ISSN

Japanese

Joumalof

TropicalMedicine

Hygiene平成元年3月15日

第17巻第1号
内原

andO304一2146

容著

ヌマカ属（アシマダラヌマカ）の幼虫および蝋の呼吸器の走査電子顕微鏡
による形態観察……・………・・…………一……・・……・…………・……………岩城

操

1−7

荘平・

9−15

丁吻αηoso㎜わ耀06プg脚6碗s召感染マウス血清中のIL−2産生抑制物質

…・陶

道，日笠

檎宮本彦四郎申田

和彦，小松

俊憲，新家

濾紙使用による微量血清中のHBsAgのELISAによる測定（英文）・・松田 晋哉，華表宏有

17−25

喀疾液化剤dithiothreito1（SputasoI）処理によるP％8㈱o釧s漉oo吻露の診断（英文）……一…・…・・…………一…・・一…………一…¶…一……・・塩田 恒三

27−31

第30回日本熱帯医学会総会講演英文抄録

目 次一…

33−36

特別講演・

37−39

シンポジウム1

熱帯性感染性と薬剤・……

40−50

11熱帯性感染症とワクチン開発の現状とトピックスー……
パネルディスカッション…

51−58
59−68

一般講演・
会

69−103

報

会則一

会員名簿・役員……………

105−108109−130

投稿規定

日熱医会誌

Japan．」．T．M．H．

日本熱帯医学会

日本熱帯医学会雑誌

第17巻

第1号

1989

1

1−7頁

ヌマカ属（アシマダラヌマカ）の幼虫および蜻の呼吸器の走査電子顕微鏡による形態観察岩城操

昭和63年8月18日受付／昭和63年12月26日受理

緒

カギ状の爪と，先端内部から一対の千鳥型の爪お言

よび薄い花冠状の膜組織が突出していること，そ孤αnson彪属の蚊は，マレー糸状虫励嘱g伽

して2令から4令幼虫の呼吸管先端部にはさらに吻吻頭の媒介蚊として知られ，疫学的に非常に重多くの爪と花冠状の騰組織とそれに包まれて特殊要な蚊である（Samarawickrema，19681Laur−なスポンジ状の構造をもった組織がみられた。一

ence，1960）。この蚊の幼虫および蠣の水中におけ方，嫡の呼吸角の最先端は鋭く尖った角状を呈し，る特殊な呼吸法に関しては，他のCπ」砿属や

先端部側面に毛細血管状の管によって作られた

・4646s属などとは異なり，幼虫や蝿の生息する水レース状組織がみられ，いずれも，水中の溶存酸域に自生する水生植物の根や茎に呼吸管，または素を呼吸するための呼吸鯛であることが認められ呼吸角を挿入して直接植物組織内に含まれる酸素

た。また，呼吸管および呼吸角内部の微細構造にを呼吸するものと考えられていた（Laurence，

ついても観察した結果から，幼虫および蝿の体内1960）。しかし，著者は，以前〃伽son彪属の幼虫の血液循環に関する経路についての知見を得たのおよび蝿を生体のままホールスライドグラス内にで考察を加えて報告する。

遊泳させ，光学顕微鏡によって観察した結果を報告した（Iwaki，1984）。即ち，水中に静止している

幼虫の呼吸管先端を静かに刺激すうと，呼吸管先材料および方法

端内部から千鳥型の爪と非常に薄い膜状組織が突観察に供した〃伽so％勿（ル勉㎜拓燃）観面撚

出すること，また，この組織が水中の溶存酸素を（Theobald）アシマダラヌマカの幼虫，および蠕

呼吸する呼吸鯉（larvalrespiratorygill＝LRG）

は，タイ国・マヒドール大学熱帯医学部医用昆虫であることを示した。さらに，蝿の呼吸角先端部

学教室において飼育した個体（Supat6！α1．，については，水生植物の根や茎に呼吸角先端を挿1982）であり，比較のために用いたC％1疏

し込み体を固定するために鋭く尖った角状を呈し渉7」如召初ozh翅6h％sていること，また，植物組織から酸素を吸収する

アカイエカおよび。4θ惚sノ ψo痂o郷（Theobald）

ための穴は開口していないばかりでなく，呼吸角ヤマトヤブカの幼虫および蝋は，京都市内で採集

先端部を包む網目状組織の呼吸角鯉（pupal

し飼育して得た個体である。試粍は，あらかじめ
70％アルコール液に保存しダー1騰成時に80％かres−piratorygill＝PRG）を有し，これによって呼吸s％郷郷ozos％s（Dyar）コガタすると考えられることを報告した（Iwaki，ら100％アルコールに置換した後，自然乾燥させ1984）。今回は，走査電子顕微鏡を用いて，幼虫のた（Matsuo召地！．，1972〉己この試料をミニポー

呼吸管および婦の呼吸角にみられる微細構造につル濾紙上に少量の接着剤を付けて固着させ，更にいて観察した。1令幼虫の呼吸管先端部の両側に

これをアルミニウム試料台に両面テープによって京都女子大学家政学部生物学教室

2

,

3
C％忽属や・46磁s属の幼虫の呼吸管は細長い固着させた。

この試料を日立E10110N蒸着用スパッター

管状を呈し，その先端は図7および図8におのおにより金蒸着し，日立S−530走査電子顕微鏡をの示されるごとく，水面に浮上して呼吸管先端を用い，加速電圧10〜15KVのもとに観察した。な

水表面に突出したときはsapを開き空気中の酸

お，幼虫および蝿の呼吸器の各部名称は，Har−素を直接呼吸管内にとり込み，また，水中を遊泳bach

する場合はsapを閉じ気管内に水が侵入するこandKnight（1980）およびIwaki（1984）とがない仕組みになっている（Gillett，1971）。し

を適用した。

かし，砿αnson毎属の幼虫の水中における呼吸法
は，Bates（1949）によれば，幼虫は水生植物の根結果および考察や茎の組織を呼吸管側面に付属するsawによってくだき，その中に呼吸管先端を挿し込み植物組

1．幼虫の呼吸管の観察結果〃伽son彪属のアシマダラヌマカ1令幼虫の織内に含有する酸素を呼吸するので，幼虫は水面

呼吸管先端は，図1のごとく，C祝傭属のコガタにはほとんど現われることはないと述べている。

アカイエカおよび・4ε48s属のヤマトヤブカの呼またGillett（1971）は，幼虫の行動について，普吸管と類似し，円筒型を呈して細長いが，呼吸管通の蚊と同様に後方向に水中を遊泳し，強く水生先端部の両側にカギ状の爪（outer

spiracular

植物の根や茎に呼吸管を挿し込む。このとき体をteeth＝OST）が一対みられる。そして，この1令リズミックに動かしsawを使って植物組織に穴

幼虫が水生植物の根や茎に固着した場合，図2にを開け細い呼吸管先端を挿し込み，同時に6対の示されるごとく，呼吸管先端内から一対の千鳥型

爪をもって体を固着させると述べている。さらにの爪（innerspiracularteeth＝IST）と薄い花冠

Kettle（1984）は，呼吸管は扁平で短かい円錐型状の膜組織（thin田mytissue＝tft）が突出する。

を呈し，堅い頑丈な弁をもち，水生植物の空気をアシマダラヌマカの幼虫の呼吸管は，2令から4含有している組織にまで呼吸管を挿し込み呼吸す

令へと令の進行にしたがって円錐型に変化するが，ることが可能な形をもっていると述べている。し呼吸管先端部には顕著な変化がみられないので，

かしながら，今回走査電子顕微鏡（SEM）を用い

本報告では，2令・3令幼虫については省き，4
令幼虫について記述する。図3は，4令幼虫の呼造について観察した結果，1令幼虫の呼吸管には吸管先端を上側面より観たところであるが，ノコ

水生植物の根や茎に穴をあけるためのノコギリ状ギリ状歯（saw）とISTがみられ，図4に示すご歯は全くみられず，また呼吸管先端が開口した場

とく水生植物の根や茎に体を固着した場合に呼吸合には内部からISTおよびtftが突出すること

管先端内から突出する多数のISTとtftが認めが観察された。このことから，1令幼虫は呼吸管られた。さらに，先端中心部には，図5に示すご先端部のOSTによって水生植物の根などに懸垂てアシマダラヌマカの幼虫の呼吸管各部の微細構

とくtftに包まれたスポンジ状組織（sponge−like

して体を固着させ，同時に呼吸管先端内部より突

tissue＝slt）がみられる。また，円錐型の呼吸管の出するLRGによって水中の溶存酸素を呼吸する

中央部を横に切断した内部には，図6の矢印に示と考えられる。さらに高令幼虫の呼吸法について

すごとく，中心部に1本の太い管（cylindricalは，水生植物の根や茎に呼吸管先端を強く触れ

tube＝ct）と両側面に2本の筋束（muscular

ISTおよびOSTを用いて体を固着させ，呼吸管

band＝mb），そして呼吸管内壁に試料を作成した

先端内部より突出したtftに包まれたLRGに際に少し扁平に変形しているが，気管状の管

よって呼吸することが示唆された。このようにし（thickてLRGから水中の溶存酸素がとり込まれた血液trachea＝tt）が左右一対あることが，それぞれ観察された。

の循環については，すでにIwaki（1984）が光学
顕微鏡による生体観察の結果について述べている

2．幼虫の呼吸管に関する考察

ごとく，左右2本に分岐したttを通り幼虫体内に

4

5
運ばれ，体の各部に酸素を供給したあとは体内の開口することを，著者は蚊の飼育中に観察し，ま中心部にあるポンプ状の管内に集められ，それにたGillett（1971）もそのことを述べている。しかつながるct内を通り再び呼吸管先端のLRGにし，孤α郷on彪属の蝋の特殊な呼吸法についてもどる循環経路をもっている。なお図6の側面には，Bates（1949）によれば蝿の呼吸角先端を水生みられるmbは，ctを前後に移動させctの先端にあるLRGを呼吸管先端内から出し入れさせる

植物の根や茎に強く挿入することによって体を植ためにあり，また，同時にISTを強く振動させて素を呼吸角先端から吸収することによって生活す

物体に固定すると同時に，植物組織に含まれる酸

水生植物の根や茎に押しあてISTを植物組織にるため，羽化するまで静止状態を続け水面には浮懸けるために使われる筋肉である。

上しないと述べている。また，Gillett（1971）は，蠣の呼吸角先端はドリル状を呈し水生植物の根や茎に穴を開け，その部分に呼吸角先端を挿し入れ

3．蜻の呼吸角の観察結果

アシマダラヌマカの呼吸角は，バナナ状で図9

呼吸するとともに体を固着させると述べている。

に示すごとく呼吸角最先端は鋭く尖った角状で，しかし，今回の著者による走査電子顕微鏡での観図10のコガタアカイエカや図11のヤマトヤブカ

察によれば，蠣の呼吸角先端には細かい毛があり，の蝿にみられるようなトランペット型とは全く異鋭く尖っていてドリル状の構造はみられないばかなった形状を呈している。そして，先端部は図12

りでなく，最先端には空気を吸収するための穴ものごとく毛細血管状の管（slender

loop＝sl）が全く開口していない。即ち，呼吸角先端は，単にレース状に組み合された鯉状組織PRGによって水生植物の根や茎に体を固着させる為に尖ってい被われている。また，呼吸角の最先端は水生植物て，実際に呼吸に関与する部分は毛細管状のs1かの根や茎に直接挿入し体を固定するための形を呈らなるレース状組織であると考えられる。このこし，図13に示されるごとく植物組織から酸素を吸とから，体内から呼吸角に送り出された血液は，収するような穴は全く開口していない。さらに，

呼吸角の中心部を通り呼吸角先端内部に達し，多呼吸管先端部を縦に切断した内部には，図14のご数開口しているshからslのレース状のPRGにとく多数の小穴が内壁に点在していることが観察送られ，そこで水中の溶存酸素を血液内にとり込され，同部分の横断面には図15の矢印に示すごと

み，再び呼吸角内のbslを通り体内にもどる循環

く，内壁の小穴から外壁のPRGに連絡している経路をもつことが示唆された。

ことが明らかとなった。また，呼吸角基部の横断面には，図16のごとく，内壁に細い管の管束（bun・dleofslenderloop；bsl）の部分と空洞の2つの部分に分かれていることが認められた。

結論〃1σnsoπ勿属の蚊の幼虫および蝿は，C認飢属や・48磁s属とは異なり，特殊な呼吸法を営むこと

4．蝋の呼吸角に関する考察
C％忽属や・4848s属などの蝿の呼吸角はトラが知られている。Bates（1949），Gillett（1971），

そしてKettle（1984）らのそれぞれの著書には，ンペット型を呈し，C％伽属では水中に潜る場合，〃伽so窺α属の幼虫および蠕は，水生植物の根や図10のごとく呼吸角先端内に細毛があり，その部

茎に呼吸器の先端を挿し込むことによって植物組

分に気泡を保ち呼吸管内に水が侵入しないように

織中に含まれる酸素を呼吸すると述べられている。

仕組まれている。そして，再び水表面に浮上し呼しかしながら，これらの記述は，いずれも肉眼的吸する際には，先端部を水表面につき出し水の表または光学顕微鏡的観察による知見にすぎない。

面張力がはたらき管内には水が流入しないように

今回，著者は走査電子顕微鏡を用いて幼虫の呼吸なっている。一方、46惚s属では，呼吸角先端部に
図11に示されるごとくバルブを有し，水中に潜る管および蠕の呼吸角の微細構造をくわしく観察しした結果，次のような結論を得るに至った。

場合はバルブを閉じ，水表面に浮上した場合には1．幼虫の呼吸法については，1令幼虫の場合，

6
呼吸管外壁に付属する気管外爪（OST）に

吸角先端部に附属する毛細血管状の管がレーよって水生植物の根や茎に懸垂して体を固着

ス状になった呼吸角鯉（PRG）によって，水し，2令から4令幼虫の場合は，呼吸管先端

中の溶存酸素を呼吸すると考えられることがを水生植物に触れさせ気管内爪（IST）を用い

構造上示唆された。

て植物体に付着して体を固定させる。そして，実際の呼吸は，すべての令を通して，呼吸管

稿を終るに臨み，本研究の資料の提供に御協力

先端内から突出する呼吸管鯉（LRG）によっ下された，タイ国・マヒドール大学熱帯医学部医て水中の溶存酸素を取り込むことによって行

用昆虫学教室のDr．Supat Sucharit教授並びにう。

教室員各位に深謝する。

2．蠣の呼吸角全体はバナナ状を呈し，キチン化して鋭く尖った最先端を水生植物の組織に

尚，本稿の内容は，第36回日本衛生動物学会大強く挿し込み，体を固着させる。そして，呼会（1983年10月）において発表した。

文
1）Bates，M．（1949）：The

natural献

history ofmosquitoes，New

2）Gillett，J．D．（1971）：Mosquitoes，Weidenfeld3）Harbach，R．E，and

York141−142

and Nicolson，London，27−55

Knight，K．L．（1980）：Taxonomists

glossaryofmosquito

and

pupal

anatomy，Plexus

Pub．，U．S．A．，130−347

4）Iwaki，M．（1984）：A

note

of

the

siphonandtrumpetinlarvalstagesof〃碗so％宛

観〃b槻お（Theobald），Japan．」．Trop．Med．Hyg．，12，33−38
5）Kettle，D．S．（1984）：Medical

andVeterinary

Entomology，Croom Helm．，London andSydney，

99−136
6）Laurence，B．R．（1960）：The

and〃4％soη砒

biology％％加ηη鉛

Culicidae），BulL
egg

two

species of mosquito，〃1αnson㍍励肱nα（Theobald）

to

the

subgenus〃lonsono痂召s（Diptera；

Ent．Res．，51，491−517

7）Matsuo，K．，Yoshida，Y．and
1．The

of

（Theobald）belonging

surfacesofKunou，1．（1972）：Scanningfive

species

of／1848s

electronand且7別㎏召名召ss幼ofmosquitoes．

z必4劾ε」．Kyoto

microscopy

Pref．Univ．

oftwo

ofmosquito，Med．，81，358−363
8）Samarawickrema，W．A．（1968）：Laboratory〃1αnso磁z（〃1伽so痂oゴ4εs）襯加 n商（Theobald）from

culture

and

life

cycle

（Theobald）andκαnson勿species（〃碗sonJo躍召s）召n％％」吻解

Ceylon，Ceylon J．Med．Sci．，17，7−199）Supat，S．，Chamnam，A．，Rachada，R．andVanida，K．（1982）：Improved

oviposition

medium for〃伽so痂αcolonization，Mosq．News，42，357−359

LARVAE
Photo1−6ル正

Photeznson凌z％n〃わプ解ゑs：l Side2view

Dorsalof

3Anteralside4

theview ofview

closedtip

theopenedoftheopenedDorsal view of the opened

5Ventral view
6ofsiphontip ofinlst

instarlarva，×1，000

siphon in lst instar

tip

of

siphontipofsiphonoflarva，×2，000

instar

in4th

instar

in4th

instarlarva，×300

larva，×1，000of the opened

tip

1nner

part

of

the

of4th

7Anterior

view

of

the

siphonof4th

instarlarva（C廊」ε究

thesiphonof4th

instarlarva（Zし召46s勉ρo初6％s），×400

siphonsiphonin4th

instarlarva，×3，000

larva，×1，000

痂磁ηわ功翅oh％s

os％s），×400
8Anterior

PUPAE

view

of

sκ御挽o処7

Photo 9 and 12‑16 Mansonia umformis:

Photo 9 Side view of the tip of trumpet, x300
10 Anterior view of the trumpet of pupa (Culex tritaeniorhynchus summorosus)
11 Anterior view of the trumpet of pupa (Aedes 'japonicus), x 400

,

x 400

12 Respiratory "gill" of the structure of a slender loop, x6,000
13 Ventral view of the tip of trumpet, x500
14 Cross section of the tip part of trumpet, x3,000
15 Vertical section of the tip part of trumpet, x3,000
16 Cross section of central part of trumpet, x 500

ABBREVIATIONSOST: outer spiracular teeth ct: cylindrical tube
LRG: larval respiratory gill tt: thick trachea
saw:
IST:
tft:
slt:

sap:

siphon mb:

inner spiracular teeth sl:

muscular band
pupal respiratory gill
slender loop

thin filmy tissue sh: small holesponge‑like tissue bsl: bundle of slender loop
spiracular apparatus fa: filter apparatus

MORPHOLOGICAL OBSERVATION OF LARVAL AND PUPALRESPIRATORY APPARATUS OF MANSONIA (MANSONIOIDES)
UNIFORMIS (THEOBALD) (DIPTERA, CULICIDAE) BY

SCANNlNG ELECTRON MICROSCOPEMISAO IWAKI
Received August 18 1988/Accepted December 26 1988

The respiratory organs of Mansonia umformis larvae and pupae were minutely observedby scanning electron microscope (SEM) . Crown‑shaped, gill‑like, thin filmy and sponge‑liketissues were observed at the tip of the siphon of the Ist and 4th instar larva. Similarly,
characteristic structure consisting of fine, slender loop (like capillary blood vessels) was

observed at the tip of the pupal trumpet. The auther suggested that immatures of Mansonia
take in water soluble oxygen with the respiratory "gills" which exist at the tip of the siphonof the larva and at the tip of the trumpet of the pupa.

Biological Laboratory, Kyoto Women's University

日本熱帯医学会雑誌

第17巻

第1号

1989

9−15頁

Tη勿％oso卿α67％06i

9gπ7nδ」伽s6感染マウス

血清中のIL−2産生抑制物質

陶道，日笠穣，宮本彦四郎
山田 和彦，小松 俊憲，新家 荘平
昭和63年9月5日受付／平成元年1月24日受理

はじめに

材料と方法

トリパノソーマ原虫は，アフリカ睡眠病を引き起こす病原体である。この原虫が感染すると，原

1）実験動物BALB／cマウスとC57BL／6マウスは日本

虫の表面抗原に対する特異抗体が産生されるが，チャールス・リバー株式会社より購入した。感染トリパノソーマは抗原変異により，この宿主の防実験には，6週齢の雌性マウスを用いた。

御反応より逃れることが知られている。また，ト

2）原虫および感染方法

リパノソーマ感染時には，非特異的免疫抑制状態

丁肋σnoso惚扉躍θ1即励ガ6πsd以下，Tbgになることが報告されている。この抑制の成因にと略する）はマウスで長期継代されたWellcome

ついて，免疫細胞の機能低下，抑制細胞の存在，株を用いた。Tbg原虫の分離は，Lanhamの方法

或はインターロイキンー2（IL−2）活性の低下等に準じた（Lanham扉α1．，1970）。すなわち，感が考えられている。最近になって，トリパノソー

染マウスより心採血し，DEAEセルロースカラム

マと同様に免疫抑制を起こすマラリア原虫に感染を通過させて血液成分より分離した後，リン酸緩すると，マウスの血清中にIL−2活性を抑制する，

衝生理食塩水（pH7．0）で3，000rpm，10分間の

いわゆる1レ2インヒビターが上昇することが報

遠沈により3回洗浄したものを実験に用いた。マ

告された（Lelchuk8∫α1．，1985）。このIL−2イ

ウスヘの感染は，Tbg原虫2×104個を腹腔内にンヒビターの上昇は，マラリア感染時のIL−2産生

投与することで行った。

能の低下の原因の1つと考えられ，したがってマ

3）脾細胞浮遊液の作成

ラリア感染時の免疫抑制に関与している可能性が示唆されている。

Tbg感染後1日，2日，3日目のマゥス，または正常マウスより，脾細胞を無菌的に摘出し，単今回の実験において著者らは，トリパノソーマ

細胞浮遊液を作成した。細胞浮遊液は，トリパン

感染による免疫抑制には，IL−2インヒビターがブルーにて生細胞数を数えて，5×106個／mlに関与しているかどうかを明らかにするため，調整した。培養液は5×10−5Mの2一メルカプト

Tη餌noso解αわ鰯6切80窺6歪6ns8の感染時におエタノール，10％牛胎児血清（FCS）を含むRPMI

ける血清のIL−2インヒビター活性，正常脾細胞−1640を用いた。

のIL−2産生に対する影響ならびに脾細胞のIL−

4）IL−2産生能の測定

2産生能を調べた。

感染マウス脾細胞によるIL−2産生能の測定は，Lelchukらの方法に準じて，脾細胞浮遊液の5×106／mJにConcanavalin A（Con A，Miles社）

を最終濃度2μg／mJになるように加え，37C，
兵庫医科大学免疫学・医動物学教室〒663

西宮市武庫川町1−1

105％炭酸ガス下に24時間培養した後，その培養上

IL−2産生能を調べた。その結果，図1で示すよう清を回収し，IL−2活性測定用のサンプルとしたにTbg感染3日目のBALB／cおよびC57BL／（Lelchuk召∫

6マウス脾細胞のIL−2産生能は正常マウスに比z！，，1984）。

IL−2の活性は，Gillisらの方法に準じてIL−2

べて低下していることが明らかとなった。しかしな

依存性のCTLL−2細胞の増殖反応を測定するこがら，感染1日目，2日目では，IL−2産生能はコンとで求めた（Gillis召∫α1．，1978）。即ち，段階希

トロールと較べて，明らかな低下を示さなかった。

釈したサンプル100μ」を96穴マルチプレートに

2）Tbg感染マウス血清中のIL−2インヒビ

入れ，5x104／mJのCTLL−2細胞を100μ1加
え，37。C，20時間培養後，3H−thymidine（アマシャ

ター活性Tbg感染3日目の血清中のIL−2インヒビ

ム・ジャパン株式会社）0．5μCi／we11を加え，さター活性は，BALB／cおよびC57BL／6マウス

らに4時間培養した後，セルハーベスタ（Semi−の何れの系統においても正常血清とほぼ同様であautomatic

り，感染によるIL−2インヒビター活性の増強は，

ence

multiplecell

harvester，Labo

Sci・Co．，Ltd．）でグラスファイバーフィルター

認められなかった（図2）。感染1日，2日目のIL−（ラボマッシュ用フィルター）上に細胞をハーベ

2インヒビター活性も3日目と同様に正常血清とストした後，3H−thymidineの取り込みを液体シ比較して上昇していなかった。

ンチレーションカウンター（Packard460CD，

3）正常脾細胞のIL−2産生能に対する影響USA）で測定した。

感染血清の正常脾細胞のIL−2産生への影響をみ

5）IL−2インヒビターの測定た。図3に示すように，感染3日目の血清は，8％

1．血清内に含まれるIL−2インヒビター活性のの濃度で正常血清より明らかな抑制作用を示した。

測定は，Lelchukらの方法に従った（Lelchuk8孟認．，1985）。即ちTbg感染1，2，3日目のマウスの血清を培養液で段階希釈し，この希釈した血考

察清50μ」を1×104個のCTLL−2細胞浮遊液100

トリパノソーマ感染によって起こる免疫抑制にμ」および100unit／mlのrecombinantIL−2は，様々な機序が存在することが多くの研究者に（rIL−2，シオノギ製薬）50μ」を含む96穴のマイよって報告されてきた（Ackerman召∫α」．，1976；

クロプレート中で20時間の培養後，3H−

Person召∫α」．，1978；Romos6∫α1．，1979；Cm−

thymidineを0．5μCi／well加え，さらに4時間

ningham6緬」．，1980；Maleckar召緬1、，1983）。

培養した後，3H−thymidineの取り込みの減少に

特に丁細胞の機能に関しては，細胞内寄生原虫でよりIL−2インヒビター活性を測定した。

ある丁珈伽oso吻αo耀認あるいは細胞外寄生原

2．IL−2産生に対する抑制は，Tbg感染3日目のマウスの血清が正常脾細胞のIL−2産生を抑制

虫の感染において，マウスの脾細胞のConAに対する増殖反応の低下や，IL−2産生能の低下が報するか否かで判定した。すなわち正常脾細胞（5×告されている（Bellan召∫認．，1983；Alcina8厩1．，

106／mZ）を最終濃度2μg／mJのConAで刺激

1985）。またIL−2をT6耀認感染マウスに投与し，同時にその培養系に上記の血清を総容量のすることによって，原虫に対する特異的な免疫反

1％，2％，4％および8％の濃度で加え，24時

応の増強，虫血症の減少，マウスの延命効果が報間後の培養上清中のIL−2

告されている（Choromanski召∫認．，1987）。こ

活性を，CTLL−2細胞の増殖度により測定することで判定した。

れらの事実は，丁細胞の機能低下が，T6剛4またはT6吻o召1感染時の免疫抑制現象の重要な要因になっていることを示唆している。しかし，T結果1）Tbg感染脾細胞におけるIL−2産生能

Tbg感染マウス脾細胞のConA刺激に対するわ．go励」伽s6感染については，いまだ報告されていない。

近年，Lelchukらはトリパノソーマと同様に免11

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Figure 1

Titration curve of IL‑2 production. Spleen cells collected fromnormal (e) and Tbg‑infected BALB/C (A) and C57B/6 (B)mice 1(O), 2(A) or 3([]) days after infection were cultured
with Con A for 24 hr and IL‑2 activity of the culture super‑

natants were assayed using IL‑2 dependent CTLL‑2 cells.
Results indicate meaniS.D. of three experiments.

12

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Figure 2

Inhibition of the activity of recombinant IL‑2.

Fifty pl of serially diluted normal mouse serum (e) or sera
sampled from mice I (O) , 2 (A) or 3 ([]) days after infection wereadded, together with 50 pl of 100 unit/ml rlL‑2 to I x 10' CTLL‑2
cells in 100 pl of culture medium. After 24‑hr culture, prolifera‑tion of CTLL‑2 cells were measured by 3H‑TdR incorporation. In
the control , culture medium was added instead of serum and c.p.m.
level were 64,600 6,578(A) or 49,758 4,127(B) . Results indicate

meaniS.D. of three experiments.

13

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Figure3 InhibitionofIL−2production．ConAactivatednormalspleencells（5x106／mJ）were incubated with serially diluted serum fromeither BALB／c normal mice（●）or mice infected for3days withTbg（○）。After24−hr−culture，the remaining activity ofIレ2inthec611−free supematants were assayed using CTLL−2cells．Results

indicate

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疫抑制を起こすマラリア感染について，マウス血かった。このことからTわ．go吻6勉s召感染にお清中のIL−2インヒビターの変動を報告した

けるIL−2抑制因子は，IL−2と競合的に作用す（Lelchukゆσ1．，1985）。彼らによると，IL−2

るものではなく，IL−2産生を抑制するものと考

インヒビターは無胸腺マウスや無菌条件下で飼育えられた。

されたマウス血清中には存在しないが，正常マウ

IL−2インヒビターはHardtら（Hardt擁α」．，

ス血清中に存在し，マラリア（jp彪s窺04伽吻

1981）の報告に，Lyt−2，3＋のsuppressorッ08」劾感染時に上昇するという。

胞より分泌されて丁細胞の機能調節の一環を担っT細今回の実験では，Tδ．gα郷6伽s召感染におけているとされ，マウス血清中では，分子量5万のる丁細胞の機能を検索するためにマウス脾細胞のタンパク質とも言われているが，その詳細はいま

IL−2産生能を調べたところ，感染3日目に著しだ不明である。ヒトでは，血清中にprostaglandin

く低下していた。そこで，IL−2産生の低下にいかの様な1レ2活性を抑制する物質も含まれておりなる抑制機構が存在しているかを明らかにするた（Rappaport8∫oJ．，1982），かならずしもIL−2

めに感染3日目のマウス血清中のIL−2インヒビ

インヒビターといわれる単一の物質が存在するかターを測定した。その結果感染3日目のマウス血については異論もある（Lelchuk6勧1．，1987）。

清は，8％の濃度で正常脾細胞のIL−2産生を明さらにこれら，いわゆるIL−2インヒビター活性

らかに抑制した。しかしながらCTLL−2細胞のは寄生虫の種や感染時期，寄生部位によって様々

IL−2依存性増殖に対しては，抑制反応を示さなな活性の変化をきたす可能性も考えられる。しか

14

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x
1 ) Ackerman, S.B. and Seed, J.R. (1976): The effects of tryptophol on immune responses and its

implications toward trypanosome‑induced immunosuppression, Experientia, 32, 645‑6472 ) Alcina, A and Fresno, M. (1985): Suppressor factor of T‑cell activation and decreased
interleukin 2 activity in experimental African Trypanosomiasis, Infect. Immun., 50 (2) , 382‑387

3 ) Bagasra, O., Schell, R.F. and Le Frock, J.L. (1981): Evidence for depletion of la+ macro‑phages and associated immunosuppression in African trypanosomiasis, Infect. Immun., 32 (1) ,188‑1934 ) Bellan, A.H., Joskowicz, M., Fradelizi, D. and Eisen. H. (1983): Modification of T‑cellproliferation and interleukin 2 production in mice infected with Tr)panosoma cruzi, Proc. Natl.

