士心

一冒口

雑囎

㏄

会㎞
学融
医劇
帯田

a
n
r
U
O

本脚

日J

第7巻第3，4号

昭和54年12月15日

内

容

著

原

亜熱帯生育者，亜熱帯生まれ温帯移住者，温帯生育者の体格
および体温の比較研究……………一辻田 純三，黛誠，田中 信雄，堀清記153−160

T7塑朋oso脚即励」6nsθの37Cにおける短期間培養
マウスレ細胞を共存させての試み（英文）……・・福間

利英，神原 広二，中林 敏夫161−169

スナネズミ腹腔より得たβ耀g毎餌h伽gゴミクロフィラリアの、464θ80εgyが」における発育（英文）∴………一荘正

國，中島・康雄，青木克己

171−189

フォーカス計数法によるデングウイルス感染価測定と中和抗体測定法の比較的感度（英文）…一…………・………・………五十嵐 章，奥野 良信，深井孝之助191−199
狂犬病ウイルスに対する種々細胞株の感受性（英文）・三舟求真人，牧野 芳大，万年三和明201−208

短報

麻疹の疫学について（英文）・・Ashitey，G．A．209−211

投稿規定

日

熱医会誌

Japan．J．T．M．H．

日

本熱帯医学会

日本熱帯医学会雑誌

第7巻

第3，4号

1979

153−160頁

153

亜熱帯生育者，亜熱帯生まれ温帯移住者，温帯生育者の体格および体温の比較研究
辻田純三・黛

誠・田中信雄・堀清記

昭和54年3月5日

受付

膚血管の緊張度が低下し，皮膚温が高く，冬は緒

低くなる季節変動を示すが（季節生理班，1952；
日

Herzman，1956），高温環境に馴化した亜熱帯人がヒトが高温環境に馴化すると，体温調節機能に長期間温帯に生活するとき，皮膚温の調節機能が適応的な変化があらわれ，一定高温負荷に対してどのように変化するかを研究した報告はない。暑体温の上昇度が減少し，より大きな高温負荷に耐熱馴化が長期に及べば，体温調節の適応的変化のえられるようになる（Dil1窃α孟，1938；Robinson

他に身体組織にも適応的変化があらわれ，体格やθ雄乙，1941；Wyndham，1967）。高温環境に曝露さ体構成が変化する。熱帯住民は痩せ型の体型をもれたときの体温調節は，・主として放熱の調節につヒ・トが多く，体重当たりの体表面積が大きく，よってなされる。放熱量の増加は発汗がないか，放熱に有利な体型をもつといわれている（Coon窃

もしあっても発汗量が少：ない場合は主として皮膚

鳳，1950）。また，高温季候下に生活すると他の組血管の拡張による皮膚循環血流量の増加と皮膚温

織より熱伝導率の低い（Keysand

上昇によって，皮膚温と気温の差を大きくするこ

皮下脂肪厚が減少して，体熱の放散を容易にするとによってなされる（Robinson，1949）。さらに高適応的変化があらわれるといわれている（堀ら，

温負荷が大きくなると発汗が発現し，体温の蒸発

1974）・本土生育者の皮脂厚は冬に厚くなり，夏調節が行われる。ヒトは発汗による体温調節能力に薄くなる季節変動を示すといわれている・（鈴木が秀れており，高温馴化時には発汗反応に大きなら，1959）。生下時より高温環境によく馴化した

変化があらわれるため，高温馴化時の発汗反応の亜熱帯生育者が本土の温帯季候下に生活すると，適応的変化についての研究は数多いが，皮膚温体型，体構成がどのような変化を示すかは興味深

Br6zek，1953）

に関する適応的変化の研究は少ない（Dil1θ孟孤，い。沖縄生育者が本土に移住した場合，本土の温1938；Kuno，1956；Bass，1963）。しかし，高温環

帯季候が皮膚温の調節機能と，体格，体構成にど境下での体温調節機能としては，皮膚温の調節能のような影響を及ぼすかを研究するために，沖縄

力は発汗反応と同様に重要である。高温馴化時に生まれの本土移住者，および本土生育者の発汗準みられる適応的変化は，適応期間が短い場合と長備状態での皮膚温の測定，および身体計測を行っ期間に亙る場合では，その様相に差異があることて，以前我々が沖縄本島で本研究と同じ条件下でが認められている。短期間の高温馴化時にみられ測定した沖縄生育者の体温と身体計測結果を比較る生理的反応は，暑熱負荷を中止すると比較的はした（辻田ら，1978）。

やく失なわれることが知られている（Williamsθ∫砿，1967）。しかし，長期間の高温馴化によって獲実得された適応的変化は容易には消失しない（Horiθ∫磁，1976）。皮膚温は同じ環境下でも，夏は皮

兵庫医科大学生理学第一講座

験 方法

沖縄生まれで本土在住期間が平均15．8年の成人

663西宮市武庫川町1−1

154

男子12名，および本土生育者の成人男子11名の測

腹：乳腺上，贋の高さ

0．162

定は西宮市で7月に行った。沖縄より本土に移住

背：肩甲骨下角直下

0．083

した群の対照群として，沖縄生育者の成人男子7上腕三角筋中央点0．082

名の測定を那覇市で7月に行った（辻田ら，1978）。

前腕

前腕内面中央線下から1／3

0．061

皮脂厚は栄研式皮指厚計を用いて，皮膚接触面積

手：手背中央線上から1／3

1mm2当たり1gの一定圧で皮膚をっまんでから大腿

大腿前面中央線上から1／2

0，172

2秒後に測定を行い，数回の測定値の平均値をそ

下腿

下腿後面中央線上から1／3

0，134の部位の皮脂厚とした。皮脂厚の測定部位と平均

足：足背中央線上から1／2

0．053

0．072

皮脂厚算出に用いた加重平均係数は次の通りである（堀ら，1974）。

部

結

係数

位

上腕：右腕背面，肩峰と肘頭の中間

0．082

果

1．身体的特徴

沖縄生育者（0群），沖縄出身の本土住民（OM

および右腕前面中央部の平均値
背部：右肩甲骨下角

0．083

群），および本土生育者（M群）の身体計測値と胸部：右前腋下窩嫉壁と右乳頭の中間0．083

腹部：右乳腺上，膀の高さ

0．081

腰部：右肋骨下縁と右腸骨稜の中間0．081

大腿：右大腿中央，前面と背面の平均0，172

OM群の本土滞在年数を表1に示した。OM群の身長，および体重の平均値はそれぞれ169．6cmと62．8kgで，M群の平均値170．2cm，および64．3kgよりわずかに小さかったが，0群の平均値166．6cm，および58．6kgよりかなり大きかっ

値

た。OM群の胸囲，および上腕囲の平均値は90．4

体脂肪含有率fは予知式（堀ら，1974）∫（％）＝28，9×

体表面積（m2》×平坂皮脂星（mm）＋3β7
体重（kg）
を用いて算出した。

体表面積の算出には，高比良の式（高比良，1925）
を使用した。

cmと28．1cmで，M群の平均値89．9cm，および27．8cmよりわずかに大きく，0群の平均値
86．6cm，およぴ26．1cmよりかなり大きかった。

OM群の大腿囲の平均値は50．1cmで，M群の平均値52．4cmより小さく，0群の平均値49．6cmよりは小さかった。これらの差のうち，0群

皮膚温の測定は発汗のない状態での乾性放熱能の上腕囲が両群の平均値より有意に小さかったが，力の指標としての比較が目的であるから，発汗準

他の差は統計的には有意ではなかった。全体とし備状態での皮膚温を測定した。体温に対する運動，てOM群はM群と比較してわずかに体格が小さ

特異力学作用および日内変動をさけるために，測い傾向を示したがほとんど差がなく，0群はOM

定日は被検者には激運動をさせず，空腹状態で午群，およびM群と比較して全ての測定項目の平後3時より実験を開始した。被検者は水泳パンッ

均値が小さく，かなり体格が小さいことが判る。

だけを着用して，30C，湿度70％の室内に30分間

2．皮脂厚

安静をとらせたのち，全身10カ所の皮膚温をパイ

皮脂厚の測定値は，表皮と真皮，および皮下脂

ロメーターを用いて測定した。舌下温は婦人体温

肪層の厚さをそれぞれ二層ずつ含んでいるが，表計を用いて測定した。皮膚温の測定部位と平均皮

皮と真皮の厚さは約1mmで個人差が少なく，皮膚温算出に用いた加重平均係数は次の通りである

脂厚の大小は皮下脂肪層の厚さに左右されるので，（Horiθ頗乙，1977）。

皮脂厚は皮下脂肪層の厚さを表わす指標として用位

係数いられる。皮脂厚の測定値を，部位別に表2に示

額：前額中央眉上2cm

O．098

した。測定された全ての部位で0群の平均値は部

胸：乳腺と第4肋間の交点

0．083

OM群，およびM群の平均値より小さく，0群

i55
Table

Gr。upNumber

Age

Height

（yr）

0

7

OM
M

12

21．0
±1．9

26．0
土3．6

22．8

11

l

±1．9

Characteristics

WeightB．S．A．

（kg）

166．6
±5．7

±5．5

169．6
±4．0

62．8

1．74

90．4

±6．6

ニヒ0．07

±5．1

170．2
±5．6

64．3
士7．9

1．76
±0．12

89．9
二L5．7

（m2）

58．6

0：Residents

ofOkinawawhowerebomandraised

who werebom andandraisedtheinraisedMain

inTable2

±

14．3

±6．9

士8．3

0，0M，M：The

same

Mean

mm）arevalues（in

as

0リノ

10．6

15．8
±7．7

竹ノ∩∠

11．1

±6．9

11．0
士5．0

31 乙⊥1

0M
M

49．6
±1．6
50．l

ofthe

27．8
士1．9

Main

52．4

ofthe
who

sur魚cearea，G．C．：Girthvaluesareand other

two

with

groups

土6．7

±3．5

land

given

l5．8

士3．7

Okinawa，OM：Residents

thickness

旦pperarm

士 士

8．0
±3．5

26．1＊

±0．8

group

anterlor posterior
3QO

0

Abdomen

Skinfbld

30

Chest

86．6

28．1

mam

（yr）

±3．3

in

O

（cm）

二k1．9

Staxinland

1．67

ofthigh。Mean

between

（cm）

G．T．

±0．10

land ofJapan，B．SA：Body

armフG．T．：Girth

G．A．

（cm）

OkinawaコM：Residents

deviations， ＊Significant dif驚rence

Group

G．C．

（cm）

land

G、A．：Girth ofupper

ofsu切ects

Mainwere．born
of

their

chest，
standard

at5％leveLof suhjects

Back

Waist

Thigh

anterior

posterior

士L2

8．6
士1．4

7．6
±3．1

10．8

16．5

11．7

13．9

圭4．0

±6．8

土4．2

士5．5

7．9
±3．3

9．8
±2．4

18．4

9．4

13．9

±9．4

±4．1

士6．5

5．5

in

Table1．

given

with theirstandard

10．5
±3．8

9．2
±3．3

11．8
±4．6

deviations．

の皮下脂肪層の厚さは他の両群よりかなり薄かっ

130．3より小さく，0群の平均値126．5より大きい。

た。OM群の平均値は胸部，腹部，背部および大OM群のBrugsch指数の平均値は53．3で，M群

腿前部でM群の平均値よりわずかに大きく，上 の平均値53．7よりわずかに小さく，O群の平均値
腕部，腰部ではわずかに小さかった。即ち，OM 52．0よりかなり大きかった。体表面積と体重の比
群とM群の皮脂厚はほとんど差がなかった。皮 は，高温環境下での体温調節能力に関係する重要
脂厚の最も厚い部位は0群では大腿後面，OM群 な指標であるが，この指標の平均値はOM群がとM群では腰部であった。全体として0群は他 278でM群の平均値276よりわずかに大きく，0の両群に比較して躯幹部の皮脂厚がうすく，皮脂群の平均値285よりかなり小さかった。体型を表厚の部位による差が少ない傾向を示している。

わすRohrer指数，Brugsch指数，体表面積と体3， 形態指数および栄養指数重の比のOM群の値はいずれもM群とO群の身体計測値を用いて算出されたRohrer指数，

間にあって，M群との差はわずかで，0群とのBrugsch指数（比胸囲），体表面積と体重の比，差はかなり大きかった。従って，OM群の体型は体脂肪含有率，および平均皮脂厚の平均値と標準0群よりも，むしろM群に近いといえる。OM

偏差を表3に示した。Rohrer指数は身体充実度，または体型を示す指数であるが，OM群のRohrer指数の平均値は128．7でM群の平均値

平均値12．8％，および0群の平均値11．4％より

群の体脂肪含有率の平均値は13．3彩で，M群の大きく，平均皮脂厚もOM群の平均値12．2mm

156
Table3

Physicalandnutritional

indicesB．S．A．（cm2）Group

Rohrer，s index Brugsch，s

一Body伝t（％）Mean

index

S．T，（mm）

Weight（kg）

OM
M
B．S．A．：Body
as

in

Mean

285
±10
278
士14
276
±16

52．0

126．5
土6．6

O

土1．7

128．7

53．3

±13．0

±2．9

130．3

53．7

士12．3

土3．7

sur魚cearea，Mean

S．T．：Mean

withstandard

9．3
士2．8

11．4
士2．4

13．3

12．2

士3．2

±4．5

12．8

11．7

±3．2

±4．6

skinfbld thickness，

0，0M，M：Thesame

Table1．

valuesare given

theirdeviations．

は，M群の平均値1L7mm，および0群の平均 部の皮膚温は3群ともに最高値を示した。OM
群の前額部の平均値とM群の平均値はともに36．1Cで，0群の平均値35．9Cよりかなり高値9．3mmより大きかった。体脂肪含有率と皮脂厚についても，OM群とM群の差はわずかであるが，OM群と0群の差はかなり大きい。

かった。躯幹部，即ち胸部，背部，腹部の皮膚温

4．体温の平均値は，いずれもOM群が最も高く，0群

口内温（島），測定部位別の皮膚温，平均皮膚が最も低かった。上肢の温度は3群で大差がなく，

温（T、）および7㌔一丁、gradientの平均値と標準下肢の皮膚温はOM群が最も低く，0群が最も高かった。平均皮膚温はOM群が34．8Cで最も偏差を表4に示した。OM群の島の平均値は低く，M群は34．9C，0群は最も高く35．OCで37．1CでM群の平均値37．2Cより0．1C低く，0群の平均値37．O Cより0．1C高かった。前額
Table437．0＊

Forehead

35，9

Chest

34．4

Abdomen

34．5

Subscapular

35．0

Upper arm

35．2＊

Forearm

35．4

Hand

35．4

Thigh

34．8

Calf

35．0＊α

Foot

34．7

T3

35．0

T。一73

2．02

S．D．

Mean

skin

temperature

OM

S．D．

37．1＊

36，1

35．3

35．0
35．1

34．7
35．5
35．6

34．4
34．0＊g

34．1

34．8
2．35

6α
80
石α
7α
50
フ0
50
5α
70
￡1
3α
60
石
α

η

0

and

Mean

M

37．2
36．1

34、8
34．5
35．1
34．4＊

35．3

35．6
34．7
34．8＊わ

34．8
34．9
2．29

S．D．

3
6
7
8
6
7
6
αα
αα
αα
αα
0α
Lα
0 797167

Mean

temperature

2
3
0
9
0
9
7
αα
Lα
Lα
00
αα
Lα
0 798278

Group

0ral

あった。7㌃T、gradientの平均値はOM群が0，0MンM：ThesameasinTableL 7も：OraltemperatureンT8：Meanskintemperature，
Meanvalues（inbctween

two

C）are

groupsgiven

at5％leveL

with

their

standard

deviations．＊Signi丘cantdi飾rences

157

2．35Cと最も大きく，M群は2．29Cでわずかするが，放熱量は体表面積に比例する。従って体にOM群より小さく，0群は2．02Cで両群よ 重当たりの体表面積の比が大きい体型のヒトは，高温環境下で体温の調節能力が大きい。この値のりカ・なり小さカ】った。

0群の平均値はOM群，M群より大きく，OM
考察

ヒトの体温調節機能は，季候の変化に応じて適群とM群の差はわずかである。以上より0群はM群より高温環境に適応した体型をもっているが，OM群はM群とほとんど同じ体型であると応的な変化をうけることはよく知られているが結論できる。しかし，季候以外にも栄養状態や身（Robinson，1949；Williamsθ孟認，1967），異なる体運動を含めた生活様式も，身体の発育に影響を季候下に長期間生活すると体格，体型，身体構成

およぼすことが知られているので，OM群とOも変化するといわれている（Lewisθ孟磁，1960）。

群の身体的差異は季候以外の因子の関与もあると亜熱帯の沖縄の季候は夏の高温多湿の期間が長く，考えられる。

気温の日内変動巾が小さい。冬は温暖で，沖縄住表2に示されたように，皮下脂肪厚の平均値は民は一年を通じて高温環境に曝露される機会が多測定された全ての部位で，0群は他の両群よりかい。一方，日本本土は温帯に属し，夏の高温期間なり小さく，OM群の平均皮脂厚の平均値はMが短く，冬は寒い。沖縄生育者と本土生育者の体群の平均値よりわずかに小さい。脂肪組織の熱伝温調節機能，体型，体構成を比較すると，沖縄生導率は，非脂肪組織の熱伝導率より低く，皮下脂

育者は高温環境下での体温調節機能が秀れており，肪層の厚さが薄くなると体熱の放散が容易に行わ

皮下脂肪厚が薄く，体重当たりの体表面積が広い

れる（Keysθ

等の，高温環境下での体温調節に有利な体構成と体型をもっているといわれている（Hori8孟磁，に関しては，OM群およびM群より有利であると推定される。30Cの環境温は，安静状態のヒ

