthe measurement of its anatomical landmarks (Iwamoto et al., 1972).

RESULTSl . The effect of teasing the skin snip on the emergence of microfilariae

Three snips were taken from each of the 6 volunteers (from T‑1 to 6). The
first snip was torn into small pieces with needles for about 30 seconds (snip P), thesecond one, coarsely torn into two pieces (snip M) and the third one (snip G) was

not teased. Skin snips were then incubated for approximately 22 hours and theMFD obtained is shown in Table I . The result of this experiment has been alreadyreported in a brief article by the present authors (Tada et al., 1973). The highest

MFD is seen almost in snip G. On the other hand, the snips P, which were torn
into small pieces due to the previously recommended technique released lessmicrofilariae than the others. When the highest MFD from 3 snips is describedas I OO the relative MFD of the other two snips are calculated by proportions.

15

The relative MFD in average is as follows: 0.92 in snip G, 0.79 in M and 0.50
in P, respectively. In contrast to the method recommended by various previous
researchers, the present data clearly show that skin snips should not be torn into
pieces to assess the accurate MFD.

TABLE I The effect of teasing the skin snips on the MFD

MFD
Snip type*G

Case No.
T‑ 1

M
27.3**

P(0.61)***( I .OO)

17.4(0.64)

1 1 .810.7(0.91)(0.54)

16.6

6.4

T‑2

( I .OO)

T‑3( I .OO)

11.7(0.55)

9.1(0.43)

T

4.0(0.89)

4.5( I .OO)

1.6(0.36)

T‑5

T‑6

21.2

1 1 1.9

84.7

89.6

( I .OO)(0.76)(0.80)

46. l

23.7

( I .OO)(0.5 1 )10.8(0.23)

0.92

0.79

0.50

Average of therelative MFD(6 cases)* Snip type : G, non‑teased; M, coarsely teased; and P, teased into small pieces** The under‑lined count shows the highest MFD among the 3 snips from the same in‑dividual*** Relative MFD: When the highest MFD of a snip among the 3 snips is determined
as I .OO, the relative MFD of the others is obtained by proportional calculations

In this experiment, as shown in Table 2, the number of microfilariae released
were counted right from the beginning every 20 minutes to 1 20 minutes, and every
60 minutes from 1 20 to the end of the incubation. The end of incubation ranged
from 8 to 22 hours depending on the emergence of microfilariae from individual
snips. The table shows the cumulative percentage of microfilariae from 3 typesof snips in association with the incubation time while cumulative percentage of micro‑

filariae was apparently the lowest in snip G at any incubation time. For example,
snip P released almost 900/0 of microfllariae in average at 80 minute incubation,
while only 67.60/0 of microfilariae emerged from snip G in average. This fact does
not contradict the above mentioned conclusion that skin snips should not be teased,
but it may indicate that the living microfilariae would emerge easily and quickly
within a short time from the teased snips. But it should be also beared in mind
that teased skin snips could lead to a wrong conclusion. The teasing would hinder
the accurate MFD of each skin snip by causing mechanical damage to the microfi‑
lariae in the skin.

16

TABLE 2 The effect of teasing on the recovery rate* ofmicrofilariae in individual snip

types arranged by incubation time
Cumulative percentage of microfilariae released
from skin snips (average of 6 cases)

Incubation
time (min.)Snip type**

G

M

P

20

36.2

50.4

61.4

40

50.0

65.5

76. l

60

60. 1

77.0

84.0

80

67.6

81.8

89.7

1 OO

72.0

85.6

92.3

120

75.6

88.9

94.2

l 80

84.6

93.8

96.0

2 40

86.9

96.5

99. l

300

91.7

97.l

99.4

360

93.4

99. l

99.9

* Recovery rate (in percent) : The ratio ofmicrofilaria count at individual incubation
time to the total count of microfilatiae obtained from the identical snip** Snip type : as shown in Table 1

From this experiment, it is concluded that skin snips should not be teased, inparticular for the quantitative assessment of MFD in human skin.
2. The distribution of microfilariae in the skin

To assess the changes in MFD for some quantitative studies, the distributionof microfilariae should be even in some small skin regions. Based on this viewpoint,

the authors examined if the microfilariae were evenly distributed in minute skinregions or not by comparing the MFD of 3 skin snips from each of 8 volunteers(from N‑1 to 1 3). The skin snips were taken in a triangular shape, I cm apart fromeach other from the left buttock of volunteers. Those snips were then incubatedfor 24 hours and the individual MFD obtained and the average MFD and percentage
deviation of the individual MFD of three snips from the average one are shown in
Table 3. The MFD of 3 different snips highly coincided with each other in most of
the cases examined. Fig. I clearly shows that in case of the subjects whose MFD

are below 10, the maximal measurement error of MFD is 200/0 or more. On theother hand, the error is markedly reduced in proportion to the increase of the average

MFD. For this reason, it is quite appropriate to use volunteers whose MFD is 20
or more for the purpose of quantitative studies in order to minimize the measurement
error within I Oo/o ' In this experiment, however, the MFD of case N‑8 unexpected‑
ly fluctuated notwithstanding its proper MFD in its average. This finding may sug‑gest the rare presence of uneven distribution of microfilariae even in closely adjacentskin regions. However, generally speaking, in onchocercal infections, it may beconcluded that the MFD is uniform in small skin regions.

17

TABLE 3 Comparisons of the MFD in minute skin regions

Case Snip Snip area Mf* count MFD
per snip (x)No. No. in mm2

N‑ I O

‑4.40
+0.91

+ 1.27+ 3.49+0.34+ I .54

8.43
7.47
6.08

l 89
l 64

22.42
2 1 .95‑o. 1 3133

2 1 .88

‑0.20

5.59
6.45
6.24

127
123
137

22.72
19.07

5.49
3.94
5.33

l 72

43.65

229

42 .96

201

373

40.04
49.74
74.30

54.69

1 92

3

5.02
3.86
5.02

4. 1 8

3

6.16

278
237
339

53.88
56.70
55.03

55.20

2

1

6.18

9.01

3.61

53
27
40

8.58

2
3

1

2
3
l

2
3

N‑9

+0.33

‑ I .60

37.61

3

N‑8

36.34

261

2

2

5.16

N‑ 1 3

2
3

3.65

7.29
7.55
6.69

* Mf: microfilaria

230

l 74
22 1
1 92

22.08

2 1 .25

42.83

10.96

23.87
29.27
28.70

27.28

+6.92

‑ ro.26

+3.34

‑0.94
+0.82
+0.rs
‑ 1 4.65‑ 4.95+ 19.61

7 .48

‑0.59
‑0.91

+ I .47

‑2.18
+0.71

2 1 .96
4 1 .89

Difference in

MFD (x‑ M) percentage* *

34.74
36.67

3

N‑ 6

MFD ¥fM¥
T1'a: 'm
! uulerence

256
238

2

N‑5

Difference of individual MFD
from the average one

7.37
6.49
6.94

N‑ 1

N‑4

Average‑2.19+ 1.91+0.30‑26.79‑9.05+35.86

‑2.39
+2.72

‑ I .32+ I .50‑o. 1 7

‑ 0.3 l

‑0.43

‑4.77

‑ I .53+ I .95= 1 6.98+ 2 1 .64

‑3.41

‑ 12.50+ I .99+ I .42+ 5.2 l

+7.29

x‑M

** Difference in percentage : M X 100("/ ). The highest absolute value of the percentage
among the 3 snips was regarded as the measurement error and graphically shown in Fig. 4

3. Relation between incubation time and the emergence of microfilariae
The authors tried to examine the relation between incubation time and the emer‑
gence of microfilariae in non‑teased skin snips, which were taken from 4 persons

whose MFD was as follows: 272.0 in Tm‑1; 63.2 in Tm‑2 ; 160.6 in Tm‑3 ; and22.0 in Tm‑4 cases, respectively. Each fresh skin snip was quickly transferred
successively to the next slide at intervals of I minute during the incubation periodranging from I to 20 minutes, and then at 60 minute intervals from I to 6 hours.
The size of the skin snip was measured after 20 minute incubation in this experiment.
The skin snips were incubated for 1 9 to 30 hours until no more microfilariae wereobseryed. In the experiment which was performed in August, only a single skinsnip was examined which was taken from 4 volunteers. Fig. 2 shows the emergenceof microfilariae from a single skin snip during an incubation time ranging from
one to 360 minutes. In November, however, a similar experiment was repeated

18
70

60

O N‑9

O N‑8

50

O N‑6
40

N‑l

A

rll

::

Q, 30'

O Nl3

tu)
a'

G,

>

<
20

O N‑4

O N 5

lO

O

:N*lO

O

¥

o *lo +20 +30 +40 *50
Measurement error in percent

Fig. l Relation between the measurement error and the average MFD of 3 biopsies fromthe same subject.

a‑M‑ X 100 (o/o)

Measurement error:M(M; arithmetic mean of MFD from 3 snips, a ; the farthest MFD from M)

lOO

q,

Tm‑ 4 J ‑'()‑

x/x" x

c!S

f x"/ x Tm‑ 3 ̲rf ' ̲̲lx/' l'l'le/e ̲dr der Ar

H

.H

' 1

O

e ̲ ar iS

L,

u

,,
,
' .' ...
O""'

" o‑

r‑

1a

ee 4f ̲.:'r"le ';::;1:! ' ‑i;' e::L .

'x 'x

L1

,d

x'

̲ ̲' ̲'‑‑ ‑'e‑‑ ‑‑ ii61'11

a

/

J't

x/ ./dls‑'dr dr Tm‑l

E:

l'

H

bOQJ
,eL,

' 4 .,i

+,qJ

C>
,uo
uu
,dqJ
q,Ll

/ 14' a'er"or"O"O,'x;p// , .O'OTln‑2 ""6

OO1:,50/ :/ .,.. O O'c "I 7 O o'O"O O

O.
q;$(:4::/:;::o ･･ O '

>

.H
,

a,

H

;!
E

,

O

O
lo

I*'*b*ti*15

ti** (

i .)20 60 120 180 240 300 360

Fig. 2 The emergence of microfilariae from the skin snips of 4 volunteers (fromTm‑1 to 4) .

by using 3 snips from one volunteer to check the previous result. The result of thelatter experiment is shown in Table 4 (group N), in which the transfer of skin snips
was made 7 times in the whole incubation only to see the general tendency of the

19

TABLE 4 The emergence of O. volvulus microfilariae from skin snips
The average cumulative percentage of microfilariae

Incubation
time (min.)releasedgroup T*
(6 cases)

15

20
30
40
60
80
1 OO

group N**L

(8 cases)
2 1 .4

36.2

35.3

50.0
60. l

46.2

67.6
72.0

120

75.6

56.5

l 80

84.6

66.8

240
300
360

86.9

72.6

91.7

78.3

93.4

* group T : volunteers examined in experiment l** group N : volunteers examined in experiment 2

microfilarial emergence. This table also shows similar observations made in the ex‑periment I . All of these experiments revealed similar results. In contrast to theresults reported by Duke (1962) and to the recommendation by WHO (1966), theemergence of microfilariae from non‑teased skin‑snips was more prolonged than
expected. In the first experiment, the recovery rate of microfilariae at 20 minuteincubation revealed that 66.90/0 in Tm‑1 , 53.60/0 in Tm‑2, 71 .50/0 in Tm‑3 and 84.90/0

in Tm‑4, respectively. In the experiment carried out in November, however, only
35.30/0 of the total microfilariae were released by 30 minute incubation. On thecontrary, the third series of experiments were quite similar to the results of the firstone. From these findings, it can be concluded that skin snips should be incubatedfor at least 5 to 6 hours at a temperature of about 20 C to assess the accurate MFD.
From a practical view point, however, it is inconvenient to incubate skin snips forsuch a long time for the mass examination of human onchocerciasis.