Acad. Sci. USA, 80, 3466‑3469
5 ) Choromanski, L. and Kuhn, R.E. (1987): Use of parasite antigens and interleukin‑2 to enhance

suppressed immune response during Tr)panosoma cruzi infection in mice, Infect. Immun., 55(2) , 403‑408

6 ) Corsini, A.C., Clayton, C., Askonas. B.A. and Ogilvie, B.M. (1977): Suppressor cells and lossof B‑cell potential in mice infected with Tmpanosoma brucei, Clin. Exp. Immunol., 29, 122‑131

7 ) Cunningham, D.S. and Kuhn, R.E. (1980): Tr)panosoma cruzi‑induced suppressor substance. I.
Cellular involvement and partial characterization, J. Immunol., 124 (5), 2122‑2129

8 ) Eardley, D.D. and Jayawardena, A.N. (1977): Suppressor cells in mice infected withTmpanosoma brucei, J. Immunol., 119 (3), 1029‑1033
9 ) Gillis, S., Ferm, M.M.. Ou. W. and Smith, K.S. (1978): T cell growth factor: parameters of
production and a quantitative microassay for activity, J. Immunol., 120 (6) , 2027‑2032lO) Hardt, C., Rollinghoff, M., Pfizenmaier, K., Mosmann, H. and Wagner, H. (1981): Lyt23+
cyclophosphamide sensitive cells regulate the activity of an interleukin 2 inhibitor in vivo, J.

Exp. Med, 154, 262‑274
11) Hatcher, F.M., Kuhn, R.E., Cerrone, M.C. and Burton, R.C. (1980): Increased natural killer cell

activity in experimental American Trypanosomiasis, J. Immunol., 127 (3) , 1126‑1130
12) Lanham, S.M. and Godfrey, D.G. (1970): Isolation of salivarian Trypanosomes from man andother mammalian using DEAE‑cellulose, Exp. Parasitol., 28, 521‑534
13) Lelchuk, R., Rose, G. and Playfair, J.H.L. (1984) : Changes in the capacity of macrophages andT cells to produce interleukins during murine Malaria infection, Cell Immunol., 84, 253‑263

14) Lelchuk, R. and Playfair, J.H.L (1985): Serum IL‑2 inhibitor in mice. I. increase during
infection. Immunol., 56, 113‑118
15) Lehchuk, R., Schmidt, T.A., Hodson, K., Aston, R. and Liew, F.Y. (1987) : Is there an Interleu‑

kin 2 inhibitor in human serum?, Cell Immunol., 104, 126‑133
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15mouse T and B Iymphocytes by bloodstream forms of Tr)panosoma cruzi, J. Immunol., 130 (2) ,908‑91117) Person, T.W.. Roelants, G.E., Lundin, L.B. and Mayor‑Withey, K.S. (1978): Immune depres‑sion in trypanosome‑infected mice. I. Depressed T Iymphocyte responses, Eur. J. Immunol., 8,
723‑72718) Romos, C., Schadtler‑Siwon, I. and Ortiz‑Ortiz, L (1979): Suppressor cells present in the
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IL‑2 PRODUCTION INHIBITORY ACTIVITY OF THE SERA IN

TRYPANOSOMA BRUCEI GAMBIENSE INFECTED MICEDAO TAO, MINORU HIGASA, HIKOSHIRO MIYAMOTO, KAZUHIKO YAMADA,TOSHlNORI KOMATSU AND SOHEI SHlNKA
Received September 5 1988/Accepted January 24 1989

African trypanosome infections result in severe immunosuppression. Previous studies
have shown that this suppression is due, at least in part, to suppressor cells (T cells andmacrophages) or impairment of IL‑2 production.
In the present study, the effects of Trypanosoma brucei gambiense infection on serum
levels of IL‑2 inhibitor and Concanavalin A (Con A) induced IL‑2 production of spleen cells
were investigated. The results showed the ability of infected spleen cells to produce IL‑2 wasmarkedly reduced and IL‑2 production of normal spleen cells was inhibited by the infected
sera. But the IL‑2 dependent CTLL‑2 cell proliferation was not inhibited by those sera.
These results suggest that depletion of IL‑2 production during T. b. gambiense infection may
be due to the increase of an inhibitor of IL‑2 production.

Department of Immunology and Medical Zoology, Hyogo College of Medicine

Japan. J. Trop. Med. Hyg., Vol. 17, No. l, 1989, pp. 17‑25

17

ENZYME‑LlNKED IMMUNOSORBENT ASSAY(ELISA) IN THE DETECTION OF HEPATITIS‑B
SURFACE ANTIGEN IN BLOOD DRIED

ON FILTER PAPER

SHINYA MATSUDA AND HIROAKI KAHYO
Received September 7 1988/Accepted December 26 1988

Abstract: In an attempt to find a convenient method for the seroepidemiologicalstudy of HBsAg in developing countries, ELISA using blood samples dried on filter paper
was evaluated. The results of this method were in complete agreement with both thoseobtained on original sera by RIA and with those obtained on sera by ELISA. We did notexperience any false positive or false negative results. In this study the sample dried on
filter paper was eluted by I ml of PBS (pH 7.2). Dilution rates were calculated by thecalibration line of each serum. Dilution rates were between 0.041 and 0.082. The
coefficient of variation of absorbance value of 10 samples of each case was between 5.0%

and 14.8%, which indicated an acceptable intratest reproducibility of this method. In
addition, storage at 20'C, 20'C with desiccants and 3'C for up to 21 days did not alter thesensitivity and specificity of this method. This method will be useful for the sero‑
epidemiological study of HBV in developing countries.

INTRODUCTION
While conducting a seroepidemiological study in developing countries, researchers often
encounter the following difficulties:

1 . Collecting blood samples
It is often difiicult to take venous blood from newborns, children, anemic patients,

pregnant women and other adults who are not accustomed to such a procedure
2 . Storage and transportation of blood samples
3 . Economical problems
4 . Technical problems
The planning method may not be available for local conditions. These difficulties are
magnified when the population being studied is spread over a large area or living far awayfrom research centers.
To overcome these di :culties, blood samples dried on filter paper have been used todetect markers of various infectious diseases like Onchocerciasis (Korenaga et al., 1983;
Tada et al., 1985) , Japanese encephalitis (Yamamoto et al., 1985) and Rubella (Sander et al.,1985). As far as HBsAg is concerned, the filter paper method has been applied to thedetection using radioimmunoassay (RIA) and reversed passive hemagglutination (RPHA)(Farzadegan et al.. 1978; Villa et al., 1980; Kitazumi et al., 1981; Zhuang et al., 1982), andDepartment of Human Ecology, School of Medicine, University of Occupational and Environmental
Health, Kitakyusyu 807, Japan18

some seroepidemiological studies have been carried out by this method (Prince et al., 1981;
Werner et al., 1985).

Enzyme‑linked immunosorbent assay (ELISA) which was invented by Engyall and
Perlmann (1971) has been used to detect various pathogenic agents because of its highsensitivity, specificity and rapidity. Ukkonen et al., (1977) demonstrated the usefullness of

ELISA to detect HBSAg. Compared to RPHA, ELISA is a more sensitive method. Also,
ELISA does not have any regulations which RIA have about radioactive agents. In this study

we intended to detect HBsAg in blood dried on filter paper using ELISA.

MATERIALS AND METHODS
1 . Collection of samples

Ten HBsAg positive cases were selected from out‑patients of the Ist Department ofInternal Medicine, University of Occupational and Environmental Health (UOEH). Ten
HBsAg negative cases were selected from student volunteers of UOEH. All cases wereretested by a commercial RIA kit (Ausria, Abbott Laboratories) on HBSAg. Twenty blood
samples dried on filter paper were collected from each HBsAg positive case and ten samples

from HBsAg negative cases.
2 . Filter paper

In this study we used a commercial blood sampling paper (Type I , Toyo Roshi, Ltd.,
Japan). This paper, which was developed by Nobuto (1970), is composed of two areas, anabsorbing area and a diffusion area (Fig. 1). The absorbing area is allowed to absorb asmuch blood as possible. An excess of blood is allowed to be diffused into the diffusion area.

In this way the amount of blood contained in the absorbing area is considered to be almost
constant (0.1 ml which is equal to 0.04 ml of serum) . Whole blood was collected by this typeof paper and air‑dried for 2 days at room temperature with the absorbing area up, avoiding
direct sunlight. The blood samples dried on filter paper were stored in small polyethylene
envelopes under 3 different conditions, 20'C, 20'C with desiccants and 3'C to be tested after
7, 14 and 21 days from collecting.
3 . Elution
The absorbing area was cut into 4 portions and placed in a test tube. Then I ml of PBS(pH 7.2) was added and the test tube was incubated at 37'C for 3 hr with vibrat ron for elution.

4 . ELISA procedure
In this study we used a commercial kit, IMMUNlS HBsAg EIA (Sanko Junyaku Co.,
Ltd.). The procedure was as follows: 1) 0.1 ml of eluate was added to each well. 2) The

30 m m

18mm
10

a

b

Ca] blood absorbing areaCb) blood diffusion area

Figure I Blood sainpling paper.

19

plate was incubated for 2 hr at 37'C. 3) After the reaction, the eluate in well was aspirated.

4) The wells were washed 5 times with wash fluid supplied by the manufacturer. 5) 0.1 mlof horse radish peroxidase conjugated antibody (mouse anti‑HBs*) was added to each well.
6) The plate was incubated for 2 hr at 37'C. 7) The wells were emptied by aspiration. 8) The
wells were washed 5 times by wash fluid. 9) 0.1 ml of freshly prepared substrate solution (o‑phenyl‑diamine, OPD) was added to each well. 10) The plate was incubated for 30 min atroom temperature in the dark. 11) The enzymatic reaction was stopped by adding 0.05 mlof 4 N H2SO*. 12) The plate was read by an autoreader (EAR340, Australia S.L.T. Co., Ltd.) .
In addition to the above tests the original sera were tested by ELISA to compare them withthe results obtained from blood dried on filter papers. The results were interpreted according

to the manufacturer's recommendation, that is, positive ELISA values were>2.5x (mean
ELISA values of negative control sera) .

RESULTS
1 . Comparison of results obtained by RIA and our method
Table I shows the comparison of results obtained by RIA and this method. The results
are in complete agreement with those obtained on original sera by RIA, indicating acceptablesensitivity and specificity of this method.

2 . Comparison of results obtaind by ELISA in serum and in whole blood dried on filter paper

Table 2 shows the comparison of results obtained by ELISA in serum and in whole blooddried on filter paper. The results were in complete agreement.
3 . Dilution rate

Theoretically speaking, the dilution rate is 0.04 when the sample dried on the absorbing
area of the filter paper was eluted by I ml of PBS (pH 7.2) . In this study we made calibrationlines of the original sera to calculate the dilution rate from the absorbance value of eachsample (Fig. 2). The results are shown in Table 3. The dilution rates were between 0.041
and 0.082.

4 . Reproducibility

The intratest reproducibility of this method was assessed by 10 blood samples dried on
filter paper 10 times for each case within the same test. The results of 6 cases are shown in

Table 4. Four cases were excluded from discussion because their absorbance values
overfiowed. The coefficients of variation (C.V.) were between 5.0% and 14.8%. According

Table I Companson of results for detection of HBsAg
obtained by RIA for sera and those obtained by
ELISA for blood dried on filter paper

Sera tested by RIA

Posrtrve Negative
Sera dried on the filter

paper tested by ELISA

Positive*200

oNegativeo100* Case in which ELISA value was>2 .5 x (Mean ELISA
value of negative controls)20

Comparison of results obtained by ELISA f or detec‑

Table 2tion of HBsAg in serum and blood dried on filter
paper
Sera tested by ELISA
Positive

Negative

Sera dried on the filter

Positive*

10O

paper tested by ELISA

Negativeo10* Case in which ELISA value was>2.5x (Mean ELISA
value of negative controls)

Diiution
rate

xl0‑3

20.0
15.0

Y = O. 038X

10.0

Dilution rate
= 3.35x l0‑3x 20‑ o.067

5.0
3.35
t
l
,

,

O

1.00

0.50
value of case6<x20)

0.1 77 * absorbance

OD

Fig.2 Calibration line(case6)

Table 3

Dilution rate calculated f rom calibration line

Case No.

l

2

3

4

5

Dilution rate

O . 069o . 082

O . 041o . 044o . 063

Case No.

6

7

8

9

10

Dilution rateo . 067

o . 069

O . 047o . 044

O . 055

Mean: 0.058 SD:

0.014

to the explanatory note of IMMUNIS HBsAg EIA, the C.V. of the original ELISA procedureare 7.7% and 9.6%. The results indicated the acceptable intratest reproducibility of thismethod.
5 . Influence of storage
Samples dried on filter paper were tested after 7, 14 and 21 days from the first test. The

21

Table 4 Intratest reproducibility

Case No.

3

5

Sample 1

1 . 814o . 287

1 . 327

O . 386

1 . 775

2 . 316

2

1 . 652o . 293

1 . 360

O . 343

1 . 782

2 . 4463

l . 686

o . 264l . 327

O . 535l . 9022 . 608

1 . 514o . 297

1 . 302

O . 539

1 . 794

2 . 8445

l . 386

O . 283

1 . 562

O . 398

1 . 924

2 . 555

6

1 . 469o . 294

1 . 355

O . 401

1 . 969

2 . 256

1 . 700

o . 253

1 . 732

O . 432

1 . 790

2 . 5618

l . 808o . 278l . 7390.416

l . 728

2 . 853

9

1 . 407

O . 294

1 . 427

O . 414

1 . 640

2 . 327lO

l . 586

O . 285l . 314

O . 393

1 . 534

2 . 294

6

7

9

10

Mean O . 283 O . 426 1 . 784 2 . 506
SD O . 014 O . 063 O . 131 O . 218
1 . 602

1 . 445

O . 155

O . 171

C.V.(%) , 4.95 14.79 7.34 8.70
9 . 68

11 . 83

results are shown in Table 5. None of the HBsAg positive samples became negative or viceversa. Also, there were no remarkable differences in the three storage conditions. These
results might indicate that HBsAg is stable at least for 2 weeks in a dried condition.

DrscUssloN
It seems to be apparent from our results that HBsAg is stable in a dried condition and
ELISA using the filter paper method is reliable and reproducible. As for sensitivity andspecificity, we have not experienced any false negative or false positive results. These results

were in concord with other reports about the filter paper method (Farzadegan et al., 1978;
Villa et al., 1980; Kitazumi et al., 1981; Zhuang et al., 1982). Farzadegan et al. (1978)
described the following remarks regarding the merits of this simple method:

1 . Blood samples may be obtained from newborn infants, children, anemic patients,
pregnant women and other adults from whom it is difficult to obtain venous blood.
2 . Samples are dry, Iight, easy to post, and cannot be broken or spilt.
3 . Samples are easy to store; they take up little space, and remain stable for a long time.
4 . The greater ease and economy of this procedure will be useful in large scale population
screening projects.

Now we are planning to detect other HBV markers by ELISA using blood samples driedon filter paper in order to apply this method to the hepatitis B surveillance program indeveloping countries. It is well‑known that hepatitis B infection is very common in develop‑
ing countries, especially Subsaharan Africa, Southeast Asia, China, and the Western Pacificislands. According to WHO Technical Report (1983) , in low endemic areas, such as North
America and Western Europe, prevalence of HBsAg is 0.2‑0.5%; in intermediate endemicareas such as Eastern Europe and Southwest Asia, the prevalence rate is 2‑7%; in highendemic areas such as those written above, the prevalence varies from 6‑15% and a highprevalence of chronic liver disease, including hepatocellular carcinoma was also shown. It
has been proven that the HBV carrier state is strongly connected with chronic liver disease.

22

Table 5 Change of absorbance values by storage under 3 different conditions

HBsAg Positive

HBsAg Negative
Days

SampleoSample I overSample 2 overSample 3 1 . 602

Sample 4 over

7

14

21

o

7

14

21

over
over
over
over
over
over

over
over
over
over
over
over

over
over
over
over
over
over

o . 039o . 024

O . 026

O . 027o . 022

O . 026

O . 031o . 025

O . 022

O . 030

o , 030

O . 030

O . 028o . 029

O . 029

O . 030

o . 028

O . 025

O . 025

1 . 636

1 . 715

1 . 756

o . 046o . 044

O . 041

O . 0362 . 214

1 . 875

1 . 881o . 039

O . 041

O . 0421 . 813

1 . 524

1 . 526o . 039

O . 031

O . 035o . 025

O . 028

O . 022o . 022

O . 020

O . 019o . 022

O . 025

O . 025o . 044

O . 041

O . 041o , 044

O . 042O . 043o . 040

O . 041

O . 039o . 029

O . 030

O . 030
O . 029

over
over
over

over
over
over

over
over
over

Sample 5 O . 283
Sample 6 1 . 445

Sample 7 O . 426

Sample 10 2.506

o , 026o . 046

1 . 709

1 . 317o , 029

1 . 555

1 . 494o . 030

O . 028l . 756

1 . 275

o . 024

O . 027

O . 026

O . 536

O . 439o , 045

O . 041

O . 0.44

O . 573

O . 477o . 044

O . 048

O . 046

O . 613

O . 439o . 044

O . 042O . 047o . 049

O . 041

O . 041o . 044

O . 044

O . 045o . 044

O . 048o . 050

Sample 8 1 . 784

Sample 9 overo . 027

O . 051o . 029

O . 025o . 030

O . 028o . 028

O . 027o . 032

O . 0292 . 259

o . 028

O . 028

O . 035

2 . 420o . 033

O . 034

O . 026

over
over
over

over
over
over

2 . 846

2 . 553

2 . 546
2 . 549

over
over
over

upper: 3'C, middle: 20'C with desiccants, Iower: 20'C‑: not tested

over: overfiowed absorbance valueo . 028

O . 031

23

Therefore, prevention of HBV carrier state will lead to the prevention of chronic liverdisease. Numerous studies have been done in developing countries to clarify the course oftransmission to provide enough data for the anti‑HBV program. Previous studies haveshown that most of HBV infection occurs in early age in endemic areas. Maternal‑childtransmission has been proven to be an important cause of HB carrier state in some areas like
Singapore (Chan et al., 1985) , Taiwan (Stevens et al., 1975) and Japan (Shiraki et al., 1977) ,but not in other areas (Johnson et al., 1980; Prince et al., 1981; Nasidi et al., 1986; Milne etal., 1987) , where horizontal transmission among children are considered to be rather impor‑tant. To develop an appropriate anti‑HB program, including HB vaccination, the large‑scale
surveillance of age‑specific prevalence of HBV markers are needed. Our method seems to besuitable for such a kind of surveillance because of its logistic, economical and technicalmerits.

But there remain some problems that must be discussed in detail. Werner et al. (1985)
mentioned that the filter paper method might not be able to detect one‑third of the HBsAg
positive persons. In our study we used high titer HBsAg involving blood, all of which wereover 30 as a cut off index value by RIA. Therefore further studies are necessary regarding
low titer HBsAg involving blood and about the quantitativity of this method.

Regarding the dilution rate, values varied from the same to twice as much as thetheoretical value. Some possibilities should be considered as reasons:
1 . Problem of filter paper

Blood volume contained in absorbing area might not be constant.
2 . Influence of other components of blood or filter paper
3 . Problem of elution
Elution period, temperature and other environmental factors might influence theconstancy of elution.

Further examination is needed to clarify the influences of such factors in order to makesure of the reliability of the filter paper method.
Although there are some problems to be solved, the filter paper method will be appropri‑ate for the seroepidemiological field study, especially in developing countries.

ACKNOWLEDGMENTS
This study was supported by a grant (to S. M.) from the Ministry of Education, Science
and Culture, Japan, No. 62770391. The authors wish to thank Prof. Yoshimasa Nishimura,
Department of Clinical Immunology, School of Nursing and Medical Technology, UOEH', fortechnical advice. We are grateful to Prof. Fumio Okuno, Department of Adult Nursing,
School of Nursing and Medical Technology, UOEH, for his help in collecting samples.

REFERENCES
1 ) Chan, S.H., Tan, K.L., Goh, K.T., Lim, C., Tsakok, M., Oon, C.J. and Ratnan, S.S. (1985):
Maternal‑child hepatitis B virus transmission in Singapore, Int. J. Epidemiol., 14, 173‑177
2 ) Engyall, E. and Perlmann, P. (1971): Enzyme‑linked immunosorbent assay (ELISA) , quantita‑
tive assay of immunoglobulin G, Immunochemistry, 8, 871‑874
3 ) Farzadegan, H., Noori, K.H. and Ala, F. (1978): Detection of hepatitis‑B surface antigan in24blood and blood products dried on filter paper, Lancet, 1, 362‑363
4 ) Johnson, D.E., Snitban, R., Scott, R.M.. Perlman, E.J. and Kenndy, R.S. (1980): Hepatitis B in
the rural tropics, Int. J. Epidemiol., 9, 123‑129

5 ) Kitazumi. T., Sakurabayashi, I. and Kawai, T. (1981): Study on micromethod for serum HBS
antigen and antibody titers using blood disc, J. J. Clin. Pathol, 12, 1259‑1262

6 ) Korenaga, M., Tada, I., Mimori, T., Sakamoto, M., Lujan. T.A., Zea. F.G., Castro, J.C. andYarzabal, L. (1983): Enzyme‑linked immunosorbent assay (ELISA) in the detection of lgGantibodies in Onchocerciasis using blood collected on filter paper, Jpn. J. Parasitol., 32, 347‑355

7 ) Milne, A., Allwood, G.K., Moyes, C.D., Pearce, M.E. and Newell, K.A. (1987): A sero‑
epidemiological study of the prevalence of hepatitis B infection in a hyperendemic New
Zealand community, Int. J. Epidemiol., 16, 84‑90
8 ) Nasidi, A., Harry, T.O., Vyazov, S.O., Munube, G.M.R., Azzan, B.B., and Ananiev, V.A. (1986) :Prevalence of Hepatitis B infection markers in representative areas of Nigeria, Int. J.
Epidemiol., 15, 274‑276
9 ) Nobuto, K. (1970): Application of filter paper strips for blood collection in the control andsurvey of animal infectious diseases, Bull. Off. int. Epiz., 74, 765‑774

10) Prince, A.M., White, T., Pollock, N., Riddle, J., Brotman, B. and Richardson, L. (1981):
Epidemiology of hepatitis B in Liberian infants, Infect. Immun., 32, 675‑680

1D Sander, J. and Niehaus, C. (1985): Screening for rubella lgG and lgM using an ELISA test
applied to dried blood on filter paper, J. Pediatr., 106, 457

12) Shiraki, K., Yoshihara, N., Kawana, T., Yasui, H. and Sakurai, M. (1977): Hepatitis B surface

antigen and chronic hepatitis in infants born to asymptomatic carrier mothers, Am. J. Dis.
Child., 131, 644‑647

13) Stevens, C.E., Beasley, R.P., Tsui, J. and Lee, W.C. (1975): Vertical transmission of hepatitis

B antigen in Taiwan, N. Engl. J. Med., 292, 771‑774
14) Tada, I., Korenaga, M., Mimori, T.. Sakamoto, M., Yoshimura, T., Recinos, C.M.M., Florfs, O.
F., Lujan, T.A., Ochoa, A.J.O., Castro, J.C. and Zea, F.G. (1985): A comparative study ofseveral diagnostic measures applied in Guatemalan Onchocerciasis, Jpn. J. Parasitol., 34, 261‑27115) Ukkonen, P., Koistinen, V. and Penttinen, K. (1977): Enzyme‑Immunoassay in the detectionof hepatitis B surface antigen, J. Immun. Meth., 15, 343‑353
16) Villa, E., Cartolari, R., Bellentani, S., Rivasi, P., Casolo, G. and Manenti, F. (1980): Hepatitis
B virus markers on dried blood spots. A new tool for epidemiological research. J. Clin. Pathol.,34, 809‑812
17) Werner, G.T., Frosner, G.G. and Fresenius, K. (1985) : Prevalence of hepatitis A and B markers

in a rural area of northern Zaire, Am. J. Trop. Med. Hyg., 34, 620‑624
18) W.H.O. (1983): Technical report series 691. Prevention of liver cancer19) Yamamoto, T. and Takagi, M. (1985): Measurement of antibodies to Japanese encephalitis
virus by Enzyme‑linked immunosorbent assay using a small quantity of blood absorbed on
filter paper disc, J. J. A. Inf. D., 59, 1135‑1141

20) Zhuang, H., Coulepis, A.G., Locarnini, S.A. and Gust, I.D. (1982): Detection of markers ofhepatitis B infection in serum dried on to filter‑paper: an application to field studies, Bull. W.

H. O., 60, 783‑787

25

濾紙使用による微量血中のHBsAgのELISAによる測定
松田晋哉・華表 宏有

濾紙法を用いてHBsAgの検出を，ELISAによって行うことを検討したので報告する。
RIA法によりHBsAg陽性を確認した10名と，同様に陰性を確認した10名の計20名について採血用濾紙（束洋濾紙・ストリップ1型）に血液の微量を吸着させ，自然乾燥後一定量を切り取り，PBS（pH7．2）中で抽出（37。C・3hr）したもの0．1mJを検体としてELISA（イムニスHBsAgEIA・

三光純薬）によりHBsAgの検出を行った。
RIA法およびELISA原法との比較では，上記の被検者より得た陽性検体200枚，陰性検体100枚を本法により分析した結果と，RIA法の結果は全例で一致した。また，血清0．1mJを用いたELISA
原法とも全例で一致した。採血用濾紙1型に採血した試料を1mJのPBSで抽出すると理論上希釈率は0．04となる。本研究では原血清について検量線を作成し，それを基に希釈率を求めたが，その値は0．041−0．082であった。1ケースより10枚の濾紙血を得，本法に於ける測定値の変動を調べた。吸光度がoverHowしなかった6ケースでみると，変動係数は7．4％一14．9％で高い再現性が得られた。採血した濾紙を，①室温（20。C），②室温＋乾燥剤，③冷蔵庫内（3。C）の3条件下で保存し7，14，21日後にそれぞれ測定を行った。陽性例ではいずれの条件下でも，21日目まで吸光度の明らかな低下は認められなかった。また，陰性検体が陽性になることもなかった。
以上により本法は熱帯地域におけるHBsAgの調査に利用可能であると考えられる。

産業医科大学医学部公衆衛生学教室

Japan. J. Trop. Med. Hyg., Vol. 17, No. 1, 1989, pp. 27‑31

27

DIAGNOSIS OF PNEUMOCYSTIS CARINIIIN SPUTUM LIQUEFIED WITH THE

MUCOLYTIC AGENT DITHIOTHREITOL(SPUTASOL)TSUNEZO SHIOTA
Received September 30 1988/Accepted October 24 1988

Abstract: l. Light microscopic investigations: Sputasol treatment did not affect thestaining properties of either the cyst wall or the parentheses‑like structures by Gomori's
methenamine silver nitrate (GMS) stain. Detection of Pneumocystis carinii was easier in

the Sputasol‑treated preparations because it was possible to prepare thin smears of thesediment, and because the cysts were concentrated. The amount of time needed for
microscopic examination was reduced to the decreased amount of sediment in the
Sputasol‑treated samples. 2. Transmission electron microscope investigations: In theultrastructure of P. carinii cysts stained with GMS stain, the heavier deposition of silver

particles on the internally thickened parts of the cyst wall corresponded exactly with thedistinctive appearance of the parentheses‑like structures seen in light microscopic
observations.

INTRODUCTION
Antemortem diagnosis of Pneumocystis carinii pneumonia is based on detecting properly
stained cysts of the parasite in sputum, tracheal aspirate, bronchoalveolar lavage (BAL) ,transbronchial lung biopsy, open lung biopsy, or lung aspirate. In the examination of sputum,
cyst concentration is very important. In a previous study (Yoshida et al., 1978) , we attempt‑

ed to achieve cyst concentration by treating sputum with acetyl‑L‑cysteine. Our initial
results were promising and encouraged this follow‑up study, in which we evaluated theusefulness of dithiothreitol (Sputasol) treatment of sputum and infected lung tissue in the
diaguosis of P. carinii. Sputasol is a protective reagent for SH groups (Cleland, 1964), andis widely used for the liquefaction of sputum samples received for bacteriologic examinations(Hammerschlag et al., 1980) .

MATERIALS AND METHODSSpecimens:

Sputum and BAL were obtained from patients who had received renaltransplants; and frozen human lung taken at autopsy of a 23‑year‑old woman renal trans‑
plant recipient.

Department of Medical Zoology. Kyoto Prefectural University of Medicine, Kyoto 602, Japan
Contribution No. 627 from the Department of Medical Zoology, Kyoto Prefectural University of
Medicine

28

Sputasol: Vials of Sputasol powder were obtained from Oxoid Limited, Basingstoke,
Hampshire, England, and were prepared as follows: To one vial of Sputasol was added 5 mlof sterile distilled water, and the contents mixed gently until the powder was completely
dissolved. To this was added aseptically an additional 95 ml of sterile water. This solution

was used immediately, or stored at 4'C for up to 48 hr. The chemical contents of each vialof Sputasol are listed in the Table 1.

Sample preparation: One volume of sputum or BAL was added to five volumes of
Sputasol. This mixture was placed in a 37'C incubator or water bath shaker for 5 min.
Following incubation, the samples were filtered through one layer of cotton gauze, thencentrifuged at 3,000 rpm for 10 min. Smears of the sediment were prepared, air‑dried, fixedin absolute methyl alcohol for 5 min, then stained by Gomori's methenamine silver (GMS)
method (Grocott, 1955). The stained smears were examined under both the high dry (400 X )and oil immersion (1,000 x ) Ienses of a compound microscope.

Transmission electron microscopy: Small pieces of lung were minced with scissors,rinsed and incubated in Sputasol in a 37'C water bath for 10 min, filtered through cotton
gauze, then centrifuged at 3,000 rpm for 10 min. The sediment was fixed in 2.5% glutaralde‑
hyde for 2 hr, then cut into I mm3 blocks. These small blocks were stained with GMS, post‑fixed in 1% osmium tetraoxide for I hr, dehydrated in a graded series of acetone, andembedded in epoxy resin. Ultrathin sections, obtained with a Porter Blum MT‑1 ultrami‑
crotome and glass knife, were stained with uranyl acetate and lead citrate and examined witha JEM 100S electron microscope.