1977）。本研究の被検者のOM群は，沖縄に生まトが発汗を発現させずに，皮膚血管の調節で体熟れて幼少年期を沖縄で過ごした後，本土に移住しの平衡を維持できる最高外気温である。このときて本土での在住期間が平均15．8年と長く，0群はのヒトの状態は，外気温が上昇するとすぐに温熱

本土での生活経験がないので，OM群と0群の差は，少年期以後のOM群の温帯季候下での生活によって，もたらされたものと考えられる。OM

性発汗が発現するので，発汗準備状態といわれて群とM群の差は，OM群が幼少年期に熱帯季候下で生活したことによる差とみなしうる。OM群
は0群と比較して，身長，体重，Rohrer指数，
Brugsch指数の平均値がかなり大きく，M群と温の差に比例するので，放熱量は皮膚温の高い方比較するとわずかに小さい。低温季候下に生活す群，およびM群より高く，このことは0群が他ると体格が大きくなり，体重が重くなって，身長の両群より放熱量が大きいことを示唆する。局所に比して躯幹部が大きくなって，体重も増加するの皮膚温を比較するとO群はOM群，M群とといわれている（Coonα認，1950）。従ってOM

比較して四肢，ことに下肢の皮膚温が高い。四肢群の体格や体型が，0群よりむしろM群に近いの表面積は体表面積の60鰯をしめ，この部位の皮ことは，OM群が温帯で幼少年期以後を過し，寒膚温の上昇が大きいことは，皮膚からの放熱量を冷季候下で長く生活した影響が，幼少年期に生育大きくする。このことは0群はOM群，M群にした熱帯季候の影響より大きいものと推定され

比し，四肢の皮膚血管の拡張度が強く，四肢の皮る。歩行や走行等の体重の移動を伴う運動を行う

膚循環血流量が増加して四肢の皮膚温が高くなっときの代謝量は，体表面積よりむしろ体重に比例たものと考えられる（Hertzman，195gl

磁，1953）。従って0群の体熱放散いる。皮膚面からの放熱量のうち，伝導，対流および輻射の機転による乾性放熱量は皮膚温と外気が大きく，皮膚温の高さは放熱能力を示すことになる。

表4に示したように，0群の平均皮膚温はOM

Rothman，

1581954）。島一丁，gradientは安静状態で代謝量にほ果を得た。

とんど差がなく体温の変化しないときは，身体の1．OM群は体格，体型ともにM群とほとん

熱貫流率と逆比例の関係にある。0群の．7㌔一丁、ど差がなく，0群より身長は高く，体重が重く，gradientの平均値は2．02CでOM群の平均値

胸囲，上腕囲，大腿囲も大きかった。OM群の形

2．35C，およびM群の2．29Cよりかなり小さい。

態指数はM群との差は少なく，0群と比較する
従って0群は，他の両群より高い熱貫流率をとRohrer指数，比胸囲が大きく，体重当たりのもっていることを示す。この0群の大きな身体 体表面積比が小さかった。

熱貫流率は，高温馴化による血液量の増加と皮膚血流量の増加による高皮膚温の維持能力の向上に

2．OM群の皮下脂肪厚，および体脂肪含有率
はM群とほとんど差がなく，0群と比較するとよりもたらされたものと推定される（Bass8∫磁，

皮下脂肪厚は薄く，体脂肪含有率が少なかった。

1936；Hertzman，1959）。OM群とM群の身体の3．30CにおけるOM群の口内温の平均値

熱貫流率はわずかである。即ち，生下時より成人は371CでM群より0．1C低く，0群よりまで亜熱帯季候下に生活したヒトは高い熱貫流率

0．1C高かった。平均皮膚温はOM群の平均値はを保ち，皮膚温を高く維持する能力を獲得して，3生8CでM群より0．1C低く，O群より0．2C

秀れた乾性放熱能をもつことができるが，幼少年低かった。口内温と平均皮膚温の差はOM群では2．35CでM群の2．29Cよりわずかに大きく，
0群の2．02Cよりかなり大きかった。

期に亜熱帯季候下に生活し，少年期以後を温帯季候下で長期間生活したヒトは，生下時より温帯に生活したヒトと同程度の熱貫流率と乾性放熱能を持っていると結論できる。

4．OM群の体格，体型，乾性放熱能力，身体の熱貫流率はM群とほとんど差がなく，0群は高温環境に馴化した熱帯人に近い体格，体型を示し，他の両群と比較して秀れた乾性放熱能力を持

約

要

ち，高い身体の熱貫流率をもっことが判った。

沖縄生育者の成人男子7名（0群），沖縄生ま5．幼少年期に亜熱帯に生活した後，温帯に移れで本土在住期間が長い成人男子12名（OM群），

住して長期間温帯季候下で生活すると体格，体型および本土生育者11名（M群）の身体計測とおよび高温環境下での耐熱放熱能力は亜熱帯生育

30C，湿度70彩の室内にて安静座位における口内者よりむしろ温帯生育者のそれに近くなる。

温と全身10カ所の皮膚温を測定して次のような結文
1） Bass，D．E（1963）：Thermorequlatory

inman，InTemperature，Its

and献
circulatory

Measurement

andadjustments

Controlinduringacclimation

to

heat

ScienceandIndustry．ed．bycompartments

to

heat

Herzfeld，C．M．Reinhold，New York，Vo1．3，pp229−305
2） Bass，D．E

andHenschel，A。（1959）：Responses

of

body

Huid

and

cold，

Physio1．Rev．，36，128−144

3）Coon，C

S・，Gam，S、M．and

formation

4）

inman，Charles

Di11，D．B．，Hall，F、G
acclimation

Birdse11，J．B．（1950）：Race：A study
C

andproblems ofrace

Thomas，11L

Edwards，H、T．（1938）：Changes

incompositionofsweat during

to heat，Am．J．Physio1．，123，412−419

5） Hertzman，A．B。（1959）：Vasomotor regulation

280−306of theofcutaneous

circulation，PhysioL

Rev．39，

159

6）

堀 清記，飯塚平吉郎，中村正（1974）：沖縄住民と本土住民の皮下脂肪厚および体脂肪含有率の比較，栄養と食糧，27，335−339
7）Hori，S．，Ihzuka，H．and Nakamura，M．（1976）：Studies
residents

inOkinawa

to

a

hot

of

physiological responses

environment，Jap．J．PhysioL，26，235−244

8）Hori，S．，Ohnaka，M．，Shiraki，K．，Tsujita，J．，Yoshimura，H，Saito，N、and

Comparison
andofJapanese

9） Keys，A．and

physicalinacharacteristics，bodyneutraltemperature

Brozec，」．（1953）：Body

10） Kuno，Y．（1956）：Human

of

men

on

a

polar

temperature

and

basal

Panata，M・（1977）：

metabolism

betweenThaizone，Jap．J．PhysioL，27，525−538

fat inadult man，Physio1．Rev．，33，245−325

perspiration，Charles

11）Lewis，H．E．，Masterson，J．P．and
ness

of

C．Thomas，Spring丘eld．

Rosenbaum，S．（1960）：Body

weight andskinfoldthick−

expedition，Clin、Sci，，19，551−561

12） 文部省科学研究費医学関係総合研究班（季節生理班），（1956）：日新医学，39，121−133
13）Robinson，S．，Dill，D．B．，Wilson，J．W．andand negroes
14）

toprolonged

work

in

Robin怠on，S．（1949）：Physiological

1ationandthe

Scienceofhumid

Nielsen，M．（1941）：Adaptations of whiteadjustment

Clothing．，

manheat，AmJ．Trop．Med，，21，261−287

ed．by

to

heat，In

Newburgh，LPhysiologyof

Temperature

Regu・

H、Saunders，Philadelphia，pp．193−

231
15）

Rothman，S．（1954）：Physiology

andBiochemistry ofskin，Univ・Chicago

Press，Chicago，

U．S．A．
16） 鈴木慎次郎，手塚朋通，大島寿美子，早川徳子，鈴木秀雄，山川喜久江，長嶺晋吉（1959）：東

北地方における高血圧症の栄養学的調査研究く第3報），日本栄養学雑誌，17，209−218

17）辻田純三，堀 清記（1978）：沖縄（亜熱帯）生育者と本土（温帯）生育者の発汗反射，能働汗
腺数および体温の比較，日熱医会誌．，6，157−165

18）Williams，C．G．，Wyndham，C．H．andin summer

and winter，エAppL19）Wyndham，C．H．（1967）：E仔ect
tionship，Morrison，工F、（1967）：Rate

of

loss

of

acclimatization

Physio1．，22，21−26

ofacclimatizationonthe

J．ApPL Physio1．，22，27−30

sweatrate／rectaltemperature rela−

1 60

COMPARISON OF PHYSIQUE AND BODY TEMPERATURE
AMONG SUBTROPICAL NATIVES, IMMIGRANTS
OF SUBTROPICAL NATIVES TO TEMPERATEZONE AND TEMPERATE NATIVESJUNZO TSUJITA, MAKOTO MAYUZUMI, NOBUO TANAKAAND SEIKI HORI
Received for publication 5 March 1979

Anthropometrical measurements and measurements of body temperature were made in July inNaha (Okinawa, subtropical zone) on 7 young male Japanese who were born and reared in Okinawa(group O), and in July in Nishinomiya on 12 young male residents of Main Islands ofJapan (temperatezone) born and reared in Okinawa (group OM) and I I young male residents of Main Islands born andreared in Main Islands (group M). The mean value of duration of residence in Main Islands ofsubjects in group OM was 15.8 years. Skin temperatures at 10 site.s and oral temperature of seatedsubjects, wearing short only, were measured in a climatic chamber maintained at 30 C and 70 per centrelative humidity after sitting in a chair at rest in the climatic chamber.

Group O showed shorter height, Iighter body weight, Iarger girths of chest, upper arm and thighthan group OM and M. Group O showed smaller mean values of Rohrer's index and Brugsch's index
and greater mean value of the ratio of body surface area to body weight. Group O showed thinner
skinfold thickness and smaller percentage of body fat content than other two groups. Differences ofanthropometrical measurement between group OM and group M were much less when comparedwith those between group O and other two groups. Mean values of oral temperature for group O,
OM and M were 37.0 C, 37. I C and 37.2 C respectively. The mean values ofmean skin temperatures
for group O, OM and M were 35.0 C, 34.8 C and 34.9 C respective]y. Group O showed considerably

smaller value of To

Ts gradient (2.02 C) than group OM (2.35 C) and group M (2.29 C). These

results indicate that physical characteristics of subtropical natives have changed and that differenceof physical characteristics between subtropical natives and temperate natives and capacity of main‑taining higher skin temperature and higher conductive‑convective heat transfer coefB:cient observed in
subtropical natives were reduced by long residence in a temperate zone.

Department of Physiolo y, Hyogo Colle e of Medicine? Nishinomiya

Japan. J. Trop. Med. Hyg., Vol. 7, No. 3, 4, 1979, pp. 161‑169

161

AN ATTEMPT FOR SHORT‑TERM CULTIVATION
OF TR YPANOSOMA GAMBIENSE IN COEXISTENCEWITH THE MOUSE L‑CELL (AT 37 C)
TOSHIHIDE FUKUMAl, HIROJI KANBARA AND TOSHIO NAKABAYASHI
Received for publication 16 March 1979

Abstract : It was attempted to cultivate Trypanosoma gambiense (Wellcome strain) ,maintained by mouse‑passages for years, in coexistence with the mouse L‑cells in vitro at

37 C. In the system of culture the population of the cultured trypanosomes increased
3‑fold in 24 hours, then decreased. The fact that the doubling time of trypanosomes
was longer in the culture system than in an infected mouse seemed to be related to the
suppressed division of parasites. It was observed that the final stage of parasite division,that is, the cytoplasmic separation into 2 individuals was much prolonged in culture,
although the kinetoplastic, nucleal and fiagellar duplication was carried through without

suppression. In the control without the L‑cells, no multiplication of trypanosomes took
place. Infectivity of the cultured trypanosomes to mice did not change throughout thecourse of culture, at least until the time of the maximum growth of parasites. The sub‑culture of trypanosomes was not successful.

INTRODUCTION
The in vitro cultivation of the trypanosomes belonging to the brucei group is
generally considered to be difEcult to establish. Many studies on the matter havebeen attempted since Novy and McNeal ( 1 903, 1 904) reported some success in culti‑
vation of Trypanosoma brucei in a diphasic blood‑agar medium called NN medium.
According to reports so far published, results of the cultivation of the trypanosomes
varied greatly depending on the species or strains of the parasites, and the tem‑
perature applied to the experiments.
For the cultivation of the African sleeping sickness trypanosomes for the purposeof diagnosis, Weinman ( 1944, 1 946) presented a culture method using a semi‑solid

medium which consisted of nutrient agar, human plasma and haemoglobin. Tobie
et al. (1950) reported a diphasic nutrient blood‑agar medium, using rabbit blood
instead of human one. In this medium T. gambiense and T. rhodesiense transferredfrom laboratory animals were found to develop only to the proventricular forms
losing infectivity to laboratory animals as usually observed in cultures tried at thetemperature below 37 C. For a valuable diagnostic procedure, a haemoculture
Department of Protozoology, The Research Institute for Microbial Diseases, Osaka University,
Yamada‑kami, Suita, Osaka, Japan
1 Present address : Department of Medical Zoology, Hyogo College of Medicine, Mukogawa‑cho,

Nishinomiya, Hyo o, Japanl 62

technique was reported on T. gambiense by Neujean and Evens ( 1 952), and Weinman( 1 960) described a reliable culture method for diagnosis and isolation of T. gambiense

and T. rhodesiense. In a defined or semi‑defined medium, T. gambiense (Nicoli,1961), T. rhodesiense and T. brucei (Cross and Manning, 1973) were found to growat the culture temperature of 25‑28 C. Although many attempts were done asstated above, at the body temperature of mammals it has not been achieved tocultivate the blood stream forms of the brucei group trypanosomes. In this con‑nection, Williamson and Rollo in 1952 observed survival in undiminished numbersof T. rhodesiense, for about 4 hours at 37 C in serum‑free synthetic medium.
In recent years, the tissue culture methods were employed in the cultivation of

T. brucei by several workers. Le Page (1967) reported that the mouse L‑cells in
NCTC 109 medium with 10 per cent calf serum could be used in cultivation of T.
brucei at 37 C. Later, Eagle's MEM medium with 20 per cent calf serum was em‑
ployed as the basal medium by Hawking (1971). It was reported in those studies
that the trypanosomes inoculated into the media were found to multiply 2‑ to 5‑foldwithin 24 hours of cultivation, but subculture was not successful. Hirumi et al.
(1977 a, b) first reported on the successful cultivation of the blood stream forms ofT. brucei at 37 C with use of the tissue culture system composed of the bovine fibroblast‑

like cell and HEPES‑buffered RPMI 1640 with 20 per cent heat‑inactivated fetal
bovine serum.
The present study was an attempt of cultivation of T. gambiense maintained bymouse‑passages for years, using tissue cultures of the mouse L‑cell. As the result, noestablishment of the culture has been achieved, but some results could be obtained ofthe multiplication, the morphological characters and the infectivity to mice on thecultured parasites during the short‑term cultivation.

MATERIALS
Trypanosoma gambiense employed in this study was the Wellcome strain which had
been maintained by serial passages through mice for years in our laboratory. Male
ddO mice, 6‑7 weeks old, were used as the doners of the blood stream forms of T.
gambiense and also brought to the infectivity test of cultured trypanosomes.

The mouse L‑cell line was kindly supplied by the Department of Pathology inthis institute. The tissue culture medium for cultivation of T. gambiense with themouse L‑cells was Eagle's MF.M medium containing heat‑inactivated cair serum atlO per cent and antibiotics (100 U/ml of penicillin G and 100 pg/ml of streptomycin
sulfate) .

In addition the miscellaneous experiments were performed as follows. As thebasal medium, RPMI 1640 or TCM 199 medium was tried in the place of Eagle's
MEM medium. As the serum component, heat‑inactivated rabbit, rat, mouse or
fetal calf serum was also tried in the same concentration as that of calf serum. For
the mouse L‑cell; another line of mouse fibroblast cell was tested.

163

EXPERIMENTAL METHODS
Collection of trypanosomes: About 48 hours after the intraperitoneal inocula‑tion of 105 trypanosomes into each mouse, more than 108 parasites per ml could befound in the peripheral blood of the mice. B]ood was drawn from the heart of theinfected mice with a syringe containing Alsever solution of almost equal volume tomouse blood to be taken, then transferred into a mixture of Alsever solution ( I O ml)

and culture medium (20 ml) in a centrifuge tube. The contents were centrifuged at
200 g for 5 min to sediment the blood cells and the supernatant was again centrifugedat I ,OOO g for 10 min to collect trypanosomes. The sediment was resuspended in
the culture medium and the parasite population was adjusted to 7.5 x 106/ml. All
the procedure to prepare the final parasite suspension was carried out at 4 C so as tokeep the fixed condition.