In August, 1971, the authors examined the inhabitants in Dedessa, Ilubabor
Province of Ethiopia. Thirty‑three microfilaria positives were found out of 54 cases

examined with 15 minutes of incubation using the non‑teased snip method. Thenthe skin snips from 2 1 negatives left were incubated again for 45 minutes longer.

Three new positives with a low MFD were found from these negative cases. This
fact may also suggest the importance of a longer incubation even for the epidemiolo‑
gical and routine examinations of onchocerciasis to pick up positives with low MFD.
Incubation for I hour may satisfy the practical purpose of mass examinations when
unteased snips are used.

20

4. Relation between skin snip size and MFD
From each of 5 volunteers (from S‑1 to 5), 3 adjacent skin snips of different size

were taken to compare the MFD. The authors called the largest snip L, the smallest
one S, and the medium one M from their relative difference in size. In this ex‑
periment, L, M and S meant the relative size of biopsies taken from the same person.
The actual size in mm2 is seen in Fig. 3. The skin snips were incubated for 8 to 29

hours depending on the emergence of microfilariae. The MFD of each skin snip
among the different sizes obtained is also shown in Fig. 3. This experiment clarifi‑
ed that the highest MFD was usually seen in the snip whose area ranged from 5 to

8 mm2. This finding suggests that one should not compare the MFD of skin snipsof extremely different size with each other.

q,F̲/ v I s‑4

l . OO,///*'

."

/

¥ . ¥

/¥ f¥¥¥･ ¥' s‑2..･･:'1
7f '¥
¥¥
¥
/ y/
s‑3
/'
'¥¥ /.¥¥.¥¥¥¥¥o s‑5 ¥¥¥

AO.SO
F
:

¥

/

¥

O

ce

¥O S‑1

o .oo

5

ro

15

Area of skin sniPs ( mm2 )Fig. 3 Relation between skin‑snip size and MFD.

5. The effect of warming the skin surface on MFD
Rodger ( 1 957) reported that when the surface temperature fell, microfilariae

tended to move more deeply into the dermis. This phenomenon will apparently
cause the reduction of MFD in the epidermis. Nelson ( 1970) stated that in East
Africa it had been common practice to apply hot water bottles to the skin to encourage
microfilariae to migrate to the 'superficial layer. These reports seem to indicate thatthe microfilariae are sensitive to changes of environmental temperature. However,according to the latter author, there are no data to substantiate the validity of thistechnique. When MFD of the skin is actually increased by warming, the warming
technique may have diagnostic validity. In order to assess this point, the present
authors examined the MFD of positives before and after warming the skin surface.
The left buttock of volunteers (from N‑14 to 20) were covered with a sheet ofblack‑colored polyvinyl immediately after the first snip was removed to exclude theeffect of light. The sheet was then heated indirectly by an electric bulb (500 W)

about 50 cm apart. The temperature of the sheet was kept at 41 C by adjusting
the distance between the bulb and the buttock. The second snip was taken adjacent

21

to the first one after 1 5 minutes of warming. The MFD of the two skin snips obtained

before and after heating were calculated and compared. The result is shown in

Table 5. In cases N‑14 and 15, MFD was markedly reduced after warming. On
TABLE 5 The effect of warming the skin surface on the MFD

N‑14 N‑15 N‑16 N‑17

Case No.

34.69

21.13

84.28

13.14

3 1 .67

37.61

24.76

22.99

12.18

88.93

2 1 .69

40. 1 1

38.54

30.41

‑ I I .70

‑8.95

+4.65

+8.55

+8.44

+0.93

+ 5.65

‑33.7

‑42.4

+5.5

+ 65. 1

+26.7

+2.5

+22.8

First snip*

MFD (MFDa)

Second snip**(MFDb)
Difference in MFD

N‑ 1 8 N‑ 1 9 N‑20

Difference inpercent * * ** before warming** This snip was taken I cm. apart from the flrst one, 15 minutes after the beginning of
the incubation.

***

MFDb ‑ MFDa x 100(o/o)

MFDathe other hand, the MFD significantly increased after warming in cases N‑17, 18
and 20. No signiflcant changes were observed in cases N‑16 and 19. From thisbrief experiment, it is not easy to reach a final conclusion whether the behavior of
the microfilariae was constant or not after warming the skin.

DISCUSSION
In onchocerciasis, teasing of the skin snips has been adopted by most of the pre‑vious workers for the quick detection of microfilariae. According to Duke ( 1 962)
it was thought essential to tear the snips with needles so that all the contained micro‑

filariae could be freed. He observed about 900/0 of microfilariae had come out
from skin snips after 5 minutes and constant total counts had been obtained at lO‑
l 5 minutes. In case of animals infected with Onchocerca gutturosa. Nelson et al ( 1966)
showed that many of the microfilariae failed to emerge into the saline unless the skinsnips were teased. Lagraulet et al (1967) also lightly teased the biopsies with 2
needles and examined them after 10 minutes of incubation. In contrast to the above
quoted results, as the result of the present study shows, the teasing procedure mecha‑

nically damaged microfilariae in the skin and caused a reduction in the MFD.
This suggests that when the unteased skin snips are incubated for 5 to 6 hours, anaccurate MFD will be obtained. Therefore, the present authors conclude that as
far as the recovery rate of microfilariae is concerned, torn snips reveal poor and in‑accurate MFD.
Some workers did not consider the individual MFD as reliable. Rodger and
Brown (1957) used IDF (Individual density figure) and DQ (Density quotient)
based on the known anatomical distribution of the microfilaria population and not
on microfilaria counts. Because the MFD was shown to be subject to wide variations

22

in the same site from day to day. Duke (1968) used multiple weighed skin snips
to assess the concentration of microfilariae. As microfilariae are not evenly dis‑tributed all over the body surface, it seems preferable to use those methods to assessthe density of infections. At the same time, it will be necessary to assess the accurate

MFD of small skin regions depending on the nature of experiments, such as theeffects of treatment, physical and chemical stimulation. Experiment 2 shows thatthe microfilariae are evenly distributed in small skin areas. This finding may help
in the future to perform quantitative studies on the changes in MFD under different
conditions.

According to Duke ( 1962), although the MFD ofadjacent snips is usually approxi‑
mately constant, the assessed densities might vary by as much as I : 3 because of theoccurrence of pocketing of microfilariae. This is reasonable because the presenceof nodules and/or adult worms would disturb the even distribution of microfilariae
in the skin. An uneven distribution of microfilariae was also reported in the caseN‑8 by the present authors. This fact, however, might not indicate the unreliabili‑
ty of MFD because of its rare occurrence. The use of multiple unteased snips willdiminish the possible errors of this kind and reveal accurate MFD.

In the present study, the authors did not weigh the skin snips, but measured
their surface area. There are apparently some advantages in measuring the areaof skin snips : 1) The snip is easily measured in a few drops of saline on a glass slide.

This enabled us to avoid time elapsing which frequently causes snips to dry before
weighing. 2) The apparatus is easily transferred. On the other hand, in case ofweighing, it needs electricity and some adjustment. This is impractical in rural
areas. 3) When skin snips are taken almost in a constant thickness, it is consideredthat the MFD reflects exclusively the 2‑dimensioned distribution of microfilariae.
This solved one of the inconveniences which are frequently shown in weighing snips
that a thin broad biopsy might weigh the same as a narrow deep one which was statedby Rodger and Brown ( 1 957). Lagraulet and Bard ( 1 969) also preferred an estima‑tion of density based upon surface area, because according to them, this method

has a theoretical advantage over a method based upon weight of a biopsy which
may include some tissue beneath that part of the upper dermis. According to Rodger
and Brown, the weighing of biopsies of such small pieces of tissue, the' rate of drying

in tropical climates and the time elapsing before weighing might all be probablesources of error. The measurement of the surface of skin snips with irregualr outline

is somewhat more difiicult than weighing them. However, as shown in the experi‑
ment 2, this method enabled us to get almost equivalent MFD of some biopsies inminute skin regions. For these reasons, mentioned bove, in order to be able tofollow the changes of MFD in several experiments, the present method is considered
to be of value.

As to the size of skin snips, Duke (1962) used bloodless circular or oval snips

about 3‑5 mm in diameter and weighing 1‑4 mg from volunteers for the skin snip
method. Further, Duke et al (1967) concluded that the concentration of mf/mg
was independent of the weight within the range from I to 3 mg snips based on theexaminations of 360 skin snips. In the experiment 4 of the authors, who examined
the relation between the size of skin snips and their MFD, the skin snips whose sur‑23

face area ranged from 5 to 8 mm2 were considered best. It can be speculated that
all the microfllariae will not migrate out in an extremely large snip due to mechani‑
cal factors. In extremely small snips, the relative area of the epidermis where micro‑
filariae are usually present might be reduced in comparison with the whole area ofthe skin snip. Furthermore, the vertical distribution of microfilaria population andthe thickness of skin snips seem to have affected MFD of biopsies in different sizes.

To overcome this kind of obstacle, the use of a skin punch method (Lagraulet andBard, 1969) might solve the problem.

ACKNOWLEDGEMENT
The authors wish to express their gratitude to Dr. Aseffa Tekle, former directorof Imperial Central Laboratory & Research Institute, to Dr. A. Sato of Kagoshima
University, and to Dr. D. Katamine of Nagasaki University for stimulating andhelpful advices on this work, and the authors are also grateful to Dr. L. R. Ash of
University of California Los Angeles for a critical reading of the manuscript.

REFERENCES
1) Duke, B. O. L. ( 1962): A standard method of assessing microfilarial densities on onchocer‑
ciasis surveys, Bull. Wld Hlth Org., 27, 629‑632

2) (1968) : The effects of drugs on Onchocerca volvulus. I . Methods of assessment
population dynamics of the parasite and the effects ofdiethylcarbamazine, Bull. Wld Hlth Org.,

39, 137‑146
3) Duke, B. O. L., Scheffel, P. D., Guyon, J. and Moore, P. J. ( 1967) : The concentration of Oncho‑
cerca volvulus microfilariae in skin snips taken over twenty‑four hours. Ann. Trop. Med. Parasit.,
61, 206‑2 194) Iwamoto, I., Tada, I. and Teferra, W. ( 1972) : Studies on onchocerciasis in llubabor Province,
Ethiopia, Japan. J. Parasit., 21 (Suppl.). , 63

5) Kershaw, W. E., Duke, B. O. L. and Budden, F. H. ( 1954) : Distribution of microfilariae of O.
volvulus in the skin. Its relation to the skin changes and to eye lesions and blindness, Brit. Med.

