ISSN

Japanese Joumal of

TropicalMedicineand Hygiene

第9巻 第1号

昭和56年6月15日

内原O304−2146

容著
高知県における大榎殖門条虫症の第4例………………熊沢 秀雄，鈴木 了司，、大倉 俊彦 1−7

免疫抑制剤およぴD班usion

Chamber移植法によるラットの日本産カンテツに対する防御免疫効果の検討（英文）………一・……・・…………・……・…一「…一…・橋口

義久

9−22

高温環境下における連続7日間の運動が高温に対する生理的反応および耐熱性におよぼす影響（英文）………一一…・一・……………・………一一…・・………一・堀清記 23−29

狂犬病ウイルス免疫マウス脾臓細胞によるウイルス螢光フォーカス形成抑制（英文）一牧野芳大，三舟求真人

31−36

コガタアカイエカの幼虫の発育と成虫の属性に及ぽす温度と日長の影響・・Niaz，Sh．and会

Reisen，W．K．37−47

報
昭和55年度第2回幹事会記録一・…………・…・・…………一…・……一…………一・…………

49

昭和55年度評議員会記録・・……一……一・……………・・…・……………・・…・………………・50

第22回会務総会記録…………………………………一…一・・
51−53
会則…………・…一・・……………………………一…・…・…………一・……………・一・・54−57

投稿規定…・……・………・…………

58−59

会員名簿………一……

日熱医会誌Japan．J．T．M．H．

61−8g

日

本熱帯医学会

The

o伍ce

of

publisher would

like

to

apologize

to makeerrorsin thetext oftwo

pub−

lications which belong to Hidco Kumazawa，Ryoji Suzuki，Toshihiko Okura，，and
Yoshihisa Hashiguchi published in voL9，No．1respectively． Please correct the
mistakes

fbllowingtheerrata．

編集事務の誤りで9巻1号掲載の著者に御迷惑をおかけしました。別表正誤表に従って御訂正下さい。

1． 高知県における大複殖門条虫症の第4例
熊沢秀雄，鈴木了司，大倉俊彦（和文論文）
訂

1頁

正

所

誤

正

左段本文下から

3行目

特続

持続

〃 上から

11行目

1571

1971

右段本文上から

1行目

判断

診断

〃

3行目

同月29日午前7時頃一

〃 下から

10行目

〃

2頁

箇

左段本文下から 9行目

同月29日午前3時頃から複鳴持続し，午前7時頃に
6彩

0彪

縦走節

縦走筋，横走筋髄層の

横断図

横断面

〃

〃

7行目

〃

〃

5行目

腔

膣

3頁 右段本文下から

11行目

1981

1891

1行目

D．9鰯n伽五）．970n4ゴs

4頁 右段本文上から 3行日

海産類

海産魚

5頁 左段本文下から 4行目

下痢腹鳴

下痢，腹鳴

2例

2列

（Cestoda；Pserdophyllidea）（Cestoda；Pseudophyllidea）

0．加1伽80がθ7σ召0．わα」α8％0ρ∫87σ召〃〃

右段〃上から

1行目

文 献

9）

12）

14）
16）

22﹃⊥2﹃⊥−

5）目目目目目目行行行行行行1）

大複殖門条虫成熟虫寄生例

大複殖門条虫未成熟虫寄生例

k892

1892

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Additional

II

￠

cases

1）ゴ1）h夕〃060渉h短％吻

Additional

ll

cases

II. Examinations on the protective immunity to the common liver fluke, fasciola inrats, using immunosuppressants or diffusion chamber techniques

YOSHIHISA HASHIGUCHI
error

correct

batachbatch

lO

Seventh line from the bottomllFifth line ll

50 minutes

50 minutes.

Twentieth line from the Top

C‑1 X 100

C‑IjC X 100

blbumin,albumin,Figure 1

C: betaglobulin,

C : beta‑globulin,

Column i.n the Table 3

without chambers

without chamber

p 16 Fifteenth line from the Top

coparedcomparedColumn in the Table 5

No. serum sampler

No. serum samples

p 17

Figure 2

B : alphaglobulin

B: alpha‑globulin

p 19

Fourth line from the bottom

theat

that

p 21

REFERENCE 25)

Fasciola hepatic

Fasciola hepatica

p 22

l

Fasciola Repatica

Fasciola hepatica// // 1l F̲..
// T ) 8 ff

Ouchterlong

Ouchterlony// // 6 ff// // 5 tEl

Dexamethazdne

p 12

p 14 Eighth line
p 14
15

p

16

p

ll

ll

ll

ll

ll

ll

!> :

ll

J

)

) I ft// 4fT// 2 T: l i

j 1 e;(Z)Dexamethasone

1 & o

ll

Ouchterlong

Ouchterlonyllr‑globulin

r‑globulin

日本熱帯医学会雑誌

第9巻

第1号

1981

1

1−7頁

高知県における大複殖門条虫症の第4例
熊沢秀雄1，鈴木了司1，大倉俊彦2
和和56年2月9日

受付した。虫体から大複殖門条虫症と判断し，駆虫を緒

大複殖門条虫勧めたが患者多忙のためその後来院しなかった。
口Z）ψあgoηψor郷

同年2月下旬より飲酒の機会多く，同月29日午前

g順n漉5

7時頃に軟便とともに虫体を排出（標本番号2）。

（BZαηchαr4，1894）mhe，1899の人体寄生例は静さらに同年3月18日午前8時30分頃，下痢便とと岡，烏取，長崎をはじめとする19府県から，すでもに標本番号3a，3b，3cの虫体断片をこの順序に85例が知られている。しかしこの中には断片的で排出した。但し標本3bは患者が持参しなかっな記録しか残っていないものも多く，感染経路をたので測定することはできなかったが，患者によ含む疫学上の諸問題や，この条虫をめぐる分類学

れぱ約20cmの長さであったという。いずれの場上の問題は未解決のままであり，個々の症例を精合も虫体排出後，腹鳴は治まったが，軟便傾向は確に記録してゆく必要性は今も変わらない。

持続した。駆虫を勧めたがその後患者からの連絡

筆者らはすでに高知県から，本条虫の未熟虫体がなく現在に至っている。

寄生の2例を報告したが，本条虫の感染源が小型

理学的所見。特記すべきことはない。

の海産魚と推定されること（Kamoθ≠磁，1571）

尿検査所見。比重1．015。pH6．0。蛋白，糖，ケなどから，高知県における本条虫症例は少なくな

トン体，潜血反応，ピリミジン，いずれも（一）。

いことが予想された（熊沢ら，1981）。最近，ひウロビリノーゲン，正常。

とりの患者が前後少なくとも3回に亘って成熟片

糞便検査は行なわなかった。

節を自然排出した1症例を得たので，ここに報告

血液学的検査。赤血球数452×104／cmm．Hb
15．Og／d1．血色素指数1．04。白血球数9，100／cmm

する。

（好中球，1核3％，2核16勉，3核21％，他20％，症例○岡O。45歳。男。高知県安芸市赤野在住。農業のかたわらで養鰻および沿岸漁業に従事。

既往歴，家族歴には特記すべきことはない。
現病歴。生来格別病気をしたことはなかったが，リンパ球25％，単球7％，好酸球8勉，好塩基球6勉）。

血液化学検査。LDH210単位。GOT7単位。
GPT8単位。A1−P6．6単位。CHE

O．65∠。ZTT70単位。黄疸指数7単位。総蛋白量6．69／d1。
患者の嗜好。自宅の前の海で捕れたアジ，サバ

飲酒後下痢をするのが常であった。1980年1月26の刺身やイワシの酢物を常食し，好物のカッオを日から連夜飲酒，同月27日，下痢便とともに虫体除き市場の海産魚介類は食べない。川魚は食べな（目測で約1m）を自然排出したが捨てたという。

い。豚肉は好食するが牛肉は特に好まない。養鰻

この頃から腹鳴が特続するようになり，時折腹痛のためイトミミズを誤まって口にすることがあっもあったが放置。同月29日早朝，虫体（標本番号た。

1）を下痢便とともに排出し，虫体を携えて受診

1高知医科大学寄生虫学教室 南国市岡豊町小蓮

2

高知学園短期大学

高知市旭天神町2
Table1

Measurements（μ）of

the

DψJo−

80吻o驚specimen＊。

虫体観察所見回収した虫体は次の4断片で，これらはすべて，

segment
length955

クロロホルム加冷水道水で弛緩させて計測したの

width

17800

ち，5％ホルマリンで固定した。

thickness600

標本番号1長さ494cm，最大幅19．5mm（前端から300cm）。

標本番号2長さ352cm．最大幅20．5mm（前端から280cm）。

標本番号3a長さ310cm．最大幅21．5mm（前端から260cm）。

標本番号3c長さ111cm．最大幅13．Omm

distance

betweentwo genital organs
genitalorgan

9・o・andthicknessnerve

excretorycanal

tegument

1．9± 0．6
44．3ニヒcortical

89．2二Lparenchyma

tranSVerSe mUSCle

よりやや小さい。いずれの断片も2列の生殖器列nerve

縦径よりも横径が大で，ストロビラの縁はやや鋸歯状を呈し，体表面には，生殖器と神経幹にそれ

±95
±141
l390 士190

2240

tCgUment Cell layCr

このうち3aと3cは連続して排出されたもの
で，3cの後端の幅は3aの前端の幅（16．Omm）

ずれの断片にも頭節はない。どの断片でも片節は

trunk

of

longitudinal

剰生殖器を含む片節（Figure1）が見られる。い

4500

and

（後端）。

を有するほか，2，3a，3cの断片では1〜3個の過

士95
士271
±35

musclemedullary parenchyma

74．7ニヒ8．4

108．〇ニヒ

21．8

trunk，diameter
dorsoventral

69．4土

6．5

horizontal

86．4土

9．1

cirrussac

length286．1土 18．1

width

240．4土

6．9

6．5±

3．4

wall thickness

117．3士

2〜4条，神経幹より外側に片側5〜7条の極めて弱い縦溝が認められる。両神経幹の間の縦溝は背

width

87．3土

wall thickness

19．2土

Table1は虫体断片1の最大幅付近の切片標本についての計測値である。片節の幅径は縦径のVeSiCletestis，diameter

18﹃︶length589

Seminal

生殖器間に5〜7条，生殖器と神経幹の間に片側

いo

7．3

35．0士

ぞれ対応する部位にやや深い縦溝があるほか，両腹で必ずしも一致せず，縦溝の数は腹側がやや多

7．0
20．5

dorsoventral

45．5士

7．5

horizonta1

62．1ニヒ10．7＊expressedasmean±standard

deviation．

18．6倍，生殖口間の距離は片節幅の25鰯となる。
虫体の断面は外側から角皮，角皮下細胞，皮質層，りに一層に配列し，横断切片では背腹に扁平な楕

縦走節，髄層の各層から成る（Figure2）。

円形で，両陰茎嚢の間に28〜31個，陰茎嚢より外1対の神経幹と2本以上の排泄管が髄層内にあり，前者の横断図は背腹にやや扁平な楕円形であ側に片側6〜31個が数えられる。精巣の配列は片節間で中断することなく連続している。

る。貯精嚢は陰茎嚢の背後方に，これと鋭角をな

卵黄腺は角皮下細胞近くの皮質層内にあり，圧して接続する。輸精管と腔は共に生殖賓に開口し，平標本では径32〜81μ（平均52μ）の不規則な後者は生殖口として片節縦径の前端から約1／3の円形をなし，その分布は陰茎嚢や子宮と重なるこ部位に開口する。子宮口はそれより約200μ後方とはなく，また陰茎嚢の前や子宮の後で左右が連にある（Figure3）。

続することはない。他の部分では卵黄腺は均一に

精巣は陰茎嚢の周辺を除く髄層内のやや背側寄分布する。子宮ループは片側で2〜5本が認めら

、‡蒙

拠

繍蕪i難

灘灘

菱

難一灘
鰻

，鑓、

轍尋翼

■

騒…

麟

難撚

1、・欝纏
譲

Figure1 Apartofthestrobi拾窺o．2ne轟ritsmaximumwidthshowingfburrowsofgenitalorgans．

Stained

w三th

alum

carm圭ne、

×23・

Figure2 Tran呂verse scction of the strobila n甑L Stained wi皇h Mayer五ematoxylin鋤deosin、

Figure3 Sagittal

×67．
section of thestrobi1包uo．L Stained with M＆yer hematoxylin and eosin．

x67．

Abb童eviations c，cortic拭1parenchyma； co，cirrus openingl csラcir罫u8言acl
ga，genital aもrium； 9P，琶eniね1pore； 1m，10ngitudinal muscle3 m，medullaryparenchyma；

sv，餐eminal ves量cle茎transversemuscle；ts｝testまsl

tうte藝ument l tc，tegument cell layer｝ tmコ

uo，uterineopenin導lvt，vitellarial

vo，vagまnal

opening，れる。子宮内には大きさ63．4〜77．9×3L2〜46、8μ（平均71，7×36、8μ）の虫卵がある。

1971）が，これらの症状と飲酒との関連は，今回の症例の場合明らかでない。今回の症例でも，本条虫の寄生が貧血や炎症その他の症状をひきおこ

考察すという証拠はない。

大複殖門条虫はヒゲクジラ類寄蛋のD，b認昭ル

大複殖門条虫の人体症例は197i年までに55例が

砂孟ぴα8（Lδ無berg，1981）と形態的に区別できな

知られていた（Kamo就認．，1971）。その後の症例いといわれる（Rausch，19641岩田，ig67）、鰭脚をT＆ble2に，主として発表順序に従って要約し類やラッ瓢寄生のDj8俄ψ≠εr欝（v、Siebold，

た。本論文の症例を日本における第85例として扱i848）が人体寄生する可能性もある（Rausch，う。このほか静岡より1例が近く報告される筈で1964）。今回の標本は虫体が大きいこと，卵黄腺（影井，私信），これを加えると全87症例となる。

の分布が子宮ループと重ならず，片節の前縁や後高知からは197工年以前には岩田・安岡（1970）の縁で連続しないこと，卵黄腺が大きいことなどの1例があるのみで，本論文の症例は高知の第4例

点でn観rヒ膨εr螂よりはD．ゐα伽塑ρ魏α8に似となる（高知の第2，3例はTable2）。
本症例でみられた下痢，腹鳴，腹痛の症状は，

従来もしぱしば報告されている （Kamo撹磁，る。人体寄生のものを後者と同一視することには未だ疑問があるので，今回の患者から得たものは
大複礁門条虫D遭灘ηぬ5と同定した。

4
条虫の成長は速く，たとえば広節裂頭条虫は犬可能性も全く否定はできない。

体内で毎日，前日の約1．4倍の長さになり，人体感染源については従来の各症例報告と同様に明内での絶対成長速度は犬でのそれの3倍を越える

らかではないが，本報告の患者が新鮮な海産類をといわれる（Wardle and Green，1941）。このこ

常食していたという事実は，過去の大複殖門条虫とから，大きな虫体断片が相次いで排出されたと症例が西南日本の海に面した府県に集中して分布しても，それらはすべて同一個体由来かもしれな