Preparation of L‑cell cultures : Cultures of the mouse L‑cell were prepared in
TD‑40 flat culture bottles. The culture medium was the same as that for parasite
culture described in the chapter of materials and 1 5 ml of the medium was used
a bottle. Each culture bottle was plugged with a spongy silicone stopper and in‑cubated at 37 C in air mixed with C02 at 5 per cent. These methods and condition

were also applied to the cultivation of trypanosomes. L‑cell monolayers wereserially subcultured at 5‑ to 7‑day intervals. For the cultivation of trypanosomes,
2 x I 06 L‑cells were inoculated into a fresh culture medium 4 days before the trypa‑
nosome inoculation, according to the results of the preliminary experiment that a
confluent monolayer was formed from 2 x 106 L‑cells within 3 days. The cultivationof trypanosomes was mamly tned m coexrstence with a monolayer of the L‑cells.
The cultivation, however, was sometimes tried with, a smaller number of the L‑cells
than in a monolayer, at the growing stage of separate colonies, in order to examine
the influence of the growth condition of the L‑cells. In preparation for this trial,
1 x I 05 L‑cells were inoculated into a fresh culture medium 4 days before trypanosome
inoculation.

Inoculation of T. gambiense into the mouse L‑cell cultures : The medium of themouse L‑cell cultures was pipetted off, 14 ml of the fresh culture medium was added
into each culture 2 hours before the inoculation of parasites. One ml of trypanosome
suspension (7.5 x 106 trypanosomesjml) prepared from infected mice was introduced
into each of the tissue cultures. Thus, the parasite population became 5 x 105jml
at the initiation of culture.

Examination of cultured trypanosomes : Usually every 4 to 8 hours, a small
amount of the liquid. portion of culture was sampled with small pipettes and al]living trypanosomes and dividing forms were counted separately with haemocytometer
under a phase‑contrast microscope. From the results, the growth curve of cultured
trypanosomes and also the ratio of dividing forms to all living trypanosomes ob‑
tained, respectively. At the same time Giemsa‑stained smears were prepared forclassification of the parasites into the following 4 types of growing stages : I ) trypa‑

nosomes with one kinetoplast and one nucleus, (1K, 1N)‑type; 2) 2 kinetoplasts andQne nucleus, (2K, 1N)‑type; 3) 2 kinetoplasts and 2 nuclel (2K 2N) type 4) no1 64

kinetoplast and one nucleus, (OK, 1N)‑type. The percentage of each type wascalculated from 1,000 parasites counted. To examine infectivity of cultured trypa‑
nosomes, 103 or 50 parasites in 0.5 ml of saline, taken from cultures at 12‑hourjntervals, were inoculated intraperitoneally into each of 4 mice, then the mice were
subjected to examination of trypanosomes in the peripheral blood. A drop of bloodfrom the tail of infected mice was placed on a slide glass and covered with a coverslip (24 mm x 24 mm), then examined for trypanosomes at x 400 magnification witha microscope.

RESULTSThe growth curves of trypanosomes which were inoculated into the L‑cell
culture under different conditions were shown in Figure l. On the L‑cell layer
which had been grown enough to cover the bottom of a culture bottle completely,
trypanosomes were found to start multiply without lag phase and the number oftrypanosomes were becarne 2‑fold between 14 and 16 hours of culture and 3‑foldwithin 24 hours, then decreased. On the other hand, over the L‑cell layer which
was just at the stage of separate colony formation, covering almost I /4 area of thebottom of a culture bottle, the growth period of trypanosomes was usually prolonged

and the number of trypanosomes decreased gradually, although the multiplication
rate was lower. In the culture of trypanosomes without using L‑cells, trypanosomes
could not multiply and decreased within 12 hours.
Growth activity of trypanosomes in culture varied greatly due to the phase ofparasitaemia in an infected mouse at the time of bleeding for parasite collection.

Trypanosomes collected from an infected mouse with the maximum parasitaemia ofover I x I 09 parasites per ml of blood, showed multiplication only during such a

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Figure I Growth curves of trypanosomes in culture,.

165

short period as 6 to 8 hours. Therefore, parasites at the peak of parasitaemia could
be regarded as inadequate materials for inoculation into this culture system.

The dividing forms of living trypanosomes shown in Figure 2 as percentage
along the course of culture time. In this study the dividing form indicated a trypa‑
nosome at the stage of incomplete separation into 2 individuals but showing the partial
separation of the cytoplasm in the anterior part. The proportion of dividing formsof cultured trypanosomes with the L‑cells were observed to increase for 1 6 hours ofcultivation, reaching almost I /3 of the whole population of cultured trypanosomes,

then decreased. Under the condition that trypanosomes could not multiply in theculture without the L‑cells, the proportion of the dividing forms remained at theinitial level for 24 hours of observation.

%40
o with L‑ceu layer

odl'o/ ' '¥o

3o
20
10

o

o

‑rf'‑̲ It'‑1'‑1',
wlthout L‑cetts

1'

8
O 4 1216 20 24
HOurs of culture

Figure 2 Proportion of dividing forms.
The proportion was determined by calculating the ratio of thedividing forms to all living parasites from the numbers counted

under a phase‑contrast microscope.

Trypanosomes in culture were counted, differentiating into 4 types of growing
stages on Giemsa‑stained smears. As shown in Figure 3, the ratio of ( I K, I N)‑typetrypanosomes apparently shifted to that of (2K, 2N)‑type, passing through the stageof (2K, IN)‑type in accordance with culture time. In the culture system with the
L‑cells the proportion of (2K, 2N)‑type trypanosomes which were thought to be at
the most advanced stage of division reached the highest level, 32 per cent in 20 hoursof cultivation, then became lower. On the other hand, in the culture without
L‑cells, in which no multiplication of trypanosomes took place, the portion of(2K, 2N)‑type increased to occupy l/3 portion ofthe total population in 8 to 12 hoursof cultivation time. From the view of the kinetics of trypanosome division, it might
be interesting that the maximum portion of the dividing forms showed similar value
to that of the (2K, 2N)‑type in the culture system with L‑cells. On the other hand,in the culture system without L‑cells, the proportion of the dividing forms stayed at
a very low value throughout the culture period, although the (2K, 2N)‑type showed
an increase in proportion. It was understood from this result that the cytoplasmic
division of the (2K, 2N)‑type parasites would be highly suppressed in the culture
system without L‑cells. Furthermore, in the absence of L‑cells, it was impossible to

1 66

%
70 e

o¥elle,,t¥"., ,

60

O¥

e
"O'b '. Ib

¥ e
>¥

50
¥O '

40

O

‑:A;‑ /'/'// / 'lA
/I
/ /¥'

20

C]

ll

10

[]/
I

O

C]
̲‑Cl‑
I
IJ¥A

/[]

30

"t
'
I

vv
v v vv
4vv

v
o 8 12Hours16
20 24
of culturev(1K,1N) (2K.1N) (2K.2N) (OK,1N)

withL‑ceu laye' ‑witho t L‑c*u= ‑‑oFigure 3 Proportion of 4 types classified by growing stages.

The percentage was determined by observation of

Giemsa‑stained smears. The proportion of the(OK, IN)‑type did not rise above 0.2 per cent.

classify into 4 types in smeared preparations after 1 6 hours of culture, because of
the rapid increase of degenerated parasites.
The infectivity test to mice of the cultured trypanosomes with L‑cells, taken
every 1 2 hours from the culture, was undertaken with I 03 trypanosomes per mouse

as inoculum size. The infectivity was measured by the prepatent period during
which no parasite could be found in the peripheral blood of mice through 20 fields
at X 400 magnification and the days after inoculation until death of mice. It wasobserved that trypanosomes within 36 hours of cultivation did not show any different
feature of infectivity to mice in comparison with the control trypanosomes inoculated
Table I Infectivity of cultured trypanosomes to mice

Inoculum size
Culture time(hours)Mice
Prepatent period(days)

Days until death

A: 103O(Control)

12

B: 50

24

36

abcd abcd abcd abcd
2222 2222 2222 2222
4555 5554 5655 5555

A : cultured with L‑cell monolayer

O(Control)52

abcd a b d
3333 3 3 3 36666 6 6 6

B : cultured with L‑cell colonies

1 67

at time zero. Furthermore, even with as few as 50 trypanosomes in the 52‑hour
cultivation no lower infectivity to mice could be found (Table I ) .
The subinoculation into fresh tissue culture with a part of the culture suspension
or cultured trypanosomes collected by centrifugation was tried at different phases of

the culture course, but the transferred parasites did not multiply, showing rapid
decrease in number.
In the miscellaneous experiments, firstly RPMI 1640 and TCM 199 as the basal
medium were examined for the supporting efficacy upon parasite growth, respectively.
Secondly, heat‑inactivated rabbit, rat, mouse and fetal calf serum was also examined
respectively, in place of inactivated calf serum. Finally, another mouse fibroblast
cell line was tried instead of the L‑cell. All these attempts gave the similar results
as described hitherto.

DISCUSSION
Establishment of the in vitro cultivation of the blood stream forms of the brucei
group trypanosomes has been expected for a long time. Hirumi et al. first in recent
years reported the successful cultivation of T. brucei, applying the tissue culture

method.
From the results obtained in this study, the presence of tissue culture cells wasthought to be indispensable to support the multiplication and survivability of T.
gambiense in vitro, though within limitation. Although many attempts were done, thedoubling time of trypanosomes in culture appeared to be much prolonged as comparedwith that in infected mice. From these facts, this culture system might be lacking

some components which would be essential for growth of trypanosomes. On thebasis of this consideration, the necessary components were probably carried over
into the culture system by trypanosomes when inocul ted from infected mice. Afterthe consumption or dilution of these components by trypanosomes in culture, theirmultiplication would be ceased and the number of them would decrease. This
would be reasonably thought from the fact that there was no lag phase until the
beginning of multiplication of trypanosomes. Those related facts would also givethe reason why the subculture of trypanosomes was impossible.
The accumulation of the dividing forms and of the (2K, 2N)‑type parasites inculture was not assumed to indicate the inhibition of the division of kinetoplast,
nucleus or flagellum, but the cytoplasmic division was probably suppressed by some
unknown factors, although other factors to promote the growth and survival of

trypanosomes would be contained in the culture medium and also supplied by
L‑cells.

This cultivation was carried out at 37 C and trypanosomes growing in culture
did not show any loss of infectivity to mice. Therefore, T. gambiense in this culture
could be regarded to maintain the characters of the blood stream forms of the para‑sites, although more detailed examinations should be necessary to determine it.
From these standpoints, in a shorter period less than 24 hours, this cultivation system
for T. gambiense was thought to be effectively applicable to some in vitro experimentsof this parasite strain.

1 68

The outline of this paper was reported at the 32nd West Japan Regional Meetingof the Japanese Society of Parasitology in Kurashiki City, November 1 1 , 1 976.

REFERENCESl)

Cross. G. A. M. and Manning, J. C. ( 1973) : Cultivation of Trypanosoma brucei sspp. in semi‑
defined and defined media, Parasitology, 67 (3), 315‑331

2)

Hawking, F. ( 1971 ) : The propagation and survival of Tr̲vpanosoma brucei in vitro at 37 C, Trans.

R. Soc. Trop. Med. Hyg., 65 (5), 672‑675
3)Hirumi, H., Doyle, J.J. and Hirumi, K. ( 1977a) : African trypanosomes: Cu]tivation ofanimal‑infective Trypanosoma brucei in vitro, Science, 196 (4293), 992‑994

4)Hirumi. H., Doyle, J.J. and Hirumi. K. (1977b) : Cultivation of bloodstream Trypanosoma

5)

Le Page, R. W. F. ( 1967) : Short term cultivation of Trypanosoma brucei in vitro at 37 C, Nature,

6)

Neujean, G. and Evens, F. ( 1952): Valeur pratique de l'h6moculture comme moyen de diag‑7)

nostic dans la trypanosomiase humaine a T. gambiense. Bull. Soc. Pathol. Exot., 45 (4), 503‑517
Nicoli, J. (1961) : Etude preliminaire sur les conditions de culture de Trypanosoma gambiense.

brucei. Bull. Wld Hlth Org., 55 (2‑3), 405‑409
216 (5120), I141‑1 142

Bull. Soc. Pathol. Exot., 54, 77‑83
8)

9)

Novy, F. G. and McNeal, W. J. (1903): The cultivation of Trypanosoma brucei. A preliminarynote, J. Am. Med. Assoc., 41, 1266‑1268
Novy, F. G. and McNeal, VV. J. ( 1904) :. On the cultivation of Trypanosoma brucei. J. Infect.
Dis., I (1), 1‑30

l O)

Tobie, E.J., von Brand, T. and Mehlman, B. ( 1950) : Cultural and physiological observations
on Trypanosoma rhodesiense and Trypanosoma gambiense. J. Parasitol., 36 ( I ) , 48‑54

ll)

Weiman, D. ( 1944) : Cultivation of Trypanosoma gambiense in vitro in cell‑free medium, Proc.
Soc. Exptl. Biol. Med., 55, 82‑83

12)

Weiman, D. ( 1946) : Cultivation ofAfrican sleeping sickness trypanosomes on improved, simple,
cell‑free medium, Proc. Soc. Exptl. Biol. Med., 63, 456‑458

13)Weiman, D. (1960) : Cultivation of the African sleeping sickness trypanosomes from the bloodand cerebrospinal fluid ofpatients and suspects, Trans. R. Soc. Trop. Med. Hyg., 54 (2), 180‑190
Williamson, J. and Rollo, I. M. ( 1952) : Stimulating effect of amino‑acids on the survival at
37 C of Trypanosoma rhodesiense in a serum‑free synthetic medium, Nature, 1 70 (4322), 376‑377

1 4)

169

lTプy鋼noso脚解幼f6％s8の37Cにおける短期間
培養マウスL一細胞を共存させての試み
福間

利英1・神原広二・中林敏夫

マゥスにより長期にわたり継代維持されてきたτ叡卿η050勉α9α励∫εη5θ（T．9．），Wellcome株を初痂ro37CでマゥスL一細胞と共存させて培養することを試みた。この培養系においてT、g．はlagphaseなしに増殖し始め，24時間でT．g．数は3倍になり，以後減少していった。T、g．数が2倍になるまで12−14時間を要したので，この系での世代時間は，感染マウス内での場合（約5時間）にくらべ非常に長くなっている。培養されたT．g．の観察結果からT．g．の分裂過程において形態上キ

ネトプラスト，鞭毛および核は倍化しているにもかかわらず，それに続いて起こるべき虫体の縦裂そして2原虫への分離の段階において時間を要していることがわかった。つまりこの分裂が部分的に抑制されているために増殖が遅くなったと考えられる。対照としてL一細胞の共存なしで，培養されたT．g。では，やはりキネトプラスト，鞭毛および核の倍化は起こっているにもかかわらず，増殖が認められなかったのでこの場合にはより高度にT、g．の分裂が抑制されていると考えられる。

従って具体的な要因は不明であるがL一細胞の存在はT．g．の生存，増殖に有用であるといえる。
この系で培養されたT、g．は少なくともT、g．数が減少し始めるまでの間，培養開始と同じ程度にマ

ウスに対する感染能を保持していた。しかしながらこの系においてT．g．を継代することはできなかった。以上のことから，本培養系ではT．g．を培養維持することはできないが，原虫数が減少し始めるまでの時間，つまり24時間以内に限定すれぱ，本原虫を用いた他の実験にこの培養法を応用する
こことができると考えられる。

大阪大学微生物病研究所原虫学部門
1現在：兵庫医科大学医動物学教室

Japan.J. Trop. Med. Hyg., Vol. 7 No 3 4 1979 pp 171 189

171

DEVELOPMENT OF BRUGIA PAHANGI MICROFILARIAE
FROM JIRD PERITONEAL CAVITY IN AEDES AEGYPTICHENG‑KUO CHUANG, YASUO NAKAJIMAI AND YOSHIKI AOKI
Received for publication I September 1979

Abstract: A new apparatus for feeding mosquitoes is described. It is constructedwithout special technique and easy of handling. The usefulness of the apparatus isdemonstrated by a report on investigations into the effects of nucleotides on mosquito
gorging and the development of the microfllariae from the jird peritoneal cavity and fromthe dog venous blood in Aedes aegypti. As a phagostimulant l0‑3M ATP is the mosteffective of the nucleotides tested. The microfilariae from the jird peritoneal cavity havebeen proved to reach st.age 111 (i.e. infective stage) as those from the dog, although therate is lower in thc former. It is also proved that the microfilariae develop to infective

larvae in the mosquitoes which have not taken blood, serum or protein meals.

INTRODUCTION
Brugia pahangi, whose normal hosts are cats and dogs (Buckley and Edeson, 1 956 ;Laing et al., 1960; Schacher, 1962 a, b), was successfully transmitted to the jird
(Meriones unguiculatus) with subcutaneous inoculation of infective larvae (Ash andRiley, 1970; Ash, 1973). When the infective larvae were inoculated into the peri‑
toneal cavity of the jird, adults and microfilariae were recovered from the peritoneal
cavity (McCall et al., 1973). The adults and microfilariae localized in the peritoneal
cavity seem to follow an aberrant mode of development, although the ease of recovery
of worms from the small rodents may serve for the studies on the biology, biochemistry,
immunology and host‑parasite relationships of filariasis. A search in the literature

failed to reveal any report on the development of the microfilariae from the peri‑
toneal cavity in mosquitoes.
There have been described several methods of feeding hematophagous arthro‑pods artificially through natural and artificial membranes. Most of the devices have

utilized a flat membrane extended in a horizontal plane. The devices with flat
membranes have been used for mosquitoes by previous authors (St. John et al., 1930;
Woke, 1937; Bishop and Gilchrist, 1946; Eyles, 1952; Kartman, 1953; Bar‑Zeev andSmith, 1959; Collins, 1963; Collins et al., 1964; Rutledge et al., 1964; Behin, 1967).
In most ofsuch devices it is diflicult to maintain the solution offered to the arthropods

at a certain temperature suitable for feeding. On the other hand, Pipkin andConnor ( 1 968) have reported a device with a pouch‑1ike membrane of rubber, sheep
caecum or pig intestine sheathing a test tube with rounded bottom and also Kitaoka
Department of Parasitology, Institute for Tropical Medicine, Nagasaki University.

l Present address : Department of Medical zoology, Faculty of Medicine, Tokyo Medical and
Dental University.