RESULTS
Light microscopy: The results can be summarized as follows: l. Detection of P. carirdi

was easier in the Sputasol‑treated preparations because the cysts were concentrated. 2.
Sputasol treatment did not affect the staining properties of either the cyst wall or theparentheses‑like structures (Fig. 1) . 3. The amount of time needed for microscopic examina‑tion was reduced due to the lesser amount of sediment in the Sputasol‑treated samples.
Transmission electron microscopy: Figure 2 shows the ultrastructure of a P. carinii cyst
stained only with GMS. The heavier deposition of silver particles in the internally thickened
parts of the cyst wall (arrows) is clearly visible. Figure 3 shows the ultrastructure of a P.
carinii cyst stained with GMS, then with uranyl acetate and lead citrate. The silver particles
are specifically deposited on the electron‑lucent middle layer and the thickened portion of thecyst wall (arrow) .
Table I Vial contents of Sputasol

Dithiothreitol O , I g

Sodium chloride O . 78 g
Potassium chloride O . 02 g
Disodium hydrogen

phosphate O . 112 g
Potassium dihydrogen

phosphate O . 02 g
pH 7.4 0.2

29

Figure l P. carinii cysts in sputum liquefied with Sputasol. The
cyst wall and the parentheses‑1ike structures (arrows; areclearly visible. (Gomori's methenamine siiver nitrate, x
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Flgure 2 pf caririi cyst in tung tissue treated with sputasol andstained only with Gomori s staind Note the heavy depusi‑tion of Sitver partides on the internany thickened Parts ofthe cyst wan (arrows) t ( x 25'ooo)30

Figure 3P, carinii cyst in luug tis.sue treated with Sputasol andstained with GOmori*8 stail' and with uranyl acetate andlead citrate.

The silver particles are specifically depasited on the elec"tron‑‑lucent l riddle layer and on the thickened portion af
the cyst wall (arrow) . ( x 25,000)

DISCusSION
The results indicate that cyst concentration in patient specimens is feasible and that goodstaining of both the cyst wall and the parel' rthese8 ‑like structures can be abtaincd following

Sputa̲ ol treatment, The great benefit of this method in the ease with whic'.h cysts in the
sputum can bc concentrated and detected. In the transmis ian electron micn sco e investiga‑
tion on cysts stained with GMS, the heaviest deposition of silver particles was seen to occur
on thickened portions of the cyst wall, areas that corre‑spond exactly with the parentheses‑
like strueture*,* seen in light microscopic observations (Shiota, 1987; Yoshikawa and Yoshida,l 987) ,Acl{:NawLEDc FiMENT
I sincerely thank Professor Yukio Yoshida, the director of the Departrnent. of Medical
Zoology, Kyoto Prefectural University of Medicine, Kyoto. Japal , for hi8 interest, guidance,and encouragement throughout this. study and his critical reading: of this mawscript,

REFERENCESl ) Cleland, W.W. (1964)

3, 480482

DithiothreitO1, a new protective reagent fc r SH g= roups, Bioch, emistry,

312）Grocott，R．G．（1955）： A

stain

for

fungi

in

tissuesections

andsmears：using

Gomoris

methenamine−silver nitrate technic，Am．J．Clin．Patho1．，25，975−979

3）Hammerschlag，M．R．，Harding，L．，Macone，A．，Smith，AL．，Goldmam，D・A・（1980）：Bacterio1・ogy

of

sputum in cystic fibrosis：Evaluation ofdithiothreitol as a mucolytic agent，J．Clin．

MicrobioL，11，552−557
4）Shiota，T．（1987）：Light
of

P％召吻30砂s

microscopic

observation

ゑs昭吻ガcysts in smearsofstained bytheso−caIledparentheses−like

Gomori s methenamine

structuresilver nitrate，Japan．

」．Trop．Med．Hyg．，15，269−273
5）Yoshida，Y．，Ikai，T．，Ogino，K．，Takeuchi，S．，Yamada，M．，Shimada，Y．andShiota，T．（1978）：Studieson

P％επ勉oCys∫∫s oσ万痂づand P％ε鋸吻oの〜s傭oα短n露pneumonia，V．Diagnosis

concentration from sputum，Jpn．」．ParasitoL，27，473−481（in6）Yoshikawa，H．and

treatedwith

Yoshida，Y．（1987）：Localization ofGomoris

methenamine

silvernitratesilverEnglishby cystJapanesewithsummary）

deposits

on、P％8祝吻oの就sωπn露

stain，Zbl．Bakt．Hyg．A，264，363−372

喀疾液化剤・dithiothreitol（Sputaso1）処理による
P％甜解磁ys漉oα7吻i歪の診断

塩田恒三

Pη顔吻ooys漉o碗ni久Pc）肺炎の患者の喀疾，および気管支肺胞洗浄液（BAL）と，Pc肺炎で死亡した患者の肺を，喀疾液化剤・dithiothreitol（Sputasol・Oxoid）で処理した後，Gomori7smethenamine

silver nitrate（GMS）染色を施し，無処理のコントロールと比較検討した。方法：①喀疾の5倍量のSputasolを加え，37。Cの水浴中あるいは艀卵器内で10分間振盟して液化。

BALでは直接3，000rpm，10分間遠沈後多量の粘液性沈渣がある場合，これの5倍量のSputasolを加え喀疾と同様の操作を行う。肺は細切後ガーゼ濾過し，充分量のSputasolを加える。②3，000rpm10分問遠沈。③沈渣をピペットでよく混和した後スライドグラスに塗抹し，風乾後GMS染色を施して400倍と油浸下で観察。また，肺の沈渣はグルタールアルデヒドで固定後，GMS染色を行い，オスミウムで後固定，ウラニル・鉛で重染し，TEMで観察。結果：嚢子壁およびPcに特異的に認められる括弧状構造物の染色性はコントロールに比べ差異はなかった。コントロールの

喀疾およびBALの沈渣塗抹標本では，厚い部位のPcの染色性は弱く，同一標本上で染色性のばらつきを認めたが，Sputasol処理標本では均一な厚さに塗抹されており，ばらつきは少なくPcを検出し易かった。Sputasol処理の沈渣塗抹量は，コントロールの直接塗抹量に比べ非常に少量になるため，標本枚数が少数で済み，鏡検時間が短縮された。GMS染色を旋したPc嚢子のTEM
像では，嚢子壁の内部への肥厚部に多くの銀粒子の沈着が認められ，この肥厚部が光顕で観察される括弧状構造物と一致するものと思われる。以上の結果から本法はPc肺炎患者の喀疾，気管吸

引物，BAL等の材料からPcを検出する場合，集シスト法として操作が比較的簡単で良い方法と考えられる。

京都府立医科大学医動物学教室

33

Japan. J. T**P. M*d. Hyg., vot. 17, N*. l' 1989, PP. 33 ro3

PROCEEDlNGS OF XXX ANNUAL MEETlNG OF
JAPANESE SOCIETY OF TROPICAL MEDICINE
ro‑n october 1988. Nagasaki

CONTENTS
Special recture:

Cooperative medical project in Kenya M. Mugambi
Symposium I: Tropical infectious diseases and drugs

1 laInsecticides
Chaird by Y. Wada
Insecticides for use in Japanese encephalitis control programmes
Y. Wadalb Insecticides for use in dengue control programmes A. Morilc Insecticides for use in antimalaria programmes T. Kurihara

2 Anti‑parasitic drugs Chaird by H. Tanaka
2a The anti‑parasitic drugs in current use Y. Otsuji

2b Neuropharmacological approach to anthelmintic drugs

M. Terada
3 Anti‑microbial agents and their developmental state
Chaird by K. Hara
3a Enteric bacteria in Asian countries and their drug resistanceY. Kudoh3b Drug resistance of Vibrio cholerae Ol isolated in tropical area
Y. Ichinose

3c Current status of respiratory pathogens and drug resistanceK. Yamaguchi
3d Present state of development of antimicrobial agents A. Imada
Symposium II: Present status and topics in development of vaccines against
tropical infectious diseases

l Vaccines for protozoal diseases Chaird by T. Nakabayashi
la Several notable achievement in recent research on malaria vaccine

M. Suzuki
lb Approaches to the development of trypanosome vaccine

T. Fukuma
2 Vaccines for bacterial diseases
2a Cholera vaccine and typhoid vaccine
2b Leprosy vaccine, present and future
3 Vaccines for viral diseases

Chaird by N. Ohtomo
Y. Takeda
Y. Mizuguchi
Chaird by K. Mifune

A. Igarashi
3b Experience with field trials of rhesus rotavirus vaccine MMU‑18006
S. Chiba

3a Dengue vaccine

34

Panel discussion: Introspective and prospective view of cooperative medical
projects experienced in tropical countries

Chaird by R. Murata and K. Matsumoto
1 Status of rural health and medical cares: human life and naturalenvironment in Sarawaku, East Malaysia I. Miyagi
2 Central and South Americas‑cultivated between wild nature and lber‑
ian cultivation

I. Tada3 Geographical pathology and medical anthropology in Africa
H. Itakura
4 Bacteriology project in Kenya Medical Research Institute‑present
status and progressive view‑
A. Ozawa
5 JICA technical cooperation project at Research Institute for Tropical
Medicine, Philippines, and its prospect

Y. Kaneko

6 Present state and some problems on the basic study of infectious
diseases by Intemational Medical Foundation of Japan

Y. Ito7 Medical cooperation activities conducted by Japan International
Cooperation Agency (JICA)
T. Kondo
8 Prospective role of Japan in international health, a view of a WHO
staff
A. Shimouchi
General presentation
1 Foreign patients with tuberculosis from tropical countries in Tokyo

National Chest Hospital H. Shishido et al.

2 Study on bacterial respiratory infections in Chiang Mai, Thailand

A. Takahashi et al.
3 The comparison of drug‑susceptibility of Haemophilus influenzae andStreptococcus pneumoniae between Philippines and Japan
K. Tateda et al.
4 Isolation of Legionella species from the environmental samples in thePhilippines K. Tomono et al.

5 Vibrio furnissii isolated from stool sample of children with diarrhea in

Negros Island and three cases of patients with diarrhea in Osaka City

E. Okumura and M. Hata
6 Characterization of Vibrio cholerae O1 isolated in Thailand in 1987
M. Iwami et al.
7 Epidemiological investigation of cholera infection in Nakuru (Kenya)in 1987 and characterization of the isolated V. cholerae

Y. Yanagase et al.
8 The analysis of the intestinal bacterial flora of the Campylobacter
enteritis in children residing in Kenya and characterization of theisolated organisms Y. Yanagase et al.

9 Microbiological study of drinking waters in tropical countries‑fre‑quency and sort of bacteria in the samples that the residual chlorine ispositive‑ M. Sugiyama et al.

35

10 Study on heat loss ability of Pika (white rabbit) G. J. Yang et al.
ll Comparison of heat loss responses induced by local heating of preoptic
area and anterior hypothalamus and medullary reticular formation

with argon laser on rabbits N. Ohwatari et al.
12 Studies on hypothalamic blood fiow changes in thermal acclimation
M. Kosaka et al.
13 Change of serum proractin due to heat load and exercise in human
T. Matsumoto et al
14 Physical performance and ambient temperature‑analysis of heart rate
M. Yamauchi et al.
15 Study on seasonal variation of thermal sweating K. Nakamura et al.
16 A native Taiwanese case of schistosomiasis japonica
C.K. Chuang et al.

17 Treatment of opisthorchiasis with praziquantel M. Tanaka et al.
18 Epidemiological survey of Leishmania in Equador and characterizationof isolates T. Mimori et al.

19 Current status of leishmaniasis and Chagas' disease in Paraguay
H. Yamasaki et al.

20 Basic studies on Mongolian gerbils of susceptible host to filarial infec‑

M. Shimizu et al.
21 Basic studies on Mongolian gerbils of susceptible host to filarial infec‑
K. Shichinohe et al.
tion (2)tion (1)22 Studies on in vitro maintenance of adult Dipetalonema viteae, withspecial reference to their survival and microfilarial release J. Maki

23 Schistosomiasis japonica and carcinogenesis‑a comparison of tumor
incidence rates in the liver and digestive organs of mice during aT. Aji et al.
mutagen (Trp‑p‑2) feeding‑
24 L ctin binding by muscle larvae of Trichinella spiralis

Y. Takahashi et al.
25 Alimentary tract associated antigen in Trichinella spiralis
H. Suzuki et al
26 Studies on antigenicity of Trichinella spiralis (7) with emphasis on thechronology of autibody response N. Mizuno et al.
27 Studies on antigenicity of Trichinella spiralis (8) with emphasis on thelocalization of antigens recognized by the patients T. Uno et al.
28 A comparative studies on the detection rate of Strongyloides stercolarisby agar method and the traditional methods R. Asato et al.
29 Enzyme‑linked immunosorbent assay for the detection of serum anti‑
body in cats and rats infected with Paragonimus heterotremus
H. Sugiyama et al.

30 Antileishmanial activity of inosine analog H. Tanifuji et al.
31 Therapeutic effect of isosine analogs loaded in liposome in mice infect‑

ed with Leishmania donovani K. Morishige et al.
32 Relations between shape and nuclear DNA contents of gametocytes in

Plasmodium vivax and falciparum H. Amano et al.

36

33 Susceptibility of Anopheles omorii to Phomodium yoelii nigeriensis

N67

K. Ichimori et al.

34 In vivo and vitro developmental conditions from ookinete to sporozoite

of Plasmodium berghei

K. Yano et al.

35 Longitudinal seroepidemiological malaria survey in an Amazonous
hypoendemic colony 11 case plotting method alming a computer graphic
H. Yanagisawa et al.
system36 Immunoreactive peptides cleaved by cyanogen bromide from Japanese
encephalitis virus envelope glycoprotein E

A.K. Srivastava et al.

37 Enhancement of dengue virus multiplication by the inhibitors of mRNA
synthesis
T. Takegami et al.

38 Enzyme‑linked immunosorbent assay (ELISA) in the detection ofhepatitis‑B surface antigen in blood dried on filter paper

S. Matsuda and H. Kahyou
39 Studies on hepatitis which occured among long‑term sojourners in thedeveloping countries, and prevention against it H. Ohara et al.
40 Purification of an antihemorrhagic factor from the serum of the

Dinodon semicarinatus Y. Tomihara et al.

41 Antitoxic effect of cephalanthin and antivenom against the Mamushi(Agkistorodon blomhoffii) venom Y. Kawamura and Y. Sawai
42 A case of severe malaria treated with ginghaosu S. Kano et al.
43 Imported malaria cases recently dealt with by us K. Yano et al.
44 One case of vivax malaria suspected to be infected in India

T. Katsumata et al.
45 The effect of haemoglobin in serum as a trigger of the transformationfrom bloodstream forms to procyclic forms of Tmpanosoma brucei invitro T. Yanagi et al.

46 Ultrastructure of transitional forms of Tmpanosoma cruzi by scanningelectron microscopy V. Sooksri et al.

47 Influence of monosaccharides on the growth of Tmpanosoma brucei

gambiense in vitro P.J. Mhando et al.

48 Studies of two enzyme immunoassays for epimastigote and trypomas‑

tigote forms of Tmpanosoma cruzi M. Iwamoto et al.

37

S pecial lecture

COOPERATIVE MEDICAL RESEARCH PRO JECTS IN KENYA
M. MUGAMBI
Director, Kenya Medical Research Institute

Introduction
Many developing countries now recognise research to be an important tool for social andeconomic development. Despite this realisation nearly all of these countries still allocate lessthan 0.5% of their GNP on research and development (R & D) activities.
In the past 10 years Kenya has been investing increasingly in R & D efforts. To this end,
apart from established Universities, Kenya in 1979 established research institutes in the fields

of agriculture, health, industry and fisheries. Medical research as a means of improving
health care delivery has received considerable attention. In 1987 4.70 million pounds wasallocated to medical research compared to 0.61 million pounds in 1980.
The above allocations, however, are far from adequate to satisfy medical research needs.
It is with this realisation that the Government welcomes increasing international and regional

cooperation in medical research.
Relevant Statistics

Kenya has a population of 22 million of which 85% is rural and 15% urban. The
population is young with 51% being 15 years and under. The mean infant mortality rate(IMR) now stands at less than 90 per 1,000 births (range 45‑220) .

The five major causes of morbidity in order of importance are:Malaria 23.6%, ARI 20.2%, Skin diseases 6.5%,Diarrhoea 5.8% and Intestinal

worms 5.5%.
Of the parasitic diseases the most important in terms of morbidity and mortality aremalaria and schistosomiasis.

Health Research
Most of the medical research activities in Kenya are located at the Kenya Medical
Research Institute (KEMRD . KEMRI has its headquarters located in Nairobi. Peripheral
centres exist in Kisumu, Busia and Mombasa. The establishment of the headquarters andcentral laboratory complex of KEMRI was generously funded by the Japanese Government
to the tune of approximately $10 million. This capital development, as we shall see later, isalso supported through technical cooperation through JICA.

The major research programmes of KEMRI are:Malaria, Schistosomiasis, Leishmaniasis, Diarrhoea disease, ARI, Viral diseases, STD

and Traditional medicines.
Since, as was stated earlier, Iocal resources are inadequate to meet research needs

KEMRI has entered into cooperative research agreements with selected Governments andagencies. The major ones of these are:38

JICA‑parasitology, bacteriology, virology

WRAIR (USA)‑malaria, Ieishmaniasis

CDC (USA)‑malaria
WHO‑TDR diseases, STD. CDD
IDRC‑dental' health, primary health careBy far the largest input in terms of resource allocation and manpower development to

KEMRI comes from JICA.
Ken a‑Ja an Coo eration
The Japanese have had interest in health related research in Kenya for many years.
However serious technical cooperation in this field started in 1979 with the establishment of

the Communicable Disease Research and Control Project (CDRCP) . This 5 year project
which terminated in 1984 identified diarrhoeal disease and schistosomiasis as the main areasof mutual research interest. The participating Japanese institution was Nagasaki University.

In 1985 a further 5 years technical cooperation agreement was negotiated. This agree‑
ment identified the following fields for joint research and control efforts:‑

Parasitology: schistosomiasis
Bacteriology: diarrhoeal diseases
Virology: diarrhoeal disease, viral hepatitis
The participating institutions were expanded to include Tokai University, Sapporo andlwate Medical Universities, in addition to Nagasaki University.
The cooperation comprises actual research, provision of research materials, training of

Kenyans in Japan and deployment of Japanese experts to Kenya.

Achievementsa ) Trainin :So far over 15 Kenyans have trained in Japan in such disciplines as parasitology, tropical

medicine, electron microscopy, medical engineering, bacteriology, virology, etc.
b ) Research:

Parasitolo : Schistosomiasis affects over 2 million Kenyans. In certain areas over 90%of the children are infected with either S. mansoni or S. haematobium. The present
research project in Kwale has looked at prevalence, diagnostic methods, cercariometrics,

malacolbgy and intervention.
So far, safe water has been provided in a pilot area and treatment with praziquantel
together with health education have been offered. Intensity of infection has been reduced

by 80%. Community awareness has increased.
Bacteriolo : The project has focussed on studies of intestinal flora, bacterial ecology
and enteric infections. So far intensive analysis of 15 healthy and diarrhoeic children has

been done. These studies complement those of viral aetiology of diarrhoea.
Virolo : Rota viruses have been shown to be significant causes of infant diarrhoea. The
Kenyan study has attempted to determine the role of rota‑virus in childhood gastroenter‑
itis. Studies have also attempted to examine the serotypes involved and factors respon‑sible for susceptibility.

A cohort of 100 children is being followed up and screening for rota‑virus using ELISA
and electron microscopy is done frequently.

39c ) Lo istics:

Equipment, supplies, transport and travel costs are provided. This greatly helps in thesmooth implementation of the project.
d ) Ex erts:

These have come and stayed for periods of 3‑12 months and some even longer. They
have been instrumental in the initiation and continuation of projects and for training
within projects.

The future
Japan has assisted Kenya in many technical fields such as forestry, agriculture, mining,energy, roads only to mention a few. In the field of health research, which is the subject of

this meeting, Japan has closely worked with Kenya to develop research and technicalcapacity. At the same time Kenya has provided the opportunities for Japanese medical
scientists to improve their skills in Tropical Medicine. The exploitation of these opportu‑
nities should be encouraged. The future is encouraging and it is my hope that more Japanese
resources can be put to this worthy effort.

40

Symposium
I Tropical Infectious Diseases and Drugs

1 INSECTICIDES

la INSECTICIDES FOR USE IN JAPANESE ENCEPHALITIS CONTROLPROGRAMMESYOSHITO WADA

Department of Medical Entomology, Institute of Tropical Medicine,

Nag saki University

The number of Japanese encephalitis (JE) human cases in Japan greatly changed inyears. Although hundreds or more than 2,000 cases appeared in a year in 1960's, the number

remarkably decreased after 1972 and less than one hundred cases only were reported every
year. It is very likely that a most important factor responsible for this reduction is thedecreased density of the vector mosquito, Culex tritaeniorhynchus, and the decrease in thevector density is due to the switch from chlorinated to organophosphorus and carbamate
agricultural insecticides sprayed in the rice field, which is the main breeding place of thevector mosquito. The slight increase of the vector in 1980's seems to be due to the develop‑
ment of resistance to the insecticides. Thus the population of the vector and therefore theepidemic of JE also are influenced by the application of agricultural insecticides in the field.

There are many insecticides that have been proven to be effective for the control of thevector mosquito in the laboratory and in the field. The application of insecticides to rice fields

is effective in controlling the vector population, but usually only for one or two weeks.
Therefore, if we wish to control vectors by larviciding, several applications will be necessary
in a year. This means that in view of a vast area of rice fields, the application of larvicides

for the control of JE will be extremely costly and probably of no operational value in usual

circurnstances. Moreover, the chemical control by using organophosphorus and carbamate
insecticides became completely ineffective in recent years due to the development of insecti‑
cide resistahce. The aerial spray of insecticides against the adult population of the vector

does not seem to be of practical value for the same reason, since the adult mosquitoes rest
in very large areas of rice fields and nearby grassy lands.

To prevent transmission of JE, the first measure to consider is vaccinating hurnans
because of its effectiveness. However, it is not easy to raise the rate of persons receiving

vaccination. The vaccination of pigs, which are the most important amplifying animal of JE
virus, is usually of little practical value in view of the cost involved. Separating pigs fromrice fields is the surest way to reduce pig‑mosquito contact, but there is a limitation to its
realization.

As mentioned in the above, most measures to control vectors by insecticides are of nooperational value at least in the present situation, however effective they are, and it is noteasy to further decrease the number of cases by measures to humans and pigs. A promising
approach would be to reduce the number of vector mosquitoes engorged with pig blood, which

41

may include mosquitoes just infected with JE virus, by using, for example, insecticide im‑preguating netting at pigsties.

lb INSECTICIDES FOR USE IN DENGUE CONTROL PROGRAMMESAKIO MORI
Department of Medical Zoology, Nagasaki University School of Medicine

Aedes aegypti, the main vector of dengue fever, breeds in various kinds of containers inand around the human dwelling, for example, ant‑traps, earthen jars, flower vases and tincans. As most of these breeding sites are artificial, it can be said that the epidemic of dengue

fever is man‑made. Only vector control are most effective measure to control dengue feverat the present time, because the vaccine against dengue viruses has not been developed forpractical application.

There are two measures against Ae. aeg)pti as the vector of dengue fever. The first one

is keeping the population of Ae. aegypti constantly in low density below the threshold
transmission level in a certain area, in order to prevent the outbreak of dengue fever. The

environmental control around houses by inhabitants themselves who receives health educa‑tion is most recommended to control Ae. aeg pti. For example, removing empty tin cans,bottles and rubber tires from their back yard, changing the water in fiower vases frequently,

putting salt into ant‑traps and covering earthen jars and tunks holding drinking water would

be easy, economical and effective methods. However, human behaviour or inhabitants' habit

may impede to dry up all disused water and put the lid on water jars completely in someareas, where we are compelled to apply the larvicide to those water containers. Many kindsof larvicide (organophosphate, IGR) may be effective to Ae. aeg pti larvae as shown bylaboratory experiment. Because most of these containers are used for holding drinking
water, we must take into consideration that the larvicide is not toxic to human and othervertebrate animals and has not a strange taste or smell.
The second measure is to suppressing the epidemic of dengue fever instantly in a certain
area. If the transmission of dengue viruses is prevented by the control of the adult populationof Ae. aeg)pti for 14 to 20 days, the epidemic will be terminated from the area. Fogging ofinsecticides (fenitrothion, permethrin and others) to houses, where this mosquito rests andfeeds on human blood, is used for this purpose. Recently ULV is recommended to control
adult Ae. aeg pti. because it is more effective to adults and also kills larvae in containers.

As larvae of Ae. albopictus occupies small containers around houses, the basic method tocontrol this mosquito is the same as that for Ae. aeg)pti mentioned above.

42

lc INSECTICIDES FOR USE IN ANTIMALARIA PROGRAMMESTAKESHI KURIHARA
Department of Medical Zoology, Teikyo Unive.rsity School of Medicine

Indoor residual spraying of DDT was a major strategy of the anti‑malaria project during

the 1950s and 1960s in various parts of the world. The effects were dramatic. It reduced

malaria incidence quickly and eliminated endemic areas in many countries. A malignant
malaria in the Yaeyama Islands of Japan which had long bothered residents was also
eradicated using this method. DDT spraying, however, experienced a crisis in the 1970s
because of environmental pollution and the health hazard. In addition, inhabitants of endemicareas often refused the spraying because it stained the interior of their dwellings. The spray

operation was also curtailed by financial difficulties. The cost of malaria control operations
has risen greatly due to increasing costs of transport, fuel and personnel. A shortage of funds

for the purchase and distribution of chemicals by national health services has resulted in an

inadequate control operation. New chemicals are now being tested and adopted in various
areas, and new application methodologies, such as selective spraying of houses and cattle
sheds, based on the resting preferences of vectors are being used in an effort to reduce costs.

Alternatives are now emerging with the involvement of communities in anti‑vector
measures. A mosquito‑net or bed‑net is a potentially excellent anti‑vector means, but it may

soon be torn. Therefore, impregnation of the net with an insecticide such as a synthetic
pyrethroid has been adopted to improve the protection efiiciency. A wide‑mesh net curtain,
which does not obstruct the ventilation of the room and even allows mosquitoes to passthrough easily, can be treated with an insecticde and may be more acceptable to occupantsin hot and humid areas. In a trial in a Toyama Prefecture pigsty, the total number ofmosquitoes collected in a light trap surrounded by a net impregnated with phenothrin wasmuch less than half the number using an untreated net. The treated net reduced the
proportion of surviving mosquitoes to a negligible level and greatly reduced the per cent of
fed mosquitoes. There is a need for further study in both the laboratory and field for accurate
assessment of the effect of insecticides not only to kill but also on vector mosquito response.

A series of laboratory tests comparing different chemicals was carried out to select an
insecticide suitable for net treatment. Results revealed that nets impregnated with pyreth‑roids such as phenothrin and cyphenothrin reduced both the number of mosquitoes passing
through and their feeding activity.

2 ANTI PARASITIC DRUGS

43

2a THE ANTI‑PARASITIC DRUGS IN CURRENT USE
YOSHIHITO OTSUJI
Kagoshima Prefectural Comprehensive Health Center

Parasitic diseases which were rampant during and just after World War 11 have rapidly
subsided these days thanks to exhaustive disease control measures, improved environmental
sanitation and a change of eating habits.
On the other hand, however, as international exchange has lately become more and more
frequent such clinically important issues as how to control imported parasitic diseases,
parasitic diseases set off due to immuno‑deficiency state and those common to both men andanimals have given rise to public attention.

Meanwhile anti‑parasitic drugs are difiicult to obtain these days because parasitic
diseases are scarce now in Japan and, in addition, some of the drugs have troublesome side
eff ects.

In order to counter these problems we instituted in 1988 a task force for studing drugs forthe imported parasitic diseases and have had 15 items of difficult‑to‑obtain drugs in our
charge for "experimental purpose", which have been dispensed on request.
Results:

1 ) The drugs had been distributed among patients for 7 years from 1980 through 1987
on 1,101 occasions (about 150‑200 cases annually). Malaria (287 cases) topped the diseaselist, followed by strongyloidiasis (210 cases) , trichocephaliasis (193 cases) , clonorchiasis (97cases) , amebic dysentery (50 cases) and pneumocystis carinii pneumonia (50 cases) , etc.

Anti‑malarial drugs of primaquine, chloroquine, fansidar, etc. had been most requested,

then mebendazole for trichocephaliasis, thiabendazole for strongyloidiasis and praziquantel
for trematodiasis (clonorchiasis) , etc., in that order.

2 ) Status quo of imported parasitic diseases: There had been 383 patients (287Japanese and 96 foreigners) with tropical parasitic diseases, 268 of them suffering frommalaria, followed by clonorchiasis (32), trichocephaliasis (30), Iambliasis (21) and various
cestoidiasis cases (7) . Such uncommon diseases in Japan as trypanosomiasis (1) , Kala azar
(1) and bilharziasis (1) were also found among the patients. Most of the presumed areas oforiginal infections with the diseases were east‑west Asian and Africal countries. Malaria
patients comprised 251 males and 17 females, most of them in their thirties.
Assortments of malaria‑originated areas, number of patients and malaria species were:
Asian countries (125 patients: 87 P. vivax. 16 P. falciparum. 2 P. malariae and 20 unknown
species) , African countries (72 patients: 27 P. vivax:. 27 P. falciparum, I P. malariae and 17

unknown species) and Oceanian countries (22 patients: 13 P. vivax and 9 P. falciparum).
The purposes of the patients' travel were sightseeing, business and academic investiga‑
tion, in that order, for Japanese citizens, meanwhile they were training, business, sightseeing
and entry into Japan as refugees for foreigners.

The task force for development and procurement of drugs against imported parasitic
diseases has made every effort since 1980 to solve aforementioned problems and, as a result,some drugs have come into the market.
The above‑described facts are the status quo of the imported parasitic diseases in Japan.

44

2b NEUROPHARMACOLOGICAL APPROACH TO ANTHELMINTIC DRUGSMAMORU TERADA
Department of Parasitology, Hamamatsu University School of Medicine

Chemotherapy is essentially important in treating patients with helminthic parasites.
Since treatments making host resistant to the infections are generally not promising inhelminthiasis, the significance of the chemotherapy in controlling the diseases should also beemphasized. A basic approach to the chemotherapy against helminthiasis is presented in thisre port.

Wh did we stud anthelmintic dru s neuro harmacologicall ?
Traditionally, many chemotherapeutic studies against helminthiasis have been aimed atenergy metabolism of the worms: i. e., effects of anthelmintics on the metabolism andmechanism of the metabolism. Although the mechanism of the energy metabolism isbasically common in nemathelminthes and platyhelminthes, there is a marked difference insensitivity to anthelmintics between these two groups of helminths. To reveal mechanismsof the difference, neuropharmacological mechanism was taken up as another target forchemotherapy. Motility of worms was regarded as the most useful parameter and an isotonic
transducer method was introduced to detect the faint motility of smaller species in vitro.
Angiostrongylus cantonensis and Hymenolepis diminuta were selected for representativemodels of nemathelminthes and platyhelminthes, respectively. Thus, effects of various
neuropharmacological drugs on the motility of helminths including the model worms werestudied by the isotonic transducer method. It was suggested from the findings reported thatacetylcholine (ACh) and y‑aminobutyric acid (GABA) in nematodes and 5‑hydrox‑
ytryptamine (5‑HT, serotonin) and ACh in platyheminthes act as stimulatory and inhibitory
neurotransmitters, respectively. It is likely that the difference in the sensitivity to anthel‑mintics between nemathelminthes and platyhelminthes is attributable to the difference in theneuropharmacological mechanism. Then, we studied effects of various anthelmintics in this
aspect. Antinematodal anthelmintics, for examp.le, were classified into three groups; anthel‑mintics including pyrantel act on cholinergic mechanism, ones such as piperazine andmilbemycin D act on gabergic mechanism, and ones like diethylcarbamazine affect bothmechanisms. Two topics on neuropharmacological actions of these anthelmintics arepresented as follows.