していることと対応している（Figure4）。感染い。特に虫体断片2，3a，3cは過剰生殖器の出現

源がカッオかアジなら初夏，サバかイワシなら秋の様子が類似しているのでその可能性は十分に存

から冬に感染する機会が多いものと思われる。感する。一方，患者の嗜好から言って，複数寄生の染から虫体排出までの期間は，広節裂頭条虫ではTable2re琵rcnceof1）ψ10goηψ01謝infヒction，recognizedpatient

after197L
ユ

age

sex

1ength

m

十

十

十十

m

5ワヘ

mff

m
m
m
m
m
m

scolex

05

m？f

m
m
m

500

m
m
m
m

width
（mm）

61
翫1
21
89751
31
41
11
081
32
1851
172
51
882
01
2771
72
557？

m？

m
m
m
m
m

15
0
4
8
8
0
0
2
0
8
6
4
6
3
0
2
76072
01
5
64
5
31
3
13
7
35
11
14
7
32
3
26
14
9
15
5
19
12
1
98
7？
3
4
1
1
1
3
1
2
1
3
1
1
2

（cm）

mf

2
3
0
8
131
8
8
P
655
8
9
0
6
302P2
63
43
44
63
43
24
35
57
44
54
53
47
75

locality

SNNNSESTTTTTTSTTSESS
713
2
3
6
6
6
6
1133
11
11
116
116
116
1 6
16
111
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1 66411

6
76
86
97
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37
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5
57
58
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50
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62
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666

灘鮮㎞藷鞭㎞羅灘㎞灘灘解

n。．

Cases

十

5

d
〃
6
2

5

1分

494

1

15

1

1レ

41

4
4

1

1

3

Figure4

Distribution by

prefbcture1

2

incidence

of

20

1

diplogonoporiasis．

出までの期間は条虫類一般にもっと短かい（An・mm，13．Ommで，いずれも2例に配列した完成
した生殖器を有し，後3者の断片では1〜3個のdersen，1978）．Kamo8君α乙（1971）は大複殖門条

過剰生殖器を備えた片節があった。どの断片にも虫の症例数のピークが南から北に次第に遅くなる

頭節はなかった。

約80日という事例があり（吉田ら，1979），卵検

4．本症例は高知県における第4例目である。

ことと関連して，魚の消費量が秋から冬に最大となることに注目している。今後も，感染源を明らかにするために，個々の症例をさらに検討する努謝

力が必要であろう。

辞本研究の逐行に当り終始ご協力いただいた高知

要

医科大学寄生虫学教室，橋口義久博士と吾妻健博

約士，症例数にっきご教唆いただいた国立予防衛生

1． 高知県安芸市在住の45歳男子より1980年1

研究所，影井昇博士，種々ご助言いただいた高知

月29日，2月29日，3月18日の3回に亘り自然排

医科大学第二内科学教室，宇賀茂敏博士に深謝す

出された4虫体断片を大複殖門条虫と同定した。

る。

2．下痢腹鳴，腹痛のほかは，特別な症状はな

本論文の要旨は第36回日本寄生虫学会西日本支かった。

部大会において発表した。

3．虫体断片は長さがそれぞれ494cm，352cm，310cm，111cm，最大幅が19．5mm，20．5mm，21．5

文1）

Andersen，K．（1978）：The

development（Cestoda；Pserdophyllidea）in

its

terns

of1》

U1一↓20献ofdefinitivethetapeworm

hosts，with

4εn4ブ露づo％ 2（Nitzsch，1824） andDψゐ郵060≠hr伽御1α如勉（L1756）

specialreferences

to

the

growthpat−Z）．4髭アr8辮％ z（Creplin，1827）ン Parasitology，77，

62）Hatsushika，R，Yamane，呈，Maejima，J．and
infectionwithD弼ogonψo欄g厩π廊，Yonago

3） Hatsushika，R．，Yamane，Y．and

Ehime

tenth

case

of humanhuman

infection

Acta Med．，18，78−83

Yoshida，R．（1977）：A

D弼ogoηqρo朋grα顧5（Cestoda）in

4）

Kanno，S．（1974）：The
case

Prefecture，Kawasaki

of

with

Med．J．，3，231−236

平井和光，酒井雅博，阿波井五郎（1976）：大複殖門条虫について，愛媛県における大複殖門条

虫寄生の第4例，農村医学，25，599−6035）

岩田正俊（1967）：大複殖門条虫1）ψZogoηqρorμ59耀n4ま3（Blanchard，1894）LUhe，1899は，鯨条虫D．わαZαηθρρ∫87αθLdnnberg，1892とすべきである，動物分類誌，3，20−24

6）岩田正俊，本田龍三，鈴木7）

岩田正俊，安岡五良（1970）：複殖門条虫の高知県における人体寄生1例，寄生虫誌，19，417−418

8）Kamo，H．，Hatsushika，R．and

cestodes9）inJapan，Yonago

木船悌二，辻卓（1972）：複殖門条虫の人体寄生1例，寄生虫誌，21（2，補），40
Yamane，Y．（1971）：Diplogonoporiasis and diplogonadic

Acta Med．，15，234−246

浩一（珍80）：大分県から見出された大複殖門条虫成熟虫寄生例，日本寄生虫学会西・南支部合同大会講演要旨，p．19
10） 熊沢秀雄，鈴木了司，近藤慶二，塩見文俊，田口博國，紙谷晋吾，井上文之（1981）：

高知県における大複殖門条虫症2例，寄生虫誌，30（印刷中）11）

望月 久（1976）：静岡県における大複殖門条虫について，静岡衛研報告，19，41−44

12）

Rausch，R、L．（1964）：Studies

of

the

on

the

helminth

fauna

of

AlaskaXII．Observations

genus Z）ψZogoηoρorμ3L6nnberg，k892（Diphyllobothriidae），

on

cestodesCanad．J，ZooL，42，1049−

1069
13）

坂口祐二，野島尚武，提 恒雄，中島昌夫，中島正敏（1974）：大複殖門条虫の人体寄生例追加，
熱帯医学，16，27−33

14）Wardle，R．A．and」

15）

Green，N．K（1941）：The

rate

of

growthofthetapeworm

Dゆhメ励o孟hr魏z伽規（L．）， Canad．J．Res．，D，19，245−251

山本真志，今岡 誠，松本慶蔵，中島康雄（1976）：小腸透視にて確認した大複殖門条虫の人体寄生例，熱帯医学，18，179−182

16）Yamane，Y．，Maejima，J．，Yazaki，S．and
gonoporiasis in

Sanin

Fukumoto，S，（1977）：Additional

districts，Yonago Acta17） 吉田幸雄，松尾喜久男，猪飼

剛，山田

ll

cases

of

diplo−

Med．，21，19−25

稔，竹内滋，荻野賢二（1979）：京都における広節裂頭条虫症の研究，寄生虫誌，28（2，補），59

THE FOURTHCASE OF DIPLOGONQPORIASIS
IN

HIDEo

A

total

offburMarch

PREFECTURE

KuMAzAwA1，NoRIJI SuzuKIl
AND TosHIHIKo OKuRA2

Received

ary29and

KOCHI

fragmented

l8in1980，fromfbrpublication9February

strobilae，evacuateda45year−old

man

l981

spontaneously

living

in

on

KQchi

January29，Febru−

Pre飴cture，werc

7
identified as Diplogonoporus grandis (Blanchard, 1894) Luhe, 1899. They were from 1 1 1

to 494 cm in length, and from 13.0 to 2 1 .5 mm in the maximum width, all with two rowsof complete genital organs, and three containing proglottides exhibiting supernumerary
genital organs. The scolex was absent. The patient suffered from diarrhea, abdominal
pain and borborygmus. Fresh raw fish was taken frequently by the patient. This is therecord of the fourth case from Kochi Prefecture, Japan.

1 Department of Parasitology, Kochi Medical School, Nankoku, Kochi 78 1‑5 1 , Japan

Gakuen College, Asahi‑tenjin‑cho; Kochi 780, Japan.

2 KochiJapan. J. Trop. Med. Hyg., Vol. 9, No. 1, 1981, pp. 9‑22

9EXAMINATIONS ON THE PROTECTIVE IMMUNITY TO
THE COMMON LIVER FLUKE, FASCIOLA IN RATS,
USING IMMUNOSUPPRESSANTS OR DIFFUSIONCHAMBER TECHNIQUESYOSHIHISA HASHIGUCHI
Received for publication 16 February 1981

Abstract: The current study was designed to obtain an information on the immune
factors which could be underlying the induction of protective immunity to the genus
Fasciola in rats, using immunosuppressive drugs and diffusion chamber technique. The
rats became resistant to challenge with the Japanese species of Fasciola following an oral

infection with metacercariae, indicating a significant degree of reduced worm burdens.
This protective effect in the immunized rats, however, was abrogated by the administrationof immunosuppressants, dexamethasone and prednisolone. In rats treated with thesedrugs, no antigen‑antibody band in immunodiffusion plate was recognized throughout
the infection, while many bands were observed in sera from the rats without treatment
with the drugs. From the results obtained, the antigen‑antibody bands in diffusion plate

would be a good indicator to estimate the degree of protective immunity to the genus
Fasciola in host animals. By intraperitoneally implanting the immature worms in diffusion

chamber, the rats were able to induce a significantly high degree of protective immunity

when compared to the natural‑immunity control. However, the degree of protective
effect was significantly high in rats which received free worm (without chamber) implan‑tation. In the experimental rats, each fraction of their serum proteins was also examinedby performing cellulose acetate electrophoresis. More detailed examination is required
to investigate the immune factors in the protective immunity to the fluke in rats, using
immunosuppressive drugs and/or diffusion chamber techniques in terms of host‑parasite
relationship.

INTRODUCTION
The Japanese common liver fluke, Fasciola sp. indicates a similarity to F. hepatica
and F. gigantica in the migration and development in the final hosts (Ono and Isoda,

1952; Mimura, 1961), though its taxonomical status has not yet been established(Watanabe, 1965). So far as is known, Iittle immunological study on the hosts
infected with the Japanese Fasciola species has been carried out in terms of the pro‑tective immunity to the fluke. The protective immunity of rats against F. hepatica.

on the other hand, has been widely recognized (Hayes et al., 1972 ; Goose andMacGregor, 1973 ; Armour and Dargie, 1974; Rajasekariah and Howell, 1977, 1978).
This immunity is induced not only by the oral infection of the normal or irradiatedmetacercariae but also by the implantation of the different ages of the liver fluke in)epartment of Parasitolo Y, Kochi Medi̲cal School, NankQku City 781‑51, Kochi, Japan.

10

rats ,(Thorpe and Broome, 1962; Eriksen and Flagstad, 1974; Anderson et al., 1975;
Rajasekariah and Howell, 1978). Moreover, the resistance ofrats to reinfection with
F. h̲ppatica is also stimulated by the transfer to naive animals with the serum
or lymphoid cells from infected ones (Corba et al., 1971 ; Armour and Dargie, 1974;
Hayes et al., 1974a, b; Dargie et al., 1974; Howell et al., 1977; Rajasekariah andHowell, 1979). Thus, so far, the previous works have provided much important
information on the irnmunological factors involved. The factors, however, shouldstill be clarified to have a better understanding of the mechanisms which could beunderlying the induction of protective immunity to Fasciola in host animals.
In the immunizing infection with the subcutaneous implantation of imlli'ature

worms, 4 week‑old, Rajasekariah and Howell (1978) have observed a significant
degree of resistance to F. h""patica in rats. According to Lang et al. (1972), moreover,the immunization with different ages (8 and 16 day‑old) of worms produced a sig‑
niflcant reduction in challenge worm burdens when compa,red to natural immunity(challenge) controls in mice. By subcutaneously implanting adult worms, on theother hand, Eriksen and Flagstad ( 1974) and Anderson et al. (1975) also showed somedegree of protective immunity to challenge infection in rats, although Rajasekariah

and Howell (1978) could not recognize any protection following subcutaneous
implantation with the adults. In this immunity, but, the duration of liver migration

by young worms may be of greater importance in stimulating the protective im‑munity , than is worm age (Lang, 1974). From these facts, anyway, there may be
several factors to induce protective immunity in relation to the age or liver migrationof immunizing worms in the host.
In the series of experiments reported hitherto, the observation seems to indicate

a complexity of the protective immune factors involved. The present study, there‑
fore, attempts to stimulate the hosts (rats) by introducing living parasite containedin a millipore diffusion chamber. In this method, a variety of soluble antigenswou]d become effective, but the inability of the liver fluke to infect the host would
reduce or eliminate all of the pathology usually associated with the infection, without

liver and/or bile duct migration of the worms. In addition, the current work is
designed to determine the effects ofimmunosuppressants on the induction ofprotective
immunity to the Japanese common liver fluke in rats.

MATERIALS AND METHODS
Animals. Male, around 250 g Wistar rats were used throughout the experiments.
They were fed a commercially prepared diet and received water ad libitum.
Parasites. A11 the Japanese species of the genus Fasciola metacercariae werefrom the same batach. They were col]ected from experimentally infected Austro‑
peplea ollula (Lymnea ollula) and administered per os to rats in normal saline, using an
injection syringe with a slender vinyl tube.

Immunosuppressive drugs. Dexamethasone (Merck Decadrone) and prednisolone(Merck Codelcortone) were simultaneously administered into alternate thigh musclesof rats beginning one day before immunizing inoculation of the metacercariae, andcontinued every five days until necropsy. The dosage levels of these drugs are11

1 mg/rat in Decadrone and 5 mg/rat in Codelcortone.
Preparation and implantation qf deffusion chambers into rats. Diffusion chambers(Millipore Filter Corp., Bedford, Massachusetts) were made of diffusion chamberring (Plexiglass U‑100, diameter: 10mm, thickness : 2 mm) and millipore filter(pore size: 14 p). They wdre closed with millipore glue after introducing the flukes
in sterile saline; the immature, 25 day‑old, worms isolated by peptic digestion fromstock‑infected rats, were introduced aseptically into the chambers. Rats that IA ere
to receive diffusion chambers A'ere anesthetized with ethyl ether during operation.

The abdominal area of the rats was washed with 700/0 ethyl alcohol and a small
incision was made either to the right or left of center. A single chamber including
two worms was then placed intraperitoneally into each rat with sterile forceps. The
incision was closed with a sterile surgical suture and then the area was washed again
with 700/0 ethyl alcohol including iodine tincture. It was observed that 100 , ofthe rat. s survived in these operations.

Experimental design. The experimental design consisted of three series. In
the Ist series of the experiment, the degree of protective immunity to the Japanese
species of Fasciola in rats was ascertained by the immunizing infection with themetacercariae per os. In the 2nd, an attempt to induce the protective immunity was

made by intraperitoneally implanting the immature worms contained in diffusion
chambers, and then the recipients were challenged with the metacercariae. In
the 3rd, it was decided t,o assess the effects of immunosuppressants on the induction ofprotective immunity to the liver fluke in the animals infected experimentally.
Student's "t" test was used to compare worm burdens between groups. Probability
values larger than 0.05 were not considered signiflcant. The standard deviation of
the mean was calculated for mean worm burdens.
Autopsy. All rats were killed 2 weeks after challenge infection with the meta‑cercariae, unless otherwise mentioned. In eac'h examination the peritoneal cavity
was opened, and the surface of the liver and other visceral organs was inspected
for haemorrhages. The peritoneal surface was flushed with normal saline and thewashings were examined for free worms under a dissecting microscope. A11 of theinternal organs were removed separately, then washed with normal saline severa]
times to recover free worms. The liver and bile ducts were examined for migrating
immature worms and/or mature ones. The liver was minced with scissors in petri
dishes. In order to obtain the penetrating worms, the minced liver in saline wasincubated at 37 C for 2 hrs; the incubation revealed that the liver was fre̲.e fromany worms.