172

and Morii ( 1 970) a feeder with a membrane of chicken crop covering a round bottom
tube, which provide sufficient surface areas of the membranes for mass feeding ofcolonized vectors. In the latter devices the temperature of feeding solution can be
maintained with ease, although relatively large quantity of feeding solution has to be
filled in the space between the membrane and the tube.
Hosoi (1959) has examined a variety of chemicals dissolved in buffered saline
offering to Culex pipiens pallens singly confined in a glass tube with a forcible feeding

technique and reported that some nucleotides stimulate the mosquito into gorging
with a blood meal, which is dispatched only to the stomach. Rutledge et al. ( 1 964)
found that adenosine triphosphate is a phagostimulant to some mosquitoes.
As the vector mosquitoes for Brugia pahangi, Armigeres subalbatus and/or Aedes
togoi were used by previous authors (Ash and Riley, 1970; Ash, 1973; Kan and Ho,
1973 ; Nakajima et al., 1976). In the present studies Liverpool strain of Aedes aegypti
has been used because of ease of maintenance and feeding as well as susceptibility
to Brugia pahangi infection.

This paper describes a new apparatus designed for feeding microfilariae tomosquitoes, the phagostimulant effect of nucleotides on Aedes aegypti and the de‑velopment of microfllariae from the jird peritoneal cavity in the mosquito in com‑
parison with those from the dog venous blood. It also discusses the effect of protein
meals for mosquitoes on the development of larvae.

MATERIALS AND METHODS
I. Feeding apparatus, solutions and mosquitoes
The feeding apparatus consisted of a glass jar about 8 cm in height with a flatbottom about 4 cm in diameter and a rubber stopper inserted with two cannulae.
The cannulae were connected to a water pump with two flexible polyvinyl tubes.
The inside of the jar was circulated with water from a thermostatically controlledwater bath whose temperature was maintained at 38 C.
Mice were sacrificed under ether anesthesia. The fur on the skin was clipped
off as c.losely as possible with a pair of electric clippers. The skin was then removed

from the carcass and the subcutaneous and fatty tissues were eliminated. The skin
was washed with saline and stored in a refrigerator or a freezer prior to use. Whenneeded, the skin membrane was washed with saline and stretched, the subcutaneousside inward, over the bottom of the jar, to which it was held by a rubber band. A
Pasteur pipette was inserted between the membrane and the side of the jar. Com‑
mercially available, salted outermost layer of sheep intestine and prophylactic rubber

condoms were also tried as feeding membrane. Their use was eventually discon‑tinued, because the former tended to be leaky after freshening and mosquitoes haddifficulty in piercing the latter.

The feeding solutions tested were physiological saline, Dulbecco's phosphate‑buffered saline without calcium or magnesium (PBS) and bovine serum. The
bovine serum was light red in color due to hemolysis. The nucleotides used asphagostimulants were adenosine 5 '‑monophosphoric acid (from equine muscle,

173

Type V, Sigma Chemical Co.) (AMP), adenosine t'‑diphosphate (from equinemuscle9 Sodium salt9 Grade r, Sigma Chemical Co.) (ADP) and adenosine 5 '‑triphosphate (disodium salt) P‑L Biochemicals Inc.) (ATP) dissolved in the final
concentration of 10‑4 I¥/1 or that of lO‑3 M. The nucleotides were added to thecold solutions just prior to the experiment and placed in an ice bath until feeding.

Controls were the solutions without nucleotides as well as dog serum and= /. *= +*.*
r ･ ･ .･･s.; .,

;,

..r".

Figure 1

Feeding apparatus for mosquitoes.
A skin membrane is stretched over the flat bottom of the glass jar. Feeding solLrtionis placed into the space between the jar bottom and the membrane thraugh Pasteur
pipette. Inside of the jar was circulated with water f'rom a temperature‑controlledwater bath through a water pump, two flexible tubcs and two cannulae.
Figure 2

Mosquitoes piercing the membrane of the apparatus hung in the cage and gorging
feeding solution.

Figure 3

Phase‑contrast microphotograph of stomach eontents of Aedes aeg,ypti dissected im‑
mediately after artificial feeding of microfilariae.

As shown in the figure, overcrowding of microfila;riae in the stomach was occasionallyobserved in the group of mosquitoes offered the microfilariae from jird peritoueal cavity
t the. density af 1,000 ta 1?SOO p, er O,03 ml Qf P

S,

1 74

deflbrinated rabbit blood. In some experiments a drop of Indian ink or a drop of
one per cent patent blue solution was added to each 2 to 3 ml solution for easy
identiflcation of the solutions in the alimentary tract of mosquitoes.

The feeding solution, 2 to 6 ml, was placed into the space between the jar
bottom and the skin membrane through the Pasteur pipette, which was then removed(Figure I ). The side of the jar was covered by a polystyrene sheet.
The mosquitoes used were Aedes aegypti (Liverpool strain) which were maintainedin our laboratory. For feeding trials 28 to 100 female mosquitoes were put into a

cage about 40 cm long, 30 cm wide and 30 cm high. Age range was 5 to 7 days
from the day of emergence. The diet, 2 per cent sucrose solution, was removed 24
hours before the trials.

The jar was hung in the cage through the top hole of the cage (Figure 2). The
ambient temperature was 1 7 to 25.5 C. The cage was covered up with a sheet ofblack cloth. The test period for each cage was 30 minutes. Immediately after
the feedrng, the mosquitoes were anesthetized with ether and dissected under anoperating microscope.
II. Infection of Aedes aegypti with Brugia pahangi microfilariae

Microfilariae were recovered from the peritoneal cavity of the infected jirds(Meriones unguiculatus) which had been inoculated intraperitoneally with the infective(i.e. stage 111) Iarvae of B. pahangi. After the injection of 10 ml of PBS into theperitoneal cavity, the syringe was removed and the 18G x I /2 " (1.2x38 mm)
needle was left to recover the injected fluid containing microfilariae. The fluid
dripping through the needle was collected, mixed with cold PBS and centrifuged
for 5 min at 6,000 rpm, O C. The sediment was resuspended in cold PBS or bovine
serum to yield 700 to 2,000 microfilariae per 0.03 ml. Just prior to the feeding,
ATP was added in the final concentration of 10‑3 M.
Controls were the direct feeding of Aedes aegypti (Liverpool strain) on an infected
dog and the artiflcial feeding of the mosquitoes with the microfilariae obtained fromthe venous blood of the same dog. The microfilaria count of the dog was 495 per
0.03 ml of peripheral blood. The mosquitoes were allowed to feed for 15 min on
the infected dog under ketamine hydrochloride (KetalarR) anesthesia. Venous
blood was obtained from the vein of the hind limb of the dog with plastic syringes
which were prepared by wetting the walls with 1,000 U/ml heparin. To each 5 mlof venous blood, 45 ml of cold one per cent saponin solution was added to attainhemolysis. The mixture was centrifuged for 5 min at 6,000 rpm, O C. The sedi‑ment was resuspended in cold saline and centrifuged under the same conditions
twice. Then the sediment was resuspended in cold bovine serum and PBS to makelOO and 600 microfilariae per 0.03 ml respectively, to which l0‑3 M ATP was added
just prior to the feeding.

For feeding, 3 ml of solution was placed in the apparatus to a cage of 89 to 140

female mosquitoes. The mosquitoes were allowed to feed for 15 min at the ambient
temperature of 25 I C. To count the number of microfilariae ingested, the mos‑quitoes were anesthetized and dissected under an operating microscope immediately
after feeding. To observe the exsheathing of microfilariaep 5 to 10 mosquitoes of1 75

each cage were anesthetized and dissected every hour from one to 8 hours after
feeding except 7 hours. The content of the stomach was smeared on a glass slide(Figure 3), dried rapidly, fixed with methylalcohol and stained with Giemsa solution.
Also 5 to I O mosquitoes per cage were dissected every day from one to 1 3 days after
feeding to observe the development of larvae. The larvae were classified into threedeveloping groups according to the morphological characteristics described by
Schacher (1962 a). Measurement of body length was done on the infective larvae
rec,overed from the mosquitoes dissected 1 1 days after feeding.

RESULTS
I. Response of Aedes aegypti to nucleotide solutions

Physiological saline without nucleotide was not detected in the stomach nor in
the diverticula of Aedes aegypti (Table I ) . Saline containing 10‑4 M AMP wasTable 1

Response of Aedes aegypti to nucleotides in saline with localization and degreeof intake of solutionst

No. of mosquitoes

Nucleotide

in stomach

dissected fed (degree of intake)tt

in diverticula(degree df intake)ft

None

30

O

O

O

AMP l0‑4M*
ADP 10‑4M*
ATP l0‑4M*
AMP l0‑8M**
ADP l0‑3M**
ATP 10‑3M**

29
29
30

5

5 (p, 3: s, 2)

1

14

14 (p, lO: s, 4)

O
O

94
83
86

18

18 (f, 4: p, 13: s, 1)

81

80 (f, 27: p, 53)80

80 (f, 27: p, 53)

O73

73 (f, 20: p, 53)O(s, 1)(,s l)t Artificial feeding at ambient temperature of 25‑25.5 C.
ft f fully distended : p; partially distended : s; slightly distended.

* A drop of I o/o Patent blue solution was added to each 2 ml.

* * A drop of Indian ink was added to each 3 ml.

found in 5 of 29 mosquitoes, in 3 of which the stomach was partially distended,in two slightly, and in one the diverticula also contained small quantity of the solu‑

tion. Saline containing l0‑4 M ADP was found in 14 of 29 mosquitoes; in 10 ofthem the stomach was partially distended and in four slightly. The phagostimulant

effect of 10‑4 M ADP was more marked than that of l0‑4 M AMP: The differencein the stomach engorgement of the solution was significant (p<0.0254) in the chisquare test. Saline containing lO‑4 M ATP was found in 1 8 of 30 mosquitoes ; infour of them the stdmach was fully distended, in 1 3 partially and i‑n one slightly.

ATP was more effective than ‑AMP: The ‑ difference in the stomach 'engorgement
between 10‑4 M AMP and l0‑4 M ATP IA'as statistically signific nt (p<0.0019).
When I 0‑3 M nucleQtides were added to saline, more mosquitoes gorged and the176rates of full stomach distension were higher than those in l0‑4 M concentrations(Table 1) : Using the chi square test the differences in the stomach engorgement

were significant in AMP (p<0.0001), ADP (p<0.0001) and ATP (p<0.0096).
PBS without nucleotides was detected in 4 of 30 mosquitoes, in one of which thestomach was fully distended, in one partially and in two slightly (Table 2). When

10‑4 M AMP or 10‑4 M ADP was added to PBS, the rate of stomach intake seemed
to be a little higher than the solution without nucleotides, but the computed va]uesof chi square were not statistically significant. PBS containing 10‑4 M ATP wasengorged by 23 of 29 mosquitoes, in 22 of which the stomach was fully distended.
As a phagostimulant, ATP was significantly more effective than AMP (p<0.0015)
or ADP (p<0.0003). On the dissection, sucrose solution was found to remain in
the diverticula of the mosquitoes offered l0‑3 M nucleotides in PBS. It mightprobably have inhibited the feeding of nucleotide solutions to a certain extent.

Only 46 of 90 mosquitoes took I0‑3 M AMP solution (Table 2). Although therate of engorgement was significantly higher than the control solution (p<0.0015),

the difference between 10‑4 M AMP and l0‑3 M AMP was not statisticallv, sig‑
nificant. On the other hand, the difference between 10‑4 M ADP and 10‑3 M
ADP was highly significant (p<0.0002). The rate of engorgement of 10‑3 M ADP
in PBS was higher than that of I 0‑3 M AMP in PBS : The difference was statisti‑cally significant (p<0.032). As a phagostimulant in PBS, 10‑3 M ATP was more
effective than 10‑3 M ADP: The difference was also significant (p<0.020).
Table 2

Response to nucleotides in PBS with localization and degree of intake of solutionst

No. of mosquitoes

Nucleotide

None

AMP 10‑4M*
ADP 10‑4M*
ATP l0‑4M*
AMP l0‑3M**
ADP l0‑3M**
ATP l0‑8M**

fed in(degree
dissected
stomachof intake)tt

30
30
30
29

90***
93***
96***

in diverticula(degree of intake)tt

4

4 (f, 1: p, I : s, 2)O

9

9 (f, 1: p, 1: s, 7)O

7

7 (f, 7)3 (s, 3)23

23 (f, 22: p, l)

3 (p, 3)46

46 (f, 19: p, 27)

O

63

63 (f, 34: p, 29)

O

80

79 (f, 34: p, 45)l (p, l)t Artificial feeding at ambient temperature of 22‑24 C.
tt f; fully distended : p; partially distended : s; slightly distended.

* A drop of I o/o Patent blue solution was added to each 2 ml.

** A drop of Indian ink was added to each 3 ml.
*** Sucrose solution remained in diverticula.

Bovine sertim was light red ih color due to heinolysis. At the ambient tempera'ture of 1 7 C, bovine serum without nucleotides was taken by 1 9 of 30 mosquitoes, inl 8 of which serum was detected in the stomach and in three in the diverticula (Table3). Only 8 of 30 mosquitoes took dog serum without nucleotides, which had been
Qbtalined without apparent hemolysis. The djfference in the Stomach engor ement

177

between bovine serum and dog serum was statistically significant. (p<0.0191). The

addition of l0‑4 M AMP or 10‑4 M ADP to bovine serum did not enhance the rateof feeding: The computed values of chi square were not statistically significant.

On the other hand, the addition of 10‑4 M ATP stimulated a little the stomach
engorgement: The difference was statistically significant (p<0.0487). At theambient temperature of 25 C, 39 of 100 mosquitoes gorged bovine serL]m without
nucleotides; only in eight of them the stomach was fully distended and in 31 partially(Table 4). The addition of l0‑3 M nucleotides to serum enhanced the rates of
feeding and full stomach distension. The differences in the stomach engorgement
were highly significant (p<0.0001) in the three nucleotides. However, the additionof l0‑3 M AMP to bovine serum was not so effective as that of l0‑3 M ADP or of
l0‑3 M ATP. When ADP or ATP was added, the rate of feeding was equal to thatin rabbit deflbrinated blood. The difference between l0‑3 M AMP and rabbit
defibrinated blood was highly significant (p<0.0012), while the computed values ofTable 3 Response to l0‑4M nucleotides in bovine serumf and to dog serumtt
No. of mosquitoes

Nucleotide

in stomach

dissected fed

in diverticula(degree of intake) ** (degree of intake) **None, bovine serum*30

19

18 (f, 4: p, 2: s, 12)

3 (s, 3)

AMP l0‑4M*
ADP l0‑4M*
ATP l0‑4M*

29

18

18 (f, 4: p, 5: s, 9)

3 (p, I :28

22

22 (f, 6: p, 4: s, 12)

3 (p, I : s, 2)

29

25

25 (f, 20: s, 5)

2 (s, 2)

None, dog serum30

88 (f, I : p, 1: s, 6)

O

t
tt

, 2)

Bovine serum was a little hemolysed.
Artificial feeding at ambient temperature of 1 7 C.

* A drop of I o/o Patent blue solutioh was added to each 2 ml.
** f; fully distended : p ; partially distended : s; slightly distended.

Table 4

Response to 10‑3M nucleotides in bovine serumt and to rabbit defibrinated bloodtt
No. of mosquitoes

Nucleotide

None, bovine serum*

AMP l0‑3M*
ADP l0‑3M*
ATP l0‑3M*
None, rabbit
defibrinated blood*

dissected fed
1 OO

39

39 (f, 8: p, 31)

o

95

74

74 (f, 40: p, 34)

o
o

l OO

90
96

90 (f, 77: p, 13)
96 (f, 75: p, 21)

o

89

85

85 (f, 68: p, 17)

o

94

tt

Bovine serum was a little hemolysed.
Artificial feeding at ambient temperature of 25 C.

*

A drop of Indian ink was added to each 3 ml.

t

in stomach in diverticula(degree of intake) **** f fully dister;ded : p. ; partially distended : s ; sli htly distended,1 78

chi square were not statistically significant in ADP and ATP. The rate of fullstomach distension was also lower in AMP than in ADP, ATP or defibrinated blood(Table 4) .

These results indicated that ATP was the most effective phagostimulant toAedes aegypti, that ADP was next to ATP and that AMP was the least of three nucleo‑

tides. It was also clear that 10‑3 M concentration was generally more effective
than 10‑4 M concentration.
II. Artificial feeding and development of larvae

The mosquitoes which fed directly on the dog with microfilaremia of 495 per
0.03 ml of peripheral blood became infected at a rate of 88.5 per cent (Table 5). At
the dissection immediately after feeding, the average number of microfilariae in thestomach of the infected mosquito was 18.0 and the range was two to 75. The mos‑quitoes fed on PBS containing the microfilariae from the dog venous blood at thedensity of 600 per 0.03 ml became infected at a rate of 92.3 per cent. The nurnberof microfilariae in the stomach of the infected mosquito was 21.5 on an average,varying from four to 47. The mosquitoes taken PBS containing the microfilariae
from the .jird peritoneal cavity at the density of 700 per 0.03 ml became infected at
a rate of 33.3 per cent. The average number of microfilariae per the infected was

38.0 and the range was five to 204. When the density was raised to 1,000 in PBS,
the infection rate was 77.7 per cent, and the average number was 204.0 with therange ()f four to 653. The microfilarial density of 1,500 in PBS produced the infec‑
tion rate of 84.2 per cent and the average number of 336.6 with the range of one to1,213. Thus, the infection rate seemed to be lower in the artificial feeding with
PBS than in the direct feeding, if the microfilarial counts were similar in both groups.