J., 25, 724‑729
6) Lagraulet, J., Robert, C. and Bard, J. ( 1967) : Technique des biopsies cutan es dans le diagnos‑

tic de l'onchocercose, Bull. Soc. Path. Exot., 60, 297‑300

7) Lagraulet, .J. and Bard, J. ( 1969) : Une m thod d' valuation du degr d' infestation dans
1'onchocercose, Bull. Soc. Path. Exot., 62, 601‑605

8) Nelson, C.. S. ( 1970) : Onchocerciasis. Advances in Parasit, 8, 1 73‑224, Academic Press
9) Nelson, G. S., Amin, M. A., Blackie, M. A. and Robson, N. ( 1966) : The maintenance of Onchocerca
gutturosa microfilariae in vitro and in vivo, Trans. Roy. Soc. Trop. Med. Hyg., 60. Demonst. 1 710) Rodger, F. C. ( 1957) : New observation on ocular onchocerciasis. Related pathological methods

and the pathogenesis of the various eye lesions, Bull. Wld Hlth Org., 16, 495l I ) Rodger, F. C. and Brown, J. A. C. ( 1957) : Assessment of the density of infection with oncho‑
cerciasis and the probable level of safety from its ocular complications, Trans. Roy. Soc. Trop.

Med, Hyg., 51, 271‑282
12) Tada, I., Iwamoto, I. and Wonde, T. ( 1973) : A preliminary report on the examination of skinsnip method used in the detection of Onchocerca volvulus microfilariae, Trop. Med., 15(2), 121‑
122

1 3) WHO Expert Committee on Onchocerciasis. Second Report (1966) : Wld Hlth Org., Geneva

24

皮膚切片からの0πoho661昭∂oJ∂ぬ5マイクロフィラリアの遊出に関する定量的研究
多田功1・岩 本功2・テフェラ・ウオンデ3

エチオピア南西部のオンコセルカ症患者にっいて皮膚切片からのマイクロフィラリア（mf）遊出を定量的に検討した。これは従来のSkin snip法が定量的でないため，精密なMFD（皮膚内mf密度）を必要とする実験のための検討である。その結果，皮膚片は細切すべきでないこと，更に生理的食塩水中でのインキュベート時間は十分長く行なうべきことが明らかにされた。この方法で近接皮膚領域のMFDを計測した結果，きわめて満足すべき値を得た。唯この際，大きさの極端に異なる皮膚片相互のMFDは多少異なることが判明した。この定量的方法を用いて，皮膚を温めた場合のMFDを測定したが加温による変動はまちまちで一定の成績を示さなかった。

1鹿児島大学医学部 医動物学教室 2長崎大学熱帯医学研究所
中央研究所

医動物学部門

寄生虫学部門

3エチオピア帝国

Jap. J. Trop. Med. Hyg., Vol 1, No. 1 1973 pp 25 29

25

ONCHOCERCIASIS, A POSSIBLE ETIOLOGY
OF ELEPHANTIASIS IN SOUTH‑WEST ETHIOPIATEFERRA WONDE1, ISAO TADA2 and ISAO IWAMOT03
Received for publication 19 July 1973

Abstract : A comparative study on dermal tissue and enlarged inguinal lymph nodes fromsubjects infected with O. volvulus is presented and the histo‑pathological findings are

discussed. The present data indicate that there could be causal relationship between
onchocerciasis and elephantiasis in South‑west Ethiopia, endemic focus of the onchocercal

disease. The authors drew particular attention to the non‑filarial etiology reported byother investigators in Ethiopia and assessed the two contradicting views known as the non‑filarial and filarial etiology of elephantiasis.

It is a well established fact that onchocerciasis is endemic in South‑west Ethiopia(Cohen, 1960, Oomen, 1969, Iwamoto et al, 1972 and Tada et al, 1972). The au‑thors observed many cases of elephantiasis, in particular that of the lower legs, in peo‑ple suffering from onchocerciasis. Although Oomen ( 1 969), Price ( 1 972) and Heather
and Price (1972) concluded that in Ethiopia filarial infection could not be the cause ofelephantiasis, the present authors tried to investigate the possible causal relationshipbetween onchocerciasis and elephantiasis. Then the inguinal and femoral lymph
nodes of many people whose skin‑snips released O. volvulus microfllariae were foundpalpable and quite often enlarged in most cases. In this paper, the histo‑path‑ological findings of the dermis and inguinal lymph nodes containing onchocercal
microfilariae are presented and onchocerciasis as a potential etiology of elephantiasis
is discussed.

MATERIALS AND METHODS
Enlarged inguinal lymph nodes were taken from I O subjects whose skin snips fromthe buttocks and legs freed numerous onchocercal microfllariae. Inguinal nodes
from affected limbs 6, palpable inguinal lymph nodes from clinically normal scrotumand limbs 3, and one inguinal node from a patient with scrotal elephantiasis. A11
surgically removed inguinal nodes were fixed in I Oo/o formalin, embedded in parafEin
1 Dept. of Medical Zoology, Imperial Central Laboratory & Research Institute, Addis Ababa,
Ethiopia. 2 Dept. ofMedical Zoology, Faculty ofMedicine, Kagoshima University, Kagoshima, Japan(Present address: , Dept. of Medical Zoology, Kanazawa Medical University, Uchinada, Ishikawa,Japan). 3 Dept. of Parasitology, Institute for Tropical Medicine, Nagasaki University, Nagasaki,
Japan (Present address : Dept. of Internal Medicine, in same Institute).
This work is supported by research grant from Imperial Central Laboratory & Research Institute,

Ethiopia and the Overseas Technical Cooperation Agency of Japan.

26

and cut at 5 to 8 p. Slides from each were stained with hematoxylin and eosin.
Skin snips from the same patients with microfilariae of O. volvulus were also sectioned

for histo‑pathological and comparative studies as described above.

RESULTSOut of 10 inguinal lymph nodes surgically removed from subjects with onchocer‑
ciasis infection, 9 were positive for the larval filariae of O. volvulus. Lymph nodematerial with microfilariae is shown in Figs. I and 2.
The criteria for the identification and differentiation of onchocercal microfi‑
lariae from those of other filarial spp. are described by the authors in another paper

which is under preparation. The tissue section of the lymph nodes shows an exten‑
sive fibrosis entangling a large part of the medullary cords and a portion of peripheral
lymphoid follicles with remarkable and wide obliteration of sinusoid spaces.
In the fibrotic lesion, blood capillaries are markedly congested and small arteries

are surrounded by dense lamellar fibrosis known as "onion‑skin" appearance. Dif‑
fuse infiltration of chronic inflammatory cells such as lymphocytes and plasma cells
as well as eosinophiles and occasional multinucleated giant cells are seen. There
are a considerable number of microfilariae embedded within the perivascular areasof the flbrotic lesion. The remaining part of the medulla being not severely collage‑

nized reveals enlarged reticulum cells and many plasma cells. Lymphoid follicles
show enlarged germinal centefs.
Despite numerous microfilariae in the dermal tissue as seen in Figs. 3 and 4, no
typical inflammatory reaction around the parasites is present. Perivascular lympho‑
cytic infiltration is observed while eosinophiles are absent.

DISCUSSIONS

Cohen (1960), Oomen (1969), Price (1972) and Heather and Price (1972) exclu‑
ded the possibility of filarial etiology of elephantiasis in Ethiopia. They reported
that both adult and larval filariae were absent from the areas investigated. Further‑more, Heather and Price (1972) reached the conclusion that silicates "play a signifi‑
cant part in establishing conditions favourable for the development of swollen leg,even possibly providing the trigger mechanism for the onset of filarial elephantiasis".
esis quoted above, as indicated in Figs. I and 2, the pre‑In contrast to theypo
h thsent authors detected a number ofonchocercal microfilariae in inguinal nodes. The
extensive fibrosis, diffuse infiltration oflymphocytes, plasma cells, fairly large numberof eosinophiles and multinucleated giant cells seem to be caused by the microfilarial

invasion. In other words, the cellular reaction could be interpreted as a responseto the parasitic infection. The absence of the inflammatory reaction of cellular ele‑ments in the dermal tissue around the microfilariae is also a common phenomenon
in other filarial infections which is not yet clearly understood. Connor et al (1970)
observed also only scattered eosinophilic leucocytes in the pretreated skin specimens.
There is a general consensus that the larval and adult stages of O. volvulus are
only restricted to the dermal and subcutaneous tissue. The presence of numerous

27

onchocercal microfilariae in the inguinal lymph nodes with and without clinical mani‑
festations shows that this opinion needs a revision.

It is quite evident that the organisms migrate from the dermis and subcutis
to the lymphatic system and cause histo‑pathological changes described by otherresearchers (Connor et al, 1970) and present authors. The finding of microfilariae
in the inguinal lymph node of a patient with scrotal elephantiasis indicates also
the causal relationship between onchocerciasis and elephantiasis.
According to the anatomical landmarks of onchocercal microfilariae described bylwamoto et al ( 1972), those of W. bancrofti are excluded in the present study. Further‑more, the examination of the peripheral blood taken by day and night interval gave
also repeatedly negative results. It is worthwhile to note that McConnel (personalcommunication) recently found W. bancrofti infection in Gambella, one of the endemic
foci of onchocerciasis. This flnding, however, does not contradict the present one.

Ouzilleau (1913), Dubois (1916) and Dubois and Forrow (1939) already reported
elephantiasis of scrotum and legs in association with onchocerciasis. Sharp (1926)
described also hydroceles, enlarged testes and lymphatic enlargement of thes crotumin patients infected with O. volvulus. Further, Connor et al (1970) reafhrmed theprobable interaction between onchocerciasis and elephantiasis in Bussinga Ubangi
territory of Zaire.

Our studies discussed above with those of previous authors suggest that O. vol‑vulus could be regarded as one of the potential causative agents of elephantiasis inSouth‑west Ethiopia, especially where onchocerciasis is mesoendemic.
We also do not deny that silicate particles observed in macrophages of inguinal
nodes by Heather and Price (1972) might possibly cause elephantiasis, although we
did not yet pay attention to the problems concerning the trace elements. In otherwords, nonfilarial elephantiasis in Ethiopia could not yet be excluded. We are also
aware of the fact that detailed investigations such as lymphangiography and others
should be performed in the near future before reaching a final conclusion that O. vol‑vulus is the etiology of elephantiasis in the regions surveyed.

ACKNOWLEDGEMENTS
The present work would have been impossible without the help of many people.
The authors express their deep gratitude to Dr. Solarov for removing the lymph nodes
surgically. We are also indebted for reviewing the histo‑pathological changes andcomments to Dr. Tokuoka, Faculty of Medicine, Kagoshima University. The
medical and paramedical personnels of Ras Desta Damtew Hospital deservealso our thanks.

REFERENCESl)

Cohen, L. B. ( 1960) : Idiopathic lymphoedema of Ethiopia and Kenya, East Afr. Med. J.,
37, 53.