Wh is rantel used clinicall onl a ainst intestinal nematodes ?
Pyrantel at 10‑5 M or less affected the motility of nematodes and isolated host tissues,
while no effect was seen in cestodes and trematodes. For the selectivity of the action ofpyrantel on nemathelminthes and platyhelminthes, it is assumed that pyrantel acts onnematodes through stimulating nicotinic ACh receptors in them, and that the anthelmintic
doesn't affect platyhelminthes since they lack the nicotinic ACh receptors. In spite of theeffects on the nicotinic receptors in host tissues in vitro, pyrantel doesn't affect host adverse‑ly, because the drug is hardly absorbed through the intestinal tract of the host and also
because nematodes are affected at lower concentrations than host tissues.

45

How are com licated actions of an anthelmintic elicited ?
Som'e anthelmintics such as levamisole are reported to act on some different sites
including the energy metabolism and neuropharmacological mechanism of helminths. On theother hand, it is reported in the case of piperazine that a secondary inhibition in energy
metabolism is derived from a primary stimulation on the gabergic mechanism in Ascaris. As
another example of the latter cases, effects of milbemycin D on A. cantonensis were shown.
Milbemycin D paralyzed the worm in vitro at 10‑*1 g/ml or more, probably through gabergic
stimulation. A single oral dose, 5 mg/kg of the drug paralyzed the worm in rats, and theeffect lasted about I week. When infected rats were treated with 9 doses of 5 mg/kg/week,

the paralysis seemed secondarily to cause the worms inhibition in food intake, energy
metabolism and reproductive function. No first stage larvae were seen in host feces persist‑
ently after the second dose, and no food materials in the intestinal tract of the worms and noeggs and first stage larvae in the lung tissues of the host were observed at sacrifice after the

final dose. Thus, as shown in the effects of milbemycin D, many complicated effects ofanthelmintics seem to be attributable to the fact that various mechanisms in the worms areclosely related each other.

Wh is chemothera a ainst helminthiasis with tissue helminths difiicult ?
Finally, anthelmintics against intestinal and tissue helminths were compared. Intestinal

helminths can be rather easily treated with anthelmintics, because there are some advanta‑
geous aspects which make chemotherapy easier. For example, even a reversible action iseffective because of the peristalsis of the host intestine by which paralyzed worms can be

expelled, and even toxic drugs like pyrantel can be useful unless absorbed through theintestinal tract of host. Thus, we have had effective anthelmintics against almost intestinal

helminths and will obtain more useful ones in respects of efficacy, safety and spectrum. On
the other hand, there remains extremely difficult situation regarding tissue helminths. It is
hard to get more effective and less toxic anthelmintics which bear irreversible or long‑lasting

action when considered their living environments. Even if we could get such anthelmintics
and kill the worms successfully, we must be faced with another problem. Adverse systemic
effects may be elicited by allergens which are released from the killed worms and distributed
through the blood syst,em. This means that many therapeutic treatments are nescessary for
effective therapy in addition to killing worms. Thus many difiicult problems remain to besolved in the chemotherapy of helminthiasis with tissue helminths.

3 ANTI‑MICROBIAL AGENTS AND THEIR DEVELOPMENTAL STATE
3* ENTERIC BACTERIA IN ASIAN COUNTRIES AND THEIR DRUGRESISTANCEYASUO KUDOH
Department of Microbiology, Tokyo Metropolitan Research Laboratory of Public

Health46

Antimicrobial agents are commonly used therapeutically and prophylactically for diarr‑
heal diseases. Resistance of enteropathogens to these antimicrobials may prevent the effectof such therapy, and thus information on distribution of antimicrobial‑resistant enteropath‑ogens are indispensable therapeutically. The information may also lead to understandepidemiology of diarrheal diseases.
In this paper we describe enteric bacteria causing diarrheal diseases, especially travelers'

diarrhea, in Asian countries, and antimicrobial resistance of the enteropathogens detectedfrom the travelers.
Enteric bacteria in travelers' diarrhea

Our surveys on travelers returning from abroad mainly from Asian countries during thelast decade (1978‑1987) showed that approximately 60% of the diarrheal cases from abroadwere possibly due to bacterial infections. The most common causal agent was enterotox‑
igenic Escherichia coli (ETEO occupying 47.2% of the total isolates. It was followed in orderby Salmonella (12.8%), Vibrio parahaemolyticus (10.9%), Shigella spp. (8.0%) andCampylobacter jejunilcoli (7.4%). Vibrio cholerae O1 was detected at a rate of 0.5%. Whendetection frequency of each enteropathogen was compared with countries where the infection

occurred, some geographical tendency were observed among Asian countries. Namely,
infection due to ETEC was common in most Asian countries, whereas V. parahaemolyticus
infection was more common in Eastern and Southeastern Asia than Western Asia. Shigellaand Campylobacter infections were prevalent in West Asia, but not so common in other Asian
countries. It is also notable that infections with multiply pathogens are very common in
travelers' diarrheas.

Antimicrobial resistance of enteric bacteria

Antimicrobial susceptibility data shown here were mainly derived from our surveys on
travelers' diarrhea or through data exchange project of Southeast Asian Medical Information
Center (SEAMIO . Antimicrobial agents tested included chroramphenicol (O , tetracycline(T) , streptomycin (S) , kanamycin (K) , ampicillin (A) and sulfamethoxazole‑trimethoprim(X) .

In Shigella, frequency of resistant strains (mostly multiple drug resistance) was thehighest among enteropathogens tested, but no marked geographical difference in incidence
was observed in incidence between Japan and other Asian countries. Among antimicrobials
tested, resistance to S, T, and C was remarkably high, followed by A and X, but K resistanceremained still relatively low. Frequency of resistance of non‑typhoidal Salmonella was about
20% both in Japan and in other Asian countries, but resistance to each drug varied bycountries and tended to increase gradually year by year. It is also noteworthy that Salmone‑lla resitance is markedly different by serovar. High resistant serovars found in Japan were
Hadar, Litchfield, Muenchen, Typhimurium and Blockley, whereas in other Asian countries

were Blockley. Hadar, Typhimurium, Krefeld and Panama. In Salmonella ser. Typhi,
incidence of resistant strain was still quite low, but it should be noted that there wereresistant strains which were mediated by transferrable R plasmid in Asian countries such as
Indonesia and Thailand. Of interest, ser. Typhi R plasmids found in Asian countries belonged

to incompatibility group H, and appeared to be identical to that in Mexican outbreak in
1970s. Resistance of diarrheagenic E. coli was about 50% , and no remarkable differenceswere observed in diarrheagenic types of organisms (ETEC, EIEC or EPEO or in places ofisolation. Diarrheagenic vibrios remained largely susceptible against antimicrobials tested.

47

However, recently resistant V. cholerae O1 strains were found to distribute in Thailand andPhilippines, and most of them were mediated by transfferable R plasmid of incompatibility

group C.

3b DRUG RESISTANCE OF VIBRIO CHOLERAE O1 ISOLATEDIN TROPICAL AREAYOSHIO IcmNosE
Department of Bacteriology, Institute of Tropical Medicine,

Nagasaki University

There have been frequent epidemics of cholera in Africa since the 7th cholera pandemic
that originated from Celebes Island in Indonesia in 1961, and reached the African continent
in 1970.

Since then, cholera is endemic in these areas and sometimes becomes epidemic. Antibi‑
otics are given routinely to patients with cholera because it decreases the duration andvolume of purging, the duration of excretion of vibrio and the amount of fluid replacement

required, although fluid replacement is the most important con ponent of therapy. Drug
resistant strains of Vibrio cholerae has been isolated sporadically in endemic areas.

However, no extensive emergence of multiply drug‑resistant strains which becomes aserious problem clinically, has occurred.

However, In 1979, Mhalu et al. reported that the strains of V. cholerae resistant totetracycline (TO , a drug considered to be very effective against cholera, were also resistant

to streptomycin (SM) and ampicillin (ABPO , were isolated in Tanzania. All isolates during
the first month of the outbreak were fully sensitive to TC, but 76% of isolates were resistantto the drugs after five months of extensive use of TC therapeutically and prophylactically. In
1980, Glass et al. also reported the emergence of multiply drug‑resistant strains of V. choleraein Bangladesh. This paper describes an outbreak of cholera and the emergence of multiply
drug‑resistant strains of V. cholerae in Kenya which we experienced during The Communi‑
cable Diseases Research and Control Project between Kenya and Japan supported by theJapan International Cooperation Agency. Two hundred and seventy two strains of V.
cholerae O1 biotype E1 Tor were isolated from six cholera outbreak and sporadic cases
during 1980 and 1981 by lwanaga et al. dispatched as the first team but no multiple drug‑
resistant strains of V. cholerae were found.

However, multiple drug‑resistant strains started to be isolated in 1982 in the South
Nyanza District near Lake Victoria and account for 41% of the strains isolated. In April,
1983 when we obtained information about cholera outbreak, we started to make a cholera
surveillance in the areas of South Nyanza, especially at Homa Bay District Hospital, Ombo
Hospital and other health centers. We isolated and collected 244 strains of V. cholerae from
inpatients, high school students, prisoners and healthy carriers. Out of 244, 235 strains of V.

cholerae were Ogawa type, nine were Inaba type. All the isolates were E1 Tor biotype except
one untypable and Celebes original type in prophage typing. All the isolates were sensitive
to nalidixic acid and chloramphenicol and 183 out of 244 strains were resistant to TC, SM,

ABPC and ST compound. A cholera outbreak occurred in Nairobi on November in 1983 and48

in Kisum in March, 1984. The number of strains isolated in Nairobi and Kisum was 70 and
23> respectively. All the strains isolated were biotype E1 Tor, Ce]ebes original in prophage

typing. The number of resistant strains to three drugs (TC, SM, ABPO were 64 (91%) and18 (78%), respectively. The resistance of these strains was transmissible. According toMorris et al., all Kenyan resistant strains contain a group C incompatibility plasmid of ca
100 MD and which is different from those in Tanzanian and Bangladesh strains in restriction

endonuclease digest pattern. Therefore, the multiple drug‑resistant strain in Kenya wasprobably not directly brought from the Tanzania border, although it occurred near theborder. In fact, close contacts of cholera patients started to be administered tetracycline by

the cholera surveillance team for prophylactic use in 1982 and the number of multiple drug‑
resistant strains was increasing in this year. An extensive chemotherapy is thought to beclosely associated with the development of drug resistance. Furthermore, inadequate
management for cholera, for example, insufficiency of dosage of drug, failure of treatment
and drop‑out cases during the treatment of cholera might have resulted in the wide spreadingof cholera and development of drug resistance. Cholera outbreaks due to multiple drug‑
resistant strains were also seen in Somalia and India.

3･ CURRENT STATUS OF RESPIRATORY PATHOGENS AND DRUGRESISTANCE
KEIZO YAMAGUCHI
Department of Laboratory Medicine, Nagasaki University
Sch061 of Medicine

Infectious diseases are still very common in the tropics and mortality rate due topulmonary infections has been the highest among infants.
In this paper, the recent situations on bacterial respiratory infections and drug resistancein the area of the southeast Asia based on data mainly from the Research Institute forTropical Medicine of the Philippines (RITM) and WHOIPRO reports were discussed.
1 . Current status of acute respiratory infections in the RITM; About 30% of theinpatient was occupied by pulmonary infections in this hospital and S. pneumoniae and H.
influenzae were the most important causative micro‑organisms among the patients withacute pneumonia. On the other hand, more than half cases autopsied had also accompanied
lethal pneumonia due to gram‑negative bacteria or measles. Although the isolation of L.
pneumophilla which is well known as a pathogen of nosocomial or opportunistic infection was
not yet reported from the clinical specimens of the patients with pneumonia in the Philippines,
we succeeded to isolate the strains from the water of cooling towers of hotels and hospitals

in Manila with a cooperation of laboratory staff of the RITM. This fact suggested thepossibility of existence of the patients with pneumonia due to Legionella in this country.
2 . Bacterial drug resistance; Resistant strain of S. pneumoniae to PCG is reported fromthe Philippines and Malaysia with the percentages of 2 and 0.2%, respectively, although thefrequency rate of resistance to erythromicin or tetracycline was lower compared to that ofour country.

49

None of resistance to PCG was yet repcrted on S. pyogenes.
More than 75% of S. aureus produces P‑lactamase and frequency of methicillin resistant

S. aureus (MRSA) varied 0‑26% according to countries.
Ampicillin resistant H. influenzae was observed with an incidence of 2‑20% and most ofthem produced p‑lactamase.

3d PRESENT STATE OF DEVELOPMENT OF ANTIMICROBIAL AGENTSAKIRA IMADA
Biology Laboratories, Central Research Division, Takeda
Chemical Industries

The rationales of the development of new antimicrobial agents are (1) progress of
medical treatment, (2) change in pathogenic organisms, and (3) increase of medication cost.
The progress of medical treatment has resulted in the prolongation of life of patients,

which is accompanied by the increase of patients exposed to the high risk of infectious
diseases. For example, the patients treated with anti‑cancer drugs on cancers or withimmunosuppressive drugs on organ transplantation become susceptible not only to a variety
of weakly pathogenic bacteria but also to viruses and fungi. For those immunocompromised
patients, antibacterial agents that have broad‑spectrum activity and strong bactericidal
action are needed. Many of the aged constrained on beds often suffer from the bed‑sore thatis regarded as the reservoir for methicillin‑cephem resistasnt Staphylococcus aureus (MRSA) .

Those factors related to the progress of medical care should be taken into account for the
future development of antimicrobial agents.
The change in pathogenic organisms is believed to be related to the usage of antimicrobial
agents. The biggest problem in this concern is the outbreak of MRSA, which is said to havesome relation to the use of the third‑generation cephalosporins having weaker activity than
the first‑ and second‑generation ones. Soon after the clinical introduction of new quinolones,
the quinolone‑resistant bacteria have rapidly increased, especially among S. aureus. Serratia

marcescens, and Pseudomonas aeruginosa. The emergence of resistance to newly introduced
antibiotics has been repeatedly experienced. The fight against resistance may continue
endlessly.

The increase of medical care cost has become so big that the national budget is threatend‑

ed by it. In order to reduce the medication cost, every effort is being made. The shorteningof the hospitalization period is one of the ways to cope with such a desire. The demand fororally active or suppository drugs is increasing in this context. As to the parenteralantibiotics, Iong half life or sustained release drugs will be needed in the future.

The major categories of antibacterial agents being persued are (1) cephalosporins, (2)new quinolones, and (3) p‑lactams with novel nuclei. The development of aminoglycosides
and macrolides is continuing but less actively. The development of penicillines, those active

until 1970s, has almost terminated.

The development of cephalosporins is directed toward the expansion of the antibacterial

spectrum covering S. aureus and/or P. aeruginosa, and the orally active compounds. The

50

development of cephalosporins has proceeded from first‑generation (represented by ce‑
phalothin and cefazolin) , second‑generation (cefotiam, cefmetazole) , to third‑generation(cefotaxime, Iatamoxef, ceftazidime) . During that process, the activity against bacteria ofEnterobacteriaceae was greatly increased. But, as mentioned before, the activity againstgram‑positive bacteria was reduced in the third‑generation compounds. Flomoxef andcefuzonam are the cephalosporins having improved activity against S. aureus. Constantefforts have been made to improve the activity against P. aeruginosa; cefpimizole andcefpiramide are recently launched compounds. E‑1040, which has potent antipseudomonal
activity, is now being developed. Cephalosporins active against both S. aureus and P.
aeruginosa besides against bacteria of Enterobacteriaceae, though never launched yet, is thenext goal to be attained. HR‑810, now under clinical evaluation, is an example of suchcompound. Increase of the bactericidal activity is another goal. Cefminox shows potent
bactericidal activity not only against growing bacteria but also against non‑growing ones;
P‑lactam antibiotics useally kill bacteria only at the growing state.

Orally active cephalosporins have been energetically persued. There are two categories
for them: one is the free form and the other ester form. Cefixine is an example of the former,

and cefteram pivoxil and cefuroxime axetil, the examples of the latter, that were launchedrecently. Several orally active cephalosporins are in the late stage of clinical evaluation:
ceftibuten (7432‑S), FK‑482, and Ly‑163,892 (KT‑3777) as free form, and CS‑807, cefotiam
hexetil, and BMY‑28271 as ester form.

The new fluorinated quinolones have opened up a new era of antibacterial therapy.
Norfloxacin was the first to be launched and ofloxacin, enoxacin, and ciprofloxacin werelaunched subsequently. Nalidixic acid and other older generation quinolones are active
mainly against gram‑negative bacteria and their clinical application was restricted tourinary tract, otolaryngological, and gastrointestinal tract infections. The introduction offiuorine into the quinolone ring not only improved the activity against gram‑negative
bacteria, but also expanded the spectrum to gram‑positive bacteria. Moreover, the phar‑macokinetic properties have also been improved, thereby enabling the new quinolones to be
used for respiratory tract and soft tissue infections also. Lemofioxacin (NY‑198) , Fleroxacin(AM‑833) , and T‑3262 are now under clinical evaluation.

p‑Lactams with novel nuclei such as carbapenem, nocardicin, clavulanic acid, andsulfazecin (monobactam) were discovered from microbial cultures in the late 1970s. Of thosecompounds, clavulanic acid, a fi‑lactamase‑inhibitor, has been clinically in combination withamoxicillin (augmentin) . Sulbactam is another p‑lactamase‑inhibitor utilized. The discov‑

ery of sulfazecin is connected to the development of new monobactam antibiotics such as

aztreonam and carumonam. They are active selectively against aerobic gram‑negative
bacteria including P. aeruginosa. The first carbapenem antibiotic clinically introduced isimipenem. Since it is susceptible to hydrolysis by the renal dehydrodipeptidase, the combina‑
tion with cilastatin, a dehydrodipeptidase inhibitor, was necessary, Since carbapenems show
broad spectrum and potent bactericidal activity, they seem to be suitable for the treatment

of infections in immunocompromised patients, and newer carbapenems such as SM‑7338 andCS‑976 are now being clinically evaluated.
At the meeting, the comparison of statistics of the use of antibacterial agents in Japan,

the United States. Europian countries, and tropical countries, which shows great contrast,
was also discussed.

51

II

Present Status and Topics in Develo pmentof Vaccines against Tropical Inf ectious
Diseases

1 VACCINES FOR PROTOZOAL DISEASES

la SEVERAL NOTABLE ACHIEVEMENTS IN RECENT RESEARCH
ON MALARIA VACCINEMAMORU SUZUKI
Department of Parasitology, Gunma University School of Medicine

Since the start of The Special Programme for Research and Training in Tropical
Diseases, studies on malaria vaccine have been one of the greatest concern in modern
biomedical science and have attracted attentions and participations of many top scientists
toward this field. Sporozoite vaccine has been studied intensively by the group of Departmentof Molecular Parasitology of New York University headed by Professor Ruth Nussenzweig
who started the study as early as in 1967. The sporozoites taken from mosquitoes are mixed
with mosquitoe tissues cells, cell debris and contaminants. Difficulties in the preparation oflarge amount of parasite materials in a purified form hampered further progress in thestudies. The problem was solved by the introduction of monoclonal antibody techniquefollowed by genetic engineering. After a successive studies, eventually repetitive peptide(NANP)3 was identified as circumsporozoite epitope which works against the attack byprotective antibodies. The repetitive peptide can be a candidate of sporozoite vaccine.
Indeed, two independent groups reported that some human volunteers who were vaccinatedwith (NANP) 3 bound tetanus toxoid did not develop malaria after being bitten by malarious
mosquitoes. (NANP) 3 works as B‑cell epitope hence unable to provoke T‑cell sensitization.
(NANP) 3 bound with lysin network makes a mass of peptide, and the mass can elicit T‑cell
activation. Gamma interferon released from the sensitized T‑cell seems to suppress thedevelopment of extra‑erythrocytic stage, which also work adversely toward parasites.
Extraerythrocytic stage of malaria parasite is very difficult to study, because this stage ofparasite only occupies really small part of the liver. Isolation and purification of liver stage

material is almost impossible. The problem was solved by using polyclonal antibody takenfrom a peculiar person who had taken chloroquine every day until 27 years old. The serum
was reactive to liver stage parasite but not to blood stage parasite. Using this serum, an
epitope of liver stage parasite was identified. It still remains to be studied if this epitope

works for protection or the epitope is only an antigenic polypeptide which does not induce
resistance to the liver stage parasite. Extensive studies have been reported on the epitopefrom blood stage parasite. Epitope pl90 and pl55 seem to be promising candidates, however,the peptides need Freunds' complete adjuvant to induce resistance in the experimentalmonkeys. Still, much studies are needed before test to humans. Synthetic hybrid polymers

reported by Pattaroyo et al. (a group in Bogoda) presented a promising result in an52experiment on human volunteers in suppressing development of parasitemia after experimen‑
tal mosquitoe bites. The experiment also supported a new principle in the production ofsynthetic vaccine by combining different effective epitopes into one substance. The resulted

large molecule eventually induced potent immune response in the given individuals, andmulti‑operative effect may be expected. An attempt to prepare highly immunogenic mutantof P. berghei has been worked by us. Such parasite derivative was obtained after giving highdose irradiation to a pool of parasitized erythrocytes. The derived parasite changed thefeature and became permanent low virulence organism which, in addition, caused a long
lasting immunity in groups of mice. Effective molecule from the obtained parasite are now
being studied. A 230 KD molecule which is unique to the derivative parasite was identified.

lb APPROACHES TO THE DEVELOPMENT OF TRYPANOSOME VACClNE
TOSHIHIDE FUKUMA
Department of Protozoology, Institute of Tropical Medicine,

Nagasaki University

The life system of the protozoa is very complicated, though they are unicellular micro‑organisms, because majority of animals of the group consist of a single cell which is refered
to a whole body of an individual. The parasitic protozoa are confined to one or more specifichost and compose a distinct life cycle changing their shape and other characteristics implied

the term "form".
It is necessary to understand the life cycle of the parasite for correct diagnosis, treatment

and prevention of the disease caused by the parasite. On vaccine development, one of animportant measure of prophylaxis, we cannot pay too much attention to life cycle of theparasite in selection of the vaccine materials.

Human trypanosomiases are roughly devided into two categories, African (sleeping
sickness) and American (Chagas' disease) . The former is caused by Tmpanosoma ( T. )brucei gambiense (Tg) and T. ( T. ) b. rhodesiense (Tr) which are transmitted by tsetse fly( Glossina) and injected into the host with saliva at the bite as is called Salivaria. Animal

tryp.anosomiases caused by African trypanosomes and mediated by tsetse, in the name of
Salivaria, are also quite important in the concequence of reduction in food supply when live

stocks and game animals are spoiled. The trypanosome speciese concerned are T. (N. )congolense (Tco). T. (D.) vivax (Tv) and T. (T.) b. brucei (Tb). Tb, Tg and Tr are
relatives included in T. ( T. ) brucei as subspecies.

The latter, Chagas' disease, is caused by T. (S. ) cruzi (Tcr) which is transmitted withfeces of reduviid bugs ( Triat(,ma) as is classified in Stercolaria. By the fact stated above andmany other differences in life cycle specific to subgenus and species, "trypanosomes" are somuch in variety as enable to mention them all together uniformly. In this occasion, African

trypanosomes are mainly discussed because they undergo characteristic antigenic variation.
Salivarian trypanosomes appear to have a simple life in mammalian host because theyexpress only one , form of trypomastigote form or blood form living in extracellular fluid

without ihvasion to host cells. However, trypanosomes avoid host immune response with

53

antigenically variable surface glycoprotein (VSG) which is a single costituent of surface coat

covering the cell. Trypanosome population expressing new VSG replaces former one, which
has been eliminated by host's immune reaction. This replacement occurs cyclically during
infection showing waving parasitaemia. The possible number of VSG expressed is estimatedto be between 100 and 1,000. It can be concluded that it is not practical to prepare polyvalentvaccines against to such a large number of VSG. Cross reactive region of VSG is detected
but is cryptic, hence acquired immunity is only VSG specific. This intricacy made blood form
assumed to be unsuitable for effective source material of vaccine and vaccine development tobe hopeless. Though, remarkable improvement in cultivation of African trypanosomes from1976 in ILRAD by Hirumi and his coworkers raised the possibility of devising a vaccine. The
life cycles of the parasites were completed in vitro. This suggested any stage of the parasites
might be produced in a desired amount, as well as metacyclics, in culture vessels. In addition,it has been reported by others confirmably that blood forms of any antigen type of VSG
ingested by tsetse lose their surface coat, at the end of cyclic change infective form of
metacyclics reacquire the surface coat and at this stage VSG would revert to restricted
metacyclic VSG (M‑VSG). If this is true, protection agaist infection by the vector appears
to be easier. Taking one more reason into account that metacyclic form is the first to be
introduced to mammal hosts in much smaller number at infection, metacyclic form turned to
be promissing as the source material of vaccine when in vitro propagation of metacyclic has
become feasible.

Though the life cycle of human infective Tg and Tr has been completed in vitro, theprogress in studies of these parasites is still behind to animal infective trypanosomes in
production of metacyclics in culture. In vitro propagation of metacyclics in semi‑large scale

has been achieved for Tc at first and recently for Tv and Tb by Hirumi and his members.
Especially in vitro produced metacyclics of Tco were broght to some practical experiments.
Calves infected with metacyclics from culture and treated with trypanocidal drugs resisted tochallenge with infected tsetse and inoculation of sonicated metacyclics from culture withadjuvant manifested protective immunity in goats.
New findings reported since late seventies proposed the reconsideration on the compe‑

tence of metacyclics for the source materials of trypanosome vaccine. Some of themdemonstrated the heterogeneity of VSG which had been supposed to be single referred to a"basic antigen". For example, at least 8 and 12 M‑VSGs were reported for Tr and Tco
respectively. The diversity found even in M‑VSG is critically unfavorable to that strategy
for vaccine. The other findings may make the situation worse because the reports on Tb

cumulatively exhibited possibility of sexual process or genetic exchange between
trypanosomes during cyclical transmission and vast antigenic variation potential.

To overcome the mechanism evolved by the parasite it is necessary to find someantigenically conservative molecules which play a role of important function on the parasite

cell surface and modify them to be immunologically exposed to host.
About Tcr, though antigenic variation is not admitted, trypanosomes change the form
from trypomastigote to amastigote and multiply after invasion to host cells. Trypomastigote
appear in bloodstream by transformation from amastigote. Infection is initiated by invasionof metacyclic trypomastigotes discharged with feces from the vector. Any trypomastigote
dose not undergo cell division. C.ultivation of trypanosomes of this species was successful invery early days compaired to African trypanosomes and stage specific antigens have been

54

detected in some number but effectively protective immunity is not experimentally demon‑
strated yet. One of the most difflcult problem in vaccine development is due to the fact thatthe disease caused by Tcr progresses by some secondary reaction of the host side rather thandirect dammage caused by parasites.

2 VACCINES FOR BACTERIAL DISEASES

2* CHOLERA VACCINE AND TYPHOID VACCINEYOSHIFUMI TAKEDA
Department of Bacterial Infection, The Institute of Medical Science,

The University of Tokyo

Recently, several effective bacterial vaccines have been developed and field trials haveyielded successful results. Those include the cholera vaccine which is a combined vaccine of

B subunit of cholera toxin and heat‑ and formalin‑killed cholera vibrios (LPS) and thetyphoid vaccine which uses a gal E mutant of Salmonella t phi (strain Ty 2la) . Field trials
of Vi polysaccharide vaccine against synthetic peptides derived from the B subunit of cholera

toxin have also proved to be protective antigens and hold promise to be used as vaccines.

2b LEPROSY VACCINE, PRESENT AND FUTUREYASUO MIZUGUCHI
Department of Microbiology, School of Medicine, Universrty

of Occupational and Environmental Health

Mycobacterium leprae is the etiologic agent of leprosy which afflicts 10 to 15 million
people in the world, mainly living in developing countries. Because M. Ieprae has not beensuccessfully cultivated in vitro, biologic and immunologic studies of M. Ieprae has been
severely restricted.

BCG has been widely used for the prevention of tuberculosis and is reported to be also
effective against leprosy to a variety extent depending on the trials. Recent work by Convit
and co‑workers indicate that the injection of a mixture of live BCG and heat‑killed M. Ieprae
induce cell‑mediated immunity to most lepromatous patients and therefore, is effective as an
immunotherapeutic agent. It is also expected that this type of vaccine is more effective than

BCG alone for the immunoprophylaxis of leprosy.
Since, the only available source of the organism is infected tissues of the nine‑banded
armadillo, the supply of the organism will be limited and costly for the large scale vaccine

production.
Using recombinant DNA technology, it now became possible to obtain different classes

55

of protein antigens of M. Ieprae from Escherichia coli.

Several studies have revealed that cell‑mediated immunity which is regulated by specificT‑lymphocyte is required for the protection to leprosy. Although, these protein antigens
were found to react with T‑cells from leprosy patients or healthy volunteers who received
BCG plus heat‑killed M. Ieprae, there will be several di :culties to produce large amount ofprotein antigens from E. coli and to induce protective immunity by injecting them. In E. coli,

it will also be difncult to synthesize complex biological molecule of M. Ieprae which might berequired for the induction of protection. The nature of protective antigen has not been
elucidated in M. Ieprae.

Stable introduction of M. Ieprae DNA into BCG or other cultivable mycobacteria whichhave been known to possess antigenically common molecule with M. Ieprae may be a solutionof such problem. It is expected that genes of M. Ieprae are expressed much more efflciently, in mycobacteria than in E. coli, and in addition, host mycobacteria will work as an adjuvant

for the induction of cell‑mediated immunity. Establishment of hostvector system inmycobacteria thus becomes a major research area in the vaccine development.
Genetic analysis of slowly growing mycobacteria has been extremely difficult since theorganism possesses thick cell wall, grows in clumps and no methods for gene transfer havebeen found.
To develop an host‑vector system in mycobacteria, it is necessary 1) to use restriction‑less organism as a host bacterium, 2) to develop an efficient means of introducing DNA into
mycobacteria, and 3) to construct a vector plasmid or a phage which possess some selectable

markers such as drug resistance within the molecule.