Table 5 Infection rates in mosquitoes and numbers of microfilariae in stomach on
dissection immediately after feeding

Sourceof mf.

D og
peripheral

count of mosquitoes
FeedingMf.No.

per 0.03 ml
medium* dissected
infected (o/o)(direct)495

26

23 (88.5)

18.0 (2‑75)

PBS

600

13

12 (92.3)

21.5 (4‑47)

bovinel OO

125 (41.7)

5.2 (2‑9)

blood

Dog
venousblood

Mean no. of
mf. per infected(range)

serum

PBS
J ird

peritoneal
cavitybovine

serum

700

27

9 (33.3)

38.0 (5‑204)

l ,OOO27

21 (77.7)

204.0 (4‑653)

1 ,50019

16 (84.2)36!6.

15‑‑(75.0)

19.0 (1‑79)
i8Q.2 (3‑505)

1;000 ‑ ‑ 20 ‑1, 00. ̲ . 20

2,000

20

* As phagostimulant l0‑3M ATP w s cQntained,

20 "(Ido)
20 ̲( I otOj)

l.L I ,2 1 3)

137.8 (1‑1,002)

1 79

The average number of microfilariae per infected mosquito was greater and theindividual count per mosquito varied more widely in the artificial feeding with PBS
than in the direct feeding. The infection rates with bovine serum were similar to
those with PBS (Table 5). The computed value of chi square was not statisticallysignificant at the density of 1,000. In bovine serum, however, the average numberof microfilariae per the infected seemed to be less and the individual count permosquito varied less widely than in PBS.
The microfilariae from the dog venous blood exsheathed in the stomach of Aedes
aegypti after the artificial feeding. Exsheathing reached the high percentage of 38.9
at one hour and that of 54.8 at 2 hours after the feeding with bovine serum (Table 6) .
Thereafter, the percentage of the exsheathed microfllariae among the ones remaining
in the stomach ranged from 26.8 to 58.4. Exsheathing of the microfilariae from thedog venous blood occurred at lower rates with PBS than with bovine serum: The
percentages were only 6.4 to 18.4 with PBS.
Table 6 Exsheathing of microfilariae in stomach after artificial feeding
No. of exsheathed mf,/total no. of mf. (o,/o)
on hourly dissection after feeding **No. ofSource Feeding dissectedof mf, medium: mosquitoes l

PBS

Dog
venousblood bovine

toneal bovine
cavity serum

3

4

5

6

8

total

50 39/610 19/156 5lj440 331399 20/140 N.D. 16j87 ‑ 178/1,832( 6.4) (12.2) (11.6) ( 8.3) (14.3)(.18.4) ( 9.7)

52 21/54 51/93 15/56 34!81 3lj70 66/‑113 21!53 239/520(38.9) (54.8) (26.8) (42.0) (44.3) (58.4) (3 *6) (4q,O)

serLtm

Jird
PBS
peri‑

2

31

21740 o!rll 51257 16j433 16/693 91312 ll988 49!3,534( 0.3) ( o) ( 1.9) ( 3.7] ( 2.3) ( 2.9) ( o.1) ( 1.4)

57

0/452 41427 3/l09 14/112 12jl30 351137 32jl74 100jl,541( O) ( 0.9) ( 2.8) (12.5) ( 9.2) (25.5) (18.6) ( 6.5)

* As phagostimulant l0‑3M ATP was contained.
** Dissection was not done 7 hours after feeding.

The microfilariae from the jird peritoneal cavity exsheathed also in the stomachof mosquito, although less than one per cent of the microfilariae exsheathed at one

hour and 2 hours after the feeding both with PBS and bovine serum. After 4 hours
offeeding the percentage of the exsheathed microfilariae varied from 9.2 to 25.5 withbovine serum, while less than 4 per cent ofmicrofilariae exsheathed with PBS. Thus,
the microfilariae from the jird peritoneal cavity exsheathed at much lower rates thanthose from the dog venous blood with both media. The differences in the overall
exsheathing between the microfilariae from the jird peritoneal cavity and those fromthe dog venous blood were highly significant (p<0.0001) in both media. ‑‑ ‑As shown in Table 7, the death rates of mosquitoes were lower 'in̲̲the grou:pstaken the microfilariae from the jird peritorieal cavity than in the groups received
the microfilariae from the dog venous blood, although the density of microfilariae in
the feeding medium and the average number of microfilariae taken up by a mosojuito

Arere higher in the fQrmer groups. The difference in the death rate between the180

Table 7

Sourceof mf.

Dog
venousblood

Jird

Feeding
medium *Total
Mf.
no. ofcount
mosqui‑per0.03 mltoes

Mean no.

No, of mosquitoes diedon days after feeding(range) * *12 3 4567 891011 total
1324 7102 OO O O 38 (42.7)

of mf.
per infected

PBS

600

89

21.5 (4‑47)

bovinel OO

1 14

5.2 (2‑9)08 822304 oo o o 45 (39.5)

700

O o

serum

PBS

peri‑

tonealcavityNumbers of deaths in mosquitoes l to 1 1 days after artificial feeding

bovineserum

132

38.0 (5‑204)

l ,OOO122

204.0 (4‑653)

O 3

4

o 2 2 Oo ooo

11 ( 9.0)

1 ,500l 05

336.6 (1‑1,213)

1

9

3

o o o 12oo

17 (16.2)

1 ,OOOl 40

19.0 (1‑79)

1

o

1

o 5 5 4 lO 3

1

1

31 (22.1)

l,500

97

180.2 (3‑505)

41o o o o1O

9 ( 9.3)

2,000

l OO

O

o o o o1O

8 ( 8.0)

137.8 (1‑1,002) 5 2

1

3

6

3 5

O o

o 34 (25.8)* As phagostimulant l0‑3M ATP was contained.
** Numbers immediately after feeding.

group received the microfilariae from the dog at the density of 600 per 0.03 ml in
PBS and the group taken those from the jird at the density of 700 in PBS was sta‑tistically significant (p<0.013). Also the difference was significant (p<0.0044)
between the group received the microfilariae from the dog at the density of I OO in
bovine serum and that taken the microfilariae from the jird at the density of I ,OOO in

bovine serum. The mosquitoes received the microfilariae from the dog most fre‑quently died 3 or 4 days after feeding. In the groups taken those from the jird at
the density of 700 in PBS and at that of 1,000 in bovine serum, most deaths occurred
5 to 7 days after feeding. When the density was higher, most deaths occurred muchearlier, within one to 3 days of infection.

On and after one day of infection, the infection rate of mosquitoes taken the
microfilariae from the dog venous blood at the density of 600 in PBS was as high asthat in the group which fed directly on the dog (Table 8). In the group offered
the microfilariae from the dog at the low density of I OO in bovine serum, the infection

rate was as low as 12.9 per cent 10 to 13 days after feeding. In the groups given the
microfilariae from the jird in PBS, the infection rates were low; although the densityof those from the jird was raised to 1,000 or 1,500, the rates at the period between 10
to 1 3 days of infection seemed to be lower than that of the microfilariae from the dog
offered at the density of 600. Also the rates were low in the groups received themicrofilariae from the jird in bovine serum. The infection rate was 30 per cent in
the mosquitoes given the microfilariae from the jird at the density of I ,OOO in bovine
serum I O to 1 3 days after‑ feeding, seemingly higher than that in the mosquitoes taken
the microfilariae at the density of 100. But the difference was not statistically sig‑
niflcant. When the ‑density of those from the jird was raised to I ,500 or 2,000 inbovine serum, the rate at the same period became significantly higher (p<0.039 at
l,500; p<0.0017 at 2,000) than that of the microfilariae from the dog given at tbc
density of I OO.

181

Table 8Infection rates in mosquitoes one to 13 days after direct or artificial feeding

No. of mosquitoes

Source Feeding Mf. countof mf. medium* per 0.03 ml

Infected/dissected (o/o) for periodsl ‑5 days

6‑9 days

10‑13 days

495

18/25 (72.0)

16/20 (80.0)

9/lO (90.0)

PBS

600

13jl5 (86.7)

12/13 (92.3)

9/lO (90.0)

bovine

1 OO

5/14 (35.7)

2/12 (16.7)

4!31 (12.9)
25/76 (32.9)

Dog

peripheral (direct)
blood

Dog
venous
blood

serum
PB S

Jird
peritoneal
cavitybovineserum

700

25j61 (41.0)

l0/42 (23.8)

1 ,OOO

42/63 (66.7)

29/42 (69.0)

51/75 (68.0)

1 ,50020/27 (74.1)

17/22 (77.3)

1 l/20 (55.0)

l ,OOO

14/28 (50.0)

2j21 ( 9.5)

12j40 (30.0)

l ,50022/27 (81.5)

ll/21 (52.4)

l0/20 (50.0)

2,000

2lj27 (77.8)

13/20 (65.0)

14/25 (56.0)* As phagostimulant 10‑3M ATP was contained.

The individual counts of larvae recovered from the mosquitoes fed directly on
the dog did not vary widely throughout the development of larvae (Table 9). In
this group, the average number of larvae per mosquito was 5.7 to 14.0 during theperiod between one and 3 days of infection and did not fluctuate widely in the sub‑sequent period of infection. In the mosquitoes given the microfilariae from the dog
venous blood at the density of 600 per 0.03 ml in PBS, the average number of larvae
per mosquito was equal to or somewhat higher than that of direct feeding group,
although the individual counts of larvae varied more widely. In the mosquitoes fed
on the microfilariae from the dog venous blood at the low density of 100 in bovine
serum, only a few larvae were recovered even in the early period of infection.
The average number of larvae per mosquito was much lower in the group offered
the microfilariae from the jird peritoneal cavity at the density of 700 per 0.03 ml in
PBS than in the group taken those from the dog venous blood at the density of 600(Table 9) : Only 2 of 1 79 mosquitoes dissected during 1 3 days of infection harbored
10 Iarvae or more. When the microfllarial density in PBS was raised to 1,000, theaverage number approached to that of the direct feeding group. In the group fed on
PBS containing the microfilariae at the density of I ,500, the average number was
higher than that of the direct feeding group during the period between one and 6 daysof infection and equal to that of the latter subsequently. In the groups given the
microfilariae from the jird peritoneal cavity with bovine serum, the average numberof larvae per mosquito was far below that of the direct feeding group. In the artificial

feeding groups, especially those given the microfilariae with PBS, the individual
counts of larvae recovered from the mosquitoes varied widely, more frequently inthe early period of infection than in the late period.

1 82

Table 9Sourceof mf.

Numbers of larvae developed in mosquitoes after direct or artificial feeding

Feeding

medium*

Dog

peri‑

pheralMean no. of larvae per infected mosquito(range) on days after feeding

Mf. countper0.03 ml2

1(direct)495

14.0(4‑26)

PB S

600

9.3

(8‑12)
4.0
(4‑4)

l .O

(1‑1)

4

3

5

5.7

9.3

(1‑13)(2‑16)( I ‑8)(4‑12)

19.3

27.5(3‑35)(4‑5 1 )

5.0
(2‑7)

(3‑16)

6.0
(6‑6)

4.0
(4‑4)

(1‑1)

8.3

4. 7

6
3.0
( I ‑6)b I oodDogvenousbloodbovine

l OO

serum

700

PBS
Jird

l ,OOOl ,500peri‑

tonealcavity1 ,OOO

bovineserum

1 ,500

2,000

3.0

4.0

( I ‑4)( 1‑8)

5.0( I ‑9)50.5( 1 6‑1 1 5)

18.6
(1‑72)
4.0
(2‑8)

l .3

l .6

( I ‑2)( I ‑3)

9.5

l .O

l .O

(1‑1)
l0.7

(2‑48)

9.8(2‑25)

2 1 .315.0( 1‑6 1 )(3‑30)( 1 3‑20)

Dog

pheralblood

Dogvenous
blood

l .O

2.0
( I ‑3)2,5(2‑3)( I ‑3)

27.0

(1‑1)
4.3

4.5

6.3

4.0

5.0

(3‑6 1 )( 3‑7 )

(1‑12)( 3‑6)(1‑11)

3.8
(2‑8)

6.3

3.8

(1‑16)
3.8(2‑ 1 2)

( 1‑7)( I ‑8)(1‑13)

2.0

5.3

on days after feeding

7

8

9

8.3
(7‑9)

4.8
(2‑lO)

6.8(4 12)

tonealcavity

12

13

9.6

l0.8

(3‑16)(5‑20)

ND

ND

ND

ND

ND

ND

10.0

6.7

14.7

l I .8(4‑16)(2‑12)(3‑2 1 )(4‑2 1 )

ND

l .O

l .O

(1‑1)

(1‑1)

l .O

l .O

(1‑1)

6.0
(3‑9)

( I ‑5)

2.7

8.5

l .O

l.l

l .8

2.7

(2‑15)( I ‑2)( 1‑3)(1‑lO)

l0.8

5.8

l .8

( I ‑44)(1‑18)( I ‑4)

ND

ND

ND
ND
ND

ND
ND
ND

8.7

(1‑1)
9.2

(1‑17)( 1‑2 1 )( 1‑25)

8.3

15.0

10.6

(3‑45)

6.4(1‑14)

6.3( I ‑24)(3‑ 1 5)

(5‑ 1 5)

l .9

peri‑

ll

17.3

( I ‑3)JirdlO(2‑4 1 )

(1‑1)

ND
3.0
(1‑7)
6.0
(4‑8)

8.2

1 .O

(1‑1)
2.6
(2‑3)
4.5
( I ‑7)

20.3(3‑57)

2.3

of mf.

peri‑( I ‑23)( I ‑4)

Mean no. of larvae per infected mosquito (range)

Source

ND

l .O

(l‑1)
5.4
(1‑19)
16.3

18.0

30.0
(2‑57)

l .O

1 .6( 1‑3)( 1‑2)

(1‑1)
3.7
(2‑5)

3.0

2.2

7.0

( I ‑6)( I ‑4)(1‑16)ND
1.5* As phagostimulant 10‑3M ATP was contained.

4.6(2‑lO)ND
2.5( I ‑4)

1 83

As shown in Figure 4, stage I Iarvae were detected in the thoracic muscle of themosquitoes fed directly on the dog and dissected one to 5 days after feeding. Only
stage 11 Iarvae were found in the direct feeding mosquitoes dissected 6 to 9 days after
feeding. In the mosquitoes dissected on 10 days of infection, 72.9 per cent of larvae
were still in stage 11 and localized in the thoracic muscle, while 8.9 per cent of larvae

had reached stage 111 and were detected in the head, and 18.8 per cent had alsoreached stage 111 and were found in the abdomen. The mosquitoes dissected onl I days harbored stage 11 Iarvae in the thoracic muscle (1 1 .60/0), and stage 111 Iarvaein the head (74.40/0), in the thorax (4.70/0) and in the abdomen (9.30/0)' The larvaein the mosquitoes received the microfilariae from the dog venous blood followed thegradual development of those in the direct feeding mosquitoes without much delay.
Only stage I Iarvae were found in the thoracic muscle of the mosquitoes which took

up the microfilariae f'rom the dog venous blood in PBS and were dissected one to
5 days after feeding. In this group of mosquitoes dissected on 6 days of infection,
3.3 per cent of larvae were still in stage I and 96.7 per cent had reached stage 11 in
the thoracic muscle. All larvae found in the mosquitoes dissected 7 to 9 days after
feeding were in stage 11 and localized in the thoracic muscle. On 10 days of infec‑
tion 47.7 per cent of larvae were still in stage 11 Iocalized in the thoracic muscle,
while 18.2 per cent had attained stage 111 in the head, 29.5 per cent had attained thesame stage in the thorax and 4.6 per cent in the abdomen. The mosquitoes dissected
on I I days had stage 111 Iarvae in the head (690/0)' thorax (11.30/0) and abdomen

G1

II

lll

ll

Ill

ND I I

lll

l

G2
G3

G4
G5

l

lll
I

ll

l

ll

tll

1 2 3 4 5 6 7 8 9 Io 11 12 13 diys
Figure 4

Development of Brugia pahangi larvae in Aedes aegypti after
direct and artificial feeding.

G 1, direct feeding on an infected dog; G 2, microfilariae
from dog venous blood fed artificially with PBS ; G 3, mi‑crofilariae from dog venous blood fed artificially with bovine

serum; G 4, microfilariae from jird peritoneal cavity fed
artiflcially with PBS (three experiments with different micro‑

filarial densities combined) ; G 5, microfilariae from jird

peritoneal cavity fed artificially with bovine serum (threeexperiments with different microfllarial densities combined) ;I, stage I Iarvae; II, stage 11 Iarvae; 111, stage 111 Iarvae.

1 84(2.70/0)' besides stage 11 Iarvae in the thoracic muscle (70/0) ' The number ofinfectedmosquitoes were small, only 1 1 , in the group which were offered the microfilariae
from the dog venous blood in bovine serum and dissected one to 1 1 days after feeding.
In this group all larvae found at one to 5 days' dissection were in stage I. Only stage
II Iarvae were detected 7 to 10 days after feeding. A11 Iarvae reached stage 111 onl I days.