2)

Connor, D. H., Morrison, N. E., Kerdel‑Vegas, F., Berkoff, H. A., Johnson, F., Tunnicliffe, R.,
Failjng, F. C.. Hale. L. N., Lindquist. K., McCormick, J. B. and Anderson, S. L. ( 1970) : On‑

28
chocerciasis：Onchocercal dermatitis，lymphadenitis and

Human
3）

elephantiasis

the Ubangi Territory，

PathoL，1，553−579

Dubois，A．（1916）： Le

r61e

pathog6nedeO．∂o伽」螂Leuckart．

4）Dubois，A．and Forrow，M．（1939）：Contributionゑ1，etude
r61e

in

de

O．∂oJ副撹5etudi6au

Nepoko，Ann，Soc．Belge

Bu1L

de

Soc．PathoL

Exot．，9，305

l，elephantiasis Congolais。Le

Med。Trop．ン19，135）Heather，c．J．andPrice，E．w．（1972）：Non一且larial elephantiasis in Ethiopia・Analytical

study of inorganic material in lymph nodes，Tr．Roy．Soc．Trop．Med．Hyg．，66，450−458
6）Iwamoto，1．，Tada，1．andWonde，T、（1972）：Studies on onchocerciasis in Ilubabor Province，
Ethiopia，Japan．」。

Parasit。，

7） Oomen，A．P．（1969）：Studies

21（supPl・），on

63

onchocerciasis

andelephantiasis

inEthiopia，Trop．geogr．

Med．，21，236
8）Ouzilleau，F．（1913）：L，elephantiasis

etles且larioses

dans

le

M，Bonou（Haut−Oubangui）：

R61edela理α吻∂oJ∂ぬ3．Ann．Hyg．M6d、Colon，16，307−321116，688−709
9）Ouzilleau，F．（1913）：Les
gaise）．

Pathogeniedenlaires

humainesl，elephantiasisdelar6gionde cette r6gion．

du

R61e

Mbomou（A倉ique6quatoriale貸an−dela躍如r彪∂oJ襯伽5，

Bull・Soc・Path．Exot．，6，80
10）Price，E．W．（1972）：The pathology of non−nlarial elephantiasis of the lower legs，Tr．Roy．

Soc．Trop．Med．Hyg。，66，150−159
11）

Sharp，N．A．D・（1926）：A

vationsonits12）Tada，1．，Iwamoto，1．andmicrofilariae貸om

contribution

prevalence

skin

in

to

Nigeria，TL

thestudy of O・∂oJ∂ぬ∫Leuckart，with someobser−Roy。Soc，Trop，Mcd．Hyg．，19，373

Wonde，T．（1972）：Studies

on the emergence of Oη

ho68綴∂oJ∂ぬ5

snips，Japan．」．Parasit．，21（supPL），65

エチオピア南西部における象皮病の一成因としてのオンコセルカ症
テフェラ・ウオンデ1・多

田

功2・岩

本

功3

エチオピア南西部には下肢象皮病患者が多く，その地域はまたオンコセルカ症の流行地でもある。従来より象皮病の成因については種々の説があるが，著者らは本症とオンコセルカとの関連を追求するため，イルバボール州に住む下肢象皮病患者10名のそけい部リンパ節の組織学的検索を行なった。その結果，9例のリンパ節についてオンコセルカのマイクロフィラリアを見出した。更に，リンパ節にはマ
イクロフィラリアないしはオンコセルカ感染に対するものと考えられる組織学的所見を認めた。こうしてリンパ系の閉塞が起こり，二次的に下肢の象皮病が成立したと考えうる所見であった。この結果から，

従来東アフリカの象皮病の成因にっいて，非フィラリア性のものとする説に対し，再検討を要すると結論する。

1エチオピア帝国中央研究所 医動物学部門
帯医学研究所 寄生虫学部門

2鹿児島大学医学部

医動物学教室3長崎大学熱懸嚢灘，懸

灘

纏

羅

叢

繭
響昏・

Ei暫l

and2Micro且1ari鼠e

shown

i旦tぬe

ingui1恨口ymI

β一 、

菱

1alltiasis

pationts（Abdel蝋11ub肌bor Provinc偽Etl
ng．3＆nd些Micでo薪1ari翫e

shown

i論the

dermaltissue負（Ab遺ella，撫b瓢b。rPrQvl紅c馬Ethi・pi訊）・

P鼠tまe騰

日本熱帯医学会雑誌

第1巻

第1号

1973頁31−3731沖縄における溶連菌の疫学
塩川優一1・村上正中1・只野寿太郎2・山田俊彦2
昭和48年6月6日

受付

連菌感染症，リウマチ熱，弁膜症という一連の疾

は

じ

め

患単位があり，従って弁膜症の発生を防ぐためににはさかのぼってリウマチ熱，溶連菌に対する対策

熱帯医学においては気候風土と疾病の関係が大が必要となる。われわれは以上の考え方に基づいきな研究対象である。われわれはリウマチ熱とそてリウマチ熱と溶連菌の調査を行なっているのでの原因となる溶血性レンサ球菌（以下溶連菌と略ある。

す）の関係について長年研究を行なっているが最溶連菌は各種の抗生剤に対して感受性を有し，

近沖縄において調査を行なったところ内地と明ら

とくにペニシリンに対してはいまだ耐性菌が発生

かな差違を認めた。これは興味ある事実であるとしていない。そこで溶連菌は至ってコントロール

考えるので以下報告する。

しやすい菌であるとの考えが拡がっている。実際一次症には罹患してもただちに化学療法により治癒せしめることが出来るのである。しかし二次症

溶連菌と疾患の関係
溶連菌は広く生物間に分布しているグラム陽性菌であり，ヒトでも咽頭，皮膚などにしばしぱ見出される。この菌によっておこる疾病は大別して二種類に分けられる。第1はその毒素によって直についてはそうではない。これは菌が直接関与せず，それに対する免疫学的な機序が原因となっているからである。この点を考える時溶連菌に対して一層の関心を持ち対策を講ずる必要があると考えるのである。

接発症する炎症性疾患であって一次症とよばれ，咽頭炎，中耳炎，副鼻腔炎，膿皮症などはもっともしばしば見られるものである。第2は二次症と溶連菌の検索とその性状

よぱれ，リウマチ熱，急性糸球体腎炎，紫斑病，

溶連菌は健康者の咽頭にしばしば証明される。

結節性紅斑などがこれに属する。これらの疾患のすなわち滅菌綿棒により扁桃上の粘液を強くこす

発症には溶連菌の菌体，または毒素に対する免疫って採取し，これを血液寒天上に培養し，β溶血学的な機序が関係するとされ，実際これらでは病を示す集落を釣菌する。最近は採取した綿棒をシ

巣より溶連菌が証明されることはない。リウマチ

リカゲル入りの試験管に入れ，密封すれば，数週熱について考えてみるとまず上気道における溶連間は保存されることが分った。そこで遠隔の地で菌感染があり，ついでその細胞壁に対する抗体が採取しても航空機などで検査機関迄送付すること産生されるが，この抗体はヒトの心臓に対してもが出来る。われわれの沖縄における調査もこの方作用する。すなわち溶連菌細胞壁とヒト心臓組織法によったのである。

は共通成分を有し交叉免疫という機序により心臓

溶連菌は上述のように健康者の咽頭からも証明

組織とも結合するのである。その結果心臓に炎症されるがその頻度は年令により地域により種々でを発生するがこれがリウマチ熱である。リウマチある。一般に学童では成人より保菌率が高い，ま熱の約半数はそのまま治癒するが残りは弁膜に傷た一年では夏には低く冬には高いことが知られて害を残す。これが心臓弁膜症である。すなわち溶いる。また密集した集団では陽性になりやすい。

1順天堂大学医学部

内科学教室

2

同

臨床病理学教室

32

しかし従来行なわれた検索は小中学校の学童，お占めており，リウマチ性心疾患の増加が始まる。

よびしょう紅熱や腎炎の発生した集団のもので一従って小中学校にお・いて心臓検診を行ない心疾患

般人の健康者集団については研究されていない。

を発見し，あわせてリウマチ熱について注意をう

溶連菌はさらに群と型により細かく分類されてながすことは意義あることである。ただしリウマいる。まずその細胞壁の成分である多糖類の性状

チ熱は急性の疾患であるから1年に1回の検診でによりA，B，C，D，Gなどの18の群に分けられる。 はその実態を把握することは困難である。そこで
溶連菌二次症の指標としては間接ではあるが成立
そのうちでヒトで感染症をおこすものは主としてA群である。A群溶連菌は沈降反応を用いてM型

したリウマチ性心疾患を取り，リウマチ熱の既往に分類される。これはLanceneldにより細胞壁歴をあわせて調査することになる。

のM蛋白質の性状を以て分類する。ただしこの方

日本循環器学会を中心とする調査では1971年，

法は免疫血清が得にくいところに問題があった。

日本全国で80万人の学童のうちリウマチ性心疾

ところが最近凝集反応を用い，細胞壁の丁蛋白質

患は0．01％に証明されたにより分類する方法が広く用いられるようになっ

0．24勉であった）3）。この出現率は1960年には（なお先天性心疾患はた。これはGri伍thによる丁型である。この方法

0．18％であったからリウマチ性心疾患はこの10年は抗血清が得やすいがその特異性に問題があった。

間に95％という著しい減少をみたことになる。な

しかし最近宮本によりその特異性が確認され，丁お先天性心疾患は当然のことであるがこの間に大型を応用した国際共同研究が行なわれている2）。

した変動を示していない。リウマチ熱の既往歴を

溶連菌の型と疾患の関係にっいては以前より研有する学童は1971年において0．49％を占めてい究が行なわれている。M型に関してはリウマチ熱

たがこの数字もこの15年間あまり変動していない。

はいずれの型でもおこる。急性糸球体腎炎は12，

リウマチ性心疾患が激減した理由はリウマチ熱

1，4，49型などでおこることが知られ，皮膚感が著しく軽症になったためと考えられる。それで染症にも特殊な型が知られている。型についてとはリウマチ熱が軽症になった原因は何かといえば

くに興味あるのはその地域的，年次的分布である。

それは医療が発達し，リウマチ熱の原因となる溶

これは1964年以来行なわれている国際共同研究

連菌感染症が起るとただちに抗生物質が投与されではじめて明らかになった2）。1964年にお・いて世るためと考える。また経済的発展により栄養がよ

界各国よりの材料では12型がもっとも多くっい

くなり，疾患に対する抵抗力がついたことも重要

で4型が多かった。日本ではこの時点では4型がと考えたい。しかしそれにもかかわらず溶連菌はもっとも多く次いで6型であった。なお・日本では依然として広く分布し，いっでもリウマチ熱，リ1938〜39年には4型が優位，1955〜57年にも4

ウマチ性心疾患が起り得る状態にあるのである。

型が主であったがやがて1958年以後6型が優位となりっいで1964年の4型の優勢に至っている。

東京都府中市の調査

第2回の国際共同研究は1968〜69年に行なわれたがこの時期には多くの国で12型が優勢となった。
そして日本もこれと同調するごとく12型が主となっぎに述べる沖縄の調査成績と比較するために対照として，われわれが1969年以来行なっている

り現在に至っている。そして12型が腎炎の原因菌

東京都府中市の某小学校の成績を述べて置く。こであることを考える時この動向は注目に値するもこでは咽頭溶連菌の検索を行なったが，1969年にのである。

は溶連菌38．2％，そのうちA群15．2％，1970年にはおのおの25．0％と19．2％，1971年には25．0男とリウマチ性心疾患の頻度

19．9％であった。すなわち溶連菌の陽性率はかな
り高い。そしてそのうちA群溶連菌の型別をみる

リウマチ熱は小中学校の学童に多い疾患である。

と12型が優勢を占めておりその他1型，3型，4型

またこの年令層では先天性心疾患がかなりの数をなどがみられ，世界およぴ日本の趨勢と一致して33いた。また血清ASO値を検査し，その333Todd
単位以上を陽性とすると・1969年10．0％，1970年
6．8％，1971年15．7％を示しており，これは溶σO連菌による感染の存在を示すものである。