Serum examination. Blood samples were collected weekly intervals by cutting

the tail part of rats. All sera were analysed with a cellulose acetate electrophoresis

cell (Model SE‑2, Toyo Kagakl4 Sangyo) supplying constant current. Serum samples
were placed on a cellulose acetate membrane (Separax: Joko Sangyo Co. Ltd.), andthen subjected to 0.8 mA per I cm membrane for 50 minutes The dried strips ofcellulose acetate membranes were scanned in a Densitordl DMU‑2 (Toyo Kagaku
Sangyo), and the relative percentages ofalbumin, alpha‑, beta‑ and gamma‑globulins

were determined.
Double immunodlffusion. Ouchterlony plates were prepared by flooding 5 by

12

1 1 cm slides with 9.5 ml of 0.90/0 agar. The central well was filled with the Fasciola

antigen and the other wells with serum samples of rats from each experirnent. The
position of precipitin bands was recorded by drawings and photograph.s.
Worm antigen. Adult worms of the common liver fluke, recovered from the bileducts ofstock‑infected rats and stored at ‑20 C, were added to 10 volumes (WjV) of

phosphate buffer, pH 7.4. The worms were homogenized fbr 5 minutes at 4 C andcentrifuged for 15 minutes at 3,000 rpm. The supernatant was stored at 20 C
as the antigen for the agar double diffusion technique.

RESULTS
Worm recovery from immunized and non‑immunized rats 2 weeks ajler challenge. At
2 weeks after challenge all experimental rats had worms from immunizing infectionin their bile ducts. In this series of the experiment, the immunized rats received
challenge infection with the metacercariae 6, 7, 8, 9, I O and 1 1 weeks after immunizing(Ist) infection. As recognized in Table 1, the experimental rats harbored fewer
challenge worms than the natural‑immunity control, apart from the rats received
challenge 1 1 weeks after immunization. The challenge (natural‑immunity) controls
harbored 3.6 worms in mean number, whereas the animals challenged during 6 to 9
weeks after immunizing infection harbored only 0.7 to I .5 worms. The percentage
immunity to the Japanese species of Fasciola in rats was calculated by the following
formula, C ‑ I x I OO, where C indicates a mean worm recovery of challenge control

and I shows a mean recovery of worms from challenge inf'ection in immunized rats .
The challenge infection from 6 to 9 weeks after immunization, revealed a higher
percenta e immunity ranging from 58.3 to 80.6 than the natural‑immunity control.

These differences m worm bu den are highly srgmficant (Student's '' ,,test, P <0.001), but the challenge at 10 weeks later had trended to indicate a gradual decreasein the degree of protective immunity to the liver fluke in rats. Even in the rats which
received both immunizing and challenging infections, there was no difficulty in the
Table I Summary of the percentage immunity to the Japanese Fasciola in the rats im‑
munized with 4 metacercariae each per os before challenge infection.

Times (wks) No.
of rats
No. worms
recovered from challenge
No. Iarvae

ercentage

challenge after used Total
challenged
rmmunity*
Mean
s.d.

immunization ( o//o )
6

5

25

5

25

8

4

20

6

l.5 l. 12

58.3

9

3

15

2

O. 7

0.47

80.6

lO

3

15

8

2.7 0.47

25.0

11

3

15

13

4･3

I .25

Challenge control 5

25

18

3.6

0.49

4
4

0.8

0.40

77.8

0.8 0.75

77.8

0.0

* Percentage immunity was calculated by the formula, C‑1/C x 100 (Miller and Smithers,
l 980)

13

differentiation of the worms between the two infections, because of autopsy at the earlyphase (2 weeks) of challenge infection. Thus, the results mentioned above havedemonstrated a significant degree of worm reduction from challenge, by the immuni‑
zation with the Japanese Fasciola metac rcariae per os.
Immunization by intraperitoneally implanting worms with and z )ithout deffusion chambers.

The induction of protective immunity which was observed by the immunizing infection
with the metacercariae per os, was examined using millipore diffusion chambers in

relation to the liver migration and/or the ages of worms. The rat immunized by
implanting worms was challenged 3 weeks after immunization, and autopsied 4
weeks after challenge. In the free worm implantation (without chambers), thedifferentiation of worms between the immunizing and challenging infections was
performed according to the size of the worms recovered. In Table 2, the protective
immunity to the Japanese species of Fasciola in rats is shown by performing im‑plantation of the worms with or without chambers. In the experimental rats, theimmunized groups indicated a significant degree of protective immunity by the
immunizing infection (implantation) of the immature (25 day‑old) worms in bothwith and without chambers. The percentage immunity obtained by the before
mentioned formula, was 50.00/0 in the immunization with chambers and 88.20/0 inthat without chambers, respectively. In the worm burden, again, the immunized
rats harbored only I .7 and 0.4 worms in the corresponding immunizations, whereas
the rats in sham operation and challenge control showed the worm recovery of 4.2
and 3.4, respectively. The differences found in worm burdens between the im‑munized, and the non‑immunized (sham) and challenge control are highly significant(P<0.001), but there is no significant difference between sham operation and chal‑lenge control. With implantation of the worms contained in diffusion chambers,
the current study demonstrated a significant degree of protective immunity to theJapanese Fasciola species when compared to the natural‑immunity control. The
degree, however, was significantly higher (P<0.001) in the immunization with free
worm implantation than that with enchambered worm ; the implanted worms could

Table 2

Results of the induction of protective immunity by intraperitoneally implanting

the immature worms, 25 day‑old, with and without diffusion chambers in rats.

Times (wks) Times (wks)Groupof challenge

of autopsy

No.
rats

No. worms fromchallenge

No. Iarvae
challengedPer‑centageimmunity

aft.

aft.

implantation

challenge

3

4

7

70

12

1 .7

0.45

50.0

3

4

5

50

2

0.4

0.49

88.2

3

4

5

50

21

4.2

0.75

0.0

4

7

70

24

3.4

0.49

used

Total Mean

s.d.

(o/o)

Immunized *With chamber
Without chamber(free worms)

Non‑immunizedSham operation(chamber only)
Challenge control* Viable 3 worms each were implanted intraperitoneally in rats.

14

survive 3 to 4 weeks or more in the diffusion chambers placed intraperitoneally inrats.

The results of serological analysis on serum samples from the rats that received
worm implantation with and without chambers are shown in Figure I , using cellulose
acetate electrophoresis. In the case, the percentage value of each fraction in serum

proteins was also determined in the samples from rats received sham operation,
taking the blood 2 weeks after operation. The values obtained were 53.5 3.450/0
in blbumin, 13.9 1.900/0 in alpha‑globulin, 6.8 0.250/0 in beta‑globulin and19.5 0.650/0 in gamma‑globulin, respectively, and the average of albumin per
globulin (A/G) ratio was I .2 0.15. In comparison with the percentage values insham operation, the values in both groups showed a remarkably higher or lower
value in each fraction, demonstrating a great fluctuation. The weekly changes inthe values, however, were similar in the serum samples from the two experimentalgroups. It was noted that the percentage value of albumin indicated an increase
S
t

e

E

o
(%)
40

, ,,,,, ,b,,, D

20

e

o

20

c

o

,

+

20
B

o

60

lllt ""I"̲"̲'̲1't"̲' f

40 't...,b

,,

A

10

o

5

3
We e ks

Figure l

Weekly changes in each fraction of serum proteins in thesamples from rats received immunizing implantation of theimmature worms with ( ) and without (‑‑‑‑) chambers.
A : albumin, B : alpha‑globulin, C : betaglobulin, D :gamma‑globulin, E : AjG ratio, S : mean value of shamoperation with empty chamber 2 weeks later.

15

after 3 weeks when the rats were challenged, showing the peak at 5 weeks after
immunizing infection. In agar double diffusion, almost all of the serum samples

showed only one precipitin band in the rats received worm implantation withchambers, while 2 or 3 bands were observed in the samples from the animals implan‑

ted worms without chambers (Table 3). It was noted that the number of bands inthe latter animals has increased gradually after challenge infection.
Table 3 Number of antigen‑antibody bands appeared in agar double diffusion plate inserum samples from rats immunized by intraperitoneally implanting the im‑mature worms with and without diffusion chambers.

No.
scrum

Weeks after immunizing implantation

Group samples
in each
test

1

2

4

5

6

7

Immunized *With 1 1 1 5 1 O 1 1 1 1 1 1 1, 1,0,0 l, l, l, l l 1, 1, l, l, 1 2, l, 1, l, 1chamber

Without 1 15 1 O O 1 1 1 1 O 2 2 1 1 O 3 3, 2,2, l 3 3, 2, 1, 1 2, 2, 2, l, 1chambers* The rats were challenged with Fasciola metacercariae 3 weeks after immunizing implantationof 25 day‑old worms with and without chambers.
** Number of precipitin bands appeared.

Effects of immunosuppressants on the induction of protective immunity. To determine

if the protective immunity observed would be influenced by immunosuppressive
drugs, the experimental rats which received immunizing infection with the meta‑cercariae per os, were treated with dexamethasone and prednisolone throughout theexperiment. The drugs were in.jected intramuscularly into the animals 5 day‑intervals. The rats received the immunizing infection were challenged 6 weeks laterwith the metacercariae, and then necropsied 2 weeks after the challenge infection.
At autopsy all the experimental rats contained worms from immunizing infection in

the bile ducts. Table 4 shows the results of this experiment. In the immunizing
infection, the non‑treated rats (Group II) with immunosuppressive drugs harbored
only 0.7 worms, whereas the animals treated (Group I) had 3.3 worms from challenge
infection. The percentage immunity of rats in Group 11 was significantly high(P<0.001) when compared to both the Group I and natural‑immunity control. No
signiflcant difference, however, was recognized between Group I and the control.
These results demonstrated that the immunosuppressive drugs, dexamethasone andprednisolone, could greatly suppress the induction of protective immunity against
the Japanese species of Fasciola in rats. In the immunizing infection (Groups I andII), antigen‑antibody systems in the animals were examined by performing Oucht‑
erlony technique. The formation of precipitin bands in agar double diffusion
plates is shown in Tab]e 5. Each test was carried out at weekly intervals from I to
8 weeks after immunizing infection with the metacercariae. All the serum samples
in Group I showed no precipitin band throughout the experiment, while all serum

16

samples taken weekly intervals, apart from those I week later, in Group 11 produced

1 to 4 bands. In non‑treated animals with immunosuppressive drugs, the bands
were first found at 2 weeks and reached to the peak in numbers at 6 weeks when thechallenge was performed, thereafter they had a tendency to decrease gradually innumbers. No stimulation was recognized in the activation of antigen‑antibody
systems in the rats by challenging infection during the period observed.
The results of serological analysis of the samples from rats treated with (Group I)

and without (Group II) immunosuppressive drugs are depicted in Figure 2. In
the examination of normal (non‑infected) rats, the percentages of each fraction were53.1 3.790/0 in albumin, 18.7 2.000/0 in alpha‑globulin, 5.3 1.620/0 in beta‑
globulin and 18.9 5.000/0 in gamma‑globulin, respectively, and the average of al‑bumin per globulin (A/G) ratio was I . I 0.16. These average normal values arealso shown in the same Figure. The gamma‑globulin fraction in sera from treatedanima]s (Group I) showed a consistently low percentage, while it represented a highvalue in the animals without treatment as copared with the average normal value.
In other globulin fractions, there was a remarkable difference of alpha‑globulin be‑
tween both groups in the early time of immunizing infection during I to 4 weeks ;Table 4 Worm recovery from rats treated with (Group I) and without (Group II)
immunosuppressive drugs ; all the rats received immunizing infection with 4
metacercariae each per os, and then challenged with 10 or 20 Iarvae each 6 weeks
later.

No. worms from challenge Percentage

Group No ratschallenged
used No.
Iarvae immunity
Total
Mean s.d. (o/o)

I*

7

80

23

3.3 0.70

0.0

II**

12

1 40

8

O. 7

0.62

79.3

Challenge control 1 7

220

55

3.2

0.8 1

* Dexamethasone ( I mg/rat) and prednisolone (5 mg/rat) were simultaneously injected into
alternate thigh muscles of rats beginning one day before immunizing infection with themetacercariae.
** Non‑treated with any immunosuppressive drugs; the rats only received both immunizing
and challenging infections with the metacercariae.

Table 5 Number of antigen‑antibody bands appeared in agar double diffusion plate inserum samples from rats in each group treated with (Group I) and without(Group II) immunosuppressive drugs.

No. serumGroup sampler in

Weeks after immunizing infection

each test l

2

3

4

5

6

7

8

I

3

O, O, O* O, O, O O, O, O O, O, O O, O, O O, O, O O, O, O

O, O, OII

5

O, O, O 3, 3, 3 3, 3, 2 3, 3, 3 3, 3, 3 4, 3, 3 3, 2, 2

2, l, 1

O, O 2, 2 2, 2 2, 2 3, 3 3, 2 2, 1

l, l

* Number of precipitin bands appeared in each sample.

17

N
,

1

E
o

(%)
f

20
o
20

,

c

o

,

20

B

o

60

'‑1‑"I‑ " '1 +

40

A
20

1 2 5 6 7 8O

Figure 2

4
Wee ks
Weekly changes in each fraction of serum proteins in the3samples from rats treated with ( ) and without (‑‑‑ ‑)immunosuppressive drugs ; the rats in both groups immunizedwith the metacercariae per os and then challenged 6 weeks

after the immunization. A: albumin. B : alphaglobulin,
C : beta‑globulin, D : gamma‑globulin, E : AjG ratio,
N: mean normal value of the non‑infected and non‑treatedrats .

in the period the value in Group I was higher than those in Group II. The per‑centage value of beta‑globulin in the two groups showed a similar trend indicating aslightly high rate of the value when compared to the normal value. In albumin
fraction, on the other hand, a remarkable difference was found in the mean percentage

between Groups I and II, during 4 to 8 weeks. A/G ratio in Group I had increased
gradually up to 6 weeks, and then decreased, whereas it had decreased with the elapseof infection in Group 11 until necropsy.

DISCUSSION
Kondo and Hashiguchi (1979) have observed, for the first time, on the influenceof primary infection with Japanese species of Fasciola metacercariae to superinfection
in rats. They demonstrated that the recovery rates of Japanese Fasciola from chal‑lenge infection were very low when compared to the natural‑immunity control.
The similar tendency, again, was also ascertained in the present study in rats, by18

carring out challenge during 6 to I I weeks following immunization. During theperiod, the percentage immunity (reduction) had a considerable variation among
the experimental groups designed. From 6 to 9 weeks after immunization, thepercentage reduction was significantly higher in the challenged groups than thecontrol, indicating the ranges from 58.3 to 80.6 per cent. It was noted, however, thatthe ability ofrats to induce the protec.tive immunity to the fluke had trended to reduce

gradually in the animals 10 weeks (25.00/0 immtmity) after immunization; theimmunity reduced to zero at I I weeks.
In F. hepatica, this protective immunity following an oral infection has been

amply documented as mentioned before. In most of these studies on the immunity
to F. hepatica worm recoveries have been done after the flukes had matured. Hayes
et al. (1972, 1973, 1974c), however, have performed a study on the ability of rats toresist a challenge infection with F. hepatica, recovering immature worms 3 to 4 weeks
after challenge, and they have obtained a high rate of percentage reduction. This
reduced worm burdens have also been recognizable during 24 to 48 hr after challenge
infection of rats with F. hepatica (Hayes and Mitrovic, 1 977; Hayes, 1 978). In thecurrent study, therefore, the markedly reduced worm burdens from challenge would
be present at the early phase of challenge infection in rats. However, exactly when
and where protective immunity is expressed still remains undetermined, though the
immunity to reinfection with F. hepatica in rats is expressed by 48 hr after challenge(Hayes, 1978).