In the group of mosquitoes received the microfilariae from the jird peritoneal
cavity in PBS at the density of 700, only stage I Iarvae were found on 5 days of in‑
fection, and only stage 11 Iarvae were detected on 6 days. On the other hand, stage
II Iarvae were found on 5 days at a rate of 3.7 per cent in the group offered themicrofilariae from the jird in PBS at the density of 1,000 and at a rate of 2.5 per centin the group offered at 1,500. On 6 days of infection, 10.3 per c̲ent of larvae werestill in stage I in the group taken the microfilariae at 1,000 in PBS, and all larvae
reached stage 11 in the group taken at I ,500. Stage 11 Iarvae were found at a rateof 12.5 per cent in the group received the microfilariae from the jird in bovine serumat the density of I ,500 on 5 days, while only stage I Iarvae were detected in the group
received at 1,000 and that at 2,000. On 6 days 6.7 per cent were still in stage I in
the group received the microfilariae at 1,500, and all larvae reached stage 11 in thatat 2.000. Thus, some of the microfilariae from the jird peritoneal cavity reachedstage 11 one day earlier than the microfilariae from the dog. On the other hand,stage I Iarvae were still found on 6 days in the groups taken the microfilariae fromthe jird peritoneal cavity as in the group received the microfilariae from the dog
venous blood. The development of the microfilariae from the jird to stage 11 seemed
to be not influenced by the density of microfilariae in the medium, nor by the natureof the medium (i.e. PBS or bovine serum).
In the groups received the microfilariae from the jird in PBS at the density of
700 and 1,000, all larvae were still in stage 11 on 10 days of infection, and stage IIlarvae were 25.0 per cent and 43.4 per cent respectively on I I days. Stage 11 Iarvae
were still found on 12 days at a rate of 1 1.1 per cent and 20.0 per cent respectively.
In the former group 7.4 per cent of larvae remained at stage 11 even on 13 days ofinfection. In the group taken the microfilariae from the jird in PBS at the densityof 1,500, stage 11 Iarvae were 96.0 per cent on 10 days and 1 7.6 per cent on I I days.
In the mosquitoes received the microfilariae from the jird in bovine serum, the rateof stage 11 Iarvae on 10 days was 33.3 per cent at the density of I ,OOO, 81 .8 per cent at

l,500 and 72.2 per cent at 2,000, and that on I I days was 28.6 per cent, 25.0 per centand 1 7.0 per cent respectively. Thus, the development of the microfilariae from thejird peritoneal cavity into the final form in the mosquito seemed to be slower thanthat of the microfilariae in the direct feeding on the dog and that of the microfilariae
from the dog venous blood (Figure 4). Also the rate of stage 111 Iarvae situated in
the head was 26.7 to 41.9 per cent on I I days in the groups taken the microfilariae
from the jird, much lower than that in the groups fed on the microfilariae from thedog.
On the dissection 10 to 1 1 days after feeding, 9 of 10 mosquitoes which fed
directly on the dog harbored the infective (i.e. stage 111) Iarvae (Table lO). The

number of infective larvae per infected mosquito ranged from one to 1 7 with an

185

Table 10 Numbers of stage 111 Iarvae in mosquitoes 10 to 1 1 days after feeding andlengths of larvae

No. of mosquitoes Mean no. of

Source Feeding stage
111 Iarvae
with stage
Mfl count
per

dis‑

495

10

9 (90.0)

5.7 (1‑17)

PBS

600

10

9 (90.0)

9.9 (1‑21)

bovinel OO

31

3 ( 9.7)l.O (1‑1)

700

76

18 (23.7)

2.2 (1‑9)

PBS I ,OOO

75

43 (57.3)

5.8 (1‑21)20

8 (40.0)

7.8 (1‑ll)

40

lO (25.0)

l.2 (1‑2)20

8 (40.0)

3.3 (1‑7)25

l I (44.0)

5.0 (1‑13)of mf. medium*0.03 ml per
sectedinfected(range)Dog

peripheral (direct)
blood

Dog
venous
blood

serum

Jird I ,500

III Iarvae (o/o)

Range oflengths(mea.n) * *l ,422‑ I ,676
( I ,464.3)

l,294‑1,627( I ,43 1 .6)

ND
l ,45 1 ‑ I , 789( I ,584. 2)

peritoneal

cavitybovine
I ,OOO
I ,500
serum

2,000

l ,235‑ I ,643
( I ,440.0)* As phagostimulant 10‑3M ATP was contained.
: * Lengths in micron on 1 1 days.

average of 5.7. The length ofinfective larva on 1 1 days varied between 1,422 p and1,676 p with an average of 1,464.3 p. In the group taken the microfilariae frorn the
dog venous blood in PBS at the density of 600, the perc6ntage of mosquitoes har‑boring the infective larvae was 90 per cent, and the average number of infective
larvae was 9.9, somewhat higher than the direct feeding group. In the mosquitoes
taken the microfilariae at the density of 100, the infection rate was low and thenumber of larvae was small. In the groups received the microfilariae from thejird, the percentage of mosquitoes harboring the infective larvae was 23.7 to 57.3
per cent, Iower than that in the mosquitoes fed directly on the dog and in thoseoffered the microfilariae from the dog in PBS. The average number of infective
larvae was also smaller in the groups received the microfilariae from the .jird than in
the group taken the microfilariae from the dog at the density of 600. The lengths ofinfective larvae in the groups received the microfilariae from the jird in bovine serum

were similar to those in the group fed on the microfilariae from the dog in PBS.
The length of infective larva in the mosquitoes offered the microfilariae from the.jird in PBS varied between 1,451 p and 1,789 p with an average of 1,584.2 p
DISCUSSION

A new apparatus for feeding mosquitoes is small and easy cif handling withouta sophisticated self‑contained heater and thermistor. Its construction is economical
and involves no special techniques, using a plain laboratory glassware, mouse skin

1 86

etc. It can be used with any mosquito cage that has a top hole. The capacity forfeeding medium is approximately I O ml. When the conservation of medium isimportant, 2 ml or less can be used. Thus, the usefulness of the new apparatus isdemonstrated in the present studies.
In the present studies ATP is the most effective phagostimulant of the nucleotides

dissolved in saline, PBS or bovine serum. Hosoi (1959) reported that AMP and
ADP were more effective than ATP to Culex pipiens pallens with a kind of forcible
feeding. The difference in the species of mosquitoes and/or feeding techniques may
have caused disagreement between the results. The fact that nucleotides in 10‑3 M
concentration are generally more effective than those in 10‑4 M concentration is inaccord with observations by Rutledge et al. (1964) that 0.005 M was near the opti‑
mum of ATP to Bangkok strain of Aedes aegypti. Thus, I 0‑3 M ATP is an adequate
phagostimulant for the artificial feeding of Liverpool strain of Aedes aegypti.

Some microfilariae from the dog venous blood become inactive within 30 min in
5 per cent glucose solution or saline both containing 10‑3 M nucleotides at 37 C,
while inactive microfilariae are detected after 90 min in PBS containing I 0‑3 M
nucleotides and after 20 hours in bovine serurn containing them (unpublished data).
So, PBS and bovine serum are suitable for the media for microfilarial feeding.
It is suggested that microfilariae are maldistributed in PBS, twining rounddebris, by the findings that the infection rates in mosquitoes are lower for the artificial

feeding with PBS than for the direct feeding and that the individual count of micro‑
filariae per mosquito varies widely in that with PBS, when the microfllarial densities
are similar in both groups. In the artificial feeding with bovine serum, the infection
rates are low as in that with PBS, although the microfilarial densities are higher thanin the direct feeding group. However, the average numbers of microfilariae per
infected mosquito are lower and the individual count per mosquito varies less widelywith bovine serum than with PBS. It seems likely that the maldistribution of micro‑
filariae is less in bovine serum than in PBS, probably due to the viscosity of serum.
The exsheathing of microfilariae from both the dog venous blood and the jird
peritoneal cavity occurs higher in the artificial feeding with bovine serum than inthat with PBS. The exsheathing may be disturbed in the mosquitoes taken up a
number of microfilariae by the overcrowding in the stomach : it is more frequentwith PBS than with bovine serum as indicated by the individual count per mosquito.
It is also possible that the viscosity of serum facilitates the exsheathing in thestomach. Aoki (1971 a, b) and Aoki et al. (1976) reported that microfllariae ex‑
sheathed well on agar pads. The findings support the view that the exsheathing isswitched on by the change of milieu toward solidification.
The death rates of mosquitoes are lower in the groups taken the microfilariae
from the jird peritoneal cavity than in the groups received those from the dog venous
blood. It is of interest to speculate whether the developing larvae inflict damage on

mosquitoes (Rosen, 1955; Wharton, 1957; Townson, 1970; Hockmeyer et al., 1975 ;Christensen, 1978), and whether some microfilariae from the jird are inadequate to
the development, which results in the low death rate of mosquitoes. It may be alsothat most deaths occurring one to 3 days after feeding in the group taken high densityof microfilariae are brought by a number of microfilariae penetrating the stomach

l 87

wall and developing in the thoracic muscle as stage I Iarvae.
On and after one day of infection, the rates of infected mosquitoes are low in thegroup taken the microfilariae from the dog venous blood at the density of 100 per
0.03 ml in bovine serum : it seems likely that the low rates of infection are broughtby the low density of microfilariae offered. In the artificial feeding groups, especiallythose with PBS, individual counts of larvae vary widely one to 3 days of infection.
It probably reflects the fact that the number of microfllariae taken varies greatly

among the mosquitoes in the same groups.
The microfilariae from the jird peritoneal cavity exsheathe in both PBS andbovine serum at lower rates than those from the dog venous blood. The rate of
infected mosquito is low in the groups received the jird microfilariae: 10 to 13 days
after feeding, the rate in the group offered the jird microfilariae at the density of I .OOO

is not signiflcantly different from that in the group taken the dog microfilariae atthe density of 100. The average number of larvae per infected tends to be low in
the group taken the jird microfilariae. These findings suggest that the microfilariae
from the jird peritoneal cavity includes the ones inadequate to the development in
the mosquitoes, such as the newborn and the aged, at a higher rate than those fromthe dog venous blood.

Some microfilariae from the jird peritoneal cavity reach stage 11 one day
earlier than the microfilariae from the dog. However, the development of the
microfilariae from the .jird to stage 111 is generally slower than that of the microfilariae

from the dog. It probably causes the low rate in the mosquitoes harboring thestage 111 Iarvae and the low average number of stage 111 Iarvae per infected in thegroup received the microfilariae from the jird and dissected 1,Q to 1 1 days after
feeding. It is clear from the present findings that the microfilariae develop to thestage 111 Iarvae in the mosquitoes which ha.ve not taken blood, serum or protein
meals, and that the infective larvae sometimes become even longer with PBS.
Brugia pahangi inoculated, Iocalized and producing microfilariae intraperito‑
neally and the microfilariae thus produced and localized in the peritoneal cavity

seem to follow an aberrant mode of development. The present studies, however,
have revealed that the microfilariae from the jird peritoneal cavity can reach stage
III in Aedes aegypti, although at a lower rate than the microfilariae produced by thelymphatic adults in a natural host.

ACKNOWLEDGMENT
Grateful acknowledgment is made to Prof. D. Katamine for his constant interest
and guidance in this investigation. Thanks are tendered to Mrs. T. Miyazaki for
raising vector mosquitoes.

REFERENCES
1) Aoki, Y. (197la) : Exsheathing phenomenon of microfilaria in vitr() (1), Trop. Med., 13 (3),13 140 (In Japanese with English summary)2) Aoki, Y. (197lb) : Exsheathing phenomenon ofmicrofilaria in vitro (II), Trop. Med., 13 (4),

1 88

1 70‑1 79 (In Japanese with English summary)3) Aoki, Y., Nakajima, Y. and Katamine, D. (1976) : Studies on Malayan filariasis in Che‑ju Is.,
Korea. 3 Microfilarial surface architecture of Brugia pahangi (Che‑ju strain) in comparison withthat of Brugia pahangi, Japan. J. Trop. Med. Hyg., 4 (2), 129‑137
4) Ash, L. R. and Riley, J. M. (1970) : Development of Brugia pahangi in the jird, Meriones un‑
guiculatus, with notes on infections in other rodents, J. Parasit., 56 (5), 962‑968

5) Ash, L. R. ( 1973) : Chronic Brugia pahangi and Brugia malayi infections in Meriones un.guiculatus.
J. Parasit., 59 (3), 442‑447

6) Bar‑Zeev, M. and Smith, C. N. ( 1959) : Action of repellents on mosquitoes feeding throughtreated membranes or on treated blood, J. Econ. Ent., 52 (2), 263‑267
7) Behin, R. (1967) : Artiflcial feeding apparatus for mosquitoes, Mosq. News, 27 ( 1), 87‑90
8) Bishop, A. and Gilchrist, B. M. ( 1946) : Experiments upon the feeding of Aedes aegypti throughanimal membranes with a view to applying this method to the chemotherapy of malaria. Para‑sitology, 37, 85‑lOO9) Buckley,J.J. C. and Edeson,J. F. B. (1956) : On the adult morphology of Wuchereria sp.
(malqyi?) from a monkey (Macaca irus) and from cats in Malaya, and on Wuchereria pahangi n. sp.
from a dog and a cat, J. Helminth., 30 (1), 1‑20lO) Christensen, B. M. ( 1978) : Dirofilaria immitis: Effect on the longevity of Aedes trivittatus, Exp.
Parasit., 44 (1), I 16‑‑123l I ) Collins, W. E. ( 1963) : Transmission of the Similiki Forest virus by Anopheles albimanus using

membrane‑feeding technique, Mosq. News, 23 (2), 96‑99
12) Collins, W. E., Jeffery, G. M., Skinner,J. C. and Harrison, A. .J. (1964) : Comparative in‑fectivity of a strain of Plasmodium falciparum from Panama to three species of Anopheles as studied

by membrane feeding, Mosq. News, 24 ( I ), 28‑3 l13) Eyles, D. E. ( 1952) : Studies on Plasmodium gallinaceum. II. Factors in the blood of the vertebrate

host influencing mosquito infection, Amer. J. Hyg., 55, 276‑290
14) Hockmeyer, W. T., Schiefer, B. A., Redington, B. C. and Eldridge, B. F. ( 1975) : Brugia pahangi:
Effects upon the flight capability of Aedes aegypti, Exp. Parasit., 38 ( l), 1‑515) Hosoi, T. ( 1959) : Identification of blood components which induce gorging of the mosquito,
J. Ins. Physiol., 3, 191L218

16) Kan. S. P. and Ho, B. C. ( 1973) : Development of Brugia pahangi in the flight muscles of Aedes

togoi, Amer. J. Trop. Med. Hyg. 22 (2), 179‑188l 7) Kartman. L. ( 1953) : F,actors influencing infection of the mosquito with Dirofilaria immitis (Leidy,1856), Exp. Parasit., 2 (1), 27‑78
18) Kitaoka, S. and Morii, T. ( 1970) : Improved artificial feeding devices for biting midges andmosquitoes, Jap. J. Sanit. Zool., 21 (1), 70‑72 (InJapanese with English summary)19) Laing, A. B. G., Edeson,J. F. B. and Wharton, R. H. (1960) : Studies on filariasis in Malaya:
The vertebrate hosts of Brugia malayi and B. pahangi. Ann. Trop. Med. Parasit., 54 ( I ), 92‑99

20) McCall, J. VV., Malone, J. B., Ah. H. S. and Thompson, P. E. (1973): Mongolian jirds(Meriones unguiculatus) infected with Brugia pahangi by the intraperitoneal route : A rich source
ofdeveloping larvae, adult filariae, and microfllariae, J. Parasit., 59 (3), 436

21) Nakajima, Y., Aoki, Y., Sakamoto, M., Suenaga, O. and Katamine, D. (1976): Studies on
Malayan filariasis in Che‑ju Is.. Korea. 4 Experimental transmission of Brugia malayi (Che‑ju
strain) to domestic cats, .Japan. J. Trop. Med. Hyg. 4 (3, 4), 163‑177

22) Pipkin, A. C, and Connor, T. J. ( 1968) : A temperature‑controlled feeding apparatus forhematophagous arthropods, J. Med. Ent., 5 (4), 507‑509
23) Rosen. L. (1955) : Observations on the epidemiology of human filariasis in French Oceania,
Amer. J. Hyg., 61, 219‑24824) Rutledge, L. C., Ward, R. A, and Gould, D.J. (1964) : Studies on the feeding response Qf

189
mosquitoes tonutritive

solutions in25）schacherラJ．F．（1962a）：Morphology
in
26）

the

a new membrane琵eder，Mosq．News，24（4），407−419

ofthe

microHlaria

ofβ川鈎加hαη9∫andofthe

larvalstages

mosquitoシJ．Parasit．，48（5），679−692

Schacher，J．F．（1962b）：Developmental

stagesofβ躍9∫αραhαη8∫in the

final

host・J・Parasit・・

48（5），693−706
27）st．J・hn，J．H、，simm・ns，J．s．andReyn・1ds，F・H・K・（1930）：Transmissi・n・fdenguevirus
fromin琵ctedtonormal漉4θ5α69卿ぎ，Amer・J・TroP・Med・，10（1），23−24

28）T・wns・n，H．（1970）：Thce価ct・fin琵cti・nwithβrμ9紳αh噸・ntheHight・f鋤」48翻∫・
Am．Trop，Med、Parasit．，64（4），411−42029）Whart・n，R．H．（1957）：Studies・nnlariasisinMalaya：thee缶ciency・fMαη5・痂」・π9紳∫5
as an experimental vector of晦 h6r8吻mα傾，Am．Trop．Med．Parasit．，51（4），422−43930）W・ke，A．（1937）：E飾ct・fvari・usbl・・d￡racti・ns・neggPr・ducti・n・fオ6485α6翻ゴLinn・・
Amcr．J．Hyg．，25，372−380

スナネズミ腹腔より得た．B鰯g毎加h㈱塵ミクロフィラリアの・セ4θs磁g塑ガにおける発育

荘正國・中島康雄1・青木克己
蚊に対する人工摂取装置を新たに考案した。製作に特殊な技術を要せず，使用に便利である。その装置を用いて，、舶漉3αθg妙房の胃内摂取に対するヌクレオチドの影響と，スナネズミ腹腔ならびにイヌ静脈血より得たミクロフィラリアの，ノ4ε漉5αεg妙孟∫における発育の比較を行った。摂食刺戟

物としては，用いたヌクレオチドの中で，10−3MATPが最も有効である。イヌ静脈血より得たミク
ロフィラリアと同じく，スナネズミ腹腔より得たものは，第III期幼虫（感染幼虫）に成長することが証明されたが，その率はイヌ静脈血より得たものより低い。また，血液，血清，蛋白を食物として摂取していない蚊においても，ミクロフィラリアが感染幼虫になることが証明された。

長崎大学熱帯医学研究所寄生虫学部門
1現所属：東京医科歯科大学医学部医動物学教室

Japan. J Trop. Med. Hyg., Vol. 7, No. 3, 4, 1979, pp. 191‑199

191

COMPARATIVE SENSITIVITY OF FOCUS COUNTINGMETHOD IN THE INFECTIVITY TITRATION
OF DENGUE VIRUSES AND IN THE
NEUTRALIZATION TEST*AKIRA IGARASHI, YOSHIOMI OKUNO AND KONOSUKE FUKAI
Received for publication 4 December 1978

Abstract: Infectivity titrations w re performed for each of the 4 prototypes ofdengue (DEN) viruses by focus counting on BHK21 cells (FFU), by plaque formation on
LLC‑MK2 cells (PFU), and by intracerebral inoculation into suckling mice (LD50)'
The FFU titers were almost similar as LD50 for all the 4 types of DEN and these titers
were also similar to PFU except type 2 DF.N (DEN‑2), which showed lower PFU than
FFU or LD50' Neutralization (N) tests were performed for standard anti‑DEN sera by
50 per cent focus reduction (FR50) and by 50 per cent plaque reduction (PR50) methods.
Both values were almost the same except anti‑DEN‑2, whcih showed higher PR50 thanFR50' The principle of these methods were also applied to Japanese encephalitis andchikungunya viruses. Hemagglutination‑inhibition (HI) and FR50 N tests on several
human sera indicated that FR50 is more specific and sensitive than HI to detect type‑specific antibodies.