Previously, we reported that a rapidly growing mycobacteria M. smegmatis ATCC607
does not possess restricition system and can be converted to protoplasts which are required

to introduce DNA into the cell by polyethyleneglycol assisted transformation. We also
succeeded to regenerate the protoplasts into original rod shaped bacterial cells. BCG, on theother hand, has some restriction enzymes. Therefore, it is necessary to isolate restriction‑lessmutants from BCG. By mutagenization with nitrosoguanidine, we have succeeded to isolate
mutants which do not restrict phage multiplication. Using the mutants, we are now trying tofind an efflcient method to introduce DNA into BCG.

With regard to the vector‑plasmid, both M. smegmatis and BCG do not possess any
plasmids. Therefore, we looked for the candidate plasmid from other mycobacterial species
and found that some strains of M. fortuitum. M. avium complex and M. gordonae harboredplasmid DNAs. However, we also found it necessary to incorporate drug resistance markers
into these plasmids because all of them were cryptic. We are now trying to incorporate drug
resistance genes of E. coli or Streptomyces plasmids into one of such mycobacterial plasmid.

In addition to the research on molecular genetics, future work on the chemical andantigenic analysis of M. Ieprae in relation to protective immunity, on the mechanism ofunresponsiveness in lepromatous patients, and on the pathogenesis and virulence of M. Ieprae,

will contribute to the control and management of leprosy.

,

56

3 VACCINES FOR VIRAL DISEASES

3a DENGUE VACCINEAKIRA IGARASHI
Department of Virology, Institute of Tropical Medicine,

Nagasaki University

Dengue virus infection has been prevalent in many tropical countries, and its spill totemperate areas and infected cases of visitors to tropical countries have been threatening thepeople in temperate areas also. The public health significance of dengue virus infection ha
been augmented since outbreak of severe manifestation of dengue haemorrahgic fever (DHF)

and increasing nurnber of patients and epidemic areas. Because environmental sanitation
requires economical support and vector control is not always reliable and long lasting, dengue

vaccine development has been required to provide "a magic bullet" to control dengue virus

mfection. Soon after dengue viruses were isolated, attempts have been made to obtainattenuated vaccines by serial passages of the viruses in mouse brains. These "classical"
dengue vaccines, althongh they were quite attenuated and conferred immunity to the recipi‑

ents, could not meet requirements by the Food and Agriculture Department in the US.
Attenuation of dengue viruses in cell culture systems without mouse brain passages was
conducted in the US and Thailand, and the vaccine viruses were characterized by several in
vitro and in vivo markers of attenuation. Type 2 dengue (D2) vaccine developed in the US
was fairly well‑attenuated and showed quite good antibody response in yellow fever (YF) ‑immune recipients, but its lower seroconversion rate and antibody titer in YF‑nonimmune
recipients along with some side reactions made the vaccinve to be regarded not an ideal one.

D2 vaccine developed in Thailand gave quite good response with slight side effects in therecipients both immune or nonimmune to Japanese encephalitis virus, although antibody titerwas higher in the former than the latter. In contrast, type 4 (D4) and type I dengue vaccines
developed by similar methods showed adverse reactions in the recipients, and in vitro or invivo markers of attenuation did not parallel to the human virulence. Because second
infection with different serotypes of dengue virus has been considered as a "risk" factor to

trigger DHF, dengue vaccine should be developed for all 4 serotypes to protect the disease.

Even when all 4 types of dengue viruses have been attenuated, they could possibly interfere
each other in the recipients.

In order to overcome these problems, development of the second generation dengue
vaccine has been planned by the WHO. The strategy consists of the following objectives. (1)ldentification of protective epitopes, especially on the virion envelope glycoprotein (E) andthe nonstructural protein (NS1). (2) Expression of these epitopes by recombinant DNA
technologies in prokaryotic and eukaryotic systems to examine their immunogenicities. (3)ldentification of virulent factors in viral genes by comparative nucleotide sequencing ofattenuated and their parental strains as well as many strains. (4) Development of geneticallyengineered attenuated vaccine by constructing infectious complementary DNAS of YF anddengue viruses and their genetical manipulation.

57

Recently, a study group at the National Institute of Health in the US reported successful

expression of D4 virus E and NSI genes by recombinant vaccinia and baculovirus systems.
They showed that the mice immunized with the extracts from mammalian or insect cells
infected with the recombinant viruses were protective against lethal intracerebral challengewith D4 virus. These findings are very promising to develop the second generation dengue
vaccine. But the gene products should be purified in large amounts for use as "vaccine" inthe future. Even when these problems are overcome, the possibility remains that the im‑munogene (s) might induce "enhancing antibodies" which could trigger DHF in the second
infection. This problem will be avoided by giving high levels of protective immunity to all

4 types of dengue viruses by massive amount of immunization, or by identifying some
protective epitopes which do not induce enhancing antibodies, or other epitope (s) whichcross‑protect all 4 types of dengue viruses.

3b EXPERIENCE WITH FIELD TRIALS OF RHESUS ROTAVIRUS
VACCINE MMU‑18006
SHUNZO CHIBA
Department of Pediatrics, Sapporo Medical College

Rotaviruses have been implicated as the major cause of viral gastroenteritis in infants

and young children under 2 years of age worldwide. Although deaths are not usuallyassociated with rotavirus gastroenteritis in developed countries, the severity and highmortality of this disease seems to be a serious problem in less developed countries according

to numerous studies in various nations. Therefore, the development of an effective vaccine
for infants is highly desirable. In this view, the WHO Diarrheal Disease Control Program has

been promoting the development of the vaccine.

Several candidate vaccines derived from human and animal rotaviruses have beendeveloped and tested for safety and immunogenicity as well as its efiicacy. Rhesus rotavirus
strain MMU‑18006 is one of those candidates. Several cross‑sectional studies of this vaccine
have been performed in order to evaluate its efiicacy and safety for infants and children.

The placebo‑controlled study of the rhesus rotavirus (RRV) vaccine MMU‑18006 strain
was conducted at an orphanage in Saporro in an attempt to observe its transmissibility an.d
immunogenicity. Twenty‑four infants under 15 months of age, Iiving in the same room, weredivided into two groups at random. Thirteen infants were given I ml of 1:100 diluted vaccine(containing approximately 104 PFU vaccine virus) while 11 infants received placebo prepara‑tions as controls. There was no significant difference between vaccinees and controls inclinical observations postvaccination.
Fecal specimens were collected daily from all infants of study groups for 3 weeks, andwere examined for viral shedding by rotary culture method of MA‑104 cells. Nine of 12
vaccinees who could be tested shed virus in their feces but none of controls was positive forviral shedding. Therefore no evidence was available to indicate the transmissibility of thevaccine among study infants.
Nine of the 13 vaccinees had a four‑fold or greater rise in neutralizing antibody response

58

against vaccine strain, and acquired the protective level of antibody titer (1 : 128) . Seven of

those seroresponders to RRV also showed remarkable seroresponses against serotype 3 ofhuman rotavirus (HRV) . However, only 2 vaccinees showed concomitant antibody responses
against serotype 2 of HRV. Additionally, acquired neutralizing antibodies gradually de‑creased within a year following vaccination.
Our results indicate that a booster dose of this vaccine would be necessary for preventionof rotavirus gastroenteritis during first 2 years of life. To cover 4 serotypes of HRVS a
polyvalent vaccine would be necessary to be developed.

59

Panel Discussion

Introspective and Prospective View of

Cooperative Medical Projects
Experienced in Tropical Countries

1 STATUS OF RURAL HEALTH AND MEDICAL CARES= HUMAN LIFE

AND NATURALENVIRONMENT IN SARAWAK, EAST MALAYSIAICHIRO MIYAGILaboratory of Medical Zoology, School of Health Sciences andResearch Center of Comprehensive Medicine, University of the Ryukyus

Borneo is the third largest island in the world and includes three different countries,

Malaysia (Sarawak and Saba States) , Brunei and Indonesia (Kalimantan) . The area ofSarawak is 124,449 km2 and 7 administractive divisions. According to the 1980 census figures

the total population of Sarawak is 1,250,000 and about 80% of them live in rural areas. On
the ethnogeographical grounds the indigenous people in the state could be divided into thecoastal groups (Malay, Melanau, Kedayan and Belait) , the lowland group (Iban, Kenyah,
Baketan, Kayan, etc.) and the upland group (Kelabit, Punan and Murut) . They usually live
in different types of long houses along the river banks. About 90% of the area is covered withtropical rain forests and there are many rivers, and most of them originated along the borderwith Kalimantan. The rivers with their innumerable tributaries form an extensive networkof waterways which are utilised for travel in the rural areas by large express (long boat) andsmall boats. Due to lack of motorable roads, overland movement is difiicult in outlying areas.

This is a major cause for the difflculty of providing medical services in the rural areas.

Prior to the second Malaysia plan period (1971‑1975) rural health services in the stateof Sarawak were provided through a chain of static dispensaries, sub‑dispensaries, travelling

dispensaries and maternal and child health clinics. The maternal and child health clinics
were built and provided by the local councils while the dispensaries and sub‑dispensaries
were administered by the medical department in Kuching. In the second and third Malaysia
plans, a new integrated system of promotive, curative, preventive health education andrehabilitative services were introduced through the establishment of Pusat Kesihatan andKlinik Desa throughout the state. A Klinik Desa serves a population of 1,500 to 2,500 covering

an area of 3‑7 miles radius and has the following staffing pattern: One hospital assistant,junior hospital assistant and rural health supervisor, and two public health nurses andattendants, one rural health supervisor. One vehicle driver and outboat driver are included.
Pusat Kesihatan is next higher unite with 3 Klinik Desa under it. It has a function providing
care for a service area of 3,000 to 25,000 population and supervising the 3 Klinik Desas under

it. Provision was made to put a medical officer into serve this centre but due to a shortageof medical doctors, it has been temporarily suspended. It consists of the following staff: one
senior hospital assistant, dental nurse, health inspector, public health nurse and junior labora‑

60

tory technician. Top priorities in rural health services were given to the expansion of rural
services to the unserved areas, static dispensaries, subdispensaries and isolated maternal andchild health clinics may be up graded into Pusat Kesihatan. Flying doctor seryices are also
operated for the serious patients in remote locations. Besides the medical care, preventive,

promotive, health education and emergency evacuation, the flying doctor services also serves
the department in strengthening the supervision of the peripheral staff, the distribution ofdrugs, vaccines and other medical supplies including facilitating diseases control activities.
In 1983, there are 8 Pusat Kesihatans, 71 Klinik Desas, 19 static dispensaries, 55 sub‑dispen‑saries, 87 village health teams and 21 travelling dispensaries in the 7 divisions of the state andthey are well functioning as an important rural medical and health service system.
A Iook at the incidence of communicable diseases in 1983 shows that gonococcal infection
is at the top with 2,572 cases. Measles and tuberculosis come next with 2,305 and 1,678 cases
respectively. Malaria (803 cases) , chicken pox (701), food poisoning (561), typhoid (337),
mumps (286) , infectious hepatitis (262) and dengue fever (171) completed the list of the top

10 communicable diseases.
I am indebted to Dr. Stalin Hardin, Director and Mr. Chang Moh Seng, State Medical
entomologist of Medical and Health Department, Kuching Sarawak, for their warm‑hearted
understanding for carrying out field research in Sarawak.

2 CENTRAL AND SOUTH AMERICAS‑CULTIVATED BETWEENWILD NATURE AND IBERIAN CIVILIZATIONISAO TADA
Department of Parasitic Diseases,

Kumamoto University Medical School

While reflecting my own experiences of investigations and technical cooperation projects

in the past in Central and South Americas, it became necessary to cast critical eyes on our

way of approach and mode of behavior in there to promote and encourage future similar
projects. From this view, I feel it primarily important to understand the culture, people andnature of these areas. In this presentation, I wotild like to introduce roughly the characteris‑tic features of Central and South American societies.

A vast territory between Guatemala, Central America, in the north and Argentine/Chile,
South America, in the south is included in this geographic category which is characterized by
the diversity of the climatic zones and topography. The residents inhabiting there also show

diversity in races and civilizations. Indigenous residents of this continent are mongoloids
which are now distributing all over the American continents particularly in the mountainousand silvatic regions. In the Venezuela/Brazil border area, for exapmle, Yanomami or
Yanomarna tribes are anthropologically well known and are residing there isolated complete‑ly from modern civilization.

The most remarkable feature of the society in this continent has been formed and held

since the era of the lberian CONQUISTADORES (conquerers) . At the present, social andpolitical uneasiness and economic instability are the common features, despite their rich

61natural resources, Iands and economic potentials. It may be considered that such problems
essentiaily derived from the traditional social structure and civilization cultivated since 16

century in this continent. Every country consists of a minority of rich caucasoides (Spanish

majority) and a majority of poor meztizos (mixed) and mongoloides/negroides. In a recent
issue of National Geographic Magazine (June, 1988) , you will read "Guatemala, a fragile
democracy" which aimed at the analysis of fragile political‑democratic‑situation seen in thissmall country standing economically on the conservative FlNCA (plantation) system of the
agriculture. Oligo‑ or mono‑culture in the agricultural industry is thus the marked featureof this continent except some industrialized countries. Higher prevalence of analphabetismand the presence of INDIGENAS (American indians) are another problems for many
countries from the viewpoints of economy, public health, and industry of the nation. Actually,

Latin America is a big mixture of wild nature and civilizations of Europe particularly of
lberia and Africa. The characteristic feature of Latin American culture is a marked individu‑
alism which derived from its history and social system. The core of the culture consists ofcatholicism, self assertion and loyalty to reliable family/friend members. Usually solidarity

with the organization or community is not so strong. They are essentially epicurean andconsider their profession as a tool in the "Corta vida" (short life) . The important concerns
in the life are honor, profit, comfort, Iove and so on.

In order to have efficient relationship with Latin Americans and its communities which
are certainly different from us, rhetoric in the communication is very important. The regions
are the so called low‑context society. Linguistically, Japanese has no similar rhetoric in themode of communication seen in Spanish and historically we passed without similar custom.
Thus, in order to communicate efiiciently, we should pay special attention on this point.
Secondly, we have to pay appropriate respect to the honor of our cbunterparts frequently andshould not criticize carelessly. Because generally they have been grown differently from thediscipline of Japanese type. Thirdly, we should pay attention to the importance of sophistica‑

tion whenever we intended to have intimate communication to each other. A plain "techni‑
cian" is not a respectable person in Latin American civilization. On the contrary, there are
frequently similar sentiment between Latin Americans and Japanese, and both of us frequent‑
ly give similar evaluation for various matters. For example, "machismo" is rather notorious
in there, while its essence is quite similar to "bushi‑do" in the definition. Machismo does not
simply imply the shortage of respect to woman, but the discipline of man. Thus, they are far

sympathetic than Anglo‑Saxons for Japanese. Further, generally speaking, Latin Americans
are with less racial prejudice. So, standing on the recognition that Japanese people have beenrather peculiar in the history and culture in the world, we will be able to maintain favorable

relation with Central and South Americas. For the present, Hispanic culture is gradually
spreading into all over the North America (United States and Canada) . For this reason, fromthe strategic viewpoint of the state, cooperations in any fields with Latin America will be
more important. Any cooperative projects in research or operation in the future may thus
require further understanding of this region from sociological and historical views.

62

3 GEOGRAPHICAL PATHOLOGY AND MEDICAL ANTHROPOLOGYIN AFRICA

HIDEYO ITAKURA
Department of Pathology, Institute of Tropical Medicine,

Nagasaki University

I . Some problems of scientific methods in field work

In the modern medicine molecular biology has developed very rapidly, and has beenemphasized as an important science in medical field. However, one must be aware thatmacroscopic scientific methods such as epidemiology and geopathology have taken a great
deal of roles in medical history. Of almost all the morbid hazard, humankind have been able
to get a thread of resolution of the problem by macroscopic scientific methods.

According to our basic philosophy, thinking way and results of our research work,
evolutions and manifestations of human diseases are quite frequently and strongly affected byexternal factors. Some of the factors are as follows: 1. geographical distribution andhistorical migration of the diseases, 2. virulence of parasites or disease agents, 3. transmission

routes of disease agents, 4. host constitutional factors on disease evolutions and manifesta‑

tions, 5. host‑parasite relationships, 6. natural environment of the life and environmental

physiology, and 7. human ecology (socio‑economical factors, Iife history, manners andcustoms, food habits, agricultural and pasturage systems and methods, etc.) .

We are conducting medical investigation on Kaposi's sarcoma and retrovirus relateddiseases in Africa. For pathological investigation into the diseases we have to get informa‑tion about geographical distribution and incidence, ethnical distribution, sex distribution, age

distribution, correlation with other diseases, natural environment including climate, andanimal and plant ecology, and human ecology. The problem is that one cannot get accurate
demography of the areas where one would plan to examine. Occasionally one can get only
an estimation of the population. One of the methods we are taking at the moment is counting
the incidence of the diseases among the surgical pathology materials at the hospitals. It isvery difficult to bet the real population. Therefore, we have to take the methods of
epidemiology and even human and animal ecology.
Some of the possible methods to use in field work are as follows:
1 ) An air photograph of the resident areas2 ) Following up the each case3 ) Observation of the disease not only we investigate but also the other4 ) Making a field inspection on foot by ourselves5 ) Operational research

6 ) Both field work and laboratory work7 ) Cooperational work with investigators in other research field
II . Contact with inhabitant poeple

There are two aspects of the scientific methods in the field of cultural anthropology;"emrc" and "etrc". The former means "the point of view" of the inhabitant people, and thelatter means "the point of view" of the researchers. The purpose of investigation researchin the field work should be how to translate "emic" into "etic". In 1960s, medical anthropol‑

ogy has been developing in the United States. And that was the new academic attempt which

63

they investigate medical systems of native people on the side of their "emic". Tropical
medicine is historically a part of western medicine itself. However, on the aspects oftreatment and therapy, tropical medicine may be their endeavor to combine two medical
systems both "emrc" and "etic" together. I think medical anthropology should not be only
treatment or health care of the inhabitants in the tropics but also "medical science" itself.

One of the another important points is how to contact with the native researchers ascounterparts in the tropics. We must have continuously collaboration work with theresearchers there. One of the best ways is to make native researchers be principal investiga‑
tors of the some research groups of our proposed projects or the first author of the papers of

the work.
Finally, it is unable to neglect the political problems of the world. It is inevitable toconsider about the international problems when you perform some projects internationally.
We must always be interested in moral of medical researchers as well as human history andsociety, and current topics of the world.

4 BACTERIOLOGY PROJECT IN KENYA MEDICAL RESEARCHINSTITUTE‑PRESENT STATUS AND PROSPECTIVE VIEW‑
ATSUSHI OZAWA
Department of Microbiology, School of Medicine

Tokai University

The most outstanding of the facilities built through external assistance in the KEMRI
Headquarters and Central Laboratories complex which was constructed through a grant aid

from the Japanese Government to the Kenya Government.
In considering the pathogenesis, prevention and treatment of bacterial diarrhea from theecological standpoint of infectious diseases, conducting an analysis of bacterial species
comprising the intestinal flora should be emphasized as a fundamentally critical objective.
Through the implementation of KEMRI/JICA technical cooperation project, it would beemphasized that good correlations between Japanese experts and their KEMRI counterparts

should be maintained. Good communication oriented direction between them is one ofimportant factors to obtain the fruitful result in KEMRI/JICA project. The communication
is a two way process and there should be mutual encouragement and effort on either side tointeract more with each other.
The success of international exchange depends on understanding different culture andhistory between us from the standpoint of common way of thinking.

64

5 JICA TECHNICAL COOPERATION PROJECT AT RESEARCHINSTITUTE FOR TROPICAL MEDICINE,
PHILIPPINES AND ITS PROSPECT
YOSHlNORI KANEKO
Emeritus Professor of Toho University

Research Institute for Tropical Medicine, Philippines, is one of the Grant‑in‑Aid Projectof JICA, which was completed in 1981 and followed by Technical Cooperation Project for 7
and half years.

The subjects for Technical Cooperation were acute respiratory infection including
immunization, diarrhoea, and others. The development of virus laboratory technology was
the most important one. The Institute had additional aids of Animal Experiment Laboratory
in 1984 and two buildings for training and residence for trainees and visiting staffs in 1988.

The annual budget for technical cooperation was 50 Million Yen for 7 years. At the time oftermination of technical cooperation project the Institute had the total of 370 staffs, theannual budget of 20 Million Peso from the Philippine Government, and getting the total ofaround 70,000 US‑dollars equivalent for research from WHO, United States, Canada, Aus‑
tralia and others.

The Institute has Departments of Microbiology, Medical Entomology, Immunology,
Epidemiology, Pathology, Biochemistry, and Clinical Research Department including 50 beds,

and Animal Experiment Laboratory.
The total of 28 JICA experts including experts for virus (7), operation of laboratory
equipments (4), Medicine (3), Pediatrics (3), Experimental animal (3) Bacteriology (2),Medical Entomology (2) , Tuberculosis (1), and others (3). Some of them cooperated two or
three times. On the other hand the total of 22 Philippine‑counterparts were sent to Japan forstudy.

Many of the senior staffs of the Institute had been trained abroad and able. However,
because of the shortage of the national budget, the corroborative study, information foraction in other words, was not enough.
The patients at the clinical research department were most important for research. The

total of 70% of the patients were pediatric age and almost 100% of the In‑patient were
infectious nature. Only 6% of the patients were able to pay for medical care.
The total of 548 deaths, 19.5%, among 2,808 In‑patients for the period of 1984‑1986 wasrecorded and 71.6% of the death among In‑patients occurred within 3 days of hospitalization.

The laboratory technology on bacteria was already developed and it was confirmed theratio of diarrhoea by plural infections to the total was 17% and very high in rainy season,suggesting the contamination of well water in the community.
In relation to EPI, the effort was concentrated on Polio, Measles, and DPT. It was
confirmed that the Schick negative rate by age strongly suggested the implementation of EPI
was not enough in depressed area of Metro Manila, suggesting the difficult situation due tourbanization. On polio the sero‑positive‑conversion rate by immunization was more than
80% and HI antibody positive rate against measles of the infant aged 6‑11 months was 36.8%
and 88.9% at the age of 4 years.

65

Some problems on the administrative prOcess of the technical cooperation were almost
the same as stated in the abstract of Dr. Ozawa. However, the author is convinced theInstitute would serve and develop as an information center for communicable disease control
in Philippines.

6 PRESENT STATE AND SOME PROBLEMS ON THE BASIC STUDY OF
INFECTIOUS DISEASES BY INTERNATIONAL MEDICAL FOUNDATION
OF JAPAN
YOHICHI ITO
Department of Parasitology, Kitasato University School of Medicine

The basic study of infectious diseases have been conducted once in a year since 1980. The
objectives of the study are to gain an understanding of the present state and the recent trendsof infectious diseases in developing countries and to conduct studies and analysis of preven‑tive measures, diagnosis and treatment of the diseases. This would lead to the formulationof the most vital projects in the fields of health and medical care, and would provide basic
data and specific recomendation. Diseases to be covered by the study are bacterial, viral,

protozoal and helmintic diseases. Following major informations are covered:
1 . Documents related to the health statistics.

2 . Factors influencing the outbreak of diseases and recent trends.

3 . Present organization of the administration for health and medical care (central andlocaD related to the control of infectious diseases.
4 . Legislative regulations for the control of infectious diseases.

5 . Control measures for infectious diseases (prevention, diagnosis and treatment, including
health and sanitary education) .

6 . Present state of strategies to improve environmental sanitation.
The team consists of four experts specializing in the fields of microbiology and virology,

parasitology, public health and health administration, and clinical medicine. Their tasks are
as follows:

1 . Microbiology, virology and parasitology; (a) Pathogenic factors (distribution, drug
resistance etc.), vector animals and insects. (b) Operational functions, physical set up

and arrangements of laboratories (including the standarization of examination methods)(c) National‑wide supply and quality control of vaccines. (d) Determinations of a future

direction towards improvements in technology and system.
2 . Public health (investigation of principal measure for controlling infectious diseases) ; (a)Preventive measures including food hygiene and management. (b) Information system(information obtained from individuals, groups, and laboratory personnel) . (c) Human

resources, training, and arrangement for assignment. (d) Vaccinations and their legal
provisions. (e) Meteorological factors, geographical factors, water supply, problems on
sewage disposal, and nutritional status of inhabitants.
3 . Present status concerning diagnosis and treatment of infectious diseases; (a) Lethality.
(b) Methods of diagnosis. (c) Methods of treatment. (d) Clinical facilities. (e) Current

66

state of medical equipment.
4 . Administrative system and its functions; (a) Information system and its functions. (b)Functions and location of clinical and laboratory facilities. (c) Human resource andtraining of health manpower.
The study have been carried out on 8 countries (Thailand 1980, Philippines 1981. In‑
donesia 1982. Bangladesh 1983, Sri Lanka 1984, Paraguay 1985, Nepal 1986, Ghana 1987, andPakistan 1988) . On the basis of our experiences I will discuss some problems.

7 MEDICAL COOPERATION ACTIVITIES CONDUCTED BY JAPANINTERNATIONAL COOPERATION AGENCY (JICA)
TAKEFUMI KONDO
Director, Medical Cooperation Department, JICA

JICA is a government agency operating under the jurisdiction of the Ministry of Foreign
Affairs. It was established in 1974 under the provisions of the Japan International Coopera‑tion Agency Law. JICA implements programs for the purpose of contributing to the eco‑nomic and social progress of the developing world. These programs cover a very wide range,

centered around government‑based technical cooperation.
Technical cooperation, one of the main activities of JICA, is aimed at helping developing

countries to develop human resources capable of playing an important role in their nationbuilding. Technical cooperation by JICA in the field of health, medicine, family planning andpopulation consists of training program, expert dispatch program, equipment supply pro‑gram. The project‑type technical cooperation is an integrated technical cooperation pro‑gram in which the three abovementioned aspects of technical cooperation are combined. This
project‑type technical cooperation is the core of the medical cooperations. In this field JICA

has 29 project‑type technical cooperation programs and 6 new projects will start this year.
Of these programs Asian countries' share is the largest (18 projects) .
JICA's other activities include a grand aid program to extend financial assistance for theconstruction of various facilities (usually as an integnal part of technical cooperation
project) and Japan overseas cooperation volunteers program (JOCV) . These two programs

cover medical cooperation. JICA has overseas emergency relief system which includes
medical relief. When a disaster takes place in a developing country, this system is expected

to be arranged immedeately upon the request from the disaster hit country.
Further expantion and change in quality in these programs are necessary in order toimprove health status in developing countries.

67

8 PROSPECTIVE ROLE OF'JAPAN IN INTERNATIONALHEALTH, A VIEW OF A WHO STAFFAKIRA SHIMOUCHI
Medical Officer in Acute Respiratory Infections, World
Health Organization, Regional Office for the Western Pacific

Manila, the Philippmes

1 . Introduction
World Health Organization (WHO) was founded 40 years ago as the first United Nations
special agency for health and has receive high requtation for its remarkable achievement, themost outstanding example is small pox eradication. The following presentation is mypersonal view on the above subject based on the experience as staff of WHO and visiting
more than 10 developing countries a year.
2 . Current Trend of International Health Activities
In the past there were many researchers and health staff engaged in tropical medicine
and public health problems in Japan such as leprosy, tuberculosis and various parasitic
diseases including schistosomiasis, filariasis and helminthiasis. They had contributed for
improvement of health status in Japan and for progress of medical science in the world. The
reason that many medical professionals worked for tropical medicine and public health was
simply those communicable diseases and parasitic diseases were endemic then in Japan.

However as those problems have become less and less important, interests in these
problems among medical professionals have also gradually been lost.
On the other hand, magnitude of health problems in developing countries remains high.
And because of development of close relation among countries in the world, expressed andfelt need of international cooperation including health field has rapidly increased. Conse‑

quently Japanese Governmental budget for overseas development aid is increasing every year
and many bilateral health projects are undertaken involving substantial number of medical
prof essionals.

3 . Appropriate Technology and Effective Programme Implementation
It can be said that when the technology was scientifically sound for example effective

vaccine or antimicrobials, and programme was feasible, such public health programme
succeeded. Therefore both components of the programme, i.e. appropriate technology andfeasibility or operationability should always be checked and studied before launching any

public health programme.
Even after starting theoritically feasible programme, many operational problems would
rise. In such cases as a clinician makes an effort to improve diagnosis and treatment of apatient, so does public health manager the health programme through giving insight tovarious aspects of problems and daily activities. To do this, he should regularly monitor
indicators of activities and outcome of health services and analyze them.

Thus it is obvious that the long term and multi‑aspects follow up of international
cooperation programme is very important to permit them to evaluate process and ultimate
outcome considering local situations. And such activities require a group of various exper‑
tise .

68

4 . Prospective Role of Japan in Intemational Health
There is no doubt about importance of international cooperation in health either fromhumanistic point of view or as one of the ideal types of contribution to the world as Japan

must seek for peace as cause given by Constitution. Therefore, Japan should further
strengthen international health activities.

However in this circumstances, how are those experiences in individual projects sharedwith each other and reviewed so that more efiicient and effective projects will be planned andimplemented? Furthermore does this situation attract more young professionals to tropical
medicine or public health? If not, some actions should be taken to change the climate.
5 . Possible Approach to Strengthen International Health Activities
In order to meet the increasing need of international cooperation in quantity and quality
in health field, the following three points should be considered; (1) organization of coordina‑tion committee at national level, (2) formulation of national policy and priorities for interna‑

tional health, and (3) establishment of the system for human resource development in the fieldof international health.

In order to build up body of medical professionals in international health, the following

system should be instituted; (a) to create scholarship programme for training in tropical
medicine/international health in other developed or developing countries, (b) to increase
research grant in this field, (c) to set up research collaboration scheme with research
institutions in developing countries.

In such a way to secure training opportunities we can encourage young medical profes‑
sionals to join and stay in this field.

Lastly WHO is collaborating with many bilateral agencies in the world including Japan
International Cooperation Agency (JICA). We expect this type of collaboration will become

more important and meaningful in the future. Besides as immediate benefit WHO can serve
as source of information including country situation, reference of technical discussions andvarious practical advice based on actual programme implementation. Therefore either inindividual cases or official mission, WHO service is ready for utilization.

69

General presentation

1 FOREIGN PATIENTS WITH TUBERCULOSIS FROM TROPICALCOUNTRIES IN TOKYO NATIONAL CHEST HOSPITALHARUMI SHISHlno, HIDEKI NAGAI, ATSUYUKI KURASHIMA,ASTUHISA TAMURA AND TEI KUDO
Department of Respiratory Diseases, Tokyo National Chest Hospital

Approximately 290 patients with tuberculosis were admitted yearly to Tokyo National
Chest Hospital. Recently, foreign patients with tuberculosis from tropical countries, espe‑
cially from Asia, have been increasing, and therefore we have discussed their clinical andsocial problems.
From January 1980 to September 1988, 8 foreign patients with tuberculosis from tropicalcountries were admitted with a tendency to increase, although the number of inpatients withtuberculosis in Tokyo National Chest Hospital were decreasing gradually. Four patientswere from the Philippines, one from Burma, one from Indonesia and one from Algeria, anda Israeli patient traveled in India and Tibet. Duration between entering Japan and hospital‑
ization was less than I year in 5 out of 8 patients. The age of these foreign patients raged
frorn 18 to 29, with the average of 23.9, the average age of all the tuberculous inpatients being

49.0. All of 8 patients had subjective symptoms, which were cough, bloody sputum, fever, etc.
Sputum smears demonstrated positive acid‑fast bacilli in 7 out of 8 patients. All the isolatesof Mycobacterium tuberculosis revealed no drug resistance. Antituberculous chemotherapy
was effective in all the patients. All of them showed to have cavities on chest X‑ray films.
A female Philippine patient's sibilings and a parent were also infected with M. tuberculosis.