The protective immunity was examined in connection with the developmental
ages of worms which were used in the immunization, and also with the liver migrationof worms which might be related to induce the functional immunity in rats.
Rajasekariah and Howell ( 1 978) have examined the role of developmental stagesof worms in the rat's resistance to challenge with F. hepatica. In their works the rats

were sensitized by subcutaneous implantation of either metacercariae, 4 week‑old
juveniles, adult worms, or eggs of F. hepatica, and then challenged with the meta‑cercariae 2 weeks later. All implanted stages, apart from adult worms, conferred asignificant degree of protection on their recipient animals. With respect to the livermigration ofworms, on the other hand. Lang et al. ( 1974) pointed out that the durationof liver migration by juveniles might be of greater importance in stimulating pro‑tective immunity in mice than was worm age. Thus, these observations suggestthat there still remains undetermined factors in the mechanisms to induce protective
immunity in the host animals against the genus Fasciola.

In order to study the immunological mechanisms which could underly theprotective immunity, the immunizing infection was conducted by implanting theimmature, 25 day‑old, worms with and without diffusion chambers in the present
experiment. The chamber allows for exchange of various materials between im‑planted parasites and their host (Hashiguchi et al., 1976). Besides, with the use ofdiffusion chambers the inability of parasites to infect the host would reduce or elim‑
inate all of the pathology associated with the infection (Despommier and Wostmann,1968) . The present study revealed that the rats immunized by the worms in chambers
showed a significant degree of protective immunity when compared to the natural‑immunity control or sham operation. The free worm implantation, however, stim‑

19

ulated a higher degree of the immunity than the worm implantation with chambers.
These results suggest that the liver migration by the immature worms implanted
may be responsible, at some extent, for stimulating the induction of protective im‑munity to the Japanese Fasciola in rats. It is of great interest that the enchamberedworms are able to induce the protective immunity by releasing a variety of soluble

antigen through the millipore chambers placed intraperitoneally in rats. Thus,
the diffusion chamber technique permits the host to come into direct contact with theliving parasites, thereby exposing it to a great variety of antigenic stimuli (Despommierand Wostmann, 1968).
In the rats sensitized by worm implantation with and without diffusion chambers,

serological analysis demonstrated that there was a remarkable difference in theantigen‑antibody systems between both implantations. During I to 2 weeks after
worm implantation, the precipitin bands in agar diffusion plate were nearly equalin numbers in the two groups. After challenge, however, Iarger numbers of the bands
were recognized in serum samples from rats sensitized with free worms than in thosewith enchambered ones. Considering the relation between the markedly reduced
worm burdens and the appearance ofmany bands in free worm implantation (withoutchambers), Ouchterlony technique may be effective as an indicator to estimate thedegree of protective immunity to Fasciola in host animals. Each fraction of serum
proteins has not indicated a remarkable difference between the two groups.

There have been many attempts to reduce or eliminate the natural immunityof hosts against various parasites; in most of these cases workers have treated the hostanimals with immuno‑suppressants and/or X‑irradiation. Recently, Hashiguchi
and Hirai ( 1 977) have examined the influence of various immuno‑suppressants on
the establishment of the lung fluke, Paragonimus rmjazakii in rats. They demonstrated

that combined treatment with dexamethasone and prednisolone or with hydro‑
cortisone and dexamethasone had suppressed the host's immune responses, while theuse of dexamethasone or prednisolone alone had a relatively little influence on the
immunity of hosts. In consideration of these results, the present work was attempted
to determine if the combined treatment of rats with dexamethasone and prednisolone
would have an effect on the protective immunity to the Japanese Fasciola or not.

The treatment greatly suppressed the induction of protective immunity in rats
sensitized by oral infection with the metacercariae, and then challenged 6 weeks later.

Both treated and control rats harbored 3.3 and 3.2 worms in mean recovery, while
non‑treated but immunized and challenged rats showed a significantly fewer worm
burden indicating 79.3 per cent worm reduction. Hayes and Mitrovic (1977) haveadministered dexamethasone into immunized rats with the metacercariae per osstarting 24 hr before challenge, and demonstrated that the drug used abrogated theprotective effect of a previous infection. These facts indicate an important role ofhumoral immunity in the protective immunity to Fasciola in rats. No precipitinband in agar plate was observed in the rats given the immuno‑suppressants throughout
the experiment. The rats, on the other hand, theat were not given the drugs showeda considerable number of bands from 2 weeks after immunization. These results
also suggest that antibody production was greatly suppressed in the animals given
immunosuppressive drugs. Among fractions of serum proteins, gamma‑globulin

20in the non‑treated rats with the drugs was remarkably high in an average after 2
weeks, when compared to that in the treated animals; the latter marked a con‑sistently lower rate than average normal value. Once again, these differences may
be caused by the suppression of the host's immune responses.
In the present study, the protective immunity to the common liver fluke, Fasciola,in rats was examined using immunosuppressants or millipore diffusion chambers.
The use of drugs and the chamber techniques would provide a good measure for theexamination on the mechanisms which could be underlying the induction of protective
immunity to the parasites in hosts. For the genus Fasciola, more detailed experimental
systems such as various ages of worms and the timing and sites of immunization, areneeded to clarify the mechnisms using these measures in relation to the host‑parasite
relationships.

ACKNOWLEDGEMENTS
The author express his deep gratitude to Professor Seiiti Inatomi, Departmentof Parasitology, Okayama University Medical School, for his kind guidance andvaluable comments to the present work. Thanks are also due to Professor Noriji
Suzuki, Department of Parasitology, Kochi Medical School, for his encouragement
throughout this study.

REFERENCES
1) Anderson, J. C., Hughes, D. L. and Harness, E. ( 1975) : The immune response of rats to sub‑cutaneous implantation with Fasciola hepatica. Brit. Vet: J., 131, 509‑5 182) Armour, J. and Dargie, J. D. ( 1974) : Immunity to Fasciola hepatica in the rat: Successful trans‑

fer of immunity by lymphoid cells and by serum. Exp. Parasitol., 35, 381‑388
3) Corba. J., Armour, J., Roberts, R.J. and Urquhart, C.. M. ( 1971) : Transfer of immunity to
Fasciola hepatica infection by lymphoid cells. Res. Vet. Sci., 12, 292‑295

4) Dargie, J. D., Armour, J., Rushton, B. and Murray, M. ( 1974) : Immune mechanisms andhepatic fibrosis in fascioliasis. In E.J. L. Soulsby (ed.). Parasitic Zoonosis. Academic Press

Inc., New York, pp. 249‑271

5) Despommier, D. D. and Wostmann, B. S. (1968) : Diffusion chambers for inducing immunity
to Trichinella spiralis in mice. Exp. Parasitol., 23, 228‑233

6) Eriksen, L. and Flagstad, T. ( 1974) : Fasciola hepatica : Influence of extra‑hepatic adult flukes on

infections and immunity in rats. Exp. Parasitol., 35, 41 1‑41 77) Goose, J. and MacGregor, M. (1973) : Naturally acquired immunity to Fasciola hepatica in therat. Brit. Vet. J., 129, xlix

8) Hashiguchi, Y. and Hirai, H. ( 1977) : Influence of immunosuppressants on the establishment of
Paragonimus rmyazakii in albino rats. J. Helminth., 51, 87‑94

9) Hashiguchi, Y., Hirai, H., Okura, T. and Hiraoka, H. ( 1976) : Effect of bithionol against
Paragonimus ohirai and Paragonimus wayazakii in diffusion chambers implanted intraperitoneallyor subcutaneously in rats. J. Parasitol., 62, 146‑147lO) Hayes, T. J. ( 1978) : Further evidence for the early expression of immunity to Fasciola hepatica
in rats. J. Parasitol., 64, 374 376

1 l) Hayes, T. J. and Mitrovic, M. ( 1977) : The early expression of protective immunity to Fasciola
hepatica in rats. J. Parasitol., 63, 584 587

2112) Hayes, T.J., Bailer, J. and Mitrovic, M. ( 1972): Immunity in rats to superinfection with
Fasciola hepatica. J. Parasitol., 58, 1 103=1105
13) Hayes, T. J., Bailer, J. and Mitrovic. M. ( 1973) : Immunity to Fasciola hepatica in rats : The effectof two different levels of primary exposure on superinfection. J. Parasitol., 59, 810‑812
14) Hayes. T. .J., Bailer, J. and Mitrovic. M. ( 1974a) : Serum transfer of immunity to Fasciola hepatica
in rats. .1･ Parasitol., 60, 722‑723

15) Hayes, T.J., Bailer, J. and Mitrovic, M. ( 1974b) : Studies on the serum transfer of immunity
to Fasciola hepatica in the rat. J. Parasitol., 60, 93C 934

16) Hayes, T.J., Bailer. J. and Mitrovic, M. ( 1974c) : Acquired immunity and age resistance inrats with chronic fascioliasis. J. Parasitol., 60, 247‑250

1 7) Howell, M.J., Sandeman, M. and Rajasekariah, G. R. (1977) : In vivo and in vitro studies on
the effects of immune rat serum on Fasciola hepatica. Int. J. Parasitol., 7, 367‑371

18) Kimura, S. ( 1961): Experimental studies on Fascioliasis. 111. Clinical and hematologicalobservation on infected rabbits. Jap. J. Parasitol., lO, 336‑341 (In Japanese with English
summary) .
19) Kondo, K. and Hashiguchi, Y. ( 1979) : Influence of primary infection with Japanese Fasciola
metacercariae to superinfection in rats. Res. Rep. Kochi Univ. (Nat. Sci.), 28, 73‑80 (InJapa‑
nese with English abstracts)

20) Lang, B. Z. ( 1974) : Host‑parasite relationship of Fasciola hepatica in the white mouse. VI.
Studies on the effects of immune and normal sera on the viability of young worms transferred tonormal recipients. J. Parasitol., 60, 925‑92921) Miller, K. L. and Smithers, S. R. ( 1980) : Schistosoma mansoni: The attrition of a challenge
infection in mice immunized with highly irradiated live cercariae. Exp. Parasitol., 50, 2 12‑221

22) Ono, Y. and Isoda, M. (1952): Studies on the Fascioliasis. 111. Experiment on the artificial
infection with metacercariae in rabbits. Jap. J. Vet. Sci., 14, 189‑203 (InJapanese with English

summary)
2a) Rajasekariah, G. R. and Howell, M.J. ( 1977): The fate of Fasciola hepatica metacercariae
following challenge infection of immune rats. J. Helminth., 51, 289‑294
24) Rajasekariah, G. R. and Howell, M.J. (1977) : Fasciola hepatica : Role of developmental stages
in the rat's resistance to challenge. Exp. Parasitol., 44, 233‑238

25) Rajasekariah. G. R. and Howell, M.J. ( 1979) : Fasciola hepatic in rats : Transfer of immunity
by serum and cells from infected to F. hepatica naive animals. J. Parasitol., 65, 481 L87

26) Thorpe, E. and Broome, A. W. J. (1962) : Immunity to Fasciola hepatica infection in albino rats

vaccinated with irradiated metacercariae. Vet. Rec., 74, 755‑756
27) Watanabe, S. (1965) : A revision of genus Fasciola in Japan, with particular reference to F.
hepatica and F. gigantica (ed. Morishita, K., Komiya, Y. and Matsubayashi, H.) Progress of
Medical Parasitology in Japan, 2, 359‑381. Meguro Parasitological Museum, Tokyo.

22

免疫抑制剤およびDiffusion

Chamber移植法によるラットの日本産

カンテツに対する防御免疫効果の検討
橋

口

義

久

カンテッEα彪o伽R砂α痂αについては，初回の感染によって，宿主動物の再感染に対する防御免疫が強く発現されることが知られている。

しかし，分類学的位置づけが未だ不明確な日本産カンテツ

に対する宿主の防御免疫についての研究は極めて少ない。今回は，日本産カンテツによる初感染後
6〜11週目にラットを再感染させたところ，6〜9週目の再感染群では，対照群（Challenge

control）に比べて，有意の虫体排除が認められた。しかし，この防御免疫能は初感染から10週以降になると次第に低下し，11週後では対照群との間に差異を示さなかった。この防御免疫効果を調べるため，幼若

虫体をD班usion

chamberに入れ，ラット腹腔内移植による感作を行い，移植3週後に再感染を試みたところ，防御効果が観察された。 しかし，虫体のみの移植群に比べると，その効果は低い傾向を示した。このことは，虫体のみ移植群において，移植虫のラット肝臓内穿入による刺激が，何らかの形で強い防御免疫の発現に関与していることを示唆するもので興味深い。また，これらのラット血清を

Ouchterlong法で調べたところ，虫体のみ移植群ではChamber移植虫群より，多くの沈降線が出現
し，抗体産生の面でも両群間に違いがみられた。一方，血清蛋白画像は両者間で大差なく，いずれも虫体移植，再感染を通して，かなりの変動を示した。次に，ラットの再感染防御免疫に対する免疫抑制剤（Dexametha茄neとPredonisoloneの併用）の影響を調べた結果，免疫抑制群では，対照群と同様の虫体回収を示した。 したがって，これらの抑制剤は今回の防御免の発現を著しく抑制することが明らかになった。また免疫抑制群では，Ouchterlongによる沈降線の出現は皆無であったのに対し，非免疫抑制群では，初感染から2週目以降に多数の沈降線が出現した。免疫抑制群の蛋白分画ではr−globulin値が著しく低下し，非抑制群との間に大差を示した。以上の結果は，ラットのカンテツ再感染防御機構を調べる上で，・一つの手がかりを与えるものといえよう。

高知医科大学環境保健医学講座寄生虫学教室

Japan. J. Trop. Med. Hyg., Vol. 9, No. l, 1981, pp. 23‑29

23

EFFECT OF EXERCISE IN A HOT ENVIRONMENTFOR 7 SUCCESSIVE DAYS ON PHYSIOLOGICALRESPONSES AND HEAT TOLERANCESEIKI HORI
Received for publication 6 March 1981

Abstract: Eight young male university students took exercise for 7 successive days ina climatic chamber at 30'C, 700/0 relative humidity. Exercise was performed on a bicycle
ergometer at a work load of 2 kp at a cycling rate of 50 r.p.m. for 60 min. Sweat tests were

performed on the day before beginning of training and on the day after the end of training.

Sweating was induced by immersion of both legs to just below the knees into circulatingwater of 42'C in a climatic chamber of 30'C, 700/0 relative humidity for 90 min. Body
weight loss induced by a certain heat load increased after heat acclimatization, while a risein rectal temperature and an increase in heat rate during sweat test decreased after heatacclimatization. Sodium concentration in sweat decreased slightly and sodium concen‑
tration at a given sweat rate decreased considerably after heat acclimatization. Heat
tolerance, assessed by our numerical indices for assessment of heat tolerance, was improvedby heat acclimatization. Improvement of heat tolerance was accompanied by greatchanges in physiological responses to heat and was induced at the cost ofincrease of physio‑

logical strain in terms of water metabolism.

INTRODUCTION
In work the metabolic heat produced in the body increases in proportion to
the intensity of work, and body heat must be dissipated into the environment at therate of its production to maintain a constant body temperature in a steady state.