IN1rRODUCTION
Infections due to dengue (DEN) viruses of 4 different serotypes are very im‑
portant diseases in Southeast Asia, causing sometimes severe hemorrhagic fevers
accompanied by shock and death ofinfected children (Hammon et al., 1960 ; Halstead,1966). Hemagglutination‑inhibition (HI) test has most routinely been used in theserodiagnosis and seroepidemiology of DEN infections (Nimmanitya et al., 1 969;
Halstead et al.. 1 969 ; Fukunaga et al.. 1 974b), because of its simplicity and rapidity(Hammon and Work, 1 964) . However, high cross‑reactivity by HI test among mern‑bers of flavivirus, or group B arbovirus (Casals, 1957; Clarke and Casals, 1965 ;Melnick, 1974) does not often tell anything but flavivirus infection in Southeast Asia,

where multiple flavivirus infections coexist. Although neutralization (N) test is
most specific among erological reactions (Hammon and Work, 1 964), the test has
not been used in a large scale, because of the difficulty in the infectivity titration of

DEN viruses. The standard method by intracerebral inoculation into suckling mice
to record LD50 requires a long observation period and a large space for animal ex‑periment. Even the more recent application of cell culture techniques require
fairly long incubation before the final results are obtained, which are, therefore, not
Presented at the 19th Annual Meeting ofJapanese Society of Tropical Medicine in Nara 1977
2 Department of Preverrtive Medjcine, ResearQh Institute for Microbjal Diseases, Osaka University

1 92

always guaranteed. In tbis laboratory we have developed a rapid titration of DEN
virus infe tivity by counting foci of infected BHK21 cells stained by indirect immuno‑

fluorescent (FA) or peroxidase‑anti‑peroxidase (PAP) technique and applied themethod to assay N antibody (Igarashi and Mantani, 1974; Okuno et al.. 1977, 1978).
In this report we describe the relative sensitivity of focus counting method in theinfectivity titration of DEN viruses compared with other standard methods, and also
the relative sensitivity of focus reduction N test, and discuss about its application tohuman serum specimens.

MATERIALS AND METHODS
Viruses : Following DEN virus strains were used : type I (DEN‑1) Hawaiian, type 2(DEN‑2) New Guinea B, type 3 (DEN‑3) H‑87, and type 4 (DEN‑4) H‑241. The
origins and passage histories of these viruses were already described as well as themethod to prepare infected suckling mouse brain (SMB) stock viruses (Igarashi,1978). The Nakayama strain of Japanese encephalitis (JE) virus was grown in
adult mouse brains, and chikungunya (CHIK) virus, African strain, was plaque‑purified and seed virus was prepared in BHK21 cells (Igarashi and Fukai, 1969).

Cells: BHK21 cells were grqwn at 37 C with 10 per cent calf serum in Eagle's(1959) minimal essential medium (MEM). LLC‑MK2 cells were obtained fromDr. S. Inoue, National Institute of Health, Tokyo, and were grown with 10 per centfetal calf serum in MEM. These cells were transferred every 4 days at cell densityof 105 cells/ml and was incubated at 37 C in 5 per cent C02 atmosphere. Mono‑layers were ready after 3‑4 days in dishes and after 2 days in 8‑chamber slides (Miles,
Ill. USA).
Infectivity titrations : Focus counting method is 'a rapid infectivity titration on BHK2 lcells prepared in 8‑chamber slides by counting the number of groups of cells contain‑

ing virus antigen which was revealed either by FA or PAP method (Igarashi andMantani, 1974; Okuno et al., 1977, 1978). In the case of CHIK virus, tragacanthgum (Wako Pure Chemicals Co. Osaka, Japan) was introduced to final I per cent in
the medium in order to prevent spreading of the secondary foci. Specimens were
harvested 16 hours for CHIK, 24 hours for JE, 40 hours for DEN‑2, 48 hours for
DF,N‑4, and 72 hours for DEN‑1 and DEN‑3 viruses, respectively, after incubation<at 37 C. Titers were shown as focus forming units (FFU) per ml. Plaque formation
on LLC‑MK2 cells was performed by the method of Russell et al. (1967) and titers
were shown as plaque forming units (PFU) per ml. LD50 titration in SMB was
performed by inoculation ofserially 10‑fold diluted specimens into the brains (0.01 ml/
brain) and titers were expressed as LD50/ml.
Human serum specimens and neutralization (N) test : Several individual human sera wereobtained from persons :ho either had been living in Southeast A, sia or had somechances tQ contact with )EN or CHIJ viruses. All the sera were hea [inactivated(56. 30i, min) before the te.st. . Serially diluted test serum was mixed with̲ the equalvolume of virus specimen diluted to 200 FFU/0.1 ml for FR5Q, or 200
for PR5a test. =After̲1 incubation at̲‑28 C. ‑fpr 2

F f/0.2 mlhoursj ̲residual virus infec.tivity ‑wasassayed by FFU (O I ml/well) or by PFU‑ ･ (O‑.2 mljdish) assay. ‑ Focu

or p:laque1 93

reduction percent was plotted on a probit chart against the logarithm of the serum
dilutions. The N titer of the test serum was shown either as 50 per cent focus reduc‑tion (FR50), or 50 per cent plaque reduction (PR50), the reciprocal of the serumc]ilution which will reduce the focus or plaque number to 50 per cent of the control
without antiserum (Russell et al.. 1967; Okuno et al., 1978).
Hemagglatination‑i,thibition (HI ) test: The method of Clarke and Casals (1958) was
followed with modification to microtiter system (Sever, 1962).

Antiserum : Anti‑DEN sera were prepared in rabbits by 2 intramuscular inoculations
with one month interval of partially purified virus with complete Freund's adjuvant(Okuno et al., 1978).

RESULTS
Standard prototype DEN viruses were assayed for their infectivity by FFU on

BHK21 cells, by PFU on LLC‑MKZ cells and by LD50 m SMB, and the results are
summarized in Table I . The FFU titers were almost the same as LD50 for all the
4 types of DEN viruses, and these titers were also similar to PFU for DEN‑1, DEN‑3

and DEN‑4 viruses. However, DEN‑2 showed significantly lower PFU than FFU
or LD50 trters. This difference was observed particularly with New CJuinea B strain
but it was less when TR1751 strain of DEN‑2 virus was used.

N test of the standard anti‑DEN rabbit sera were performed either by FR50
or PR50 method against the homologous DEN viruses, and the results are shown in

Table 2. Both the FR50 and PR50 titers were almost the same for anti‑DEN 1,
anti‑DEN‑3 and anti+DEN‑4 sera, however, PR50 titer of anti‑DEN‑2 serum wassignificantly higher than its FR50' This may reflect the relative insensitivity I t)f

PFU assay on LLC‑MK2 for DEN‑2 virus, but detailed mechanism is not known yet.
Table I Relative infectivity (log/ml) of

Table 2

Neutralization titers of' anti‑

dengue sera

different dengue viruses

Virus BHK21 LLC‑MK2 SMB

Antiserum

FR50 on

BHK2 1

LLC‑MK2

940
820

620
2500

1,lOO

l,380

l 90

320

FFU LD50
PFU

6.2

6.6

6.8

Anti‑DEN‑ 1

DEN‑2
DNE‑3

8.3

7. 1

8.3

5.7

5.8

6,7

D E N‑4

6.3

6.2

6.4

Anti‑DEN‑2
Anti‑DEN‑3
Anti‑DEN‑4

DEN‑ l

PR50 on

Limitted numbers of human sera from persons possibly exposed to DEN or
CHIK virus were assayed for their antibody titers by HI test and the results areshown in Table 3. A11 the sera from residents of Southeast Asian countries had HI
antibodies broadly reactive to all the 4 types of DEN as well as JE antigens.' OrreJapanese who had previously been immunized with JE and yellow fever vaccine andwho had suffered from an apparent DEN infection (No. 18)‑ also showed the broadly
reactive pattern. On the other hand, another Japanese who had received JE vaccine
and who experienced an apparent DEN‑ symptQm howed monospecific reaction '‑t )1 94

Table 3 HI titers of human sera

Serum Permanent
IN*o. residence
1

2
3

Thai
Thai
Korea

13

Japan
Japan
Thai
Thai

17

18

4
5

Age
Sex DEN IAntigen
DEN 2 DEN 3 DEN 4 JE CHIK
40 M

20

39M
34M
41M

40

40

20

40

40

80
80

20

80

20

40
40
20

40

32F

28M

40

80
1 60

Burma

40F
32F

1 60
1 60

320

80
80
80

36M
28M

80
80

20

20

40

20

19

Japan
Japan

23

Philippines

29F

l,280

320

l 60

80

80

24
25

Japan
Japan

55M
54M

26

Indonesia

30F

40

20

20

20

20

9

20

80

* Iess than 20.

DEN‑1 antigen (No. 19). No other Japanese or Korean had any positive HI anti‑
body against DEN or JE antigen.
These same sera were assayed for their N antibody titers by FR50 test and theresults are shown in Table 4. Compared with the results in Table 3, two character‑istic aspects could be pointed out. (i) The N test is more specific than HI, forexample, serum No. I reacted broadly by HI, however the FR50 assay clearly indi‑cated speficity to DEN‑2 virus. Also, serum No. 18, though reacting broadly by HI,
showed more specific reaction to DEN‑‑1 virus by N test. (ii) The N test is, ingeneral, more sensitive than HI to detect the presence of type‑specific antibodies.

There are 2 sera for DEN, 4 sera for JE and 6 sera for CHIK virus, which were
negative by HI but positive by N test. Because of the broad reactivity of HI test
and higher specificity of N test, several sera showed negative or lower N titer evenwhen HI test showed positive or higher titers (Serum No. 13 for JE; No. 18 for

DEN‑2, 3 and 4; No. 23 for DEN‑1). Serum No. 3 was obtained from a person
working on DEN‑4 virus in the laboratory without any obvious symptoms, and sera
Nos. 4 and 5 were obtained from investigators dealing with CHIK virus. The dataon these sera indicate some chances of laboratory infection by these viruses and also
the importance of N test to detect such accidental infections because HI did not tellpositive results for these cases. Serum No. 25 was obtained from a person who had
been experimentally infected, more than 35 years ago, with dengue virus which wasisolated during the dengue epidemic in Osaka in 1 942 and was passaged several times
in experimental animals (Taniguchi, 1 943 ; Taniguchi et al.. 1 951). Since he did

not experience any overt DEN symptom afterward, the presence of anti‑DEN‑1 N
antibody in the serum No. 25 indicates that the epidemic in Osaka, 1942, was pre‑

1 95

Table 4

Neutralization titers (FR50) of human sera

Previous history

Serum

Virus

VaccineNo. JEOvertLab
YF Dis * Exp* * DEN‑ l DEN‑2 DEN‑3 DEN
2

4
5

+
+
+

CHIK

18

960

16

64

45

270

l 50

42 O

350

84

44
88

4, OOO

4,000

2,600

l,200

92

950

***

D E N‑4

3

JE

l 05

CHIK
CHIK

33

9

55

230

770

l,450

330
580

205

13

17

460
460

l,150

250

1 OO

33

80

420
280

18
19

23

24
25
26

+ + +
+
+

+

15

72

5,400

7 40

78

76

820

230

125

l ,200

16

10

12

94

21

570

l,550

26

1 10

82

l,750

2 ,400

32

200
16

16

* Typical dengue fever.

** Any experience with laboratory experiments using viruses indicated.
*** less than I O.

sumably due to type I DEN virus. Long‑lasting presence of anti‑DEN N antibodies
has been described (Halstead, 1974; Fujita, 1977).
DrscussroN

Considering the public health importance of dengue hemorrhagic fever inSoutheast Asia (Chow et al., 1966), it is necessary to establish a simple, rapid andreproducible and also economical infectivity assay for all the 4 types of DEN viruses.
Since the introduction of cell culture systems, many people have developed various
assay systems for DEN viruses. The principle of these methods is either by plaqueassay (Schulze and Schlesinger, 1963; Miles and Austin, 1963 ; Georgiades et al..
1965 ; Hotta et al., 1966; Westaway, 1966; Russell et al., 1967; Bergold and Mazzali,

1968; Banerjee, 1969; Yunker and Cory, 1975) with its adaptation to micromethod(Skuhavachana et al.. 1969; DeMadrid and Porterfleld, 1969; Fujita et al.. 1975) or
to hemadsorption negative plaque (Makino and Mifune, 1 975), or by interference
method (Halstead et al., 1964) . We have developed the focus counting method forthe rapid titration of DEN viruses (Igarashi and Mantani, 1974; Okuno et al., 1977,
l 978). As shown in this paper, this method has almost the same sensitivity as thestandard SMB‑LD50' or PFU assay on LLC‑MK2 except ,DEN‑2. The principleof focus assay was originally used for rabies virus (Smith et al., 1 973) and recently for

rapid detection of poliovirus (Katzenelson, 1 976). The method was used for the1 96

cloning ofDEN virus which did not form plaques (Lubinski et al.. 1973). As alreadydiscussed (Igarashi and Mantani, 1974), this method has several advantages to give
rapid results using comparatively small amounts of cells and medium and compara‑tively small space for incubation.

The application of focus counting method for the N test (FR50) also gave quitesatisfactory results with comparable sensitivity as PR50 titer by plaque reduction(Russell et al., 1967). Although microplaque methods were reported for dengue N
tests (Sukhavachana et al., 1969; Kanamitsu et al., 1973; DeMadrid and Porterfield,
1974; Fujita et al., 1975), which give fairly rapid results, the numbers of plaques
observed in a single microtiter well are rather limited in contrast to our focus counting

which could give a good dose‑response up to 200 foci/well. The practical applicationof the FR50 assay has already been reported (Quina et al., 1978) and now in this paper.
Compared with HI test, N test is more sensitive to detect type‑specific antibodies as
has been described for JE virus (Kobayashi et al., 1967; Minamitani et al., 1974;
Fukunaga et al., 1974a), and also is more type‑specific than HI. Considering thepossibilities of multiple flavivirus infections prevalent in Southeast Asia, N test is very

important to detect type‑speciflc antibodies in the serodiagnosis and seroepidemiology
ofdengue infections in this area (Kanamitsu et al., 1973).

ACKNOWLEDGMENTS
We are grateful for the people who have willingly supplied their sera, and for

Mr. Kul Boranintr and Mr. Panya Polpruksa from Thailand who joined us asparticipants in the Promotion of Provincial Health Services Project operated underJapan International Cooperation Agency.