この集団におけるリウマチ性心疾患は1969年，70年，71年の3力年にわたり0．of％であり，これ

Main
100km

Is．αo

（type12）・♂

は同期間の日本全国の成績と一致していた。また・08

リウマチ熱の既往者は1969年は0．53％であったが1970，71年はおのおの0，18お・よび0．11％と減少を示した。

00kinawa4

Is． 宅（type12）》

沖縄波照間島における調査

IshigakiIs．

以上のような日本内地の状況，とくに東京都府（type11）D

中市の成績と比較するために沖縄において1971年

な4∠2Hateruma

以来調査を試みた。まず沖縄の最南端にある波照

Is．

（type11）

間島を選び調査を行なった。

Taiwan

この島は石垣島より海上約70kmの位置にある孤島で行政上は沖縄県八重山郡竹富町に属する。

面積15km2人口約1，000，本土との交通は1週

Fig．l

2回の定期船によるのみである。

TABLE1

Incidence

ofhemolytic

secondaryschoolchildren

Fuchu（Tokyo）

Group

A

（％）

Total Str．
（％）

Dominant
T−Type22（15．2）

38（38．2）

188

36（19．2）

47（25．0）

1971

176

35

（19、9）

44（25．0）222

90（40．5）

135 （60．8）

ll

332

83（25．0）145（43．7）11ll2

58（5L7）70 （62．5）

11

110

39

56

（50．9）

11

55（7L4）

12

1971

July
1972

Ishigaki1972（R．yukyu）

March（Ryukyu）

NumberExmined

1972

March

77

（35．4）

41（532）islands

and

145

March

Koza

streptococci庁om throat culture among primary

1970

1971

Hatemma

Ryukyu1969

March（Ryukyu）

and

222

Year

T types ofhemolytic streptococci isolated

位om main island
in Japan．

34
Incidence of heart disease, rheumatic fever history, and high ASO tit.er

TABLE 2among primary and secondary schoolchildren

Number( o/oCHDExamined)

RHD

16.162 50 (0.31)
18.187 47 (0.25)
19.300 44 (0.23)

2 (0.01)86 (0.53)

3 (0.01)32 (0.18)

9/132 (6.8)

2 (0.01)22 (0.ll)

25jl59 (15.7)

t

Year
1 969

Fuchu

1970

(Tokyo)1971*High ASO

RF History
('/.)

('/*)

('/.)

(10.0)

1 4/ 1 40

H Lteruma(Ryukyu)

1971

334

2 (0.60)o?

lO!273 (3.7)

Koza

1971

2.200

2 (0,09)

4 (0.18)

p?

(Ryukyu)* over 333 Todd Unit

35
l 97 l

15

5

6

l

so

5

4

4

2‑

I 972
T

56

March
,,

t 970

IO

9
,

15

2

5

t2

3

6

f

a8

3

2

l 97 l

I 2 3
s

e

4

5
:

.*

2a
!

Ju I y

t 969

2 I

22
12

5

I

GROUP A
3TYPE
I

3 5i.Ia

2

30

9
l

2

I

5

A B c G4 6 12 18 22 528 OT

1971
March

Ty pe

Group

Group and type of hemolytic streptococci
from throat culture in Fuchu (Tokyo).

4 S

e 4
'.

*'

24

s s

s

z

:

( A Primary and Secondary School
in Fuchu'
Fig. 2

9

13 49

ll

12 13 14 22 25 s/a7'4s,g ; {ae e4 UT

:

Total

ABCD G sP

A

Fig. 3 C.roup and type of hemolytic streptococci

from throat culture in Hateruma (Ryukyu) .

41

l 972

23

March
( xoza )
Il

2

I

5

4 4 f

,

2

2

l

39

l 972
4

March

12

B

Ishigaki

5

Ty pe

Group

Fig. 4I

5

3

3 4 6 9ll

l 2 f

12 14 22 23 28 4 a7

A

7

I

UT

3

Total

A B

CD G sP

Group and type of hemolytic streptococci from throat culture in Ryukyu Is.

35

医療施設として薬局1軒，医師はなく，医介補50．9％，A群35．4％とやはり著しい高率を認めた。

1名である。従って住民は重病に罹ると石垣島そそしてその型分類では11型が優勢であった。またの他に行かなければならない。そして抗生物質も9，14型をみること，12型をみないこと，G群がほとんど使用されていない。

かなり多いことなど，波照間島によく似た傾向をこの島にある唯一の小中学校の学童にっいて溶連菌の検索を行なった。第1回は1971年3月，第示した。なお心臓検診や血清ASO価の検査はこ
こでは行なっていない。

2回は同7月，第3回は1972年3月に施行した。

つぎに沖縄本島にっいて調査を行なった。コザ

その成績を述べると，溶連菌およびA群溶連菌は

市の小学校では1972年の調査の結果，溶連菌71．4

第1回60．8％および40．5％，第2回43．7％およ％，A群53，2％という成績であった。これも著し

び25．0％，第3回62．5彩および51．7％に陽性でい高値である。そしてその型別をみると12型が優あった。すなわち対照である東京都に比すれば2勢であった。ここでは11型をみないし9型もみら

倍前後の高い率であった。

れない，など東京都に近い傾向があるが一方14型

っぎにA群の型をみると著しい所見として優勢
なのは11型であってこれはこの3回を通じて同じがありG群が多いことは波照間，石垣島に近い。

そしてここでは23，28型などもみられた。

であり，12型が優位である日本内地および東京都

コザ市にお・ける心臓病については渡慶次寛氏のとは明らかに異なっていた。また12型は第3回以御教示を受けた。その結果はリウマチ性心疾患

外にはみられず，東京都にはない9，14型をみる

0．18％ということで内地に比して明らかな増加をことがあるなどの差違を認めている。さらに群にみている。なおコザにおいては1973年4月にふた診いてG群が多いことも注目すべきであろう。

たび検診を行なったがその最終的な成績は得られ

このように咽頭溶連菌の出現率が高いのに拘らていないがリウマチ性心疾患は0．1％と推定され，ず血清ASO値の333Todd単位以上は3．7％とやはり内地より多いようである。また血清ASO

低い。すなわちこのように保菌率は高いが感染は価，リウマチ熱既往も今回は調査しているので次少ないことを示している。

の機会に報告する予定である。

それではここでリウマチ性心疾患の頻度はどの位かが間題になる。1971年心電計，心音計を用い以上の調査よりの結論てこれらの学童の心臓検診を行なった。その結果
は先天性心疾患0．6％，リウマチ性心疾患は0という成績であった。ただし対象学童の数は334名であるからこれで内地より低いということは推計学上いえないが，しかし著しくリウマチ性心疾患が多いであろうという予想は裏切られたのである。

すなわち血清ASO値が上昇していないことと相まってこの島の溶連菌は健康保菌者の状態で存在している。その理由が11型という特殊な型のためであるかどうかはなお未解決である。

われわれは，日本内地と沖縄各地を溶連菌の面より比較しこれによりその後遺症であるリウマチ
熱とリウマチ性心疾患の発生要因を探ろうという考えで以上のような研究を行ないつつある。その結果得られたことはまず日本内地と沖縄の咽頭溶
連菌の陽性率における大きな差違である。すなわ
ち溶連菌は日本内地よりも沖縄にはるかに出現率が高い。その原因は衛生状態，医療とくに抗生物質使用の普及状況などにあると考えるが，その他に沖縄の高温，高湿も関係するであろう。ただしその他の沖縄における調査

波照間島における3回の調査では3月の方が7月より陽性率が低かった。そしてそれに伴ってリウ

内地と波照間島の明らかな差違が知られたので

マチ熱，リウマチ性心疾患の陽性率も高いわけでその中間はどうなっているかがつぎの研究課題とあるが，確かにコザでは府中よりも高率であった。

なる。まず石垣島の石垣市における小学校で調査

しかしこの咽頭に高率に存在する溶連菌はすべを行なった。その成績は例数は少ないが溶連菌て感染しているわけではない。まず咽頭菌陽性で

36

あってもかならずしも血清ASO値は上昇してい

約3日，悪感戦標で発病し，高熱，頭痛，四肢痛，ない。そしてこの点は府中に比べて波照間の方が数ヵ所の皮膚に蜂窩織炎を生じ，その他筋炎，関血清ASO値ははるかに低値であったことでも明節炎，リンパ節炎，腹膜炎，胸膜炎，眼球炎，顔らかである。しかしわれわれは陽性率が高いこと

面丹毒などをみる。そして敗血症または心臓麻痺

はそれだけ感染の機会が多いと考えている。

で急速に倒れた。患者数は1，000，死亡300余名

最後に興味のあるのは証明された咽頭溶連菌のに及んだ。翌1915年二木，宮川の出張研究によリ

群，および型である。まずA群溶連菌の丁型におレンサ球菌を分離し，その感染症が流行したもの

いて内地は12型が優勢であるのに沖縄では波照間とされた。当時のことであるから抗生剤がなく，島，石垣島では11型が優勢であり，さらに沖縄本

このようなはげしい流行をみたものと思われる。

島では内地と同じく12型が優勢であった。すなわ

さらに沖縄の高温，高湿な気候，風土がこのよう

ち石垣島と沖縄本島の間には溶連菌の型が変化すな流行を起すに関与したものであろう。

この奇病の記録は非常に興味深いものであるがる所があると考えられる。われわれはさらにこの両島の間の小島を調査しこの変換する線を明らか

当時この菌の群，型を分類することはなく終ってにしたいと考えている。この型の変化の原因は分おり，現在の溶連菌の保菌状態といかなる関係にらない。しかしおそらく住民の移動により溶連菌あるかは分っていない。しかし沖縄と溶連菌の関が運搬されそれによりこのような変化が生じたも係が古いものであり，また密接であることを物語のと考えたい。最近沖縄の急速な発展と観光客をるものである。

含めた人口の移動は著しいものがあり，この情勢は当然溶連菌の型の分布に影響を与えていると思

お

わ

り

に

われる。われわれはこの研究を続行しているがそ

1．沖縄の数カ所において小中学学童の咽頭粘の中で，とくに波照間島の菌型が内地と同じにな

液より溶連菌を分離した。そしてその出現率，群，る時点はいっか，その時もっとも大きな要因は何型（丁型）を内地（東京府中市）のそれと比較しかということに興味を集中している。すでにこのた。また同時にリウマチ性心疾患の罹患率，血清

島でも1972年にはじめて12型が出現しており，

ASO価，リウマチ熱既往などを調査した。

1973年に予定される調査の結果が期待されるので

2．沖縄の学童では内地のそれに比し咽頭溶連ある。

菌の出現率は高く，リウマチ性心疾患の頻度も高いが血清ASO価の異常率は低い。大部分は保菌

沖縄における奇病の報告状態にあるもので何らかの機会に感染しリウマチ熱が発症すると考える。

この研究に従事している途中で東京大学医科学研究所沢井芳男教授よりひとっの重要な示唆を受3．沖縄の波照間島，石垣島ではA群溶連菌のけた。それはかって沖縄に奇病の流行がありそれうちで11型が優勢であった。一方沖縄本島は内地

は溶連菌感染症によるということである1）。文献と同じく12型が主力を占めていた。この型の分布によると大正3年（1914年）7〜8月より沖縄本 状態は興味ある問題であろう。
島において一種の奇病が流行した。本症は潜伏期

文

献1） 二木謙三，宮川米次（1918）：沖縄県に於ける一種の流行性連鎖状球菌疾患の細菌学的研究，日2）3）

本衛生学会誌，11，277
宮本 泰（1972）：菌型のもっ意義とその変遷，診療と保険，11，1230
Shiokawa，Y．（1973）：Past，present
diseaseinandJapan，Jap．Circul．J．，37，171

futureofrheumatic

fever

and

rheumatic

heart

37

EPIDElvIIOLOGY OF STREPTOCOCCI IN RYUKYU ISLANDSYUICHI SHIOKAWA1, MASANAKA MURAKAMll
JUTARO TADAN02 and TOSHIHIKO YAMADA2
Received for publication 6 June 1973

Primary and secondary school children in Ryukyu Islands were subjected to throat
culture for hemolytic streptococci (HS) to study the incidence of HS as a whole, group andtype distribution, and the prevalence of rheumatic heart disease. The results werecompared with those obtained in school children in Fuchu City of Tokyo Metropolis by
the similar procedure.