Since the amount of heat dissipation from the body decreases by increasing theambient temperature, capacity for heat dissipation or tolerance to heat stress is oneof the factors determining work capacity in a hot environment (Robinson, 1 963;
Piwonka et al., 1967). It is known that physiological responses to heat change as
unacclimatized individuals are repeatedly exposed to a hot environment. After
successive heat exposures it has always been found that subjects sweat more readily
and more profusely and heat tolerance is improved by increased capacity of heatdissipation (Dill et al., 1938; Adolph, 1946; Belding and Hatch, 1963; Hori et al.,
1 975). It is known that the temperature regulatory responses of sweating andcardiovascular changes are involved during exercise (Robinson, 1 963). Therefore,
the acclimatization to heat may be facilitated by physical exercise in a hot environ‑ment. Thus it is of interest to study the effect of daily exercise in a hot environmenton the physiological reponses to heat and heat tolerance of men. Recently we
Department of Physiology, Hyogo College of Medicine, 1‑1 Mukogawacho, Nishinomiya City, 663,

Japan. Tel. (0798) 45‑6S85

24.

proposed a numerical index for evaluation of human heat tolerance (Hori et al.,
1 974). We reported that this index could follow improvement of heat tolerance
during short‑term heat acclimatization and long‑term heat acclimatization suchas superior heat tolerance of subtropical natives to that of temperate natives (Horiet al., 1975, 1978). In the present study, we atternpt to compare the physiologicalresponses of heat and heat tolerance of men before physical training with thoseafter physical training to study adaptive changes induced by physical training in a
hot environment for 7 successive days.

MATERIALS AND METHODS
Eight young male Japanese, aged 18‑23, were exposed to a combination of
muscular exercise and environmental heat for 7 successive days in autumn. Muscularexercise was performed on a bicycle ergometer at a constant work load of 2 kp at thecycling rate of 50 r.p.m. for one hour in a climatic chamber of 30'C with 700/0 rela‑tive humidity. A sweat test was performed on the preceding day of beginning ofphysical training and on the first day after physical training for 7 successive days.

The sweat test was performed as follows. Each subject dressed in shorts only andsat in a chair at rest in the climatic chamber of 30'C with 700/0 relative humidity
for 30 min. Then the subjects, sitting in chairs, dipped their legs to just below theknees into a stirring water bath of42'C and stayed there for 90 min. Rectal temper‑

ature was measured continuously by copper‑constantan thermocouples before andduring exercise, and heart rate was recorded continuously by electrocardiography.
Body weight was measured with an accuracy of 5 g before and after the experimentand the net body weight was calculated by subtracting the weight of the dry andwet shorts from body weight measured before and after experiment respectively.
Local sweat samples from the chest and back were collected by the filter paper

method during each 15 min. period (Ohara, 1966). The skin areas for samplingof sweat were washed with distilled water and an area of the skin covering 12.6 cm2
was covered with a sweat capsu,1e by using collodion solution. Sweat was absorbed
by a prewashed filter paper, Iocal sweat rate was determined by the difference between
weight of filter paper after absorption of sweat and that of dry filter paper. The
filter paper was diluted with distilled water and sodium concentration in the sweat

was determined by flame photometry. The magnitude of physiological strain
induced in the body by heat exposure was expressed by the combination of relativerise in rectal temperature, relative water loss and relative salt loss. He̲at to]erance
indices I and S are calculated as follows :Heat tolerance index I =VA2+B2 + C2

Heat tolerance index S= VA l B + C2

A =O AW07 x W C= AT

Q.
'
40.6‑T'
B=
7 W
where W=Body weight before the experiment (Kg)
AW=Weight loss at the end of the experiment (Kg)
T. =Rectal temperature before the experiment ('C)

25

AT=Rise in rectal temperature at the end of the experiment ('C)

Q =0.058 >< AW X C
C=The mean sodium concentration in local sweat (mEqjL)
Smaller value of heat tolerance index I indicates s̲Tr!̲'all physiological strain induced

by a certain heat load and superior heat tolerar̲ce.

RESULTS
Table I shows body wei̲ :ht, body weight loss, mean Na concentration in local
sweat, rise in rectal temperature and increase in heart rate observed in sweat test
before and after physical training in a hot environment for 7 successive days. The
mean value of body weight loss a,fter training Aras considerably greater than that

before training, while mean Na concentration in sweat decreased slightly after
physical training. However, these differences were statistically not significant. The
mean value of rise in rectal temperature after training was significantly smaller thanthat before training, and that of increases in heart rate after training was considerably
less than that before training though this difference was statistically not significant.

Thus the ratio of sweat volume (body weight loss) to rise in rectal temperature
increased after training. Changes in heat tolerance indices and their three c,orn‑
ponents during physical training in a hot environment are shown in Table 2. The
mean value of component A after physical training was greater than that before
training. The mean values of component B, component C, heat tolerance indices
I and S decreased after physical training. Among these diff'erences, differencesof component B and heat tolerance index S were statistically significant. Contri‑
Table lDay

Changes in physiological responses to heat induced by daily exposure to amoderate work and environmental heat.

W*ightWeight loss

Mean Na conc.

(kg)

(kg)

(mEq/1 )Rise in rectal

temp.
(eC)

o

61.7 4･1

0.588

O. 1 41

51.2

15.4

0.63

O. I O*8

6 1 .2

0.658

O. 1 52

49.9 1 5.8

0.50

O. I O

4.0

Increasein H.R.
(beatsjmin)

15.5 4･9
12.6 4･6

lvlean values are given with their standard deviations.

* Significant difference between days of experiment at l o/o level.

Table 2

Changes of heat tolerance indices and their components induced by acclimati‑
zation to heat.

Day
o
8

A

C

B

S

I

0.139 0.038 0.193 0.030** 0.045 0.015

0.244

0.039

l .48

0.50*

O. 1 550.226 0.039

l .07

0.4 1

0.04 1 O. 1 53

0.029 0.042

0.0 1 6

Mean values are given with their standard deviations.
* Significant difference between days of experiment,* at 50'
7Q Ievel ** at lo/o level,26bution of component A to heat tolerance indices I and S was the greatest after training,

while that of component B was the greatest before training. Since the values ofcomponents A and B represent the magnitude of strain induced by sweat volume
and rise in core temperature respectively these results indicate that the pattern ofphysiological responses to heat after trainin'. differs greatly from that before training.

DISCUSSION

Considerably greater mean value of sweat volume and slightly smaller meanvalue of Na concentration i.n sweat after physical training in a hot environment for7 successive days than those before training are shown in Table I . Many investi‑

gators have already reported that unacclimatized subjects tend to sweat moreprofusely as they are repeatedly exposed to hot environments (Adolph, 1946; Bass,
1963; Dill et al., 1938; Hori et al., 1976). It is known that the Na concentration
irii w; hincreases progressively as the s Areat rate inc.reases (Dill et al., 1938; Kuno,

1956). Thus, changes in sweating reaction induced by acclimatization to heat
were chafacterized by a hiegher sweat rate and lower Na concentration in sweat at
a given, sweat rate (Dill et al., 1938; Kuno, 1956). As shown in Table 1, rise inrectal temperature cluring exposure to a hot environment decreased significantly
and increase in heart rate during heat exposure tended to decrease after heat ac‑

climatization. Thus it may be assumed that direction of change in correlation
between rise in rectal temperature and sweat volume during heat acclimatizationdiffered from that in correlation between rise in rectal temperature and increase in

A P ats; 1:min)AW(k g)

'
,
: O )

0.70

os5

oSo

8

̲̲̲ )¥ 1 5///'LV:.b,L
I /

0.55

8

Q50

¥¥ /'

/

//

/

' , '

13

l

12

..

05 0.6 OS5 AT ( oC )055

Figure l Changes in correlation between body weight loss, increase in heart rate and rise inrectal temperature.

dW: Body weight loss, AP: Increase in heart rate, AT: Rise in rectal temper‑
̲=‑ ature, O : Correlation between dW and AT, O : Correlation between dP and
AT, Circles : Drawn around the means with radiuses of standard errors. Arrows
indicate direction of changes induced by physical training. Figures in circles
indicate time of expetimen.t in day.

27

heart rate (Fig. I ) . This figure indicates a decrease in rise of rectal temperature
under a certain heat load induced by heat acclimatization tended to be accompanied

by an increase in sweat volume and a lessening of increase in heart rate. Since
rectal temperature is a dominant factor in determining sweat volume and heart
rate at rest (Benzinger at el., 1963; Newburgh, 1949) the sweat center became more
responsive, and sensitivity of the vasoregulatory center against rise in core temperaturechanged slightly during heat acclimatization. As shown in Table 2, values of B,
relative rise in rectal temperature, and index I, the magnitude of physiological strain
induced in the body, decreased after heat acclimatization, while value of A, relative
water loss increased after heat acclimatization. A smaller value of B (rise in rectal

temperature) after heat acclimatization might be due to increased heat dissipationby profuse sweating. A smaller value of index I indicates superior heat tolerance(Hori et al., 1974). Thus, it can be said that heat tolerance was improved at thecost of increase of strain in terms of water metabolism. The value of index S indi‑cates the ratio of relative rise in recta,1 temperature to relative water loss and salt
loss i.e. pattern ofphysiological responses to heat. As shown in Fig. 2, the magnitudeof decrease in value of index S was greater than that in value of index I after heatacclimatization when compared with those before heat acclimatization. These
results indicate the pattern of physiological responses to heat changed greater thanthe magnitude of physiological responses during short‑term heat acclimatization.
Changes in relationship between indices I and S are shown in Fig. 3. In this flgure,circles are drawn around the mean values of indices I and S with radiuses of their

S

I

o.co
f

/,
L

¥

,

O t
¥b ‑‑¥
,

/

¥

0.25

1 .5

IL

o

/

¥

,

¥

14
¥

¥

¥

13

¥

¥

¥

12
¥
'l": ...‑‑..1' ".Ib'b

I" 8 /

0.2 o

1.1

10

O1 4 O J 6 A
O j 5

Figure 2

Changes in correlation between heat tolerance indices and relative water
loss during physical training.

I and S : Heat tolerance indices. A : Relative water loss, O : Correla‑tion between I and ,A : : Correlation between S and A, Circles :Drawn around the means with radiuses of half value of standard errors,
Arrows indicate direction of changes induced by physical training. Figuresin circles indicate time of experiment in day.

28

a.OJ8

I

a::016 a=0J4

O a=012

0.2 5

0.20

075
Figure 3

1()

1.2 5

1 .5

S

Changes in correlation between heat tolerance indices I and S during physicaltraining in a hot environment for 7 successive days.

/ C2
a : Parameter indicating the points of same value of
AV ICrrcles
+ '
A2 '

Drawn around the means with radiuses of standard errors, Arrow indicatesdirection of change induced by physical training. Figures in circles indicate
time of experiment in day.

standard deviations and the family of iso‑sweating lines are drawn by connecting
the points of the same values of parameter "a". By calculation and transfourLation,
Eq. I =a(1 S2) : can be derived as follows (Hori et al., 1974) :

) 1=

A2

x ( I +S2) 2 a( I +S2)

where a=A( I + )A2Thus, the value of parameter "a" runs parallel to change in sweating reaction.
Mechanism of changes in physiological response to heat induced by physical training
for 7 successive days might be as follows. The increases in sweat rate after repeatedheat exposure is attributable to an increase in the activity of the sweat glands andincreased excitability of the sweating center (Kuno, 1956). Lower Na concentration
at a given sweat rate after short term heat acclimatization may be caused by increased

reabsorption from precursor sweat in the duct of sweat glands. The cooling power
ofsweat is dependent on the degree ofwetness of the skin and the difference in vapor

pressure between the skin surface and the air. Lower Na concentration in sweat
increases difference of vapor pressure between the skin surface and the surrounding
air (Hori et al., 1978). Therefore, Iess rise in body temperature after short‑termheat acclimatization might reflect increased capacity of evaporative heat loss due to
profuse sweating with a lower Na concentration.

REFERENCES
Adolph, E. F. (1946)
Bass; D. E. ( 1 963)

The Initiation ofsweating in response to heat. Am. J. Physiol., 145 : 710‑715.

Ther̲ moregulatory and circulatory adjustments during acclimation tQ heat in29
man．In temperature，its measurement
Belding，H．S．and
toand

Hatch，T．F．（1963） Relation

heat，Federation

control．Vol．3，Rheinhold，New

of

skin

temperature

toYork，229−305、

acclimationandtolerance

Proc．フ22：881−883．

Benzinger，T．H．，KilzingerコC．and Pratt，A．W．（1963）The human thermostat637−665in tem−
perature，its measurement and control in science and industry，part3，In：Biology andMedicine

ed．byHardy，J．D．，Rheinhold Publication co・，New York
Edwards，H．T、（1938） Changes in composition of sweat during accli−

Dil1，D．B．，Ha11，F．G．and

mation to heat．Am．J．Physiol．，123：412−419．
Hori，S．，Inouye，A．and Ihzuka，H．（1974） Indices andheattolerance．

Hori，S．，Nakamura，M．，Sugawara声K，Inouye，A．and
sweating

pattemandheattolerance．A

neld

Int．J．Biometeror．，19：154−193・
Hori，S．，Ihzuka，H．and Nakamura，M．（1976）Studies

Okinawa

to

a

hot

afしer

pattemsfbr

theassessment

ofIhzuka，H．（1975〉A

comparative

study

of

on

on

residents

physiological

Okinawa

responses

of

study
in

on

summer．

residentsinenvironment．Jap．J．PhysioL，26：235−244・Hori，S．，Tsujita，J．，Tanaka，N．and
nativessweating

Jap．J．PhysioL，24：263−275・

migrationtoaMayuzumiフM．（1978）Studies
temperate

zone・

on

heat

toleranceofsubtropical

Int・J・BiometeoL，22：82−93・

Kuno，Y．（1956） Human perspiration．Charles C．Thomas，Springfield．
Newb皿gh，L．H．（1949） Physiology of Heat Regulation and Science ofClothing，Philadelphia，

Saunders．
Ohara，K．（1966）

and

some

Piwonka，R．W．，Robinson，S．，Gay，V．L．and Manald，R．S．（1963）Preacclimatization to

work

other
in

hot

Chloride concentration

variations，andclimates．Am．J

Robinson，S．（1963）

interrelations

insweatlbetweenIts

individual，regional，seasonal

them．Jap、J．PhysioL，16：274−290・

Physiol．，140：168−176・

Temperature

regulationin

exercise．Pediatrics，32：691−702．

高温環境下における連続7日間の運動が高温に対する生理的反応および耐熱性に及ぼす影響堀清記

8名の男子大学生に7日間毎日30。C，湿度70％の室内で労作を行わせた。労作は負荷2kg，毎分50
回のペダル踏み運動を60分間行わせた。発汗テストを訓練開始前日と訓練終了の翌日に行った。発汗
テストは30。C，湿度70％の室内で両下腿を42。Cの水槽に90分間温浴させて行った。高温環境下の労働の馴化により発汗量の増加，発汗テスト中の直腸温上昇の減少と心拍数の減少が認められた。高温

馴化によって同じ発汗量に対する汗のNa濃度はやや減少した。我々の開発した半定量的耐熱性の指標で判定すると，耐熱性は高温馴化で向上したことが示された。耐熱性の向上は高温曝露時の生理的変化を著しく変えて水分損失による生体内に生ずる歪を犠牲にしてもたらされている。

兵庫医大，第一生理学教室

Japan. J. Trop. Med. Hyg., Vol. 9, No. l, 1981, pp. 31‑36

31

FLUORESCENT FOCUS SIZE REDUCTION BY SPLEEN CELLSOF MICE IMMUNIZED WITH RABIES VIRUS
YOSHIHIRO MAKINol AND KUMATO MIFUNE2
Received for publication 20 March 1981

Abstract: The spread of rabies virus between adjacent mouse neuroblastoma cells
was inhibited in the presence ofspleen cells from mice sensitized to rabies virus as measured

by the size of fluorescent focus formation. This activity of focus size reduction was ap‑
peared to be closely related to the development ofcell‑mediated cytotoxic activity. Rabies

virus inactivated by beta‑propiolactone was also capable of generating such an activity aslive virus.