Most patients had past histories of tuberculosis and/or family histories of tuberculosis.
Tuberculosis is a very important infection in their home countries.

Immigrants and students from tropical countries have been increasing rapidly andtuberculosis prevails in these countries. Therefore, foreign tuberculous patients immigrated

from tropical countries to Japan should be taken account of prevalence of tuberculosis inJapan. International cooperation between a tropical country and Japan would be importantmeasures to improve in tuberculosis control in both countries.

2 STUDY ON BACTERIAL RESPIRATORY INFECTIONSIN CHAING MAI, THAILAND

ATSUSHI TAKAHASHI, NAOTO RIKITOMI, TOSHIAKI YOSHIDA,KAZUNORI OISHI AND KEIZO MATSUMOTO
Department of Internal Medicine, Institute of Tropical Medicine,

Nagasaki University

70

Respiratory infections are common diseases not only in tropical area but in temperateareas. And causative bacteria show different phases according to environmental changes
including climates, antibiotics, socio‑economic conditions. In the present work, the causative

bacteria for respiratory infections in Chiang Mai area (Thailand) were investigated tocompare them with those in Nagasaki (Japan) . To recognize the difference of causative
bacteria and their antibiotic susceptibilities between these two countries will be useful tomanage respiratory infections.

Methods:
Place: Chaing Mai Hospital, Thailand
Sample: Sputa from patients with respiratory infections
Technique: Quantitative culture method by using serial ten fold dilutions of the sputum

Period: From December 8, 1987 to January 7, 1988
Antibiotic Susceptibility: Agar dilution method
Results:

Ninety one specimens were obtained from 90 patients with respiratory symptoms in
Chaing Mai University Hospital. Of 90 patients, causative bacteria were demonstrated in 37
patients.

Eight strains of H. influenzae, 6 strains of S. pneumoniae, 5 strains of B. catarrhalis andKlebsiella sp., 4 strains of S. aureus and p‑streptococcus, 3 strains of Pseudomonas sp. and 1
strain of S. enteritidis w. ere isolated more than 107 cfu/ml. Susceptibility of those clinicalisolates was examined. All of S. pneumoniae showed good sensitivity for ampicillin. Fourout of 5 strains of B. catarrhalis produced p‑lactamase. Two of 8 strains of H. influenzae
demonstrated P‑1actamase. High resistance to ampicillin was found in two strains which wasresistant to causative organisms
1 ) Main pathogenic bacteria of bronchopulmonary infections in Chaing Mai area are alittle different from those in Japan. We found B. catarrhalis. H. influenzae, and S. pneumoniae

to be common pathogens in Chaing Mai area as well as Japan. However K. pneumoniae wasmore frequently isolated in Chaing Mai area.
2 ) Unexpectedly S. enteritidis was isolated as causative agent of lung abscess in acompromised host.
(2) Antibiotic susceptibilities

1 ) All the isolates of S. pneumoniae were sensitive to ampicillin.

2 ) The rate of p‑lactamase producing strains of B. catarrhalis isolated in Chaing Mai

area is approximately as same as Japan. However they were rather sensitive to p‑lactam
antibiotics.

3 ) None of the H. influenzae isolates produced p‑lactamase. Of the eight strains, twoshowed resistance to ampicillin while ampicillin resistant H. influenzae in Janan was mostly

associated with p‑lactamase.

71

3 THE COMPARISON OF DRUG‑SUSCEPTIBILITY OF HAEMOPHILUS

INFLUENZAE AND STREPTOCOCCUS PNEUMONIAE BETWEEN
THE PHIL̲TppINES AND JAPANKAZUHIRO TATEDAl, KAZUNORI ToMONOl, CHIKAKO MOCHIDA1, KAZUYUKI SUGAHARAl,
KEIZO YAMAGUCHI*, TOSHIAKI USUl*. TOSHIAKI HAYASH12, SHIGERU KoHN02,
MASAKI HIROTA2, KOHEI HARA2, L. T. SOMBRE03, I. P. LIVEL03,
M. C. SANIEL3, YoSHINORI KANEK03 AND T. E. TUPASI'
Department of Laboratory Medicine, Nagasaki University School of Medicine*,
2nd Department of Internal Medicine, Nagasaki University School of Medicine',
Research Institute for Tropical Medicine, Manila, The Philippines' and

Makati Medical Center, Manila, The Philippines'

H. influenzae and S. pneumoniae are important pathogenic organisms causing respiratory
tract infections and septicemia in the Philippines as well as in Japan. Recently, the appear‑
ance of drug‑resistant strains of H. influenzae and S. pneumoniae, especially to ampicillin(ABPO , are becoming a therapeutic problem in some of the countries. For this reason, we
have performed drug‑susceptibility tests for these two species isolated in the Philippines andJapan.
The strains used for MICs measurement were H. influenzae 98 strains and S. pneumoniae
59 strains provided by Research Institute for Tropical Medicine in the Philippines and H.
influenzae 356 strains and S. pneumoniae 179 strains isolated in Japan. Micro dilution brothmethod with inoculum size of 2 x 105 CFU/ml was used for MICs measurement. The drugs

used were ABPC, CEZ, CTM, CZX, OFLX, EM, MINO. p‑1actamase production of H.
influenzae was detected by the nitorocefin solution method.
Results and discussion are as follows;
H. influenzae:(1) Two of 98 strains (2.0%) in the Philippines and 63 of 356 strains (17.6%) in Japan wereP‑lactamase positive and those MICs to ABPC were all over 1.56 mcg/ml.
(2) Five of 356 strains (1.4%) isolated in Japan were resistant to ABPC (MICs>1.56 mcglml) with resistant mechanisms other than p‑lactamase production but none of H.
influenzae isolated in the Philippines were resistant to ABPC with this mechanism.
(3) Among other fi‑lactam antibiotics used in this report, CZX was the most potent and thegrowth of most H. influenzae in both countries were inhibited at < 0.025 mcg/ml of thisdrug.

S. pneumoniae:(1) Among antibiotics used for S. pneumoniae, ABPC was the most potent and there wereno resistant strains in both countries, those MICs were all below 0.2 mcg/ml.
(2) Thirty four (19.0%) and 108 (60.3%) of 179 strains isolated in Japan were resistant to

EM and MlNO respectively, those MICs were over 1.56 mcg/ml, but there were no
resistant strains isolated in the Philippines.

We have reported that there were few resistant strains isolated in the Philippines,
especially to p‑lactam antibiotics in H. influenzae and to EM, MINO in S. pneumoniae.
Recently some of the papers reported the appearance of ABPC resistant strains of H.

72

influenzae without any P‑lactamase production. This resistant mechanism was considered,in part, due to the changes of PBPS of this strain. Considering the increase in the use ofvarious kinds of antibiotics in the Philippines as well as in Japan, we should pay carefulattention to the appearance and increase of resistant strains in these two organisms.

4 ISOLATION OF LEGIONELLA SPECIES FROM THE ENVIRONMENTAL
SAMPLES IN THE PHILIPPlNESKAZUNORI TOMONO*, KAZUHIRO TATEDAl, KEIZO YAMAGUCHll, TOSHIAKI UsUl*
TOSHIAKI HAYASH12, SHIGERU KOHN02, MASAKI HIROTA2, KOHEI HARA'AKIHIRO HAMAMOT03. I. P. LIVEL04, O. T. MoNzoN', M. C. SANIEL'

AND YoSHINORI KANEK04
Department of Laboratory Medicine, Nagasaki University School of
Medicine*, 2nd Department of Internal Medicine, Nagasaki University

School of Medicine', Nagasaki Municipal Hospital' and ResearchInstitute for Tropical Medicine, Manila, The Philippines'

Legionella infections have been recognized as one of the important cause of pneumoniae

among immunosuppressed patients, and it was appeared that Legionella spp. were widelydistributed in environment.
October 1987, we investigated distribution of Legionella spp. in environment in Metro

Manila, the Philippines, where there had been no reports about isolation of Legionellaorganisms either from environment or clinical specimens.
We tried to isolate Legionella spp. from 9 samples of cooling tower water and I sampleof water supplying tower water from 2 hotels and 4 hospitals.

Samples for isolation were inoculated to intraperitoneum of guinea pigs, and onto

selective medium (MWY or WYO agar) after low pH (pH2.2, 0.2 M, HCl‑KCD or heat
treatment (50'C, 20 min) . Only 2 of 9 samples were positive in low pH treatment, on the other

hand 5 of 9 samples and 6 of 10 samples were positive in heat treatment and guinea pig
inoculation method, respectively. Out of 6 strains of Legionella spp., 4 strains of which
belonged L.' pneumophila serogroup 1, one L. pneumophila serogroup 3, and one unidentified.

Legionella organisms can grow even in tap water between 25'C and 42'C, and clearlytemperature is one of the important amplification factors for the growth of Legionellaorganisms. In the Philippines, the average temperature for through the year is 27'C which
may be optimal temperature for their growth. So it is likely that Legionella organisms are
more frequently isolated from environmental samples in the Philippines than in Japan.
Additionally from the fact that 3 out of 4 hospitals were positive culture of Legionella spp.,

and all of two hotels were also positive, there should be Legionella infections. So further
research are required to isolate Legionella spp. from clinical specimen in the Philippines.

73

5

VIBRIO FURNISSII ISOLATED FROM STOOL SAMPLE OF CHILDRENWITH DIARRHEA IN NEGROS ISLAND AND THREE CASES
OF PATIENTS WITH DIARRHEAIN OSAKA CITYETSUSHI OKUMURA* AND MITSUTAKA HATA'
Department of Medical Technology, Kohchi Gakuen Junior College*
and Department of Clinical Laboratory, Minami Osaka General Hospital'

Bacteriological examination of stool from 34 children with diarrhea living in Neglos
Island revealed the following results,

1 . Four kinds of enteropathogenic bacteria were isolated from 6 cases (17.7%).
2 . The species of bacteria isolated were Enteropathogenic Escherichia coli, 2 cases (5.6% ) ,Vibrio furnissii, 2 cases (5.6% ) , Enterotoxigenic Escherichia coli, I case (2.9%), andAeromonas hydrophila, I case (2.9%).
And V. furnissii was isolated from three subjects of children with diarrhea (11 and 13
years old female and 9 years old male subject) in Osaka City in June 1987. They were treatedby Lactbacillus btfidus and Fosfomycine that suspectibity was admitted against the bacteria.
It was suggested that the enteritis may be associated with over‑eating seafood, especiallyshrimp, ark‑shell, turbo and other kinds of shellfish.

6 CHARACTERIZATION OF VIBRIO CHOLERAE O1 ISOLATED IN
THAILAND IN 1987

MAMORU IWAMI, YOSHIO ICHlNOSE, MASAHIKO EHARA,AKIYOSHI UTSUNOMIYA AND TATSURO NAITO
Department of Bacteriology, Institute of Tropical Medicine,

Nagasaki University

A total of 100 strains of Vibrio cholerae O1 were isolated from diarrheal patients
hospitalized in Thailand in 1987 and were characterized biochemically. All the strains of V.

cholerae O1 tested were serotype Inaba, biotype E1 Tor and Celebes original type in prophage

typing. Hemolytic activity to sheep red blood cells was detected in 92% of isolates in Graig

test but all were positive in heart infusion broth containing 1% glycerol by the method ofBarua and Mukerjee. Out of 100 strains of V. cholerae O1, 97 were positive in chicken red
blood cell agglutination test and all strains with HA activity were sensitive to D‑mannose
except one resistant strain to D‑mannose. Ninty‑nine strains of V. cholerae O1 were sensitive

to all drugs tested (CP, TC, SM, ABPC, EM and NA). One strain was resistant to ABPC,
showing MIC of more than 100 pglml and to 10 pglml of 2, 4‑diamin0‑6, 7‑diisopropyl‑
pteridine phosphate (0/129). The MICs against TC and CP were 3.13 and 12.5 pglml,
respectively. Multiply drug‑resistant strain of V. cholerae O1 was not isolated.

74

7

EPIDEMIOLOGICAL INVESTIGATION OF CHOLERA INFECTlON IN
NAKURU（KENYA）IN1987AND CHARACTERIZATlON
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CAMPYLOBACTER ENTERI皿S IN CHILDREN RESIDING IN KENYA AND
CHARACTERIZA皿ON OF THE ISOLATED ORGANISMS
YAsuo YANAGAsEエ，MITsuo KAKu2，Kozo FuJITA3，ATsusHI OzAwA4，
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75

microorganisms, such as Bacteroides. Blfidobacterium and Lactobacillus, were lower in diarr‑

hoeal samples than in recovery samples. It is essential to maintain a balanced normal
intestinal fiora, especially anaerobic flora, to prevent or recover from diarrhoeal diseases.

Campylobacter jejuni and Campylobacter coli were isolated from bloody diarrhoeal faeces.
Three different serotype strains (TCK2, TCK19, Lior20) of Campylobacter were isolated fromone patient.

Isolated strains showed PenicillinR, ABPCs, SMs, and TCs.
Two patients seemed to be affected by the family contacts.

9 MICROBIOLOGICAL STUDY OF DRINKlNG WATERS IN TROPICALCOUNTRIES‑FREQUENCY AND SORT OF BACTERIA IN THE SAMPLESTHAT THE RESIDUAL CHLORINE IS POSITIVEMASATOSHI SUGIYAMA , YOSHIYUKI OKUWAKI', SETSUKO TSUKIDATEAND KOICHIRO FUJITA*
Department of Medical Zoology, Faculty of Medicine, Tokyo
Medical and Dental University* and Department of Microbiology,

Kagawa Nutrition College'

We have carried out the examination of drinking water in tropical countries for 10 years.

This survey reports the number of sample which has both residual chlorine and bacteria, anddetermined the species of bacteria. Water samples were placed into gas‑sterilized containers

directly. One of water sample was used for water examination and another water sample wasused for bacterial test using URICURT which contained CLED and MacConkey medium. We
counted the colonies number of general bacteria and coli‑form groupes. This survey was
based on the data from 1980 to 1987.
The area finding the sample that has residual chlorine is Middle East and this counts for

20%. We found much less samples in the other areas.
This percentage cannot tell you whether bacteria positive level is high or low in thetropical area. The bacterial species were determined by isolated five samples which was
found 1987.
We found Ps. fluorescens and Ps. maltophilia in two samples and Ps. maltophilia in one
sample and gram positive bacillus in one sample. E. cloacae and Cit. freundii were found in

tap water sample.
According to survey by Dr. K. Takeuchi, there is a possibility that Ps. fluorescens,
Staphylococcus aureus and Serratia marcescens can survive in drinking water of 0.5 PPM
residual chlorine. Therefore this implies of possibility that some bacteria can survive indrinking water which has low levels of residual chlorine.

76

10 STUDY ON HEAT LOSS ABILITY OF PIKA (WHISTLE RABBIT)
GUO‑JIE YANG, TAKAAKI MATSUMOTO, MITSUO KOSAKA,
MASAKI YAMAUCHI AND NOBU OHWATARI
Department of Environmental Physiology, Institute of Tropical Medicine,

Nagasaki University

Pikas ( Ochotona rufescens rufescens, Whistle rabbits) are old‑fashioned rabbits which
had survived the Age of lce, and which are called "Living fossil". They have characteristic
short rounded ears and weigh less than 300 g. We have reared and breeded pikas since 1985

and reported their higher metabolic rate, higher body temperature and pyrogenic responses
to intra‑venous lipopolysaccharide (exogenous pyrogen) and to intra‑venous recombinanthuman interleukine‑1 (endogenous pyrogen) previously. In the present study, pika's heat loss
ability was examined and discussed from the view point of thermoregulation. During general
heating (30'C‑33'C‑40'C, 60% rh) , thermal panting which is one of the main heat dissipa‑
tion mechanism for rabbits was observed in rabbits, however, no increase in respiratory rate
w s observed in pikas. In 40'C rabbits did not seem to be exhausted, however, pikas lay downon the cage floor with their limbs stretched and sometimes they jumped to the cage wall as
if they were confused in a range. Noteworthily, pikas rapidly increased the rectal tempera‑ture at heat exposure (40'C, 60% rh) and then finally died after 60‑90 min, of which rectal

temperature was 44.0'C and 43.1'C, respectively. Labolatory albino rabbits survived thisexperiment. Though the radiation from the ear surface is another main heat loss mechanism
for rabbits, the ear surface area ratio to body surface area in pikas was small compared torabbits. It was 7.2% in pikas and 17.0% in rabbits. These results revealed the poor heat loss
ability in pikas constituted by smallness of the ear surface area and lack of thermal panting.

11

COMPARISON OF HEAT LOSS RESPONSES INDUCED BY LOCAL HEAT‑ING OF PREOPTIC AREA AND ANTERIOR HYPOTHALAMUS AND
MEDULLARY RETICULAR FORMATION WITH ARGON LASERON RABBITSNOBU OHWATARI, MITSUO KOSAKA, TAKAAKI MATSUMOTO, GUO‑JIE YANG,
KOICHI NAKAMURA AND CHI‑MlNG CHEN
Department of Environmental Physiology, Institute of Tropical Medicine,

Nagasaki University

It is well known that the capability of heat loss in temperature regulation is enhanced byrepetitive heat exposure.

In this study, heat loss responses such as cutaneous vasodilation and thermal panting
were induced by local heating of the preoptic area and anterior hypothalamus (PO/AH) andthe medullary reticular formation (MRF) in rabbits. PO/AH and MRF were locally heatedby an argon laser irradiation guided with a glass fiber 0.4 mm in diameter. Then irradiation
powers of argon laser (ILP) were within 10 mW to 25.4 mW, and irradiation time was for 10

77

min at each power. The minimal ILP enough to induce heat loss responses and thentemperaiure changes in the rectum, brain and ear skin as well as in respiratory and heart

rates were quantitatively compared in between PO/AH and MRF heating.
The minimal ILP to induce heat loss responses in PO/AH and MRF were 14.5 mW and
19.0 mW, respectively. And there was statistically significant difference in both values (p<0.01) . Then maximal rising temperature and volume risen over 0.5'C in the heated area were

1.1'C and l0.2 mm3 at 14.5 mW, and 1.5'C and 24.3 mm3 at 19.0 mW. Heart rate (HR)markedly decreased by local heating of MRF compared with local heating of PO/AH. Thisdecrease in HR may be an infiuence of medullary local heating on the cardiac nerves andcardiovascular center in the medulla oblongata. On the other hand, there were no significant
differences in various indicators of heat loss responses except the minimal ILP and change inHR.
Furthermore, heat loss responses were always induced by local heating of PO/AH at 45mW in the same rabbit, therefore, the present findings are available for research on themechanism of heat acclimation in central neurvous system.

12 STUDIES ON HYPOTHALAMIC BLOOD FLOW CHANGES IN
THERMAL ACCLIMATIONMITSUO KOSAKA, NoBU OHWATARI, TAKAAKI MATSUMOTO, GUO JIE YANG
KOICHI NAKAMURA AND CHl‑MING CHEN
Department of Environmental Physiology, Institute of Tropical Medicine,

Nagasaki University

Hypothalamic blood fiow as well as thermal inputs from several thermosensores plays arole in temperature regulation at a neural level in the hypothalamus. Thermal acclimation
causes some alteration in the mechanisms of temperature regulation. Therefore, the present
study was designed to clarify the influence of thermal acclimation on hypothalamic blood

flow. Cerebral blood flow (CBF) of preoptic area and anterior hypothalamus (PO/AH)
during general heating and cooling in the heat‑acclimated, cold‑acclimated and thermallynon‑acclimated (controD rabbit was measured by hydrogen clearance method. The change
in CBF was closely associated with ambient temperature as well as other thermoregulatory
parameters. Not only the less increase in CBF due to heating but also less decrease due tocooling was observed in the heat‑acclimated rabbit compared to those of control rabbit, andsimilar results were obtained in the cold‑acclimated rabbit. The reduction of gain inthermoregulatory responses occurred during general heating and cooling in both heat‑ andcold‑acclimated rabbit. The present finding suggests that the reduced change in CBF of POl

AH in the process of thermal acclimation might cause habituation phenomenon of thermo‑
regulatory responses to heat and cold stimulation.

78

13 CHANGE OF SERUM PROLACTIN DUE TO HEAT LOAD
AND EXERCISE IN HUMAN
TAKAAKI MATSUMOTO, MASAKI YAMAUCHI, MITSUO KOSAKA, GUO‑JIE YANG,
KOICHI NAKAMURA AND NoBU OHWATARI
Department of Environmental Physiology, Institute of Tropical Medicine,
N agasaki University

Serum prolactin concentration is known to be modulated by various physiologicalconditions i.e. pregnancy, Iactation, stress, exercise, sleep, feeding. Especially, hyper‑
prolactinemia due to exercise attracts much attention, which is considered to be a reason of

amenorrhea in athletic women. On the other hand, heat load also increases serum prolactin
level. In the present study, changes in serum prolactin during heat load and exercise (under
20'C and 28'O were examined and discussed. Ten athlete students, 5 male and 5 female, 19‑
23 years old, were subjects. Significant increse in prolactin, 16.0:!12.3‑19.3d:2.1 (MeaniSE)

ng/ml p<0.03, due to 40‑45'C, 20 min heat load by sauna box was observed. 50% V02max
bicycle ergometer exercise under neither 20'C nor 28'C condition increased serum prolactin

level. There was considerable discrepancy between the results in former reports. It isconsidered to be due to lightness in exercise intensity, 50% of V02max, in this study
compared to those in former reports. There was no statistical correlation between serum
prolactin and oral temperature. Adrenocorticotropic hormone (ACTH) did not alter by heatload and exercise, which supported 50% V02max exercise was light and stressless. Increasein growth hormone (HGH) Ievel due to exercise was observed under 20'C and 28'C. No
significant changes in luteinizing hormone (LH) and follicle stimulating hormone (FSH)
were observed in any experiment. And additionally, significant decrease in serum prolactin
level, 21.7 2.3‑11.6 1.8 ng/ml p<0.0001, was observed during 2.3 km, 160 min swimmingat 28.5'C of water temperature, which was considered cold stimulus and exercise. These
results indicate that light exercise less than 50% V02max may not affect serum prolactin
concentration and that thermal factor play a role in prolactin regulation mechanism.

14 PHYSICAL PERFORMANCE AND AMBIENT TEMPERATURE‑ANALYSIS OF HEART RATE‑MASAKI YAMAUCHI, TAKAAKI MATSUMOTO, MITSUO KOSAKA, GUO‑JIE YANG,NoBU OHWATARI, KOICHI NAKAMURA AND CHl‑MING CHEN
Department of Environmental Physiology, Institute of Tropical Medicine,

Nagasaki University

Physical training enhances thermoregulatory function and suppresses body temperatureelevation during exercise. Among athletes, considerable interest is attracted to the problemof the relation between thermal stress and physical performance. High ambient temperature
above 20'C has an influence on the record in a race and worsens it. In the present study, the79

influence of ambient temperature on changes in heart rate during exercise and recovery phase
was examined. Ten athletic students, 5 male and 5 female, 19‑23 years old, were subjects.
50% V02max, 30 min bicycle ergometer exercise was applied under 60% rh, 28'C and 20'C.
Initial values of heart rate were 63.4 2.9 (MeaniSE) beats/min at 28'C and 58.8i 2.6 beats/
min at 20'C, respectively. The values after 30 min exercise were 139.0i 4.4 at 28'C and 132.8i
5.0 at 20'C, respectively, in which values there exists no statistical significance. However, thevalue (101.8 :5.5) at 31 min (first I min in recovery phase) at 28'C was significantly highcompared to that (92.3i6.4) at 20'C (p<0.05). The value at 60 min (30 min in recovery
phase) was 74.8 4.6 at 28'C and 70.6d:3.9 at 20'C, respectively, which was significant
difference (p<0.05). Significant increase in oral temperature due to exercise, 36.78'C‑
37.39'C at 28"C (p<0.01), 36.65'C‑37.19'C at 28'C (p<0.01) , was observed, however, there
was no significant difference in increases of heart rate between under 20'C and 28'C.
Summarizing the present results, there was no significant differences in the fluctuation ofheart rate during exercise between 28'C and 20'C, however, in recovery phase, heart rate at

28'C was higher than that at 20'C. These findings suggest that ambient temperature may
affect physical performance.

15 STUDY ON SEASONAL VARIATION OF THERMAL SWEATING
KOICHI NAKAMURA, TAKAAKI MATSUMOTO, MITSUO KOSAKA, MASAKI YAMAUCHI,GUO‑JIE YANG, NoBU OHWATARI AND CHI‑MING CHEN
Department of Environmental Physiology, Institute ofTropical Medicine, Nagasaki University

It is known that sweat volume and threshold temperature for sweating are altered bythermal acclimation. We have reported that thermally acclimatized tropical inhabitants have
longer sweat‑onset time and smaller sweat volume compared to Japanese. In Japan seasonalvariation of ambient temperature is distinct, therefore, it is suspected that thermal sweating

response may alter season by season. In the present study, analysis of thermal sweating was

performed in both summer and winter, in order to clarify the seasonal variation of thermal

sweating. Six healthy men living in Nagasaki City were the experimental subjects. Local
sweating at the chest and abdomen induced by local heating (43'C water bath) on lower legsfor 30 min in an environmental control chamber (25'C, 60% rh) , which was determined byusing of capacitance hygrometer‑sweat capture capsule method. Mean ambient temperatureof experimental period was 26.1'C in summer (July to September) and 7.9'C in winter(January to March) in Nagasaki. Sweat‑onset time at the chest in summer was 10.8 1.2 min(Mean SE) . While that in winter was 16.3d: 2.0 min, which was significantly long comparedto summer (p<0.005). Sweat‑onset time at the abdomen was 11.9 1.3 min in summer and16.4 1.9 min in winter (p<0.03). Total sweat volume induced by 30 min heat load at thechest was 121.23 20.90 mg/capsule in summer. And that in winter was 36.59ill.46 mg/
capsule, which was significantly small compared to summer (p<0.01) . Total sweat volume
at the abdomen was 48.46i 10.83 mg/capsule in summer and 19.20 i 5.88 mg/capsule in winter(p<0.04) . These results is presumable that distinct seasonal variation may exist not only in80

thermal sweating but also in various kinds of physiological system such like metabolic,cardiovascular and endocrinological system which is closely associated with comformation oflong term thermal acclimation and thermoregulatory habituation seen in tropical inhabitants.

16 A NATIVE TAIWANESE CASE OF SCHISTOSOMIASIS JAPONICA
CHENG‑KUO CHUANGl, WEI‑JEN CHEN2, WEN‑HSIUNG HUANG3 AND YASUO NAKAJIMAl
Department of Parasitology, Yamanashi Medical College', Department of Pathology,

Chang Gung Memorial Hospital, Kaohsiung' and Department of Parasitology,
National Cheng Kung University Medical College3

It is generally accepted that Schistosoma japonica in Taiwan is zoophilic. The reported
cases of Schistosomiasis japonica in Taiwan were all mainlanders who came from Continen‑

tal China after World War II. In this paper we report a native Taiwanese case complicatedwith colonic carcinoma.

Case: A 66‑year‑old male, ex‑policeman and bywork farmer. Chief Complaints: Bloodystool, anal bleeding and anal pain. Family History: Noncontributory. Past History: An
appendectomy 5 years ago. Present lllness: The patient noted difflculty in the bowel
movement with occasional bloody stools in January 1986. Anal bleeding developed soon.
Anal pain was noted in April. He came to the Chang Gung Memorial Hospital, Kaohsiungon 7/4/86, and was admitted under suspicion of rectal cancer on 7/21/86. The patient camefrom South Taiwan. He was said to have never traveled outside Taiwan. Admission Note:
The patient had malaise, emaciation and anal pain with bleeding. The abdomen was flat andsoft. The liver and the spleen were not enlarged. An ulcerative, ill‑defined mass was

palpated 4 cm above the anal verge. The chest X‑ray film was normal. BP 130/90.
Hematology, blood chemistry and liver function test were within normal limits, except CEA
26.4 ng/ml.

Rectectomy and jejunal tumor enucleation were made on 7/23/86. The patient wasdischarged on 8/5/86.

Pathological Findings: Sections showed a moderately differentiated adenocarcinoma of therectum invading the anus, and deeply into the perirectal soft tissue. The jejunal tumor wasa leiomyoma. There are also many calcified S. japonica ova in the submucosal region andmuscular wall of the rectum.
Follow‑up: The patient expired on 8/2/87.
The patient was born in Taiwan and said to have never been abroad. It seems likely thatthe patient was infected in Taiwan. However, previous epidemiological studies on schis‑tosomiasis in Taiwan indicated that S. japonica in Taiwan is zoophilic. Neither S. japonica
nor Oncomelania snail has been reported from the patient's native place, Pingtong. Thereremains a possibility that he was sent as a soldier of Japanese Army to the Philippines orContinental China during World War II, and infected with Chinese strain or Philippine strain.

The rice field which the patient had farmed was sold, and changed to a coconut farm. Anysnail except Ampullarium canaliculatus has not been found there.

81

17 TREATMENT OF OPISTHORCHIASIS WITH PRAZIQUANTELMANAMI TANAKA, HIROSHI OHMAE TAKESHI NARA HIROMI UTSUNOMIYAHlROYUKI TAGUCHI, YUJI IRIE AND KAZUO YASURAOKA
Department of Medical Biology, Institute of Basic Medical

Sciences, University of Tsukuba

One case of opisthorchiasis treated with praziquantel is reported with clinical manifesta‑

tions, Iaboratory data and liver ultrasonography findings. The patient is a 27‑year‑old Thai

woman from northeastern Thailand and used to eat raw freshwater fish in her birthplace. She

was admitted to the hospital because of continuous right quadrant pain, nausea and epigas‑
tralgia. Since abdominal X‑ray and liver ultrasonography revealed the stone‑1ike shadow in
the gall bladder, cholecystectomy and choledochostomy were performed under the diagnosisof choledocholithiasis. During the operation, over ten of adult O. viverrini were found in thebile and ectomized organ. Fecal egg examination detected 350‑500 eggslg of feces of thepatient. Laboratory investigation revealed eosinophilia (22%) just before the operation, andtransient elevation of serum alkaline phosphatase and ZTT, which was slightly suggestive of
the obstruction of the biliary duct. A test for hepat'ocellular and renal function was whithin

normal limits. She was orally administered with 50 mglkg of praziquantel in 3 doses for 3
consecutive days. Side effects as nausea and abdominal pain were observed, but they weremild and disappeared after treatment. Within I week after administration, complete clinicalcure was obtained and a stool examination revealed no eggs in feces.