The incidence of HS as a whole was much higher in Hateruma, Ishigaki and Koza
of Ryukyu Islands than in Fuchu City. In typing of group A streptococci by T‑aggluti‑
nation it was interesting to see that the dominant type in Hatetuma and Ishigaki was
T‑1 1, while in Koza it was T‑12, which was similar to the dominat type in Tokyo andother part of Japan. The incidence of rheumatic heart disease in Koza, Ryukyu, waslO times as high as Fuchu, Tokyo Metropolis, as expected. The discrepancy of typesof streptococci in Ryukyu Islands is worth mentioning, and further investigation is con‑tinued by us.

l Dept. of Internal Medicine, 2 Dept, of Clinical Pathology, Juntendo University, School of
Medicine, Hongo. Bunkyo, Tokyo, Japan.

Jap. J Trop. Med. Hyg., Vol 1, No. 1, 1973, pp. 39‑50

39

SEASONAL VARIATION IN HEMOGLOBIN CONCENTRATION
AND HEMATOCRIT VALUE*SHOTARO NERIISH12, KAZUKO FUKUSHIMA3 AND LEONARD A. SAGAN4
Received for publication 1 3 June 1973

Abstract: The observation that hemoglobin concentration and hematocrit value varywith the season has not been generally accepted but studies made in Japan support this.

Reported here are the results of analysis on the month‑specific mean hemoglobin
concentration and hematocrit values from 29,482 blood samples collected during the 5

years from 1958 to 1963 from ABCCIJNIH Adult Health Study sample members in
Hiroshima and Nagasaki. The values show definitely that seasonal variation is present.

Both hemoglobin and hematocrit values showed a pronounced negative correlation totemperature and humidity.
It was found on grouping the subjects by sex, age, and relative weight that the rangeof seasonal variation was greater in older persons than in younger, and in obese persons
than in those of light weight. It was characteristic that the range of seasonal variatio,nwas small in the groups with high levels and, contrarily, Iarge in the group with low levels.

The two extremes were presented in males under 40 years of age of the light relative weightgroup, and in obese males aged 40 and over.

There have been a number of reports that hemoglobin concentration varies
seasonally, but this conclusion has not been generally accepted. Wintrobe (1967),
in his text book of hematology, denies the existence of seasonal variation.
Engelbreth‑Holrn and Videbaeck ( 1 948) reported in 1948 that determinationsof hemoglobin concentration and red blood cell counts in 69 medical students in Jan‑uary, March, June, and October showed seasonal differences with somewhat lower
values noted in June. According to Wilson ( 1 953) who made monthly examinationsfor 2 years between 1949 and 1952 of 1 5 members of an Antarctic Exploratio,n Party,
no variation was seen in red blood cell counts, but there was an evident seasonalvariation in hemoglobin concentration. Further, Christie (1958) observed in six

members of a party exploring the Central Greenland lcecap a rapid decline inb blhemoglobin level during the first 2 weeks, and thought it was proa yattributableto physical labor.

In a I ‑year follow‑up study of the hematocrit value in 24 young Japanese menl Adult Health Study, 1958‑63, Hiroshima and Nagasaki by Atomic Bomb Casualty Commission
and Japanese National Institute of Health. 2 Dept. of Clinical Laboratories, ABCC, Sakurababa,

Nagasaki, Japan. 3 Dept. of Statistics, ABCC, Hijiyama Park. Hiroshima Japan. 4 Dept. of
Medicine, ABCC (Present address : Palo Alto Medical Clinic, 300 Homer Av., Palo Alto, California,
U.S.A.).

40

and women, Watanabe (1958) observed a decline in the summer, and ascribed it
to probable blood dilution. The results of Wadsworth ( 1 954) did not support theinference made by Watanabe. He could not demonstrate any statistical differencein hemoglobin value between inhabitants of tropical Singapore and of temperate
Europe.
These reports indicate that opinions are divided regarding seasonal variationof hemoglobin and hematocrit values. Numerous factors might induce seasonalvariation. For example, the amount of physical activity fluctuates during a year
according to occupation, which could be reflected in seasonal variations in thesevalues. They could be affected by seasonal changes in food variety and intake.
The presence or absence of seasonal variation in hemoglobin, hematocrit, andother blood constituents and the relationship of such variation to sex, age, and somato‑

type are examined here. Analysis was made of monthly variation in approximate‑ly 30,000 blood samples obtained during the 5 years from 1 958 to 1 963 from a large
fixed sample of adults in two middle‑sized Japanese cities.

MATERIALS AND METHODS
The individuals on whom blood tests were regularly conducted are voluntary
participants in the joint ABCC‑JNlH Adult Health Study. This population sample
comprises A‑bomb survivors and nonexposed comparison subject residents in Hiro‑shima and Nagasaki Cities (Freedman et al.). This fixed population is divided into
24 subgroups so that each month one subgroup will undergo medical examination
in a biennial cycle. The actual visit of the subjects in each subgroup for examina‑tion may deviate 2 or 3 months from the scheduled month, but the subjects under‑

going examination each month can be considered to be randomly representative of
the entire sample.

Analysis was made of five items of the regular examination : hemoglobin, hema‑

tocrit, mean corpuscular hemoglobin concentration (MCHC), white blood cellcount, and specific gravity of urine.

The number examined each month is approximately 350‑400 in Hiroshima
and I O0‑150 in Nagasaki, and the total blood samples obtained in examinations
during 1 958‑63 was 22,404 in Hiroshima and 7,078 in Nagasaki ; a total of 29,482
for the two cities combined. Since the results of blood tests employed in this study
showed no significant difference in mean values between the exposed groups and thenonexposed groups (Hoshino, 1 964) , data for all groups were combined in this study.
First, the year‑speciflc mean values for each item classified by city, sex, and age
were studied to determine the levels for each group and the presence or absence ofa specific trend during the 5 years. The month‑specific mean values were then exam‑
ined by group to determine the presence or absence of seasonal variation. Month‑
specific mean values were obtained by pooling results for the 5 years.

Relative body weight was used as an index of somatotype. Those of the same
sex, age, and stature were classified into three groups according to body weight:
those under the 25th percentile were designated as the light weight group (L‑group),
those between the 25th and 75th percentile as the middle weight group (M‑group),

41

and those at the 75th percentile or more as the heavy weight group (H‑group) (Siegel
et al, 1958‑60).

Standard methods were used in the blood tests (Freedman et al.). The cyan‑

methemoglobin method was employed for hemoglobin determination ; for hema‑
tocrits the Wintrobe tube was used in Hiroshima and the capillary tube in Nagasaki.

The month‑specific mean values for temperature and humidity were basedon records of the weather stations of the two cities.

RESULTS
The mean hemoglobin and hematocrit values by city, age ( <40 vs 40+), andsex in each year during the 5 years demonstrate a slight but not significant increase
from 1 958 to 1 963 (Table I ) . However, Iarge differenqes are noted by sex and age.

TABLE I Number examined and mean values of Hb and H‑crit by year, sex and age( 1958‑63, Hiroshima and Nagasaki)

40 +

< 40

Year

N

H‑critHb(g/ I OOml) ( '/* )N

Hb(g/ I OO^1)

Total

H‑crit N(o/o)

Hiroshima

Male 1 958‑59

60 l

l 4. ll 4.5

60‑6 l680
603

6 1 ‑62

56 1

62‑63

59‑60

595
3040
919
1597

6( 6 1l 1 156 1 ‑62

1 162

62‑63

l 056

14.5
12.0
12.2
12.2
12.4
12.3

Total

5849

12.2

59‑60

Total

Female 1 958‑59

14.5
14.7
14.7

43,9
44.6
44.7

888
1 157

13.6 42.5
13.8 42.5

l 489

13.9 42.6

l 596

1 3.8 42.8

8137

12. 1 37.5

3497
2747
2904
2873

1 3.8 42 .9

44. 5

990
1035

45 . 2

1 02 7l 4. 1 43.4

44.6
37,7
37.7

509 7
1 900

38. l

1632

38, l

1 742

l 2 .4 38. l

38.4
38.0

1817
84 1 812.4 38.4
12.2 38.0

1327

12.0 37.8

12.2 38.0

1837
l 593
1 622

2 246

l 4267

Hiroshima total :

22404
311
931

NagasakiMale 1 958‑59

167

14.2

44, l

1 44

l 3.6

42.5

59‑60

46 7

14.5

44. l

464

14.0

42 . 7

60‑6 1

1 94

14.7

45 . 3181

14.0

43 . 2

61‑62
62 53

378
428

14.5
14.6

44.7
44.2

46 5

13.9

43 . 2452

14. 1

42 . 7

Total

l 634

14.5

44. 5

l 706

13.7

Female 1 958‑59

l 1.9

1 1.8

464

12.2

60‑6 1

32 l

12.3

503
524

12.2

62‑63

787
690

12.2
12.1

2 06

6 1 ‑62

Total

2876

37,6
37.6
38.0
38.0
37.5
37.7

165

59‑60

263
815

42.9
37.2
37.5
38.2
38.0
37.6
37.7

12.2

12.3
12.1

l 862

12.4
12.2

Nagasaki total :

375
843
880
3340
42 8

1279
527
l 290

1214
4738
7078

42

Therefore, the study of monthly changes was made by sex and age. Data weremaintained by city since seasonal temperature and humidity variation are somewhat
different in each city.

oo

Hiroshima 1960‑63 age 40 and over

¥

o

14

Hemoglobin

12

c

s=
" ::

HColo

30 ,'¥̲.
90 Humidity
20
80)¥,
,/
'‑
'¥'v¥
¥¥,/;/'+‑*jv'/'
* .)
('('
lO 70
(7
y
O 60
mperature. f:"'

.'//

¥+ *

‑ . ¥

'1/

/ "

‑‑¥

¥ *¥.‑

44

Hematocrit40

ro 1 4 7 Io 119624 7 Io 1 4 Month1960 ro61 1963 Year

Fig. I Monthly changes in hemoglobin, hematocrit, temperatureand humidity.