The role of cell‑mediated immunity either in the recovery from primary rabies
infection or in the postexposure immunoprophylaxis of rabies is poorly understood.
Miller et al. (1978) have suggested the contribution of cell‑mediated immunity in therecovery from rabies by demonstrating the potentiation of HEP rabies infection bothin immunosuppressed and thymectomized mice. Kaplan et al. (1975) describedsimilar observations. Recently, we have established the mouse model system bywhich the protection mechanisms of postexposure vaccination against rabies might
be studied. The study has demonstrated that although interferon (IF), which isinduced early after vaccination, is important, the postexposure prophylaxis of rabies

cannot be elucidated simply by the IF nor neutralizing (NT) antibody produced by
the vaccine and suggested the requirement of some other mechanisms including cell‑
mediated immunity (Mifune et al., 1 980).
Evidences indicating the development of cell‑mediated immunity in rabies
immunized animals have also been demonstrated by several in vitro and in vivo studies(Wiktor et al., 1974; Tignor et al., 1976; Wiktor et al., 1977; Lagrange et al., 1978;

Tsiang & Lagrange, 1 980). However, whether these cell‑mediated immune re‑sponses really play a role in the recovery or in the postexposure prophylaxis has notclearly been shown. Simmons et al. (1974) demonstrated that the in vitro control of
the growth of herpes virus was quantified by determining plaque size in monolayers
to which sensitized spleen cells were added as opposed to nonsensitized spleen cells
and this method was a sensitive and more direct parameter to measure a cell‑mediatedimmunity.
Present paper briefly describes that this technique is applicable to rabies virus
and immune spleen cells are capable of limiting the spread of virus in the infected cell
cultures.

C3H/He strain mice were immunized by intraperitoneal inoculation with ap‑
l Department of Virology, Institute for Tropical Medicine, Nagasaki University 2 Depart‑
ment of Microbiology, Medical College of Oita

32

proximately 5 x 107 focus forming units(FFU) of live or the corresponding amount ofbeta‑propiolactone(BPL) inactivated HEP‑Flury strain of rabies virus (Kondo,
1965 ; Mifune et al., 1980) . Spleens were harvested from the mice at various intervals

after immunization. They were pressed gently through stainless steel mesh (#200)
and single cell suspensions were prepared in RPMI 1640 medium supplemented with
10 per cent heat‑inactivated fetal calf serum(FCS). The single cell suspensions werethen fractionated into the cells nonadherent to plastic plate and the effluent cells
from nylon wool column as described by Julius et al. (1973). Cloned N‑18 strain ofmouse neuroblastome(MNB) cells were grown in Dulbecco's modified Eagle's medium

supplemented with 5 per cent FCS, 5 per cent calf serum, 3 g/1 of NaHCO; andantibiotics and was plated into Lab‑Tek 4‑chamber slides (Miles lab.). When thecells became confluent, they were infected with 100 FFU of plaque purified CVS
strain of rabies virus (Buckley & Tignor, 1975) and i.ncubated at 37'C for 4 hrs.
VVhole or fractionated spleen cells from nonimmune or immune mice were carefully
overlayed onto the infected MNB cells at a spleen cell to monolayer cell ratio of
50: l, and further incubated for 26 hrs at 37'C. Preliminary experiments indicatedthat during the 30 hrs ofincubation period, secondary fluorescent focus was not formedby the progeny virus from the primary infected focus. At the end of the incubation

period, the culture media were harvested and the supernatants after low speed
centrifufation were assayed for the activities of antiviral NT antibody, IF and lymp‑

hotoxin(LT) which might be produced in the co‑cultivated culture. NT antibody
was assayed as described by Smith et al. (1973) using CER cel]s (Smith et al., 1977),
IF was assayed in L cells with vesicular stomatitis virus and LT was assayed as de‑scribed by Shimizu et al. (1977) using L cells and MNB cells. On the other hand,
infected MNB cells on the chamber slides were washed in phosphate buffered saline,
fixed with cold acetone and then stained with FITC‑conjugated anti‑rabies globulin(BBL, #40604). The number of cells in each immunofluorescent focus was conuted
under a fluorescent microscope. At least, one hundred fluorescent foci were examined
and the statistical significance of the focus size reduction was estimated by the analysis
of variance.

Infected MNB cells co‑cultivated with non‑immune spleen cells (Fig. IA) showedlarge fluorescent foci, usually consisting of 5‑20 cells, whereas when co‑cultivatedwith immune spleen cells (Fig. IB), fluorescent foci were small, usually consisting of
1‑5 cells.

The effect of immune spleen cells harvested on day 7 after immunization on thefocus size of rabies virus was shown in Table I . The average focus size of the cultures

co‑cultivated with non‑immune whole spleen cells was 6.2 cells, while that withimmune whole spleen cells was 3.2 cells and the reduction rate of the focus size was
48.4 per cent which was statistically significant (Exp. I ). Depletion of plastic plate‑adherent cells from immune spleen cells did not influence the activity of immune
spleen cells to limit focus size (Exp. 2). Effluent cells from nylon wool column
consisted mostly of T cells as tested by rosette formation and this T cell‑rich fraction
also suppressed the virus spread by 47.3 per cent (Exp. 3) . In contrast, pre‑treatmentof immune nonadherent spleen cells with anti‑Thy 1.2 serum (OLAC, clone F7D5)
plus complement before co‑cultivation resulted in the abrogation of the activity

33

Figure 1

Immunofluorescent focus of rabies virus‑inf'ected murine neuroblastoma c.ells. The cells

in the 4=chamber slides were infected 1'vith 100 FFU per chamber of plaque purified
CVS strain of rabies ¥'irus. FoLlr hours later, the cells were ('.arefully Overlaye,.d with (A)non‑immune spleen cells or (B) rabies immune spleen cells and further incubated at 37 C

for 26 hours. Ai'tert he incubation period, the cell monolayers were washed with PSB
fixed with c.old acetone and stained with FITC‑conjugated anti‑rabies gamma‑globulin.

(Exp. 4) . No aignifica,nt inhibition of focus size was observed in the cultures c0=

cultivated with spleen cells from mice immunized with Sindbis virus (Exp. 5), In
additlon, neither NT antibody, IF nor LT was detected in each of the c0=cultivated
MNB cel]. culture medium. These results appear to suggest that the suppression ofvirus spread by immune spleen cell, s is mediated by virus specific immune T cells andnot by the NT antibody, IF nor LT,
The k, inetics of the development of f'ocus size limiting activity after immunizationis shown in TabJe 2. High activity was observed in the spleen cells obtained on day
6 after immuni2:ation. However, sucll high activity was no longer observed in thesp].een cells harvested on day 14. In parallel with this experiment, cell‑mediatedcytotoxic activities of spleen ce].1 suspensions prepared at intervals after immunization

were examined against virus inf'ected MNB cells by 51Cr‑release method. ':1'heresults showed that the cytotoxic activity was first detected on day 4 and reached amaximum level on day 7 and then declined to a low level by day 14 (Data not shown)as already described, by us and other investigators (Tignor et al,, 1977; Mifune eial., 1979).

These results seemed to suggest that the suppression of virus spread by immune
spleen cells is closely related with the development of the cytatoxic activity of immune

34
Table 1

Fluorescent focus slze reduction by immune spleen cells in rabies virus‑infectedmurine neuroblastoma cells.
Effector cell population

Expt.
l

2

3

Fluorescent Reduction Ab, IF andrate (O/o) LT assayb)

focus sizea)

Normal whole spleen cells

6.2

Rabies immune whole spleen cells

3.2

Normal non‑adherent spleen cells

9.3

Rabies immune non‑adherent spleen cells

4.9

Normal .nylon wool column effluent cells

1 1 .OAb (‑)LT (‑)Ab (‑)

47.3 (sS)

LT (‑)Ab (‑)

effluent cells5

IF (‑)IF (‑)

Rabies immune nylon wool column

4

IF (‑)

48.4 (SS)')

5.8

Rabies immune non‑adherent spleen cells
treated with anti‑Thy 1.2 +complement
Rabies immune non‑adherent spleen cells
treated with medium + complement

6.6

Normal non‑adherent spleen cells
Sindbis virus immune non‑adherent spleen

7.9

47.3 (sS)

LT (‑)Ab (‑)IF (‑)

2. 1

cells

5.6

68.2 (ss)29.1 (NSS)d)LT (‑)NTe)a) Average number of cells an fluorescent focus consisted.

b) Ab (NT antibody), IF (Interferon) and LT (Lymphotoxin) in cocultivated culture media
were assayed as described in the text.

c) Statistically significant, P<0.01 by analysis of variance. d) Not statistically significant.
e) Not tested.

Table 2Development of fluorescent focus size reduction activity of spleen cells from mice

immunized with live or BPL‑inactivated rabies virus.

Fluorescent focus sizea) Reduction

Days spleencells harvested
after

wi th

Stimulant non‑immune rabies immune rate

Effector cells

effector ( %)immunization
6
14
6
14

effectornylon wool column
effluent cells

nylon wool column
effluent cells

non‑adherent
spleen cells

non‑adherent
spleen cells

57.4 (SS)b)l ive

1 4. l6.0

live

1 4. l

1 O. l

BPL‑inactivated

19.8

7.6

61.6 (SS)BPL‑inactivated

19.8

12.3

37.8 (SS)28.4 (NSS)")a) Average number of cells an fluorescent focus consisted.

b) Statistically significant, P<0.01 by analysis of variance.
c)

Not statistically significant.

spleen cells. It should be noted that BPL‑inactivated rabies virus is also capable ofgenerating the activity of spleen cells to limit the virus spread as live virus. Thisobservation might be of interest in terms of the possible role of cell‑mediated irn‑
munity in the postexposure treatment of rabies by BPL‑inactivated vaccine.

35

ACKNOWLEDGEMENT
A part of the study was supported by the grant from the .Iapan‑United States
Cooperative Medical Science Program.

REFERENCESl) Buckley. S. M. & Tignor, G. H. ( 1975) : Plaque assay for rabies serogroup viruses in Verocells, J. Clin. Microbiol., l, 241‑242

2) Julius, M. H., Simpson, E. & Herzenberg, L. A. ( 1973): A rapid method for the isolationof functional thymus‑derived murine lymphocytes, Eur. J. Immunol., 3, 645‑649
3) Kaplan. M. M., Wiktor, T.J. & Koprowski, H. ( 1975): Pathogenesis of rabies in immuno‑
deficient mice, J. Immunol., I 14, 1761‑1765

4) Kondo, A. (1965) : Growth characteristics of rabies virus in primary chick embryo cells,
Virology, 27, 199‑204
5) Lagrange, P. H., Tsiang, H., Hurtrel, B. & Ravisse, P. ( 1978) : Delayed‑type hypersensitivity
to rabies virus in mice : Assay of active or passive sensitization by the footpad test, Infect.

Immun., 21, 931‑939
6) Mifune, K., Nakamura, M. & Mannen, K. (1979) : Persistent infection of avirulent strain ofrabies virus in immunosuppressed mice, Trop. Med., 2 1, 131‑138
7) Mifune, K., Schichijo, A., Makino, Y., Takeuchi, E., Yamada, A. & Sakarnoto, K. (1980) :A mouse model for the pathogenesis and postexposure prophylaxis of rabies, Microbiol.
Immunol., 24, 835‑845
8) Miller, A., Morse, H. C. 111., Winkelstein. J. & Nathanson, N. ( 1978) : The role of antibody
in recovery from experimental rabies. I. Effect of depletion ofB and T cells, J. Immunol., 121,
32 1 ‑326

9) Shimizu, F., Hanaumi, K.. Shimizu, Y. & Kumagai, K. ( 1977) : Antibody‑dependent cellular
protection against herpes simplex virus dissemination as revealed by viral plaque and infectivity

assays, Infect. Immun., 16, 531‑536
lO) Simmons, R. L., Centifanto, Y. & Kaufman, H. E. (1974) : Plaque size reduction as a measureof viral cell‑mediated immunity, Infect. Immun., 10, 1034‑l039
1 l) Smith, A. L., Tignor, G. H.. Mifune, K. & Motohashi, T. (1977) : Isolation and assay ofrabies serogroup viruses in CER cells, Intervirol., 8, 92‑99

12) Smith, J. S., Yager, P. A. & Baer, G. M. ( 1973) : A rapid reproducible test for determining
rabies neutralizing antibody, Bull. W.H.O., 48, 535‑541
13) Tignor, G. H., Mifune, K. & Smith, A. L. ( 1976) : The immunopathology of rabies, pp. 13‑14.
In Symposium on advances in rabies research. Atlanta, U.S. Department of Health, Educationand ¥ relfare.

14) Tsiang, H. & Lagrange, P. H. (1980) : In vivo detection of specific cell‑mediated immunity instreet rabies virus infection in mice, J. Gen. Virol., 47, 183‑191

15) Wiktor, Y. J.. Doherty, P. C. & Koprowski, H. (1977) : In vitro evidence of cell‑mediatedimmunity after exposure of mice to both live and inactivated rabies virus, Proc. Natl. Acad.
Sci., 74, 33 i338

16) Wiktor, T.J.. Kamo, I. & Koprowski. H. ( 1974) : In vitro stimulation of rabbit lymphocytes
after immunization with live or inactivated vaccines, J. Immunol., 1 12, 2013‑2019

36

狂犬病ウイルス免疫マウス脾臓細胞によるウイルス螢光フォーカス形成抑制

牧野芳大1）・三舟求真人2）
狂犬病ウイルス（HHp−Flury株）で免疫されたマウス脾細胞は，マウス神経芽細胞腫（MNB）細胞上でのウイルスの螢光フォーカス形成を有意に抑制した。脾細胞のこのウイルス伝播抑制作用は，

脾細胞からプラスチック面付着細胞を除去しても活性が消失しないこと，ナイロン綿カラム通過細胞に認められること，脾細胞を予め，抗Thy−1．2血清と補体で処理すると活性が消失すること，MNB細胞と免疫脾細胞の混合培養液中に，ウイルス中和抗体，インターフェロン或いはリンホトキシンがが検出されないこと等から，Tリンパ球によることが明らかであった。また，この活性は，同時に行なった免疫脾細胞のウイルス感染MNB細胞に対する細胞障害活性の発現と時期を同じくして発現
し，脾細胞の細胞障害活性と密接な関係があることが示された。また，β一プロピオラクトンで不活化されたウイルスで免疫されたマウス脾細胞にも，生ウイルスと同様にこのウイルス伝播抑制作用が認められ，不活化ワクチンによる狂犬病のいわゆる感染後投与による発症防御機構に，細胞性免疫が一つの役割を果たしている可能性を示唆するものとして興味深い。

長崎大学熱帯医学研究所ウイルス学部門1）及び大分医科大学医学部微生物学講座2）

Japan.J. Trop. Med. Hyg Vol 9 No I 1981 pp 3 7‑47

37

C. ULEX TRITAENIORHYNCHUS GILES = SOME EFFECTS

OF TEMPERATURE AND PHOTOPERIOD ONLARVAL DEVELOPMENT AND SELECTEDADULT ATTRIBUTESSHAHEEN NIAZ AND WILLIAM K. REISEN1
Pakistan Medical Research Center
International Health Program
University of Maryland, School of Medicine
6, Birdwood Road, Lahore, Pakistan
Recieved for publication 25 March 1981

Abstract: A Pakistan strain of Culex tritae'tiorhyllchus was exposed to conditions of

18, 22, 28 and 34'C at 16L: 8D and 18 and 22'C at 9L: 15D in an incubator. Cooler
temperatures reduced immature survival from eclosicn to emergence and slovL'ed the rateof development resulting in larger and heavier aclults at ernergence with increased energy
reserves in the form of triglycerides. The rate of mating, blood feeding and the synthesisof triglycerides from sucrose increased as a function of temperature. Short day length
further slowed the rate of developrr!ent increasing the size and weight, but not triglyceride

content, of adults at emergence. The insemination rate also decreased under shorter day
length. These results suggested that cold‑induced quiescence, but not necessarily dia‑pause, was induced by the present experimental regimens.