18 EPIDEMIOLOGICAL SURVEY OF LEISHMANlA IN ECUADOR AND
CHARACTERIZATION OF ISOLATESTATSUYUKI MIMORll, YOSHIHISA HASHIGUCH12, MASATO FURUYA3, MASATO KAWABATA4EDUARuo E. GOMEZ5, VICENTA V. DE CORONEL5, GABRIEL GRIMALD16, RICHARD D. KREUTZER7DIANE MCMAHON‑PRATT8 AND ROBERT B TESH
Department of Parasitic Diseases. Kumamoto University Medical School*, Departmentof Parasitology. Kochi Medical Scho012, Institute for Laboratory Animals,
Kochi Medical Scho013, Department of Clinical Pathology, Nihon University Schoolof Medicine', Departamento de Parasitologia, Instituto Nacional de Higiene y Medicina

Tropical, Ecuador5, Department of Immunology, Oswaldo Cruz Instituto, Brazil6

Biology Department, Youngstown State University, USA' and Department of
Epidemiology and Public Health. Yale University School of Medicine, USA8

New World leishmaniasis is widely distributed in Ecuador, where it is a considerable
health hazard. According to the articles published in Ecuador to date, there may be three orfour clinical forms of the disease: cutaneous cases, ca. 93% of the total; mucocutaneous, ca.

6 or 7% and visceral and diffuse cutaneous ones. The last two forms have not yet been

82

parasitologically proven in the country. The identification of parasites have been done bosedon clinical manifestations in human patients, together with the epidemiological features, thelesion developments in hamster infections and the growth patterns of parasites in vitro
cultures. We are studying the transmission of leishmaniasis in this country since 1982. In
addition to the two known vector species, Lutzomyia trapidoi and Lu. hartmanni (Hashiguchi
et al., 1985) , Lu. gomezi was added to the list of Ecuadorian leishmaniasis vectors in thissurvey. With regard to reservoir hosts, Tamandua tetradactyla was newly implicated, in
addition to Potos favus. Sciurus vulgaris and Choloepus hoffmani didactylus had already been

listed as leishmaniasis reservoirs. Six strains of Leishmania, isolated from wild mammals
and humans, were identified by isoenzyme electrophoresis and by their reactivity patterns toa crosspanel of specific monoclonal antibodies using a radioimmune binding assay. Single
isolates from S. vulgaris, P. favus and T. tetradactyla were identified as Leishmania amazonen‑sis. Three other strains, isolated from cutaneous lesions of humans, were identified as
Leishmania panamensis.

19 CURRENT STATUS OF LEISHMANIASIS AND CHAGAS' DISEASE

IN PARAGUAYHIROSHI YAMASAK11, OFELIA ARIAS2, JORGE ALONS03, MYRIAM MoRAN2,BALBINA PAV6N2, MARTA VERA2, PEDRO SACHER02, RYOZO OKADA4,JUAN IRRAZABAL2, LEONALDO LopEZ5, ANDR s AMARILLA2, FEDERICO VILLASBOA2,
TAKASHI AoKll, KIYOSHI KITAI AND HIROSHI OYA1
Department of Parasitology* and Cardiovascular Pathology', Juntendo University Schoolof Medicine, Tokyo, Japan, Laboratorio de Investigaci6n, Laboratorio Central e Instituto de

Medicina Tropical (LACIMET)2, Secci6n de Leishmaniasis, Departamento de Salud Rural3and Departamento de la Enfermedad de Chagas, Servicio Nacional de Erradicaci6n del Paludismo(SENEPA)5, Ministerio de Salud Ptiblica y Bienestar Social (MSPBS), Asunci6n, Paraguay

Leishmaniasis and Chagas' disease are serious medical problems in the Central and SouthAmerican countries. From 1983 to 1987, we participated in a cooperative medical project inParaguay and carried out epidemiological studies of these tropical infectious diseases.
(1) Leishmaniasis: This disease is found in the eastern parts of the country, among

which three regions, San Pedro, Caaguazu, and Alto Parana, are highly epidemic. These
regions accounted for about 90% of 1,093 reported cases (MSPBS, 1985) . The major clinicalsymptom was cutaneous leishmaniasis characterized by ulcer formation on the arms, hands,
face, Iegs, and body trunk. Mucocutaneous leishmaniasis was also observed in which thepatients exhibited severe damage of the nasal septum, Iips, and larynx. These observations
suggest that the Paraguayan pathogen belongs to the Leishmania braziliensis complex. The
damaged foci frequently came in contact with bacteria and/or fungi causing further advance‑
ment of the damage. Treatment by repeated injections of Glucantine requires long durationand large amounts of the drug, yielding high‑cost treatment for these patients. In addition,
there was limited supply of the drug. The spread of endemic areas of leishmaniasis has
become an increasing problem. The reasons for this are considered to be as follows: a) the

83

rapid, vast deforestation for the development of agricultural fields, b) disappearance of
natural habitats for animal hosts, e.g., forest rodents, and c) increase in infection among
forest workers, immigrants, and seasonal workers who become available as hosts due to thedecrease of resevoir animals.
(2) Chagas' disease: This disease is distributed throughout Paraguay, including thewestern areas (Chaco). We conducted an epidemiological survey of Chagas' disease in La
Colmena (a population of about 5,000) Paraguari. Out of 884 samples collected, 82 sera were
judged as positive in IHA and IFA. The seropositivities varied from a lower rate of 0‑4.6%
in the community of Fatima, in the center of La Colmena, and in the Japanese colony to ahigher rate of 16‑21% in the communities of Cerrito, Mbocayaty, and Yajhapety. The
average seropositivity (9.3%) in La Colmena was lower than that in other highly endemicareas (23%) (SENEPA, 1984). Sixty four of the 82 seropositive cases were examinedelectrocardiographically, and 20 cases exhibited some abnormalities. On the basis of theabnormal findings of the ECG {complete right bundle brance block, ventricular prematurebeats (multiform) , QRS Ieft axis deviation} combined with echocardiography (leftventricular dilatation) and other clinical symptoms, 4 cases, including three who were past

the age of 40 years old, were diagnosed as having chronic Chagasic heart disease.

20 BASIC STUDIES ON MONGOLIAN GERBILS OF SUSCEPTIBLE HOST
TO FILARIAL INFECTION (1)MASUMI SHIMIZU*, KAZUHIRO SHICHINOHE*, MITSUO ASAKAWA2SETSUKO TSUKIDATE3 AND KOICHIRO FUJITA3
Department of Experimental Animal Science*, Department of Anatomy',
Nippon Medical School and Department of Medical Zoology, Faculty of
Medicine Tokyo Medical and Dental University*

The mongolian gerbil is used as laboratory animal in the fields of virology, cerebral
neurology, endocrinology and parasitology.
Especially, in the parasitology, the mongolian gerbil is very useful as the susceptible hostto filarial infection. But, there is almost no report about biological characters in this animal.

We estimated biological characteristics of the animal such as growth curve and reproduc‑tion, in order to analyse the infection kinetics of the filarial infection.

We obtained the coat color mutant male gerbil called white spotting type. The animal
was covered brown back with white spot on nose, head, neck and abdomen, so we attempted
to make an increase in the scale of the colony, white spotting male gerbil mated with agouti

female gerbil as parents. After the litter of this parents as F* , they were mated withlittermate.

In 1978, Waring et al. reported that coat color mutant gerbil was semidorninantautosomal mutant (gene symbol Sp) and homozygotes (Sp/Sp) were prenatal death.
However, in our laboratory, albino type with white hair and red eyes advented in F3. So

we thought albino mutants were autosomal homozygotes.
The weight of new borns was about 3 g, body weight increased to 3 months age and after

84

that, they reached plateau.
Number of new borns are 1‑9 heads per one parents and sex ratio is about I : 1. Conditionof suckling had better in the case of the housing female with male after delivery.
In the ratio of organ to body weight, thymus is 2 times and adrenal is 4 times larger thanthose of mice or rats, and this is remarkable character in the mongolian gerbil.

21 BASIC STUDIES ON MONGOLIAN GERBILS OF SUSCEPTIBLE HOST
TO FILARIAL INFECTION (2)KAZUHIRO SHICHlNOHE1, MASUMI SHIMIZU1, SETSUKO TSUKIDATE2
AND KOICHIRO FUJITA2 'Department of Experimental Animal Science, Nippon Medical School*

and Department of Medical Zoology, Faculty of Medicine Tokyo Medical
and Dental University'

The mongolian gerbil is representative animal to experimental filarial infectio,n. There

are many reports to agouti type gerbil infected with filaria. Estimation of these data,
however, are not standardized.
Five conventional animals were housed in one cage at 25'C room temperature and 70%
moist. Ten adult animals of each sex and each coat color were used in this study.
Blood samples were obtained from the anesthetized animal with ether from the retro‑
orbital venous plexus by heparinized capillary pipettes. Total red blood cell (RBO andwhite blood cell (WBO counts were determined with microscope. Packed red‑cell volume(PCV, Ht) were examined by means of microcentrifugation. Hemoglobin (Hb) Ievels were
determined spectrophotometrically by the cyanmethemoglobin technique. Dry film bloodsmears were stained with Wright and Giemsa stain and subjected to a 100 cell differential
count. The data were subjected to statistical evaluation using the Student's t test.
RBC counts of the spotted and albino gerbils were siguificantly (p<0.05) Iower than the
agouti gerbil, indicating the presence of a slight anemia. In this phenomenon, Waring et al.

were discussed that expression of lowered red blood cell (RBO counts was not sex limitedor sex linked. WBC and 100 cell differential counts were not significant among coat colors
and between sexes, but in each animal, WBC seemed to varied with physiological factors.
Eosinophil counts are a few in dry film blood smears, and this is thought to be possible markerof the infection model with parasite.
Now, we observe to compare the change of that 3 coat color gerbils infected with Brugia

pahangi.

85

22

STUDIES ON刀Vγ1TRO MAINTENANCE
yl7E4E W皿H SPECIAL
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female worms of various

ages were maintained in NI medium（a1：1mixture of NCTC135
Dulbecco s Medium）or NI−20FBS（NImediumsupplemented with20％

andIscovefetal

bovine serum）withchangedsmodified

every

three

the

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days．Thepeakofof95％N2−5％CO2．Themicrofilarial

releasemedium

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the

gas

were

of2，000−18，000microfilariae

perfemaleinNImediumper24hroccurredabout10daysafterthestartofculture．Micro丘larial
releaseinNI

culture．The

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declinedandfemales moved

ended

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more

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days in NI medium．The females in NI−20FBS showed active movement for about twomonths．Some of the worms survived more than83days in NI−20FBS．Addition of fetal
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sed．Inaba6厩乙（1977）haveofof

microfilariae released andwithDepartment

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feedingofout

OF TUMOR INCIDENCE RATES IN THE LIVER AND DIGESTIVEORGANS OF MICE DURING A MUTAGEN（TRP−P−2）FEEDING一TosHIKI AJI1，HIROYuKl

mice

numbercarried

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SCHISTOSOMIASIS

SON

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Japan．Therearereportsinincreased thejaponicaarea

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liver

of Trp−p−2which

is known

asaspecific

CDF1（female，8weeks old）were
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in

has

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been
that

discus−experimen・

susceptibility of liver tissue to carcinogen．We

cancerintraperitoneally

University2andanddividedthe intestine

carcinogen

into

for

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is

facilitatedthe liver．One

inlong−term

hundredninegroups．Group1（41mice）wereof Sノ勿on廊π別and provided with carcinogenic dietcontaining O．02％Trp−p−2．Group2（29mice）were not infected and kept with the same
carcinogenic diet．Group3（20mice）was infected with Sノのo雇6％吻，and group4（19mice）

86

was not. They were provided with basal diet as references to group I and 2, respectively.
Untill the 57th week of infection, tumor incidence on the digestive organs in group I was 13
in 19 mice died. On the other hand, that in group 2 was 4 in 6. There was significant difference

between the incidence of the two groups. Histological examination of hepatic tissue at 57
weeks showed the same tendency. Hepatocellular carcinoma was detected in 9 out of 13 micedied with tumor in the digest̲ive organs in group I and 2 in 4 mice died with tumor in group
2. All of survived mice were killed at 58 weeks. Hepatocellular carcinoma had developed inall 11 mice in group I and 21 mice of 23 mice in group 2 in the liver. Only two mice in group
2 showed hepatocellular adenoma. We could not find significant difference in both groups.
The facilitation on the incidence of liver cancer was not observed finally in group 1. It wasconsidered that Trp‑p‑2 was too strong as carcinogen to examine a weak risk factor ofschistosomiasis or observation period was too long to examine the difference.

24 LECTIN BINDING BY MUSCLE LARVAE OF TRI CHI NEL LA SPIRA LISYUZO TAKAHASHl, TAKAKO UNO, YOSHIRO YOSHIKAWA
AND TSUNEJI ARAKI
Department of Parasrtology, Nara Medical University

The topographycal distribution of lectin binding sites on the muscle larva of T. spiralis

was investigated by means of fluorescen‑tagged lectin and colloidal gold‑tagged lectin
aiming to gain chemical information of each cellular components. Staining with fluorescen‑
conjugated Con A was granular and the location resembled that described for glycogen.
WGA gave similar results but far less intense staining. Under the electron microscope Con
A binding sites were observed on the midgut occupying substance, brush border, cord
granules, hemolymph and glycogen aggregates all over the worm body, but not on the cuticle
and stichocyte granules; WGA binding sites were on hemolymph and glycogen aggregates but
not on the cuticle and stichocyte granules. No effective staining was observed with Lentil
lectin. GS Iectin stained the inner layer of the cuticle especially a region close to the cuticle
surf ace.

25 ALIMENTARY TRACT ASSOCIATED ANTIGEN IN
TRICHINELLA SPIRA LISHIDEKAZU SUZUKI, YUZO TAKAHASHI, TAKAKO UNO,
NAOTO MIZUNO AND TSUNEJI ARAKI
Department of Parasitology, Nara Medical University

By means of immunocytochemical staining we show the occurrence of antigenic sub‑stance in the lurnen of the esophagus and the midgut, the antigenicity can be recognized by

Wistar rats, Fischer rats and humans. The esophagus‑ and midgut‑occupying substances and87brush border in the midgut are responsible structures for that antigenicity. By reacting
ultrathin sections of LR White‑embedded muscle larvae of Trichinella with a panel of sera

taken from patients with non‑Trichinella helminthiasis, including anisakiasis, par‑agonimiasis, gnathostomiasis, fascioliasis, dirofilariasis and trichuriasis, we demonstrate afairly good specificity of alimentary tract associted antigens for immunodiagnostic use; they

had no, or insignificant, cross‑reactivity among the serum pool with the only exception ofsevere trichuriasis serum.

26 STUDIES ON ANTIGENICITIES OF TRICHINELLA SPIRALIS (7)WITH EMPHASIS ON THE CHRONOLOGY OF ANTIBODY RESPONSE
NAOTO MIZUNO, TAKAKO UNO, YUZO TAKAHASHIAND TsUNEJI ARAKI
Department of Parasrtology, Nara Medical University

A spectrum of antibody production by Fischer rats with time after T. spiralis infection

was investigated by means of immuno‑gold staining.
Muscle larvae were fixed with a mixture of paraformaldehyde and glutaraldehyde,
dehydrated with alcohol, embedded in LR white resin, ultrathin sectioned and treated with
infected sera. Those included sera taken 1, 2, 4, 6, 8, 12, 16, 32 weeks after infection. The

specific reaction was visualized by subsequent labeling with biotinated anti‑rat lgG andavidin‑colloidal gold complex.

Specific lgG antibodies against cuticle, hemolymph, midgut occupying substance,
hypodermis, glycogen granules and genital primordium began to be detected from 2 weeks
after infection; antibodies against EOS and stichocyte al, a2 granules from 6 weeks; anti‑
bodies against stichocyte fi granules from 8 weeks. The time of seroconversion, an essential
information to design immunodiagnostic method as well as the specificity of antigen, was thus

demonstrated.

27 STUDIES ON ANTIGENICITIES OF TRICHINELLA SPIRALIS (8)WITH EMPHASIS ON THE LOCALIZATION OF ANTIGENS RECOGNIZEDBY THE PATIENTSTAKAKO UNO, YUZO TAKAHASHI, NAOTO MIZUNOAND TSUNEJI ARAKI
Department of Parasitology, Nara Medical University

In situ localization of Trichinella antigens recognized by the patient was investigated.
Sera were collected from trichinosis patients at the time of the outbreak in Mie Prefecture

in 1982, and reacted with ultrathin sections of LR White‑embedded muscle larvae. A protein

A‑gold complex was used as a second layer of the staining; therefere detected specific88

antibodies are supposed to be lgG and lgM class. The immunostaining positive structures
included the cuticle surface, the inner layer, hypodermis, hemolymph, intestinal gland cell
granules, the esophagus occupying substance (EOS) , the midgut occupying substance (MOS) ,stichocyte aO‑ and al‑granules. Immunonegative structures included the esophagus cuticle,
nucleus, nucleolus, mitochondria, muscle fibers and endoplasmic reticulum. While certain
worm components, such as stichocyte a2‑, fi‑, y‑granules, cord granules and discrete areasin the genital primordial cells, gave positive and negative results depending on each individ‑

ual examined. Staining results as to glycogen aggregates was chaos devoiding any specificpattern.

28 A COMPARATIVE STUDIES ON THE DETECTION RATE OFSTRONG YLOIDES STERCOLARIS BY AGAR PLATE METHOD
AND THE TRADITIONAL METHODSRYUJI ASATO , TAMIO NAKASONE1, TAMIKI ARAKAK12
AND TSUYOSHI IKESHIR03
Okinawa Prefectural Institute of Public Health*, Departmentof Bacteriology, University of the Ryukyus School of Medicine' andCenter for Preventive Medicine, Okinawa

Although test tube culture method for diagnosis of Strongyloidiasis has been widely used,

it has recently been recognized that the inethod is not so satisfactory as has been believed.
A new method to detect Strongyloides larvae was reported by Arakaki et al. in 1987 (Japan.

J. Trop. Med. Hyg., 16, 11‑17) . In the new method, nutrient agar medium for bacteriologicalexamination was used.
In the present study, the authors examined 780 Okinawan inhabitants to detect Stron‑
gylbides larvae by using this new method and 2 traditional methods (MGL and test tube
culture) .

The furrows left by crawling larvae on the agar plates were found in 135 cases (17.3%)

out of 780, and they are regarded as positive findings of Strongyloides larvae. On thecontraly, 24'cases (3.1%) and 34 cases (4.4%) were positive for the larvae in test tube culture

method and MGL, respectively.
Fifty‑four stool samples, known as positive for Strongyloides, were examined by theabove 3 methods. The characteristic furrows appeared in all 54 cases (lOOo/o ) . The detectionof larvae by the traditional methods did not reach to 100%. In a single examination of the
54 samples, the furrows were found in about 90% , but the traditional methods revealed about
20 to 40% of positive strongyloides. In the subsequent field work, the furrow positive plates
were directly examined by low magnification microscope, and the crawling larvae were foundin about 80% of the plates.

In conclusion, the agar plate method was excellent to detect Strongyloides from thestools especially in the cases with small number of larvae.

89

29 ENZYME‑LINKED IMMUNOSORBENT ASSAY FOB THE DETECTION OF
SERUM ANTIBODY IN CATS AND RATS INFECTED

WITH PARAGONIMUS HETEROTREMUS

HIROMU SUGIYAMA*, JUN KATAHIRA , TEIJI HORlUCHI*, TAMOTSU TOMIMURAl,
TOSHlYUKI SHIBAHARA2, TAKESHI AGATSUMA3, SHIGEHISA HABE4, KENJIRO KAWASHIMA5,PUNSIN KETUDAT6 AND SODSRI THAITHONG7
Department of Veterinaty Pathology, University of Osaka Prefecture , Laboratory Animal
Research Center, Tottori University School of Medicine', Department of Parasitology,

Kochi Medical School', Department of Parasitology, School of Medicine,
Fukuoka University', School of Health Science, Kyushu University*, Department of Biology,

Faculty of Science, Srinakharinwirot University, Bangkok' and Department of Biology,
Faculty of Science, Chulalongkorn University, Bangkok'

Applicability of P. westermani (diploid type, collected in Mie Prefecture, Japan) antigen

was examined for the detection of serum antibody in cats and rats experimentally infectedwith Thailand P. heterotremus. Cats served as experimental definitive hosts and rats served

as experimental paratenic hosts of Thailand P. heterotremus. The antibody titers (inabsorbance) of infected animals detected by ELISA with P. westermani antigen were as highas those with P. heterotremus antigen. These findings indicated that P. westermani antigen
was available for immunodiagnosis of paragonimiasis heterotremus in both the definitive
hosts and the paratenic ones.
The antigens recognized with serum samples of cats and rats infected with P. heter‑
otremus were also characterized. In SDS‑PAGE with P. heterotremus antigen, 3 major bands
with molecular weights of about 27,000, 17,000 and 15,500 and a few additional faint bands

with molecular weights of about 26.000 were detected. P. westermani antigen also migrated
into 3 major bands with molecular weights of about 27,000, 17,000 and 15,500. In immunoblot‑
ting, serum samples of infected cats and rats reacted strongly with the 27,000‑dalton band inboth P. heterotremus and P. westermani antigens. The 27,000‑dalton band in both lung fluke
antigens was also recognized with a monoclonal antibody (A4‑1; Sugiyama et al.. 1988)
against adult P. westermani. These findings indicated that P. heterotrem s possessed common
antigen to P. westermani and the molecular weight of the common antigen was 27.000. As theinfection with P. heterotremus strongly induced antibody against the common 27.000‑daltonantigen, it was suggested that P. westermani antigen could be substituted for P. heterotremus

antigen in immunodiagnosis of paragonimiasis heterotremus.

30 ANTI‑LEISHMANlA ACTIVITY OF INOSINE ANALOGSHISATO TANIFUJI, HIROYASU SATAKE AND YUSUKE WATAYA
Department of Pharmaceutical Chemistry,
Faculty of Pharmaceutical Sciences, Okayama University

90

A promising approar̲h for developing chemotherapeutic agents that possess selective
toxicity toward parasites is the one based on certain unique features in structure or functionof the parasites that differ from those of the host cells. L. donovani is incapable of synthesiz‑ing purines de novo and is thus dependent on the host for the source of purines. Over two
hundred nucleoside analogs were tested in anti‑Leishmania assay. The results show that and3'‑deoxyinosinel) and carbocyclic inosineare2) potent inhibitor for the growth of promas‑.

tigotes of L. donovani and L. tropica. In the present experiments, we show that 3'‑fluor‑
oinosine (3'‑fluor0‑3'‑deoxyinosine) inhibits the in vitro growth of L. donovani promas‑
tigotes and amastigotes. In culture, EC50 Value is 1.0X 10‑6 M for the promastigotes. On theother hand, it is less toxic towards mouse mammary tumor FM3A cells: EC50 value is 1.9 x
10‑4 M. J774.1 cells, a mouse macrophage cell line, cultured in GIT medium containing
lipopolysaccharide. The adherent cells were exposed to promastigotes of L. donovani for 1day, and treated with the drug. Drug activity is then assayed by counting the number of
infected host cells/100 host cells. The results indicated that 3'‑fluoroinosine is active against

amastigotes of L. donovani in vitro.
1 ) Wataya, Y. et al. (1984): Biochem. Biophys. Res. Commun. 123, 677

2 ) Hiraoka, O. et al. (1986) : Biochem. Biophys. Res. Commun. 134, 1114

31 THERAPEUTIC EFFECT OF INOSINE ANALOGSLOADED IN LIPOSOME IN MICE INFECTED

WITH LEISHMANIA DONOVANIKAZUHISA MORISHIGE TOSHIKI AJI AKIRA ISHn*, YUSUKE WATAYA2
AND TATSUJI YASUDA3
Department of Parasitology, Okayama University Medical School*, Departmentof Pharmaceutical Chemistry, Faculty of Pharmaceutical Sciences, Okayama
University' and Institute for Environmental Medicine, Okayama University'

We reported last year that inosine analogs (carbocyclic inosine and 3'‑deoxyinosine) had

therapeutic effect in mice infected with Leishmania donovani. Those drugs and F‑2B as a
new inosine analog loaded in liposome were examined in vivo by the same method.
BALB/C mice infected with L. donovani were treated with inosine analogs loaded inliposome for appraising the therapeutic effects. The mice infected with L. donovani promas‑
tigote 2S‑15M strain (1 x 108) were treated with 5 different doses of each drug adrninisteredby intravenous injection on alternate days. A group of mice administered with saline or
liposome without drug was prepared as negative control and a group with Pentostam as
positive control. At the end of the treatments for 2 weeks, impression smear of the liver was
prepared to determine the parasite load which was expressed as L.D.U. by counting parasites

per 1,000 hepatic cell nuclei. +In the mice infected with L. donovani, carbocyclic inosine (10 mglkg) Ioaded in liposome

showed 89% effect of inhibition as compared with the group of mice administered only
liposome, while carbocyclic inosine (100 mglkg) showed 92% effect of inhibition in theprevious experiment. 3'‑deoxyinosine (5‑10 mglkg) Ioaded in liposome showed 61‑73%

91effect of inhibition, while 3'‑deoxyinosine (100 mglkg) showed 63% effect of inhibition in theprevious experiment. F‑2B (5 mglkg) Ioaded in liposome showed 67% effect of inhibition
compared with the group of mice administered saline, while F‑2B (50 mglkg) showed 73%
effect of inhibition. The inosine analogs loaded in liposome showed 5‑20 times high activity
as the free inosine analogs.

32 RELATIONS BETWEEN SHAPE AND NUCLEAR DNA CONTENTS OFGAMETOCYTES IN PLASMODIUM VIVAX AND FALCIPARUM
HIROYUKI AMANol, TOSHIMASA NISHIYAMA2YUZO TAKAHASH12 AND TSUNEJI ARAK12
Department of Overseas Medical Services, Tenri Hospital*and Department of Parasitology, Nara Medical University'

The relationship between the shape and nuclear DNA contents of malarial parasites by

microfluorometry on DAPI‑stained thin‑blood‑films was studied with emphasis on
gametocytes of P. vivax (Pv) and P. falciparum (Pf) . In brief, thin blood films were preparedfrom 4 cases with Pv malaria and I case with Pf malaria, fixed with methylalcohol andstained with I pg/ml DAPI solution for 15 min. DNA contents were expressed in arbitrary
fluorescence units (FU) . The FU of ringforms (R) of Pv (n=77) and Pf (n=19) were 10.12
1.43 and 10.07i2.57, respectively. Macrogametocytes (MG) (n=40) and microgametocytes(mG) (n=30) of Pv were distinguished from the size and nuclear position, and the DNAcontents were more than that of R (15.35i:4.82 and 29.07

17.15, respesctively) . Thus mG of

Pv have 1‑6 times as much DNA value as ringform which is supposed to contain haploid

DNA, probably refiecting a various stage of DNA synthesis up to octoploid. Young
gametocytes of Pv (n= 11) were recognized by the comparatively round shape and sufficientmalarial pigments. However it was difiicult to distinguish males from females. Nearly all
young gametocytes had the diploid value of DNA (20.86 4.23) . Exflagellating gametes of Pv(n=10) showed the haploid value (11.26 3.13). MG (n=10) and mG (n=16) of Pf were
equipped with more DNA contents than those of R and distinguishable each other by the
degree of nuclear DNA condensation. And there was no difference between the amounts ofDNA (16.63d:4.18 and 15.30i4.66, respectively) in male and female. Young MG of Pf (n=8) were distinguishable from young mG of Pf (n= 11) by the nuclear shape. It was suggested

that the both young gametocytes of Pf were diploid DNA value (male: 20.59 8.11, female:
18.95 2.85) as well as those of Pv. Although our results closely fits the hypothesis by Sinden(1982) and the results of experimental study by Janse (1987) on P. berghei and P. falciparum,

futher observation on clinical materials using our method will be recomended.

92

33 SUSCEPTIBILITY OF ANOPHELES OMORII TO PLA SMODI UM
YOELII NIGERIENSIS N67

KAZUYO ICHIMORI*, TAKESHI KURIHARA , RYO ARAKAWA2
AND KIYOSHI KAMIMURA'
Department of Medical Zoology, Teikyo University School of Medicine*and Department of Parasitology, Toyama Medical and Pharmaceutical University'

Anopheles omorii (TM) was examined for its susceptibility to Plasmodium yoelii niger‑
iensis (N67) under laboratory conditions. An. omorii is one of the rarest mosquitoes in Japanand the larvae are found in tree holes in mountains (Tanaka et al., 1979). Recently, TM
strain of An. omorii has been colonized in the laboratory (Arakawa et al.. 1988). In paired

feeding experiments, Beech strain of An. stephensi from India, as a standard was fed
simultaneously on the same mouse whenever TM strain of An. omorii was fed. It was foundthat An. omorii (TM) was less susceptible than An. stephensi (Beech) .

34 IN VIVO AND VITRO DEVELOPMENTAL CONDITIONS FROMOOKINETE TO SPOROZOITE OF PLASMODIUM BERGHEIKENYICHI YANOl, KARL MARAMOROSCH2 AND ALBINA KOZLOWSKA2
Department of Protozoology, Research Institute for Microbial Diseases,

Osaka University

and Department of Entomology and Economic Zoology,
Cook College, Rutgers University'

Anopheres stephensi was infected with Plasmodium berghei by bite of an infected hamster.

Temperature shift of infected mosquitoes from 21'C for 24 hr to 25'C and 28'C increased
degeneration rate of oocysts and inhibited the maturation of oocysts. P. berghei ookinetes
were cultured from hamster blood and purified by discontinuous Percoll gradient (0‑36%‑45%) . Purified ookinetes provided very stable infection to A. stephensi through membrane
feeders. Mosquitoes fed purified ookinetes in phosphate buffered saline (PBS) and thenmaintained only with 5% sugar could grow infective sporozoites. Fetal bovine serum (FBS) ,mammalian cell medium, Ham's F‑12, and hamster red blood cells supplemented with PBS
improved infectivity of ookinetes to mosquitoes and decreased degenerative changes duringoocyst development.
Purified ookinetes were resuspended in the culture medium, F‑12 supplemented with 10%
FBS and subsequently incubated at 21'C for 2‑3 days in a candle jar. Microscopic examina‑tion showed a few ookinetes with swollen posterior regions like a bacterial spore in better
culture conditions. They were considered intermediate forms in the development frorn
ookinete to oocyst.