As shown in part in Fig. I , the curves of the mean hemoglobin and hematocrit

values for the 5‑year period and the mean curves for temperature and humidity
vary inversely. That is, the hemoglobin and hematocrit curves are elevated during
the cold season between December and February and are depressed in the warm
season between July and September. This phenomenon, though differing in ex‑
tent, is noted in all sex and age groups, and the monthly variations are all signifi‑
cant at the I o/o level by the F‑test.

Since the yearly increment was negligible for the hemoglobin and hematocrit
values, an index of seasonal variation could be obtained by merely averaging all
results over the 5‑year period for each specific month, and it was not necessary toemploy more sophiscated methods, such as link relatives.
The levels for each month are shown in Fig. 2. The range of annual varia‑
tion (the difference between the highest and the lowest values) together with the an‑nual mean derived from the mean values for the 5‑year period are shown in Table 2.

43
Hiroshima and Nagasaki 1958‑63
Hemoglobin

< 40

15

40 +

Male

14

*,

Male

S' 13
o

12

FemaleFemalell

Hiroshima‑‑ ‑‑‑ Nagasaki

Hematocrit

o/o < 40 40 +

46

44

..

'+' ,M l4038= ' 'Female

2 4 6 8 10 12 2 4 6 8 10 12Fig.

TABLE 2Month Month

2 Seasonal variation in hemoglobin and hematocrit by age and sex.

Mean and range* of variation in year of Hb and H‑crit by sex, age andrelative weight groups (Average of 1958‑63, Hiroshima and Nagasaki)
H‑crit (o/o)

Hb (g/lOO*1)

Male I Female< 40 40 + <0 40 +

FemaleMale< 40 40 + < 40 40 +Hiroshima
Total

Mean

14.5
0.7

13.8

12.2

12.2

44.6

42.8

38.0

l .O

0.8

1 .O

1 .9

2.8

2.2

3. 1

L‑group

Mean

15.0
0.5

14.4

12.5
0.9

45.7
2.0

44.3

l .O

12.4
0.9

38.3
2.6

38.7
2.8

37.9
2.3

37.8

37.6

37.7

Range

Range

3. l

37.9

M‑group

Mean

13.8
0.8

12.2
0.7

12.2

44.4

Range

14.4
0.7

42 . 7

1 .O

2.3

2.3

H‑group

Mean

14.2
0.9

13.5

12.l

43.9

41 .8

1 .4

12.2
0.9

1.l

3.3

4.0

37.9
2.8

Total

Mean

14.5
0.7

13.9
0.4

12.2
0.6

12.2
0.5

44.4

42.9

37.7

Range

2.5

2.0

2.3

l .9

L‑group

Mean

14.9

14.4
0.7

12.3
0.5

12.6
0.9

45.4

44.0

38.8

2.4

2.3

38.0
2.4

37.3
2.2

Range

3. l

3.9

Nagasaki

Range

1 .O

M‑group

Mean

13.9
0.5

12.2
0.5

12.1

42.9

37.7

Range

14.5
0.6

44 . 4

0.6

2.4

2. 1

2.l

H‑group

Mean

14.2

13.6
0.9

12.2
0.7

12.1

43.5
4.4

41.9

37.6

Range

3. 1

3.0

l.l

* Difference between the highest and lowest

0.9

3.0

37.5
2.0

44

Between the summer and the winter season a difference of 0.5‑1 glIOO ml in hemo‑
globin and 20/0 30/0 in hematocrit is seen, and this difference appears more marked
in the age group of 40 years or over in both sexes in Hiroshima. The trend was not
as consistent in the Nagasaki data which are based on smaller numbers.
The hemoglobin and hematocrit values not being of the same level for the twosexes and age groups, they have been presented in the form of relative month‑specificindex numbers with I OO as the annual average (Table 3). The month‑specific index

numbers for hemoglobin and hematocrit are very close and the range of variationof each is about 60/0 'TABLE 3

Seasonal index number of Hb and H‑crit by sex and age(Annual average= 100, Hiroshima and Nagasaki)

Hb
Male

Male

Female

40 +

< 40

40 +

< 40

40 +

< 40

lOl.2

l02.3

101.9

102.7

1 02 .O

102.4

l02.3

103.2

2

l02.6

l04.0

l03.5

104.5

l02.2

l03.6

l03.2

l 04. l3

lO1.9

1 02. 1

lOl.8

l02.8

l02.2

102.9

l02.3

l03.7

4

99.7

99.6

99. l

l OO. l

100.4

lO0.6

100.3

101.2

5

lO1.3

99.6

99.7

99.9

lO1.1

99.7

99.7

1 OO. l

6

100.2

99.9

lO0.5

100.0

98.9

99.3

99.3

99.3

7

97.6

97.0

98.2

97.7

98.0

97.2

97.9

97.2

8

98.3

97.9

97.8

96.6

98.2

97.l

97.3

96.0

40 +

9

97.8

97.3

97.2

97.0

97.9

97.0

97.5

96.8

lO

99.9

1 O0.2

99.5

99.3

98.9

99.2

98.6

97.8

ll

99.4

98.8

99.6

99. l

99.8

99.3

lO0.2

99.7

12

99.3

lO0.6

lO0.4

99.3

99.5

101 .O

lO0.9

lO0.3

5.0

7.0

6.3

7.9

4.3

6.6

5.9

8. l

Range *

Range*

Female< 40

Hiroshima

NagasakiH‑critll O0.9

101.l

lO1.7

99.9

lOl.O

l 02 .8

l02.7

101.8

2

lOl.7

lOl.O

l 02. llO1.5

l 02 .4

lO0.9

l02.5

102.0

3

lO0.6

101.6

101.4

lO1.9

lO0.9

lO1.8

lOl.7

lOl.9

4

lO0.2

l OO. 7100.5

1 O0.899.8

l OO. 7

lO0.7

100.9

5

lO0.5

98.8

1 OO. l

98.7

100.3

98.4

lO0.2

98.7

6

100.2

99.5

99.2

99. l

l O0.6

lO0.2

99.6

99.8

7

96.6

99.4

98.5

lOl.O

96.9

99.4

98. 1

lO0.0

8

98 . 9

99.7

98.2

97.8

98.6

98.7

97.6

97.0

9

98.4

99. 1

97.4

98.5

97.4

98 . 2

96.7

97.9

10

100.2

99.5

98.9

99. l

l OO. l99 . 3

98.3

98.5

11

99.9

99.0

1 O0.8

101.5

99.3

98.5

99.9

lO0.9

12

99.7

lO0.5

99.4

99.7

100.2

lO0.6

99.6

99.4

5. l

2.8

4.7

4. l

5.5

4.6

6.0

5.0

* Difference between the highest and lowest in the year

45

Further, to consider the factor of somatotype, the mean hemoglobin and hemato‑
crit levels during the 5‑year period were compared by sex, age, and relative weightgroup, and the results are shown in Table 2. Generally the H‑group shows lower
values for both hemoglobin and hernatocrit than the L‑group in all sex and age groups.
Except for Nagasaki females under 40 years of age, the difference is significant at
the I o/o level. The difference between the two groups is more marked in males andbecome more pronounced with age in both sexes. The M‑group presents values
intermediate between the L‑ and H‑groups. Accordingly, the highest hemoglobin
and hematocrit values were observed in males under 40 years of age in the L‑groupand the lowest values in females of 40 years of age or over in the H‑group.

The monthly changes in mean values for the 5‑year period in these relative
weight groups are shown in Figs. 3‑1, 2. The range of annual variation is given

under each mean value in Table 2. These show for both sexes and age groups,
excluding Nagasaki females under 40 years of age, a smaller range of annual varia‑
tion in the L‑group, who have high hemoglobin and hematocrit levels, and a greater
range of variation in the H‑group, with low levels. This tendency is particularlymarked in Hiroshima, where males under 40 years of age in the L‑group with thehighest levels presented the smallest range of variation annually (0.5gjIOO ml andHiroshima and Nagasaki 1958‑63 (Average)
Hemoglobin Hiroshima^ ̲ 4.40 40+

15+¥ M a I e

‑ 14

Male

S

e
o

¥

ec 13

12

Relative

FemaleFemale‑ L group
Bodv Weight‑‑‑‑ M group

H group

Hemoglobin Nagasaki

Male

15
,

‑ 14

Male

S

o
o

'*

¥

eo 13

r +

12

FemaleAV
Fig. 3‑1

i Femal e

6 8 10 12
2 4 Month
6 8 10 12 AV 2 4 Month
Seasonal variation in hemoglobin by age, sex andrelative weight group.

46
Hiroshima

and Nagasaki1958−63（Average）
Hematocrit

Hiroshima％464442403836

40十

＜40
ノヤ

ノ

、一r、

！

、＼、！

＼、／

︑﹄．

￥、＿

Male

一ノ

Male

FemaleFemale％46444240383

Hematocrit Nagasaki

〆へ、
！

6
AV 2 4

6 8

10

12

AV

2

Month

Relative

4

6

8

10

12

Month・

Body・Weight

一…一L

group−M group一H groupFig．3−2 Seasonal variation in hematocrit by age，sex and relative weight group．

2．1％）．Contrarily，in those40years of age or over in the H−group，in whom thelevels−are quite low，both males and免males present ranges of annual variation ex−

cceding19／100ml

TABLE4fbr

hemo910bin

Rangc＊inseasonal

index

numberage

relativeweightgroup

and（Annual

7．2

7．0

5．6

5．9

7．9

10．5

7．7

9．5

5．5

7．6

10．0

6．7

7．3

6．1

8．1

7．3

10．2

﹃ノFD5

4．8

40十

884

5．2

＜40

6FDリノ

the highest and lowest

7．4

40十

6∠69

between

6．9

＜40

−Q﹂4

＊Difference

40十Female5生乳

H−group

〈40

04只∪
乙■45

M−group

Male

40十

Q﹂4﹃︸

L−groupH−critFemale436

Nagasakihemoglobin

and Nagasaki）

Q﹂46
6ドD9

H−grouptheH−crit by sex，

F
Dス26
生義

M−group

＜40

84
30
60﹃
6
8
3
pD6

L・groupof Hb andaverage＝100，Hiroshima

MaleweightHiroshima

hematocrit，andHb

R．elative

9「oupand3％fbr47

TABLE 5

Correlation* of Hb and H‑crit to temperature or humidity bysex, age and relative weight group ( 1958‑63, Hiroshima)

To Temperature To Humidity
Sex, Age andRW Group

p

b

Total

‑.41
‑.Ol

Test forH0: b=0pb‑ .02 l**‑ .30

‑ .022‑ . 009

N.S.

Test forHo: b=0

Hb
Male

/̲ 40

N.S.