The climate of Punjab Province, Pakistan, presents an extremely varied temporalseries of ecological regimens to its mosquito fauna. To persist, species must with‑stand temperatures ranging from 45'C in midsummer to O'C in mid‑winter, seek
more ameliorated microhabitats or evolve physiological meclrLanisms to altermetabolism during periods of stress. Although aestivation has yet to be reported,
several Culex species, including Culex tritaeniorhynchus Giles, appear to undergo acessation of reproductive activity in response to shorter days and/or cooler temper‑
atures and overwinter as inseminated, unfed and nulliparous females; males andlarvae are not collected during mid‑winter (Reisen, 1978 and unpubl.). However,on warm days during winter, Cx. tritaeniorhynchus females occasionally are collected
biting bovid baits (Reisen, 1978), suggesting that if a diapausing state is indeed
realized, it must not be obligatorily governed by photoperiod or require an elongatedperiod of cold exposure prior to reactivation. Under slightly colder winter con‑ditions in Japan, overwintering diapause is induced during autumn by a combinationof cooler temperatures and shorter day lengths (e.g., Omori, et al., 1965a, b; Wada,l Address reprint requests to W.K.R., Arbovirus Field Station, University of California School of

Public Health, PO Box 1564, Bakersfield, CA 93302

38

et al., 1973) and persists through the first warm days of spring. Some gonotrophic
dissociation occurs in autumn (Oda and Wada, 1973) ; however, the accrual of fat
reserves prior to diapause seems to be accomplished mainly through sugar feeding.
The purpose of the present study was to examine the effects of different temper‑

atures and long and short day photoperiods on immature development and adultcharacteristics under controlled laboratory conditions. Emphasis was placed on
studying those attributes related to overwintering behavior to determine if diapause
could be induced in strains of Cx. tritaeniorhynchus collected from̲ Punjab Province,
Pakistan.

METHODS AND MATERIALSStrain : The Balloki strain from Punjab Province, Pakistan, was used throughout,and was well‑adapted to mating in 3.8 Iiter carton cages (Reisen, et al., 1979) andfeeding on restrained laboratory mice. The females used to produce the cohorts forexperimentation were held under normal insectary conditions of' 28'C, 600/0 R. H.

and 16L: 8D photoperiod with I .5 hr crepuscular periods simulated by graduallychanging the illurnination produced by 60 watt incandescent bulbs from 1 8 to O Lux.
Biological methods : Egg rafts, within 1 2 hrs of oviposition, were placed in an

incubator to hatch under the test temperature and photoperiod regimens. On themorning of eclosion, 216 Ist instar larvae were counted into each of 4 replicate,
16 x 27 cm enameled pans (density=0.5 Iarvae/cm2 of pan surface area) filled withwell‑water to a depth of 1.5 cm. and were fed a total of 1.075 gm of finely sifted
liver powder (Siddiqui, et al., 1976). The addition rate of the food was adjusted forthe larval development rate at each temperature. Pupae were counted daily into
plastic jars and the numbers of males and fernales successfully emerging recorded.
Adults unable to leave the water surface were considered dead. The length of thewing, from its insertion at the mesothorax to its most distal margin excluding wing
scales, and dry weight were determined for the first and last 5 females and 5 males

emerging from each of the four replicate pans. The triglyceride content of theweighed females was determined using the colorimetric method described by Shahid,
et al. (1980).

Ten females per pan were released into plastic jars (vol. = 1200 cm3) where
they were continuously offered a 100/0 Sucrose solution in vials with sponge wicks.
After four days, the females were killed by freezing, clried for 2 days at room temper‑

ature in a dessicator, weighted and the triglyceride content determined. The
remainingr males and females were pooled by date of emergcrence and released into
3.8 Iiter carton cages, in ¥A'hich they were continuously offered 30/0 sucrose in vials

with sponge wicks, and, at night, a restrained laboratory mouse as a blood source.
Each morning all blood eng,orged females were isolated individually in vials withdilute straw‑infusion water as an oviposition substrate (Reisen and Siddiqui, 1978).
Dying females as well as some unfed females were dissected to determine if they
were inseminated, and during the short‑day photoperiod, if 06genesis had progressedbeyond Stage I (Clements, 1963). Females ovipositing hatching egg rafts wererelea5ed i.ntQ a separate ca Gre and were Qffered a mQuse as a bIQQd source. ThQse

39

refeeding were re‑isolated for a second oviposition. The number of eggs in eachraft was counted under a microscope to determine fecundity.
The entire experiment was done in an incubator set at 18, 22, 28 and 34'C,

with a 16L: 8D photoperiod and at 18 and 22'C with a 9L: 15D photoperiod.
The lowest temperature at which restramed mrce survrved was 18'C. The photo‑
period was established by turning the incubator fluorescent lights off and on, witha I to 2 hr dusk simulated at light‑off by allowing the used cells of a flashlightto dim gradually from about I OO to O Iux. Cx. tritaeniorhynchus had been found to
mate readily at 16L : 8D and 28'C in 3.8 Iiter cages under these illumination regimes(Reisen, et al., 1979).

Statistical methods : Median developmental time (E50) was calculated by regress‑
ing the cumulative proportion ofadults emerging, transformed to probits; as a functionof time in days from eclosion, transformed to logr.. Temperatures within the 16L : 8D

photoperiod were compared by model I analysis of variance (ANOVA) withindividual means compared by posteriori least significant range tests (Sokal and Rohlf,

1969). Measurements of the adults subsampled from each pan were considered
to be nested within pans, and the amcng‑pan mean square was used to test themain effects and flrst and second order interaction terms. The effect of shorter
photoperiod was tested individually at each temperature using Student's t‑tests or

ANOVA. Observed sex ratios were compared using 2 X 2 contingency Chi Square
adjusted by Yates' correction factor (Sokal and Rohlf, 1969). Non‑replicatedobservations were tested over temperature using a Model I regression analysis withthe regression coefElcients tested for significant departure from O by ANOVA (Sokal
and Rohlf, 1969).

RESULTS
L!:tfects of temperature at 1 6L : 8D S urvival from eclosion to pupation did not
significantly vary among temperatures, but pupal to adult survival was significantly

lower at 18'C when many of the adults, especially the males, did not successfully

shed the pupal integument and/or leave the water surface (Table 1). Overall
survival from eclosion to emergence was highest at 28'C. The sex ratio at emergence(males/total) was lowest at 18'C and significantly departed from I :1 at 18 and 28'C
(p < 0.05) .

Median developmental time (E50) varied inversely with water temperature(Table I ) . Males developed significantly faster than females, and this disparity
varied disproportionately over temperature, since the sex by temperature interaction

term in the ANOVA was significant (p<0.01). The relation of temperature (t)to the velocity of immature development (V=1jE50) was expressed by the law ofheat summation, V=(t‑to)/K, where to is the developmental point and k=athermal summation constant (Mogi, 1978). V varied linearly as a function oftemperature for both males and females, with t0=8.3 and 9.2'C for males and females,

respectively (Fig. I ) . The thermal summation constant of 158.7 for females and1 64.2 for males implied the number of degree days above to required for the com‑pletion of immature development.

40
Table lAttribute

The effects of temperature and photoperiod on selected immature attributes.

16L: 8D1

Photoperiod :Temperature ('C) : 18

22

9L: 15D2

28

34 group

18

22

Survivorship3

L to P.8 1 3"

.767

.866a

.647a

.598*

.899nsP to A.600b.83 1 "

.9 1 3.806.629"s.886ns

L to A. 488b

. 646

.79la

.52 1 b.380 s.796ns

.356b

.520

.448

.486

. 442 *

Sex ratio (E50 (days)4

itotal)jgroup x.493ns

17.67

1 3.087.59

6.85

1 1 .30" 18.62*

l 3.82 *1 7.5 1

1 1.977.55

6.85

l0.97b 18.03

13.25*

l 7.59*12.53b

7.57"

6.85d

i Means compared within 16L : 8D; means in each row or group means followed by the same
least significant range test (P>0.05).

2 Means compared between photoperiods at the same ternperature, ns : P>0.05 ; * : P<

0.05; **: P<0.01.
3 L=Larval stages I to IV; P=pupae; A=adult.
4 E50 =median developmental time to adult emergence in days.

80

o/oB=‑36.76t 3. 4 3 t

r2 =.gg8 ¥.
o

60
.2

v J:d'= ( T‑ 8.3 ) / 1 6k . 2

r2 ‑

>
5 ･1

OJ
>
UJ

.951 9t 1v* (T‑ 9.2 )/1 58.7

z

UJU 40
o,

l

UJ

20

I

r2 . .gk8.o

8 TEMPERATURE
18 22 ('C28, 34(velocity, v= E50 1, where E50 Is mediantion of water temperature, T, in Co.r2= the coefficient of determination in‑
dicating the goodness of fit.

olol= ‑ 37.05t2 .A 2 t
r2= ' g35

8 18 TEM22PERATU28
RE (oc3a)

Figure l The velocity of immature development
emergence in days) for males (squares)
and females (circles) plotted as a func‑

D

O

¥

Figure 2

The percentage of emerging females
imbibing a blood meal (B) and mating(1) plotted as a function of holding

temperature, t, in "C for long day( 16L : 8D=filled squares and circles)

and short day (9L : 15D= open squares
and circles) photoperiods. Regressionfunctions calculated from long day
photoperiod data ; r2=coefficient ofdetermination indicating the goodness
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42

The proportion of females imbibing a blood meal and mating (i.e., the numberof dissected females that were inseminated or that oviposited hatching egg rafts)
decreased as a linear function of decreasing temperature (Table 2 and Fig. 2).

The extrapolated temperatures at which no blood feeding or mating would occur
were l0.7 and 15.3'C, respectively.

There was no signiflcant difference (p>0.05) between female fecundity (etgg/.female) in the first and second gonotrophic cycles (Table 2). Fecundity at 18 and

22'C was signiflcantly lower than fecundity at 28 and 34'C. The mean durationfrom emergence to oviposition (GC 1) signiflcantly decreased with increasing
temperature; however, the length of the second gonotrophic cycle, the night ofre‑feeding to oviposition 16 (2), did not differ significantly among temperatures. At
18'C, no female laid more than I egg raft, and none of the rafts hatched or contained

embryonated eggs.
Mosquitoes reared at 18 and 22'C had longer wings than those reared at 28

and 34'C (Table 2). Fema]es had longer wings than males and this difference
varied disproportionately over temperature, since the sex‑by‑temperature interaction

term in the ANOVA was signiflcant (p<0.05). Adults emerging first did not havesignificantly longer wings than those emerging last (p>0.05). Similarly, the dryweight ofadults reared at 18 and 22'C was heavier at emergence than the dry weightof adults reared at 28 and 34'C (Table 2). Females were heavier than males, andthe difference remained proportionate, since t.he sex‑by‑temperature interaction term

was not significant (p>0.05). The mean weight of adults emerging first was sig‑
nificantly greater than adults emerging last, suggesting some form of larval intra‑
specific competition for food as described previously by Siddiqui, et a]. (1976).
The significantly greater weight of females emerging first was attributed to fat
reserves at emergence, since the triglyceride content was also significantly greater(Table 2). Adults reared at 18'C had significantly more triglycerides at emergencethan did those reared at 22, 28 and 34'C.
The dry weight of females offered 100/0 sucrose solution for four days increased
markedly from estimates made at emergence and the percent gain ranged from 135.3

to 216.00/0 (Table 2). The mg of dry weight (dw) and the percent gain was sig‑
nificantly higher at 28 and 34'C than at 18 and 22'C and increased as a linear functionof temperature (t), (dwo/0=35.75+5.32 t, r2=0.982). Weight increases were
attributed to triglyceride synthesis from the imbibed sucrose, since the greatest
percent triglyceride increases were also realized by females held at 34 and 28'C.
The rate of triglyceride synthesis (ts) was temperature dependent, i.e., the percent
of triglyceride gain increased as a linear function of temperature (t) : O/ots= ‑201.19+21 . 18t, r2=0.955.

Effects of photoperiod at 18 and 22'C: Under a "short day" illumination, there
was no difference in survival from larval eclosion to adult emergence ; however, themedian develop.mental time was generally slower (Table I ) . The sex ratio of theimmat res reared at 1 8'C increased significantly, but the proportion of males
remained loweir than the expected I : I ratio when tested by Chi square (p<0.05).
None of the females held at 180 and significantly fewer females held at 22'm,ated under the shorter photoperiod (Table 2). In addition; significantly less43

and more females at 18 and 22'C, respectively, imbibe,d blood meals. Females
taking blood meals matured eggs at all photoperiods and temperatures studied,although under 9L: 15D, 1 p‑/16 L
dissected (3.20/0) at 22' exhibited gonotrophic
disassociation, i.e., took a blood meal, but after death 2 or more days later, ovariandevelopment had not progressed beyond Stage lb.
Fecundity at 9L: 14D did not differ significantly at either 18 or 22'C (Table 2).
The duration of the first gonotrophic cycle at 18' did not differ signiflcantly, althoug'hat 22'C the duration of both the first and second gonotrophic cycles were significantly
shorter.

At 9L: 14D the wing lengths of adults reared at 18' and 22'C and the dryweight at 18'C, but not 22'C, were significantly longer and heavier than adults
reared at 16L: 8D, respectively (Table 2). Females were agian larger and heavier
than males. At 18', but not 22'C, the wing lengths of the adults emerging first weresignificantly longer ; no significant differences in the dry weights of the adults emerging

first and last was found. At 22'C, there was signiflcant among‑rearing l 'an varia‑

bility (P<0.05) in both the wing length and dry weight ANOVAS which contributed
to the inability to detect significant differences in the time of emergence effects.
The triglyceride content of the emerging females was significantly less at the 9L : 15D

photoperiod and 18'C, while no significant difference was detected at 22'C. Thiswas unexpected, since at 18'C the dry weights were significantly greater at emergenceand usually the triglyceride content was significantly correlated with dry weight(Shahid, et al. 1980).