93

35

A LONG皿UDINAL SEROEPIDEMIO1。OGICAL MALARIA SURVEY IN
AN AMAZONOUS HYPOENDEMIC COLONY ll CASE PLOTTINGMETHOD ALMING A COMPUTER GRAPHIC SYSTEM

HIRosHIYANAGlsAwA1，MILToN M．HIDA2，SHIGEYuKI KANo3，RoMwALDo E SHIBATA4，ELIzA IwAsHITA4，ALFRED HONDA4，RuMIKo KosuGl3，EIIcHI NAKANol
AND MAMoRu SuzuKI3
Department

Faculty

of

Gunma

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diseaseinof

PublicUniversity

controla target community．Whenisadmittedthat

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dynamics ofvisit．Personal

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methodHealth，Kyorin

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test．Totaling

Daini Tom6−Agu by dotting positive cases on the map．Westem area showed P吻砿prevalence，and in the central area of Daini Tom6−Agu Pヵ1吻αn槻prevalence wasdemonstrated
dotting

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36 1MMNUOREACTIVE PEPTIDSES CLEAVED BY CYANOGEN BROMIDEFROM JAPANESE ENCEPHALITIS VIRUS ENVELOPE GLYCOPROTEIN E
AsHoK
Department

KuMAR

Purified JapanesethroughsucroseSRIvAsTAvA，KoulcHIof Virology，Instituteencephalitis

gradient．Envelope

virus

MoRITAof

Tropicalwasdisrupted

glycoprotein E

AND

AKIRA

IGARAsHI

Medicine，NagasakibyTritonUniversity

X−100andcentrifuged

was precipitated by trichloroacetic

acid

94

and dissolved in formic acid and cleaved with cyanogen bromide (CNBr) , followed by SDS‑
PAGE. Multiple bands of molecular weight ranging from uncleaved 54 K to cleaved 8 K wererevealed by Coomassie blue staining. Bands corresponding to 39 K, 22 K and 8 K, as well as
uncleaved 54 K, were cut out and immunized to mice by 6 weekly intraperitoneal injection
with Freund's adjuvant. Anti‑JE antibody titer of individual mouse serum was tested by
ELISA and neutralization (N) test. Geometrical mean titer of ELISA was highest for anti‑
54 K, with gradual decrease for anti‑39 K, anti‑22 K, and anti‑8 K, in this order. N‑test at
1 : 10 serum dilution was positive in 1/2 anti‑54 K, 0/4 anti‑39 K, 5/9 anti‑22 K and 4/4 anti‑

8 K sera. Reactivity of these antiserum to each fragment was tested by SDS‑PAGE andWestern blotting in order to show their interrelationship. Anti‑54 K as well as anti‑22 K sera

reacted with 54 K, 39 K, 22 K and 8 K bands, while anti‑39 K sera reacted with 54 K, 39 K and

22 K, but not with 8 K bands. On the other hand, anti‑8 K sera reacted only with 22 K and

8 K bands. The results indicate that 8 K fragment, which may carry some neutralizing
epitopes, is a part of 22 K but not included in 39 K fragment, while, 22 K and 39 K fragments

share overlapping part. From this reasoning and position of methionine residues on the Eprotein deduced from its nucleotide sequence (Sumiyoshi et al.. 1986) , a hypothetical positionof these fragments on the E protein was postulated.

The 8 K fragment was purified from CNBr‑cleaved E protein by reverse‑phase high‑pressure liquid chromatography and its N‑terminal amino acid sequence was determined.
Eleven residues showed complete agreement with amino acid sequence deduced from thenucleotide sequence of postulated position of the 8 K fragment.

37 ENHANCEMENT OF DENGUE VIRUS MULTIPLICATION BY THE
INHIBITORS OF MRNA SYNTHESISTSUTOMU TAKEGAMI, MASAKO SAHARA, ZULKARNAIN EDWARD
AND SUSUMU HOTTA
Institute for Tropical Medicine, Kanazawa Medical University

Recently the genome structure of dengue Virus (DV) has been clarified. However, thepathogenesis of DV infection and the mechanism of viral replication have remained to beelucidated. Here we will report an enhancing effect of actinomycin D (Act‑D) upon the
reproduction of DV.
Dengue Virus type I (DIV, Mochizuki strain) was used for the tests. When DIV‑infected
cells were treated with I pg/ml Act‑D, D‑1 viral reproduction was significantly enhanced.
HA titers of DIV released from infected C6/36 and Vero cell cultures increased to twice to
4 times of those from control untreated cells. The amounts of viral genomic RNA extracted
from Act‑D treated materials were significantly larger than those from untreated one. Dataof 35S‑labeled experiments also indicated that more amounts of DIV‑structural proteins wereobtained in the Act‑D treated cultures. Ten pg/ml of alpha‑amanitin, an inhibitor of RNApolymerase 11 in eukaryotic cells, showed a similar enhancing effect upon D1 Virus reproduc‑tion as Act‑D. It appeared that the virus yields became higher when the synthesis of cellular

mRNA was inhibited by substances such as Act‑D or a‑amanitin.

95

These data suggest a possibility that certain factor (s) produced in host cells retard theviral production processes, and that the synthesis of such factor (s) is inhibited in the presenceof Act‑D or a‑amanitin. There must be several mechanisms for the enhancement of DV
multiplication that may lead to the severity of clinical manifestation of dengue, and thephenomena we here describe is probably one of the cases.

38 ENZYME‑LINKED IMMUNOSORBENT ASSAY (ELISA) IN
THE DETECTION OF HEPATITIS‑B SURFACE ANTIGENIN BLOOD DRIED ON FILTER PAPER
SHINYA MATSUDA AND HIROAKI KAHYO
Department of Human Ecology, School of Medicine,
University of Occupational and Environmental Health

In an attempt to find a convienient method for the seroepidemiological study of HBsAg
in developing countries, Enzyme‑Linked Immunosorbent Assay (ELISA) using blood samples
dried on filter paper was evaluated. The results of this method were in complete agreementwith both those obtained on original sera by Rla and with those obtained on sera by ELISA.
We did not experience any false positive or false negative results. In this study the sample
dried on filter paper was eluted by I ml of PBS (pH 7.2) . Dilution rates were caliculated by

the calibration line of each serum. Dilution rates were between 0.041 and 0.082. The
coeficient of variation of absorbance value of 10 samples of each case were between 5.0% and14.8% , which indicated an acceptable intratest reproducibility of this method. In addition,
storage at 20'C, 20'C with desiccants and 3'C for up to 21 days did not alter the sensitivity andspecificity of this method. This method will be useful for the seroepidemiological study ofHepatitis‑B virus in developing countries.

39 STUDIES ON HEPATITIS WHICH OCCURRED AMONG LONG‑TERM
SOJOURNERS IN THE DEVELOPlNG COUNTRIES,AND PREVENTION AGAlNST IT
HIROSHI OHARA1, ISAO EBISAWA2 AND HIROSHI NARUT03
Department of International Cooperation, National Medical Center

Hospital , Keihin Kyuukou Kawasaki Clinic' and Japan International

Cooperation Agency'

Investigations were made on acute hepatitides which were contracted during stay indeveloping countries. The subject of this investigation was members of the Japan Overseas
Cooperation Volunteers who stay in developing countries for two years. The age of thesubject was between 20 and 35, and mid year population in 1987 was 1687.
For prevention against hepatitis, inoculation of human immune serum globulin (ISG) and96

hepatitis B (HB) vaccine (medical staffs only) was started in 1980 and 1982, respectively.
Before inoculation of ISG was started, hepatitis A (HA) was showing the highest frequency
among hepatitides, however the incidence decreased remarkably after the inoculation of ISG.
In 1986 the inci ence of HA was less than 0.1%. No cases of HA was confirmed among those

who were inoculated with ISG although there were many among who were not. The
possession rate of HA antibody before departure was 12.0% in 1982 and 3.5% in 1986.
Twenty‑nine cases of HB have been confirmed during the past 7 years. 27 cases wereregarded as those contracted during stay in developing countries, while 2 cases were onset
from HBe antigen carrier. The rate of sero‑conversion of HB marker was 4.5% in 1986. 194
out of 221 who were inoculated with HB vaccine became HBS antibody positive, and no casesof HB occurred among them. Statistical significance in occurrence of HB was recognized
between male and female.
Eight cases of non‑A non‑B hepatitis was confirmed during the past 7 years. Most cases
were regarded as infected orally.
When Japanese, especially young people, stay in developing countries, the risk of contrac‑

tion of acute viral hepatitis is high. For prevention against it. ISG and HB vaccine isimportant as well as precaution against foods, water, blood and sexual intercourse. In thepresent investigation, the prophylactic efficacy of ISG and HB vaccine to long‑termsojourners in developing countries was proved.

40 PURIFICATION OF AN ANTIHEMORRHAGIC FACTORFROM THE SERUM OF DINODON SEMICARINATUS
YASUHIRO TOMIHARAl, KAZUO YoNAHA2, MASATOSHI NOZAKll,

MASANOBU YAMAKAWAl, TAKAO KAMURAI AND SEIZEN TOYAMA2
Department of Habu, Okinawa Prefectural Institute of Public Health*and Department of Agricultural Chemistry, University of The Ryukyus'

Purification of an antihemorrhagic factor from the serum of Dinodon semicarinatus. An
antihernorrhagic factor was purified from the serum of Dinodon semicarinatus by a series ofhigh performance liquid chromatographies with a TSK gel DEAE‑5PW column. The purified
antihemorrhagic factor showed a single band on polyacrylamide gel disc electrophoresis.
The factor inhibited the hemorrhagic activity of HRI and HR2, the hemorrhagic factors of
Trimeresurus flavoviridis Okinawa. The purified antihemorrhagic factor was stable from O to

60'C and at the pH values between 2.0 and 11.0. The molecular weight of the factor was
estimated to be 59,000 and 52,000 by a gel filtration and SDS‑disc electrophoresis, respective‑
ly, suggesting that the antihemorrhagic factor consists of a single subunit. No precipitin lines

were found for the purified antihemorrhagic factor with the venom of T. favoviridis Okinawa

and its hemorrhagic factors, HRI and HR2.

97

41 ANTITOXIC EFFECT OF CEPHALANTHIN AND ANTIVENOM AGAINSTTHE MAMUSHI (AGKISTRODON BLOMHOFFII) VENOMYOSHIHARU KAWAMURA AND YOSHIO SAWAIThe Japan Snake Institute

Varying dilution of mamushi venom (0.1 ml) was mixed with 0.2 ml (1 mg) of Ce‑phalanthin or 0.1 ml of anti‑mamushi antivenom, incubated at 37'C for I hr and injected
intramuscularly into the thigh of mice. The results indicated that 0.1 ml of the antivenom
neutralized 600 pg (2 mlds) of lethal activity and 75 pg (129 mlds) of hemorrhagic activityof the venom, whereas any significant antitoxic effect of Cephalanthin were observed ascompared with the control of venom alone.
The same results were obtained when the venom was previously injected intramuscularly
into the thigh of mice before the administration of Cephalanthin or antivenom.
From those results, it is elucidated that Cephalanthin is not effective to neutralize thevenom in vitro and in vivo. Thus, the idea to substitute Cepharanthin for antivenom on
medical treatment of envenomation due to mamushi is unacceptable.

42 A CASE OF SEVERE MALARIA TREATED WITH QINGHAOSUSHIGEYUKI KANO1, MASACHIKA TSUJ12, JUN‑ICHlRO HosOYA3

AND MAMORU SUZUKll
Department of Parasitology, Gunma University School of Medicine*, Department of
Infectious Disease' and Clinical Laboratory', Tokyo Metropolitan Ebara General Hospital

The herb Artemisia annua has been used for many centuries in Chinese traditional
medicine as a treatment of malaria. The substance resp6nsible for its medicinal action iscalled Qinghaosu (artemisinin) . Artemether is one of the derivatives of qinghaosu and we

attempted chemotherapy of a case of severe falciparum malaria.
A Japanese 28 years old male complained of a high fever during the round trip in Africa
one week before coming back to Tokyo. On the day of admission into Tokyo Metropolitan
Ebara General Hospital, he developed fever at 41'C, and asexual forms of P. falciparum wereobserved in the peripheral blood of the patient at a density of 17% of erythrocytes. His
consciousness was turbid; cerebral malaria was suspected. Liver was palpable by two finger

breadth but spleen was not palpable. Ultrasonography confirmed hepatosplenomegaly.
Slight jaundice was observed. On the laboratory findings, anemia (RBC 270 X 10', Hb 7.9 gl
dl, Ht 23%) , Ieukocytosis (15,300/mm3) and the decrease of the number of platelets (32,000/mm3) were observed. A considerable degree of dysfunction of liver and kidney was also
noted.

Two hundred mg of artemether on first dose was administered intramusculary followedby 100 mg per dose at intervals of 12 hours to the amount totaling 1,000 mg. The asexual
stage parasites from blood films were eliminated in 66 hr after starting treatment. Therefore,

98

artemether amounted to 600 mg, which is the standard total dosage, saved the patient fromseverity. When the clinical signs were away, the patient was supplemented by chloroquine(1,500 mg) , because recrudescence rate of artemether within one month is recorded higher
than 10% . No resistance of the isolate to chloroquine was confirmed beforehand by in vitro
chloroquine susceptibility test.

Quinine has been very effective in treating severe malaria. However we considered
artemether as the antimalarial of higher priority than quinine especially in the treatment ofa patients with dysfunctions of liver, kidney and CNS, because side effects by quinine
occasionally involve those organs. In addition, the half‑life of quinine in the blood is 2.5 times

as long as that of artemether. Because of the short half‑life and rapid action of thederivative, artemether is documented to be remarkably well tolerated in man and also
appears to be safe in the cases complicated by heart, Iiver, and renal disorders, effectivenessagainst both chloroquine‑sensitive and resistant strains of Plasmodium falciparum is anotherstrong quality of artemether.

Most Japanese are non‑immuners to malaria, hence tend to develop severe malaria if
treatments delay. Artemether seems to be a promising new antimalarial with high potential‑
ity to cure such patients.

43 IMPORTED MALARIA CASES RECENTLY DEALT WITH BY US
KENYICHI YANO TOSHIO NAKABAYASHll, EI‑ICHIRO NISHIMOT02TADASUKE ONO1 AND YOSHITAKA OMATA1
Department of Protozoology, Research Institute for Microbial

Diseases, Osaka University* and Kinan Hospital2

There were six malaria cases (4 falciparum and 2 vivax malaria) diagnosed by the
Department of Protozoology, Research Institute for Microbial Diseases, Osaka University
from January to July, 1988. A patient (41‑year‑old male) suffered severe falciparum malaria
with a maximum parasitemia of 35.4 x 104/mm3. All forms including developing trophozoite,
young schizonts and gametocytes were observed in the peripheral blood. He and three otherengineers visited Yercaud, a local town in India on March 18, 1988 and stayed there for aweek to teach how to manage machineries used in the processing of stone. They did not haveany knowledge about malaria. Three slept with open windows but without mosquito nets.
Two of the three including our case suffured from severe falciparum malaria. Small to
medium Japanese enterprises trading abroad are increasing these years and are sending theiremployers to malaria endemic areas without informing them about malaria and othertropical diseases.

A relapse case of vivax malaria occurred with a Japanese worker at a forestry company

in Papua New Guinea. The forestry company provides antimalarial drugs but workers andtheir families do not want to take adequate prophylactic chemotherapy and rather prefermalaria infections. Therefore, almost everybody including children suffured from malaria
during their 5 years stay there. It seems that there are a lot of Japanese malaria patientsabroad, most of which are not investigated in statistic studies on malaria incidence in Japan.

99

It is necessary that all the enterprises trading abroad organize an eff ective antimalarial

campaign to decrease of imported malaria.

44 ONE CASE OF VIVAX MALARIA SUSPECTED TO BE INFECTEDIN INDIATATSUYA KATSUMATA, KEIJI INOKO, TOSHIAKI HAYASHI,
SHIGERU KoNO, KEIZO YAMAGUCHI, MASAKI HIROTA

AND KOHEI HARA
2nd Department of Internal Medicine, Nagasaki University School of Medicine

One case of vivax malaria, which was treated with sulfamethoxazole and trimethoprim,is presented. In December 1987, a 24‑year‑old Japanese male came back from India where
he had been on voluntary service. He had got high fever and treated with chloroquine inAugust 1987 and there had been no such symptom later in India. In the evening of April 5,
7, 9 and 11, 1988, shivering and headache associated with high fever developed periodically.

On April 12, he admitted to Nagasaki University Hospital. Blood smears on admission
revealed the presence of Phomodium vivax. The patient was successfully treated withsulfamethoxazole and trimethoprim and primaquine, and there was no sideeffect. Plasma
concentrations of sulfamethoxazole and trimethoprim were determined, which reachedmaximum at 45, 3.5 mcg/ml respectively 4 hr after prescription of the drugs and thendecreased gradually. From 1972 to 1988, we have experienced 6 cases of maraliasis, whichwere successfully treated with sulfamethoxazole and trimethoprim. We are encouraged touse sulfamethoxazole and trimethoprim to maraliasis in Japan, where chloroquine andfansidal are not easy to get.

45

THE EFFECT OF HAEMOGLOBIN IN SERUM AS A TRIGGER OF THE

TRANSFORMATION FROM BLOODSTREAM FORMS TO PROCYCLICFORMS OF TRYPANOSOMA BRUCEI IN VI TRO
TETSUO YANAG11, HIROYUKI HIRUM12, S. K. MoL002,

M. SHAW2 AND KAZUKO HIRUM12
Department of Protozoology. Institute of Tropical Medicine, Nagasaki
University*, presently a visiting scientist at International

Laboratory for Research on Animal Diseases, Nairobi, Kenyaz

A further study of the factors which trigger the transformation from bloodstream forms(BSFs) to procyclic forms (PCFs) of a pleomorphic clone (GUTat 3.1) of T. b. brucei was
carried out using an in vitro system in which BSFS continued to grow without transforming
to PCFS at 27'C as reported earlier.

A total of ten sera obtained from various suppliers were examined and resulted in their100classification into four groups (G‑1, II, 111 and IV). G‑1: Four sera supported the continuous

growth of BSFS Without transforming to PCFs. G‑II: One serum induced a small number of
PCFS among many BSFS after two weeks of cultivation, but they disappeared in the following
week resulting in a BSF population in the final culture. G‑III: Three sera supported thecontinuous growth of BSFs, however, also induced a number of PCFS by day 10 resulting ina mixed population in the final culture. G‑IV: Two sera transformed all BSFS to PCFS Withina week and the final culture contained only rapidly proliferating PCFs.
One of the sera in G‑III was fractionated into 10 equal parts by freezing, thawing andseparating by means of gentle pipetting and their effects were examined in the same manner.
Fractions of the Ist to the 5th layers were equivalent to the sera in G‑1. The 6th layer was
equivalent to the serum in G‑II and the 7th to the 10th layers to the sera in G‑IV. Further‑more, the Ist fractions of another serum in G‑III and two sera in G‑IV were also found to beequivalent to the sera in G‑1 while the 9th and the 10th fractions of the serum in G‑IItransformed all BSFS to PCFS (equivalent to G‑IV) .
An addition of various amounts of bovine haemoglobin (BHb) in three groups, (1) 0.05‑
0.2, (2) 0.4 and (3) 0.6‑40 mg % , to the culture medium supplemented with a serum in G‑I
resulted in the same transformation as induced by the sera in G‑II, 111 and IV, respectively.

An addition of 3 or 6 mM of cis‑aconitate (CA) in the same manner did not transformthe BSF to the PCF.
These results suggest that (1) BHb or its degradation products, most probably trigger
the transformation, (2) CA may not play a direct role in the transformation, (3) mostcommercial sera may contain the factor varying in amount from lot to lot, (4) the factor canbe easily removed (or at least minimized) by the natural gravity separation method and (5)sera containing low amounts of the factor may be suitable for the cultivation of BSFS while

those containing high amounts may be used for the propagation of PCFs.

46 ULTRASTRUCTURE OF TRANSITIONAL FORMS OF TRYPANOSOMACRUZI BY SCANNING ELECTRON MICROSCOPYVARUNEE SooKSRI, HIROJI KANBARA, TOSHIHIDE FUKUMA,

SHUSUKE NAKAZAWA AND HARUKI UEMURA
Department of Protozoology, Institute of Tropical Medicine,

Nagasaki University

The ultrastructure of transitional forms between amastigotes and trypomastigotes ofTr)panosoma cruzi was studied by scanning electron microscope. Two samples wereobtained. Sample I was the extracellular trypanosomes collected from LIT medium culture
after 48 hr of inoculation which was initiated from trypomastigotes from fibrobrast cell (L‑celD culture and passed through C.M. cellulose column. Sample 2 was the intracellulartrypanosomes collected from destructive fibroblast cells which were mechanically disrupted
by homogenization after 72 hr of incubation. These two samples were fixed in 2% glutaralde‑
hyde for 2 hr for scanning electron microscopy.
The number of each forms of T. cruzi in both sample was different as in sample 1, thelO1

percentage of amastigotes, trypomastigotes and transitional forms was about 10, 30 and 60
while in sample 2 is 80, 10 and 10 respectively. Dividing amastigotes were found also in both

sample around 0.1% and 0.7% in sample 1, and 2 respectively and were counted in theamastigotes. The free and attached flagella were clearly seen in these amastigotes.
Therefore, here we propose the new concept of the amastigote that it is one form of T.
cruzi with rather oval in shape, convex at one side and concave at the another, 2.25 x 1.50 pm

in average size and possesses the fiagellum both attached and free parts. The attachedflagellum originates near the posterior end, runs straight along the convex curve toward theanterior end then protrudes out freely and shortly to become the free flagellum.
Because it is quite impossible to observe the attached flagellum by light microscope oreven the short, free flagellum is also very difiicult to be seen with improper staining. That

is why the amastigote is described with no flagellum.

The amastigote gradually developed to trypomastigote through the transitional forms bystraightening combined with the helical movement of the body together with the change inflagellar structure. The transitional forms could be concluded into c‑Form, s‑Form and sc‑
Form as following.

c‑Form: The body was oval about 2.73 x 1.51 pm the posterior end was broadly conical with''c', shape of the attached fiagellum and short free fiagellum. Some showed coiling movement
at the anterior end.

s‑Form: The body was slightly elongate about 2.85 x 1.47 pm. The helical twist appeared,
the attached flagellum became ''s ,'shape with free flagellum as short as the c Form

sc‑Form: The body was slightly elongate about 3.14xl.30 pm, the posterior end wastaperingly conical with "sc" shape of the attached fiagellum and longer free flagellum.

By twisting or coiling with straightening of the ody, it formed the trypomstigotes inwhich the shape and size varied depending on how they moved. Usually the slender trypomas‑
tigote was about 15.25 x 1.42 pm. Whether the shape or size they would appear, the structureof the flagella showed more advanced in shape and length than those of the transitional form.

To become the amastigote, the trypomatigote might round up the entire body together
with coiling and hiddening of the flagellum.

47 INFLUENCE OF MONOSACCHARIDES ON THE GROWTH OF
TR YPANOSOMA BRUCEI GA MBIENSE IN VITRO
PETER J. MHANDO, TOSHIHIDE FUKUMA, HARUKI UEMURA
AND HIROJI KANBARA
Department of Protozoology, Institute of Tropical Medicine,

Nagasaki University

Tmpanosoma brucei gambiense adhere on the surface of feeder layer cells and form
clusters between these cells. The failure of adherence will always lead to elimination of theparasites. To investigate this phenomenon, the effect of nine monosaccharides among whichconstitute cell surface carbohydrates, mannose derivatives, Iectin, mannan, anti‑mouse brain

cell rabbit serum and other reagents on the growth of Tr)panosoma brucei gambiense over102

new born mouse brain cells and feeder layer free system was assayed.
Different concentrations of each of the monosaccharides { a‑D‑(+)‑glucose, D ‑ ( + ) ‑galactose, D‑ (+)‑mannose, a‑D‑ (+)‑fucose, D‑(‑)‑ribose, D‑ ( +)‑xylose, D ‑(‑)‑
arabinose, N‑Acetyl‑D‑glucosamine and N‑acetyl‑D‑galactosamine} and various dilutionsof antiserum were added into culture medium which consists of Eagle's Minimum Essential
Medium (MEM) with Earle's salt supplemented with 5% fetal bovine serum and 5% new born
bovine serum and used in feeder layer system while, a mixture of equal volumes of MEM and
Leibovitz's L‑15 medium supplemented with bathocuproine sulphonate and lO% fetal bovine
serum was used in feeder layer free system.
In feeder layer system 24 hr prior to parasite inoculation tissue culture dishes wereseeded with new born mouse brain cells. Parasites used were Tr)panosoma brucei gambiense
Wellcome strain (Tbg W) , obtained from blood of infected ICR mouse and separated fromblood components by chromatography method. Procyclic forms Tmpanosoma brucei gam‑biense TH‑1/78E were obtained from cultures. Other trypanosomes used for testifying theexperiments were Tmpanosoma brucei gambiense ILRAD 1582 and Tr)panosoma brucei
rhodesiense ILRAD 1501.
Initiation of the cultures was carried out by either replacing half of the old medium by

fresh one or by centrifuging the supernatant and return the pelleted trypanosomes. The
parasite density was determined from the supernatant fiuid.
Among the monosaccharides tested only mannose inhibited the growth of bloodstream
forms of trypanosomes but had no effect on the growth of procyclics. Mannan, Iectin andanti‑mouse brain cell rabbit serum had no effect on the growth of the parasites. This
inhibitory effect of mannose was presumed to have affected the parasites rather than FLC.
Thus, competitive inhibition test of glucose and mannose was performed, but even at the ratioof I : 40 mannose/glucose could not eliminate the inhibitory effect of mannose. The effect ofmannose and 2‑deoxy‑D‑glucose was also detected in feeder layer free system. Furthermore,derivatives of mannose were tested but most of them did not have any effect on the growthof the parasites. However, D‑mannosamine hydrochloride and a‑D‑mannose pentaacetate
inhibited the growth of the trypanosomes, but it was speculated that their mode of actions

were different from those of mannose and 2‑deoxy‑D‑glucose because they inhibited thegrowth of both bloodstream and insect forms and regardless of that, toxic effect was detectedon the FLC.
Electroh micrographs of trypomastigotes cultivated in culture medium mixed with 5 mM
mannose or 2‑deoxy‑D‑glucose showed degeneration of the glycosome, nucleus and kineto‑
plast.

Taking into account, the same mode of trypanosome growth inhibition in vitro and thesame morphological structure changes of trypomastigotes after treatment with mannose and2‑deoxy‑D‑glucose, it is assumed that inhibitory activity of mannose on bloodstream forms
resembles that of 2‑deoxy‑D‑glucose which inhibits metabolism of trypanosomes partly byaffecting some enzymes in Embden‑Meyerhof‑Parnas pathway.

103

48 STUDIES OF TWO ENZYME IMMUNOASSAYS FOR EPIMASTIGOTE
AND TRYPOMASTIGOTE FORMS OF TRYPANOSOMA CRUZIMASAYUKI IWAMOTO1, HIDEAKI TANlMORll, TSUNEHIRO KITAGAWA1,
MARCUS L. B. BARROS2 AND HIROJI KANBARA3
Department of Microbiological Chemistry, Faculty of Pharmaceutical Sciences,
Nagasaki University*, Department of Infectious Diseases, Institute of Manaus

Tropical Medicine, Amazonas University, Manaus, Brazil' and Departmentof Protozoology, Institute of Tropical Medicine, Nagasaki University'

This study was undertaken to develop new tools to study Tr)panosoma cruzi, thecausative agents of Chagas' disease. Every three forms in the life cycle of the Tulahuen strain

of T. cruzi, epimastigotes (EPD the living form in the vector Triatomine reduviid bugs,
trypomastigotes (TRP) the intertransmitting form between the bugs and mammalians orvarious tissue cells in their hosts, and amastigotes (AMA) the living form in mammalian cells

was separately cultured and then isolated. Each antiserum specific to EPI, TRP or AMA waselicited in rabbits. Three sensitive and accurate enzyme‑linked immunosorbent assays(ELISA) for EPI, TRP and AMA were developed using the corresponding antiserum with p‑
D‑galactosidase‑labeled anti‑rabbit lgG as the common second antibody and cell fragmentsof the corresponding parasites as antigens attached on Amino‑Dylark solid‑phase balls. The

working range of each ELISA was 5‑320 x 103 parasites/ml of EPI, and 1‑100 X 10' for both

TRP or AMA form parasites/ml. Accuracy of the assay results of each ELISA wasdemonstrated. It was found that ELISA for AMA was highly specific to AMA and contamina‑tion of various tissues of mice in assay samples gave no interference in the assay, proving thatthe direct assay of AMA present in various tissue cells was possible. Thus, mice were
inoculated with 10 parasites of TRP, and time courses in the increased numbers of AMA in
three tissues of the infected mice were assayed by the newly developed ELISA for AMA,
which was possible to apply starting from the 5th day after the infection to the 18th day when

all the infected mice were dead. Usual microscopic detection of the parasites in blood of theinfected mice was possible at the 8th day. The mice were sacrificed and organs werecollected. Thus, the first observations for time courses of infected parasites in heart (2‑400 X
10' parasites/g tissue) , Iiver (2‑350 X 106 parasites/g tissue) and spleen (8‑80 X 107 parasites/

g tissue) of the mice were successfully performed using the new tool.

JAPANESE JOURNALOF
TROPICAL MEDICINE AND HYGIENE
March 1989

Vol. 17 No. 1

CONTENTS
Original article

lwaki, M.
Morphological Observation of Larval Pupal Respiratory Apparatusof Mansonia (Mansonioides) unlformis (Theobald) (Diptera, Culicidae)

by Scanning Electron Microscope (in Japanese) """""""""""""""""

1‑7

Tao, D., Higasa, M., Miyamoto, H., Yamada, T.,
Komatsu, T. and Shinka, S.
IL‑2 Production Inhibitory Activity of the Sera in Tmpanosoma

brucei gambiense Infected Mice (in Japanese) """"""""""""""""""

9 ‑15

Matsuda, S. and Kahyo, H.
Enzyme‑1inked Immunosorbent Assay (ELISA) in the Detection of
Hepatitis‑B Surface Antigen in Blood Dried on Filter Paper " " " " " " " "17‑25

Shiota, T.
Diagnosis of Pneumocystis carinii in Sputum Liquefied with
the Mucolytic Agent Dithiothreitol (SputasoD ･･････････････････ ‑27‑31

Proceedings of XXX Annual Meetings of Japanese Society of Tropical
Medicine

Contents """""'

Special lecture """""""""""""""""""""""""""""""
Symposium I Tropical Infectious Diseases and Drugs """"""""""""II Present Status and Topics in Development of Vaccines
against Tropical Infectious Diseases """ """"" " " " """

33‑36
37‑39
40‑50

Panel discussion " "" """ '

51‑58
59‑68

General presentation " " " '69‑l03Published byJAPANESE SOCIETY OF TROPICAL MEDIClNE
c/o Institute of Tropical Medicine, Nagasaki University

12‑4 Sakamoto‑machi, Nagasaki 852, Japan