.39

‑ .020

*:s

‑.O1
‑.27

‑.014

M‑group
H‑group‑ .02 l*‑ .42

‑ .028**‑ .30‑ .030

Total

‑ .63

‑ .032***‑ .47‑ .036**

L‑group‑ .57‑ .034

**

*

‑ .48

‑ .02 5

**

‑.31
‑.31

‑0.28

M‑group
H‑group‑ .024*‑ .52‑ .035**‑ .49‑ .049

**

Total

‑ .59

‑ .023***‑ .39‑ .023**

L‑group‑ .53‑ .028**‑ .28‑ .023*M‑group
H‑group

‑ .52

‑.019

**

‑ .39

‑ .022**‑ .54‑ .029**‑ .39‑ .032**Total‑ . 63

‑.030

***

.46

‑ .033**

L‑group‑ .50‑ .025**‑ .35‑ .02 7**

M‑group
H‑group‑ .59‑ .030**‑ .41‑ .03 1**

‑ .59

‑ .033***‑ .49‑ .04 1

**

Total

‑.61

‑ .078**‑ .41‑ .079**L‑group‑ .26‑ .038*‑ .26‑ .056*

M‑group
H‑group

‑ .57

‑.077

**

‑ .38

‑ .078**‑ .54‑ .096**‑ .33‑ .090*Total‑ .3 1‑.115‑‑ . 1 3 1

‑ .50

‑.131
‑.130

**

‑ .76

***
**

‑.61

L‑groupM‑group
H‑group‑ .66‑ .089**‑ .43‑ .088

‑ .69

‑.137

**

‑ .62

‑ . 185

**
**
**

Total

‑ .82

‑ ,090‑ .54‑ .089**L‑group‑ .69‑ .099

***
***

‑.37

‑ .082**

M‑group
H‑group‑ .74‑ .082**‑ .54‑ .090‑.71‑ ,097**‑ .44‑ .09 l

**
**

Total

‑ .82

‑.115

***

‑ .59

‑.125

**

L‑group‑.71‑ . 102**‑ .2 7‑ .058*M‑group
H‑group

‑ .76

‑.114
‑.123

**

‑.51

‑.116

***

‑ .63

‑ . 148****

L‑group

40 +Female< 40

40 +H‑crit

Male

< 40

40 +

Female

< 40

40 +

‑ .79

* Linear correlation : Y= a + bX

X' monthly records of temperature or(.
humidityY :monthly average of Hb or H‑crit48

and hematocrit levels become especially low in the summer.
The range of annual variation becomes more impressive if expressed as a rangeof monthly index numbers with 100 as a base (Tables 3, 4). In groups in which
variation is marked the range reaches I OO/o of the annual average.

Lastly, the correlation of monthly mean hemoglobin and hematocrit values withmean temperature and humidity values, by relative weight group is shown in Table 5.
For both hemoglobin and hematocrit high negative linear correlations with tempera‑ture and humidity were noted (with the exception of Hiroshima males under 40
years of age of L‑group, all groups presented a correlation significant at I O/0 50/0
level with Ho : b=0). Generally the correlations tend to be low in the L‑group andhigh in the H‑group. The group with especially low correlation is the L‑group of
Hiroshima males under 40 year of' age, in which there apparently is hardly any cor‑
relation with seasonal change. Further, Fig. I suggests a time‑lag of about lmonth between the observed values of hemoglobin and hematocrit and those of
temperature and humidity. If this was taken into account, the correlation would
probably have been higher, but calculations have not been made.
With reference to MCHC, WBC, and specific gravity of urine, no correlationof their monthly mean values with temperature and humidity was noted in any of thegroups.

DISCUSSION

Analyses made in this study of the numerous data collected over many years
confirm that hemoglobin and hematocrit values in Japanese tend to be high in thewinter and low in the summer.
AdultsChi I dren‑‑‑‑‑ Temperature

%

45

Male
*
.

*

Female 30
‑ 25

20
15

9 10 11 12 1 2 3 4 5 61959 1960

Fig. 4 Monthly changes in hematocrit for children and adults Nagasaki 1959‑60.

49

The presence of seasonal variation of hemoglobin and hematocrit has not general‑ly been accepted in foreign countries. However, in Japan the presence of such varia‑
tion has been noted in studies made by Japanese scientists over a period of some

20 years from around 1940, including those of Watanabe (1958), Kuroda (1953),
Nunoyama (1956), Nishimura (1961) and others.
The result of the present study support these previous reports. This has been
confirmed also by an observation of the seasonal variation of hematocrit in a separate

population of children made in the same laboratory at the same time as this study(Neriishi S., 1969, Fig. 4). Due to multiple reasons no attempt will be made to
explain the mechanism that produces the seasonal variation which is so character‑istically observed in Japan.

The presence of seasonal variations in hemoglobin and hematocrit values it‑
self supports and confirms the findings of a number of other studies, but further ob‑

servation by sex, age, and relative weight group revealed a number of interesting
facts. Though the seasonal variation of the different sex, age, and somatotype
groups may be ,of a common pattern, each differs in extent. Thus, it may be in‑terpreted that the influence of season does not affect all people uniformly. The
most characteristic example is seen in males under 40 years of age in the light weightgroup. In this group, showing the highest hemoglobin and hematocrit levels, therange of seasonal variation is the smallest, and contrary to the general tendency forthe levels to lower with age, the range of seasonal variation becomes greater. Further,the H‑group as compared with the L‑ and M‑groups has lower levels of both hemo‑
globin and hematocrit, and moreover the range of variation is greater. An extreme
example is seen in males 40 years of age and over in the H‑group. In this group,

both hemoglobin and hematocrit levels are markedly lower than those in the L‑
group and the range of seasonal variation is greater than that of any other group,

extending to as much as I Oo/o of the annual average. Thus, each age group wasshown to have its own hemoglobin and hematocrit levels and range of seasonal varia‑
tion.

It is highly probable that the results of clinical laboratory tests may be affectedby such natural conditions as temperature and humidity. However, the fact thatthe seasonal variation differed between the young and old and, especially, between
subjects ofrelatively light or heavy weights, implies that seasonal laboratory variability

cannot possibly represent the whole explanation. .
No detailed analysis of the health condition of the participants was attempted,
but as most of them were ambulant the observations made can be considered to havegeneral applicabilities. Recently, the relationship between obesity and health has
become the subject of much discussion (Fredman et al, 1964), and also a study on
seasonal variation in cardiovascular functioning was reported (Schneider and Cos‑
tiloe, 1972).

The flnding of definite differences in blood parameters such as hemoglobin
and hematocrit is felt to be significant.

This study was based on data collected during 5 years frorn 1958 to 1963, but
the mode of living of the Japanese people is rapidly changing (Kagan et al. 1972),
so that it would be interesting to continue observations hereafter to see whether this

50

phenomenon ofseasonalvariation willtend to disappear．

REFERENCES
1）Christie，R・w・（1958）：Arctic anemia，New Engl．J．Med．，259，605−610
2） Engelbrcth−Holm，J・and videbaek，A・（1948）：Normal blood counts in di佃erent
Blood，

3，seasons身612−616

3） Freedman，L．R．et aL（1963）：ABCCJNIH Adult Health Study Report4，1960−62，ABCC
T．R．，No．20
4）一 （1964）：ABCC−JNIH Adult Health Study，Hiroshima，1958−60、Mortality and bodyweight，ABCC T．R．，No．20
5）

Hoshino，T・et

al・（1964）：Blood

pictureof

aged

A−bombsurvivors，J．Jap．Geront．soc．，1ラ

190−197
6）

Kagan，A．etaL（1972）：Epidemiologic

studiesofcoronary

heart

disease andstrokein

Japmesemen living in Japan，Hawaii，and califbmia−Demographic physical dietary，and biochemical
characteristics，ABCC T．R．，No．16
7） Kuroda，K．（1953）： Seasonal variation of water content in blood，Nisshin Igaku，40，409
8） Neriishi，S．（1969）：Seasonal variation of hematocrit value in childhood，Acta Paediatrica
Japonica，73， 1517
9） NishimuraラY。（1961）：Some studies on change of hemoglobin concentration in human blood
with re琵rence to dynamic constitution，Niigata

Igaku

10）

Nunoyama，T．et32，33711）

schneider，R・A・and Costiloe，J．P・（1972）： Seasonal

Zasshi，75（1），1315−1336

aL（1951）：Environmental temperature and erythrocyte volume，Rodo Kagaku，

12） Seige1，D．（1962）：ABCCJNIH Adult

Hcalthvariation，Arch．Environ．Health，24，10−16

Study，Hiroshima

andNagasaki，1958−60身ABCC

T．R．No．19ラ13）Wadsworth，GR，（1954）：｝laemoglobin
climate，Brit．Med．J．，2，910−911

levelsofnormal

men

and

women

livinginatropical

14）watanabe，G・（1958）：climatice価ctonthepackedredcellvolume，Brit．J．Haemat．，4，108−ll2
15）Wilsonラ0．（1953）：Physiologicalchanges

in

blood

in

the

Antarctic．A

preliminary

report，Brit。Med．J．，2，1425−142816）Wintrobe，M．M．（1967）：Clinicalhematology，90，Lea＆Febiger，Philadelphia

ヘモグロビン濃度およびヘマトクリット値の季節的変動
錬石昇太郎1・福島和子1・Leonard

A．Sagan2

ヘモグロビン濃度ならびにヘマトクリット値が季節的に変動するという観察は一般には容認されていないが，日本で行なわれた研究はこれを肯定している。

ここに報告するものは，広島市および長崎市におけお成人健康調査対象者にっいて1958−63年の5年間に収集した29，482件の血液標本からヘモグロビン濃度ならびにヘマトクリット値の月別平均値を求め解析した成績である。これらの値が季節的に変動することが明確に認められた。ヘモグロビン濃度もヘマトクリット値も，気温および湿度に対して明らかな負の相関を示した。

調査対象を性，年齢および相対的体重値別に分類すると，高年齢層は若年齢層に比べ，また，肥満群
は軽体重群に比べ，季節的変動の幅が大きいことが認められた。季節的変動の幅は，検査で高値を示す

群では小さく，低値を示す群では逆に大きいことが特徴的であった。その両極端は，男子における40歳未満の軽体重群と40歳以上の肥満群とに認められた。

1 原爆傷害調査委員会

長崎

2

同

広島

JAPANESE JOURNALOF
TROPICAL MEDICINE AND HYGIENE
Vol .

August, 1973

l No. lCONTENTS
ITo, Y., ITAGAKI, H. AND WONDE, T.
Studies on the Susceptibility of Biomphalaria pfelfferi rueppellii (Dunker) andB. sudanica (Martens) to Schistosoma mansoni in Ethiopia . . . . . . . . . . .

l‑ 5

TANAKA, I., INouE, Y., TADA, I., IWAMOTO, I. AND WONDE, T.
Simulium damnosum, Naturally Infected with Onchocerca volvulus in South‑west Ethiopia

7‑1 1TAl)A, I., IWAMOTO, I. AND WONDE, T.
Quantitative Studies on the Emergence of Onchocerca volvulus Microfilariae

13‑24

from Skin Snips . . . . . . .

WONDE, T., TADA, I. AND IWAMOTO, I.
Onchocerciasis, a Possible Etiology of Elephantiasis in South‑west Ethiopia . . . . . . . . . . . . . .

25‑29

SHIOKAWA, Y., MURAKAMI; M., TADANO, J. AND‑YAMADA, T:
Epidemiology of Streptococci in Ryukyu Islands . . . . . .

NERIISHl, S., FUKUSHIMA, K. AND SAGAN, L. A.
Seasonal Variation in ‑Hemoglobin Concentration and Hematocrit Value .

Published byJAPANESE SOCIETY OF TROPICAL MEDIClNE
clo Institute for Tropical Medicine, Nagasaki University

12

Sakamoto‑machi, Nagasaki, 852, Japan

31‑37

39‑50