The dry weight of females after being offered 100/0 Sucrose for 4 days increased

markedly from the values estimated at emergence, but the weight attained and thepercent weight gain did not vary between photoperiods (Table 2). Unexpectedly,
the mg of triglyceride accrued at 9L : 15D was significantly less and greater tha,nthe mg of triglyceride accrued at 16L : 8D and 18 and 22'C, respectively; however,there were no significant differences in the percentage of triglyceride gained. Thus,
although the actual values of triglyceride at emergence and after 4 days of being
offered 100/0 sucrose were signiflcantly lower at 18' under the short day photoperiod,

the relative quantity of triglycerides synthesized was comparable. Discrepancies
in the absolute values were attributed to variability among the groups of mosquitoes
measured.
DISCUSSIONAs defined by Clements (1963), "Quiescence is a state of inactivity induced byan unfavorable environment and it ceases on amelioration of the environment.
Diapause is terminated only after reactivation, i.e., exposure to a factor suc.h as cold

for a period of time." Faculative diapause in the adults of multivoltine species ischaracterized by the accrual of fat reserves usually in response to an exogeneous cue
such as photoperiod. The influence of photoperiod in some mosquitoes is mediatedby temperature which reduces the rate of development, so that a critical number ofshort‑day photoperiods are experienced and diapause is induced (e.g. Beach, 1978).
In Punjab, differences between the parity rate of biting and resting Cx, tritae‑

44

niorhynchus females during autumn suggested that the population bifurcates into
gonotrophically inactive overwintering and active non‑overwintering segments(Baker, et al. 1979; Reisen, et al. 1980). Parallel observations have been recordedin Japan by Fujimiya and Yajima (1976). In Punjab, the rate of insemination
typically decreases in November, as indicated by an accrual of uninseminated restingfemales; however, by January all females were inseminated (Reisen, et al., 1977),

suggesting that the uniseminated females either mated or did not overwinter.
Females collected during December and January are typically without blood in thegut, nulliparous and have Stage̲ I ovaries, although blood fed and gravid females

may be collected during every month of the year. These data suggest that an
overwintering state is perhaps achieved, but loosely maintained due to mild wintertemperature regimens.
In the present study, cooler temperatures and shorter day length elongatedlarval development and resulted in the emergence of larger adults with more energy
reserves. These results agreed with studies in Japan by Yoshida, et al. ( 1 974) w'hofound that cooler temperatures and shorter photoperiods elongated immaturedevelopment, and by Nakamura, et al. (1968) and Fujimiya and Yajima (1976) who
found that the diapausing generation was always significantly larger in size thanfemales that remained gonotrophically active during autumn.
The mating and blood feeding rates were slower and the time from emergence
to initial oviposition was greatly elongated at cooler temperatures, which, throughtime, could result in an accrual of uninseminated, unfed nullipars under field con‑ditions. At 18' the shorter photoperiod decreased the proportion offemales feeding,while at 22' the proportion feeding actually increased. These observations contrasted
with the findings of Eldridge ( 1 962) who reported that a Japanese strain of Cx.
tritaeniorhynchus was reluctant to feed on a shaved chick at 28' and 8L : 1 6D. In
addition; in the present study, Iittle gonotrophic dissociation was observed at 9L :l 5D and 220C, whereas this phenomenon was commonly reported under similar
experimental conditions with strains of Cx. tritaeniorhynchus from Japan, althoughrarely observed under field conditions (e.g. Harada, et al., 1968, Takahashi, 1970,
and Oda and Wada, 1 973). The rate of triglyceride synthesis from sucrose did notincrease under the shorter photoperiod, but rather decreased with temperature, as
was expected from similar experiments with other species ofmosquitoes (Van Handel,
1966a and b).
Survivorship and longevity did not appear to be greatly enhanced, and adults

at all temperatures and photoperiods were dead 6 weeks after emergence. In
contrast, Omori et al. (1965a), using semi‑natural conditions in Japan, induced
Cx. tritaeniorhychus females to hibernate in October and more than 200/0 Of females

fed with sugar survived for more than 20 weeks, November through March. In
Pakistan, females would have to survive at least 1 5 weeks to overwinter from Nov‑
ember through late February or early March, when the first overwintering females
carr'‑ be collected at bovid baits. These data suggest that a true diapause state wasprobably not induced during the present study.
The results of the present study indicated that some of the attributes charac‑
teristic of diapausing Cx. tritaeniorhynchus were induced, although these attributes were45

not separable from quiescence, or cold‑induced physiological changes, and did not
impart marked longevity. Speculative explanations included :1 . Incubator conditions were not condusive for diapause; i.e., metabolic
processes were not slowed sufficiently.

2. The photoperiod regimens were too extreme and "not recognized" as thediapause induction cue.

3. The exposure of the immature stages to a critical number of "short day"photoperiods does not induce diapause.
In Japan, Omori, et al. (1965a) were able to induce diapause experimentallyand keep females alive throughout the winter. They found that survival wasenchanced in hibernacula that were continuously dark, had little air movement andwere thermally stable at about I I 'C. Conditions in the present study retained adiel photoperiod, were warmer (min. temp. = 18'C), but had reduced air movementin paper carton cages with a wet cotton pad on the gauze top to increase humidity.
However, the habitats of diapausing females appear to differ considerably. In Japan,
Cx. tritaeniorhynchus females have been collected from brush piles and caves (Bullock,l 959), but crevices and rodent burrows in the stone walls supporting permanent
terraced rice fields seemed to be the preferred microhabitat (e.g. Omori, et al. 1965a,

Wada, et al. 1973). In Punjab overwintering females can be recovered throughout
the winter in closely spaced agricultural crops such as clover ( Trifolium spp.) andin dense clumps of elephant grass (Imperlata spp.). Hibernacula in the Lahore areaof Punjab are thus more subject to diel temperature changes, which ranged in 1978,

for example, from a minimum of 2'C in January to a maximum of 25'C in February,
and are exposed to diel illumination changes. Perhaps, the constant incubatortemperatures of 1 8'C may have been to warm.
Natural photoperiods in Lahore area range from 14: 1 7L: 9 : 43D on 2 1 June
to 10: 09L: 13: 51D on 21 December, with 1 1 : OOL : 13: OOD occurring on I Nove‑ember. Photoperiods in the incubator were markedly longer and shorter than thenatural condition. Some "long day" insects such as Acronycta enter diapause only
after exposure to critical photoperiods; days which are either too long or too shortfail to induce diapause (Lees, 1 955). Possibly, the 9L: 1 5D photoperiod presented

in the present study was not recognized as diapause induction stimulus by Cx.
tritaeniorhync/tus.

Bifurcation of overwintering and non‑overwintering populations may begin
as early as October in Punjab. At this time, afternoon water temperatures at
breeding sites were 25'C, dropping to 15 or 16'C by early morning; however,developmental time in nature remained at 6 to 7 days (Reisen and Siddiqui, 1 979).
In the present study at 9L: 15D and 22'C, E50 exceeded 13 days, and thus if theimmature stages were receptive to the induction cue, they should have experienced
a sufficient number of "short‑day" photoperiods to induce diapause. Our results
suggest that the exposure of the immature stages to a given number of short‑day
photoperiods was not the triggering stimulu responsible for diapause induction.
In agreement, Omori, et al. ( 1 965b) used adults reared from their laboratory colony(no details given) for their diapause experiments, and Eldridge ( 1 962) found that46

blood feeding activity of females reared at 1 4L: I I D decreased after exposure to 2
to 4 " short‑day" (8L : 16D) photoperiods.

ACKNOWLEDGMENTS
We thank Ms. Khawar Azra and Messrs. J. Ahmad, S. Amar, S. H. Chishti,
T.J. Rathore and especially A. A. Shahid for their technical assistance. Dr. R. K.
Sakai kindly provided the Balloki strain eggs for experimentation, and Drs. R. K.

Sakai and C. G. Hayes, PMRC, and T. Oda, Nagasaki University School of Medi‑cine, offered advice and comments during the preparation of the manuscript. This
research was funded by Grant No. AI‑10049 from the U. S. Institute of Allergy
and Infectious Diseases, N. I. H. The administrative assistance of the U. S. Agency
for International Development in Pakistan is acknowledged.

REFERENCES
Baker R H Reisen, W. K., Sakai, R. K., Hayes, C. G., Aslamkhan. M., Saifuddin, U. T., Mah‑mood, F., Perveen, A. & Javed. S. ( 1979) . A field assessment of mating competitiveness ofmale Culex tritaeniorhyhchus carrying a complex chromosomal aberration. Ann. Entomol.
Soc. Amer. 72 : 751‑758.
Beach, R. ( 1978). The required day number and timely induction of diapause in geographic strainsof the mosquito Aedes atropalpus. J. Insect Physiol., 24, 449 55.

Bullock, H. R.. Murdoch, W. P., Fowler, H. W. & Brazzel, H. R. ( 1959). Notes on the over‑
wintering of Culex tritaeniorhynchus Giles in Japan. Mosq. News, 19, 18

l88.

Clements, A, N. (1963). The physiology of mosquitoes. Permagon Press, New York.
Eldridge, B. F. ( 1963). The infiuence of daily photoperiod on blood‑feeding activity of Culex tri‑
taeniorhynchus Giles. Am. J. Hyg., 77, 49‑53.
Fujimiya, Y. and Yajima, T. ( 1976). Seasonal changes in the wing length, head width and proboscis
length of the mosquito Culex tritaeniorhynchus summorosus Dyar in relation to the physiologicalage and overwintering. Ann. Sci., Kanazawa Univ., 13, 91‑lOO.
Harada, F.; Moriya, K. & Yabe, T. ( 1968). Observations on the habits of feeding and ovipositionof Culex tritaeniorhynchus summorosus Dyar (II). Jap. J. Sanit. Zool., 19, 230‑236.

Lees, A. D. ( 1955). The physiology of diapause in arthropods. In: Chapman, R. F. ( 1971) The
insects structure and function. American Elsevier Publ. Co. pp. 721‑723.
Mogi, M. ( 1978). Population studies on mosquitoes in the rice field area of Nagasaki, Japan, es‑
pecially on Culex tritaeniorhynchus. Trop. Med. (Nagasaki), 20, 1 73‑263.

Nakamura, H., Yoshida, M. & Ito, S. ( 1968). Seasonal change in the wing length of Culex tritaenior‑hynchus Giles in relation to overwintering of Japanese encephalitis virus. Jap. J. Ecol., 18,

259‑263.
Oda, T. & Wada, Y. ( 1973). On the gonotrophic dissociation in Culex tritaeniorhynchus summorosus
females under various conditions. Trop. Med. (Nagasaki), 15, 189‑195.
Omori, N., Ito, S., Taketomi, M., Mifune, K., Shichijo, A. & Hayashi, K. (1965a) . Experimental
hibernation of Culex tritaeniorhynchus in Nagasaki, Japan. End. Dis. Bull. (Nagasaki), 7,
288‑295.
Omori, N., Wada, Y., Kawai, S., Ito, S., Oda, T., Suenaga, O. & Nishigaki, J. (1965b). Preliminary
notes on the collection of hibernated females of Culex tritaeniorhynchus in Nagasaki. End. Dis.

Bull. (Nagasaki), 7, 147‑153.

47
Reisen, W. K. ( 1978). A quantitative mosquito survey of 7 villages in Punjab Province, Pakistan,with notes on bionomics, sampling methodology and the effects of insecticides. Southeast
Asian J. Trop. Med. Publ. Hlth., 9, 587‑601.
Reisen. W. K. & Siddiqui, T. F. ( 1978). The influence of conspecific immatures on the oviposition
preferences of Anopheles stephensi Liston and Culex tritaeniorhynchus Giles (Diptera : Culicidae) .
Pak. .J. Zool., 10, 31‑32.

Reisen. W. K. & Siddiqui, T. F. ( 1 979). Horizontal and vertical estimates of immature survivorshipfor Cu!ev tritaeniorhynchus (Diptera: Cullcidae) in Pakistan. J. Med. Entomol. 16: 207‑218.
Reisen, W. K., Baker, R. H., Sakai, R. K., Aziz‑Javed, A., Aslam, Y. & Siddiqui, T. F. (1977). Ob‑
servations on the mating behavior and survivorship of Culex tritaeniorhynchus Giles during lateautumn (Diptera : Culicidae) . Southeast Asia J. Trop. Med. Publ. Hlth., 8, 537‑545.
Reisen. W. K., Sakai, R. K., Baker, R. H., Rathor, H. R., Raana. K., Azra, K. & Niaz, S. (1980).
Field conrpetitiveness of Culex tritaeniorhynchus Giles males carrying a complex chromosomal

aberration: a second experiment. Ann. Entomol. Soc. Am. 73: 479‑484.

Rersen W K Siddiqul T F Khan A Q Mahmood, F. & Parveen T ( 1979) The time ofmating of the arbovirus vector, Culex tritaeniorhynchus Giles(Diptera : Culicidae), underlaboratory conditions. Entomol. Exp. Appl., 25, 267‑278.
Shahid, A. A., Parveen, T. & Reisen, W. K. ( 1980) . Changes in weight, calories and triglyceride
content of Culex tritaeniorhynchas Giles and Anopheles st"･phensi Liston fed sucrose. Pak. J.
Zool., 12, 163‑169.

Siddiqui, T. F., Aslam, Y. & Reisen, W. K. (1976). The eff;ects of larval density on selected im‑mature and adult attributes in Culex tritaeniorhynchus Giles. Trop. Med. (Nagasaki), 18, 195‑
202.

Sokal, R. R. & Rohlf, F. J. ( 1969). Biometry. W. H. Freeman & Co., San Francisco.
Takahashi, M. (1970). Appearance of the gonotrophic disassociation in Culex tritaeniorhynchus underseml expenmental condrtions. Jap. J. Sanit. Zool., 21, 18‑23.
Van Handel, E. ( 1966a). Temperature mdep ndence of the composmon of trrglyceride fatty acids
synthe lzed de novo by the mo quito. J. Lipid Res., 7, I 12‑115.
Van Handel, E. ( 1966b). The thermal dependence of tbe rates of glycogen and triglyceride synthesisin the mosquito. .J. Exp. Biol., 44, 523‑528.

Wada. Y., Oda, T., Mogi, M.. Suenaga, O., Miyagi, I.. Omori, N., Ito, S. & Nishigaki, J. (1973).
Ecology of vector mosquitoes of Japanese encephalitis, especially o' Culex tritaeniorhynchus

summorosus. 5. Overwintering of Culex tritaeniorhynchus summorosus and Anopheles sinensis.
Trop. Med. (Nagasaki), 15, I 1‑22.
Yo hida. M., Nakamura, H. & Ito, S. (1974) . E fects of temperature and photoperiod on the larval
development in Culex tritaeniorhynch, s su'n710rosus Dyar. Jap. J. Sanit. Zool., 25, 7‑1 1 .

JAPANESE JOURNALOF
TROPICAL MEDICINE AND HYGIENE
Vol. 9.No. lJune, 1981

CONTENTS
Original article

Kumazawa, H., Suzuki, N. and Okura, T.
The Fourth Case of Diplogonoporiasis in Kochi Prefecture. (in Japanese)

Hashiguchi, Y.
Examinations on the Protective Immunity to the Common Liver Fluke, Fasciola inRats. Using Immunosuppressants or Diffusion Chamber Techniques.

l‑ 7

9‑2 2

Hori, S.

Effect of Exercise in a Hot Environment for 7 Successive Days on Physiological
Responses and Heat Tolerance.

.23‑29Makino, Y. and Mifune, K.
Fluorescent Focus Size Reduct on by Spleen Cells of Mice Immunized with
Rabies Virus.

.31‑36

Niaz, Sh, and Reisen, W. K.
Culex tritaeniorhynchus Giles : Some Effects of Temperature and Photoperiod on
Larval Development and Selected Adult Attributes. . . . . . . .

Published byJAPANESE SOCIETY OF TROPICAL MEDICINE
c/o Institute for Tropical Medicine, Nagasaki Univcrsity

12‑4 Sakamoto‑machi, Nagasaki, 852, Japan. 3 7‑47