REFERENCES
1) Banerjee, K. ( 1969) : Observations on certain parameters for the growth and assay of dengue
viruses in tissue culture, Ind. J. Med. Res., 57, 30‑38

2) Bergold, G. H. and Mazzali, R. ( 1968) : Plaque formation by arboviruses, J. Gen. Virol., 2,

273‑284
3) Casals, J. ( 1957) : The arthropod‑borne group of animal viruses, Trans. New York Acad.
Sci. (Ser. No. 2), 19, 219‑235

4) Chow, C. Y., Grats, N. G., Halstead, S. B., Hammon, W. M., Ann, L. K., Marshall, I. D.,
Montoya, J. A., Rudnick, A., S enz, A. C. and Tuchinda, P. (1966) : Memoranda: Mosquito‑
borne haemorrhagic fevers of South‑east Asia and the western Pacific, Bull. WHO, 35, 1 7‑3 15) Clarke, D. H. and Casals, J. ( 1958) : Techniques for hemagglutination and hemagglutination‑
inhibition with arthropod‑borne viruses, Amer. J. Trop. Med. Hyg., 7, 561‑573
6) Clarke, D. H. and Casals, J. ( 1965) : Arboviruses; group B, pp. 606‑658 In F. L. Horsfall, Jr.
and I. Tamm (ed.) Viral and rickettsial infections ofman, J. B. Lippincott Co. Philadelphia andMontreal
7) DeMadrid, A. T. and Porterfield, J. S. ( 1969) : A simple micro‑culture method for the study ofgroup B arboviruses, Bull. WHO, 40, I 13‑12 1
8) DeMadrid, A. T. and Porterfield, J. S. ( 1974) : The flaviviruses (Group B arboviruses) : A
cross‑neutralization study, J. Gen. Virol., 23, 91‑96

1 979) Eagle, H. ( 1959) : Amino acid metabolism in mammalian cell cultures, Science, 130, 432‑437lO) Fujita, N. ( 1977) : On dengue and dengue hemorrhagic fever, Abstr. 19th Annual MeetingofJapanese Society of Trop. Med., p. 18l I ) Fujita, N., Tamura. M. and Hotta, S. ( 1975) : Dengue virus plaque formation on microplatecultures and its application to virus neutralization, Proc. Soc. Exp. Biol. Med., 148, 472‑475

12) Fukunaga, T., Rojanasuphot, S., Wungkorbkiat, S., Thammanichanon, A., Kan, T.; Otsu, K.,
Tuchinda, P., Jatanasen, S., Chiowanich, P. ( 1974a) : Japanese encephalitis vaccination in
Thailand, Biken J., 1 7, 2 1‑3 l13) Fukunaga, T., Rojanasuphot, S., Pisuthipornkul, S.. Wungkorbkiat, S., Thammanichanon. A.,
Chantrpenkul, P., Tuchinda, P., Jatanasen, S., and Fukai, K. ( 1974b) : Seroepidemiologic
study of arbovirus infections in the north‑east and south of Thailand, Biken J., 1 7, 169‑182

14) Georgiades, J., Stim, T. B., McCollum, R. W. and Henderson, J. R. (1965) : Dengue virus
plaque formation in rhesus monkey kidney cultures, Proc. Soc. Exp. Biol. Med., I 18, 385‑388
15) Halstead, S. B. ( 1966) : Mosquito‑borne haemorrhagic fevers of South and Southeast Asia,

Bull. WHO, 35, 3‑1516) Halstead, S. B. (1974) : Etiologies of the experimental dengues of Siler and Simmons, Amer.
J. Trop. Med. Hyg., 23, 974 982
l 7) Halstead. S. B.. Sukhavachana, P. and Nisalak, A. ( 1964) : Assay of mouse adapted dengue
viruses in mammalian cell cultures by an interference method, Proc. Soc. Exp. Biol. Med., I 15,
l 062‑ I 068

18) Halstead, S. B., Udomsakdi, S.. Scanlon, J. E. and Rohitayodhin, S. (1969) : Dengue andchikungunya infection in man in Thail.and, 1962‑1964. V. Epidemiologic observations outside

Bangkok, Amer. J. Trop. Med. Hyg., 18, 1022‑1033
19) Hammon, W. McD., Rudnick, A. and Sather, G. E. ( 1960) : Viruses associated with epidemic
hemorrhagic fevers of the Philippines and Thailand, Science, 1 31, I 102‑1 103

20) Hammon. W. McD. and Work, T. H. ( 1964) : Arbovirus infection in man, pp. 268‑311 In
E. H. Lennette and N.J. Schmidt (ed.) Diagnostic procedures for viral and rickettsial diseases,

American Public Health Association, New York21) Hotta, S.. Fujita, N. and Maruyama, T. (1966) : Research on dengue in tissue culture. I.
plaque formation in an established monkey kidney cell line culture, Kobe J. Med. Sci., 12,
1 79‑ 1 8722) Igarashi, A. ( 1978) : Isolation of a Singh's Aedes albopictus cell clone sensitive to dengue andchikungunya viruses. J. Gen. Virol., 40, 531‑544
23) Igarashi, A. and Fukai, K. ( 1969) : An intracellular component associated with chikungunya
virus‑specific RNA, Biken J., 12, 107‑1 1824) Igarashi, A. and Mantani. M. (1974) : Rapid titration of' dengue virus type 4 infectivity bycounting fluorescent foci, Biken J., 1 7, 87‑93

25) Kanamitsu, M., Fukuhara, M., Tamura, H., Ishizawa, F., Yoshida, I., Ogata, R.. Yoshioka. I.
and Saroso. J. S. (1973) : Geographical distribution of arboviruses in Southeast Asia. II.
Surveillance on group B arbovirus antibodies among the people. Virus, 23, 353 (in .Iapanese)
26) Katzenelson, E. ( 1976) : A rapid method for quantitative assay ofpoliovirus from water with theaid of the fluorescent antibody technique, Arch. Virol., 50, 197‑206

27) Kobayashi, N., Endo, S., Rokugo, M.. Oya, A. and Kan, G. ( 1967): Antibody surveillanceagainst Japanese encephalitis virus among the people in Yokohama city, Igaku‑no‑ayumi, 60,
135‑136 (in Japanese)

28) Lubinski, A. S., Tarr, G. C. and Hammon, ¥Af. McD. (1973) : Cloning of dengue virus type 2
by the direct fiuorescent antibody technique, Proc. Soc. Exp. Biol. Med., 144, 70‑72

29) Makino, Y. and Mifune, K. (1 975) : Sensitivity ofrapid assay method ofdengue viruses, Trop.

Med., 16, 163‑170

198
30)

Melnick, J. L. ( 1974) : Classification and nomenclature of viruses, Progr. Med. Virol., 1 7,

290‑294
31)

Miles, J. A. R,;nd Austin, F. J. (1963) : Growth of arboviruses in BHK21 cells, Austral. J.

Sci., 25, 466Jl;67

32)

Minamitani, M., Shinozaki, T.. Torii, M., Tokimatsu, A., Tabata, E., Sakamoto. H., Takayama,N., Oya, A., Ogata. R., Takeda. H. and Shimizu, A. ( 1974) : New findings about Japanese
encephalitis vaccine, Shonika Rinsho, 49, 8ld 823 (in Japanese)
34)

35)

Nimmanitya; S., Halstead, S. B., Cohen, S. N. and Margiotta, M. R. (1969) : Dengue andchikungunya virus infection in man in Thailand, 1962‑1964. I. Observations on hospitalized
patients with hemorrhagic fever, Amer. J. Trop. Med. Hyg., 18, 954‑971
Okuno, Y., Sasao, F.. Fukunaga, T. and Fukai, K. ( 1977) : An application of PAP (peroxidase‑anti‑peroxidase) staining technique for the rapid titration of dengue virus type 4 infectivity,
Biken .1･, 20, 29‑35

36)

Okuno, Y., Igarashi, A. and Fukai, K. ( 1978) : Neutralization tests of dengue and Japanese
encephalitis viruses by focus reduction method using PAP (peroxidase‑anti‑peroxidase) staining,
BikenJ., 21, 137‑147

37)

Quina, M. A., Thein, S., Auvanich, W., Okuno, Y., Igarashi, A. and Fukai, K. (1978) :Changes in dengue and Japanese encephalitis (JE) antibody after JE vaccination, Biken J., 2 l,
1 49‑ 1 59

38)

Russell, P. K., Nisalak, A., Sukhavachana, P. and Vivona, S. ( 1967) : A plaque reduction testfor dengue virus neutralizing antibodies, J. Immunol., 99, 285‑290

39)

Schulze, I. T. and Schlesinger, R. W. (1963): P]aque assay of dengue and other group B

40 )arthropod‑borne viruses under methyl cellulose overlay media. Virology, 19, 40‑48
Sever, J. L. ( 1962): Application of a microtechnique to viral serological investigations, J.

41)

Smith, J. S., Yager, P. A. and Baer, G. M. ( 1973) : A rapid reproducible test for determining

42 )Sukhavachana, P.. Yuill, T. M. and Russell, P. K. ( 1969) : Assay of arbovirus neutralizing anti‑

Immunol., 88, 320‑329
rabies neutralizing antibody, Bull. WHO, 48, 535‑541

45)body by micromethods, Trans. Roy. Soc. Trop. Med. Hyg., 63, 446‑455
Taniguchi, T. (1943) : Studies on the etiology and immunity of dengue fever. I. Studies on
animal experiments̲ and cultures of dengue fever, Study report, of Preventive Med. in Japan,
Army Med. Sch., part 2, No. 632 (in Japanese)
Taniguchi, T., Fujino, T., Inoki, S. and Okuno, Y. ( 1951): Studies on the experimental
inoculation of dengue fever, Med. J. Osaka Univ., 2, 1‑36
Westaway; E. C. ( 1966) : Assessment and application of a cell line from pig kidney for plaque46)

Yunker, C. E. and Cory, J. ( 1975) : Plaque production by arboviruses in Singh's Aedes albopictus

43 )

44)

assay and neutralization tests with twelve group B arboviruses, Amer. J. Epidemiol., 84, 439 :56

cells, Appl. Microbiol., 29, 81‑89

199

フォーカス計数法によるデングウイルス感染価測定と中和抗体測定法の比較的感度
五十嵐

章・奥野

良信・深井孝之助

4つの異なる血清型のデングウイルスの感染価をBHK−21細胞でのフォーカス計数法（FFU），
LLC−MK2細胞でのフ。ラック形成（PFU），および乳呑マウス脳内接種によるLD5。によって測定した。

すべての型のデングウイルスについてFFUはLD50とほぼ同じ値であり，2型以外はPFUともほぼ同じであった。2型ではPFUはFFU又はLD50よりも低値を示した。デングウィルスの各型に対する抗血清の同型ウイルスに対する中和抗体価を50％フォーカス減少法（FR5。）と50彩プラック減少法（PR50）で求めた。2型以外の抗血清ではFR50とPR50はほぼ同じ値であったが，2型ではPR50
がFR50よりも高い値を示した。この方法は，また，日本脳炎ウイルスおよびチクングニアウイルスにも応用できた。数名のヒト血清の抗体測定から，中和抗体測定は血球凝集抑制抗体測定よりも一般に感度が良く，型特異的である事が示された。

大阪大学微生物病研究所防疫学部門

Japan. J. Trop. Med. Hyg., Vol. 7, No. 3, 4, 1979, pp. 20 1 ‑208

201

SUSCEPTIBILITY OF VARIOUS CELL LINESTO RABIES VIRUS

KUMATO MIFUNEI 3 YOSHIHIRO MAKINol AND KAZUAKI MANNEN2
Received for publication 2 March 1979

Abstract: Twelve established cell lines including 7 mouse‑derived cell lines andprimary c.hick cmbryo fibroblast cells were examined for their susceptibility to rabies
virus. The results indicated that, in addition to CER cells and BHK‑2 1 cells, murine
neuroblastoma cells and human neuroblastoma cells (SYM cells) are highly permissive toinfection with both fixed and field rabies strains and suggested the usefulness of theseneuronal cell lines in various immunologic aspects of rabies studies in which histocompati‑
bility requirements play a significant role.

INTRODUCTION
Rabies virus has been successfully propagated in many tissue culture systems
in the past (Kissling, 1958; Wiktor et al., 1964; Kondo, 1965 ; Diamond, 1967; Hum‑meler et al.. 1967; Sedwick and Wiktor, 1967; Wiktor et al., 1969). These include
primary cell cultures such as hamster kidney cells and chick embryo fibroblast cells,

and human diploid cells which have been used mainly for the purpose of vaccine
production (Kissling, 1958; Wiktor et al., 1964; Kondo, 1965 ; Wiktor et al., 1969;
Grandien, 1977). Of established cell lines, BHK‑21 cells and their sublines havebeen used by several investigators for the production of high yields of virus (Sokol
et al.. 1968, 1969; Schneider et al.. 1971 ; Cox et al., 1977; Kawai and Matsumoto,1977). CER cells, recently established by Dr. Motohashi, Japan, have also been
demonstrated to be highly permissive to rabies virus infection and to have a potential
for isolation of field strains of rabies virus (Smith et al.. 1977, 1978).

One aspect of the search for susceptible cells in tissue culture systems is to de‑velop appropriate tissue culture cells for use in in vitro tests of iuununologic.al respon‑
siveness to rabies in laboratory animals and in human. In these sorts of studies, it

has been demonstrated that a histocompatible system (H‑2 in the mouse) is required

for the full expression of the interaction between immune lymphocytes and thevirus‑infected target cells (Doherty and Zinkervagel, 1975). The present study was
initiated to examine the susceptibility of mouse‑derived cell lines to rabies virus, withthe ultimate goal of studying the immunological responses to the virus in mice. In
addition, some commonly used cell types derived from other animals were simultane‑
ously tested for permissiveness to the virus.
l Department ofVirology, Institute for Tropical Medicine, Nagasaki University. 2 Animal Quaran‑tine Service, Nagasaki Office. 3 The copy should be ordered to Medical College of Oita, P.O.
Box 37, Oita 870‑91, Japan, after April 1980

202

MATERIALS AND METHODS
Virus : Three strains of rabies virus were used. The HEP‑Flury strain of rabies
virus was supplied by Dr. Kondo, National Institute of Health, Tokyo, Japan, andused at passage levels of 106 to 1 1 1 in chick embryo fibroblast cells. HEP‑Flury of

2nd to 3rd passage level in CER cells was used in some experiments. The mousebrain‑adapted CVS strain was used as a 10 per cent infected mouse brain suspension
at the 56th passage level. In some experiments, the CVS strain, plaque‑cloned in

Vero cells (Buckley and Tignor, 1975) obtained from Yale Arbovirus Research
Unit (YARU), Yale University, was used after one additional passage in murine
neuroblastoma cells. The 1088 rabies strain, originally isolated from a woodchuckby the Center for Disease Control, U.S.A., was obtained from YARU and used atthe 3rd passage level in suckling mouse brain.
Cells and culture media : Seven mouse‑derived cell lines and 6 cell cultures derived fromother animals (5 continuous lines and primary chick embryo fibroblasts) were tested
for their susceptibility to rabies virus. Growth media for these cells were shown in
Table I . After virus infection, the percentage of heat‑inactivated fetal calf serum
was reduced to 2 or 5 per cent.
Table 1

Cell lines tested for susceptibility to rabies virus

Other animal‑derived

Mouse‑derived
Cell

Growth medium

Growth medium

Cell

Vero
BSC‑l

Eagle MEM+ lO'/* FCS
Eagle MEM+ lO'/* FCS

L 51 78Y

Eagle MEM+ 10'/. FCS")RPMI 1640 +20"/. FCS
RPMI 1640 +20'/. FCS

BHK‑2 lEagle MEM‑Hanks salts

Neuroblastoma

Dulbecco's modified

CER

Eagle MEM+ 10"/* FCS

SYM')l /2 Eagle MEM+ l/2 RPMI 1640
+20?/ FCS
Eagle MEM‑Hanks salts

L
L 1210Eagle MEM+ 10'/. FCS
SR‑CDF I ‑DBTb) Eagle MEM+ lO"/* FCS(N‑ 1 8)

(glioma)(Human neuro‑

blastoma)

Mastocytoma

Eagle MEM+ lO"/. FCS

CEFd)+ 10"/. FCS(P‑8 1 5)

Ehrlich ascites+ lO'/ FCS

Eagle MEM+ 10'/. FCStumora) Heat‑inactivated fetal calfserum. All culture media contained antibiotics.

b) Established by Dr. T. Kumanishi (1967), Tokyo University> Japan.
*)

Established by Dr. M. Sekiguchi (personal communication), Tokyo University, Japan.

d) Primary chick embryo fibroblast cells.

Parameters of susceptibility : Infection of the cells with virus was carried out in thepresence of 50 pg/ml of DEAE‑dextran for I h at 37 C. Infected cultures were thenrefed with maintenance media or overlay media and incubated at 37 C. At least
2 tests using the following parameters were performed to examine permissiveness.

203a) Fluorescent antibody staining. The cells capable of adhering to glass were culturedin 4‑chamber Lab‑Tek chamber slides (Lab‑Tek Products, Miles Laboratories,
Naperville, Ill.) and infected at a multiplicity of infection (MOI) of approximately

0.1 focus forming unit (FFU)jcell and incubated in a 50/

C02 mcubator. Other

cells not capable of adhering to glass were packed by low speed centrifugation andinfected at an MOI of 0.1 FFU/cell. The cells were then transferred to test tubes
at appropriate cell densities and incubated.
On days 1, 2, 3 and 4 after infection, these cells were examined for cytoplasmic
fluorescent antigens using anti‑rabies virus FITC‑labelled goat serum. The percent‑age of cells containing specific cytoplasmic fluorescent antigens was determined byexamining at least 300 cells. The amount of cytoplasmic fluorescent antigens in thecells was estimated as large, intermediate and minute, respectively, depending on
the relative size and distribution of inclusions in the cells.

b) Cytopathic ffect. Monolayers in culture flasks and suspension cultures in test
tubes, both infected with virus at a MOI of 0.1 FFUjcell, were observed for visible
cytopathic effect under an inverted microscope for 8 days.
c) Plaque formation. The ability of some cells to support plaque formation was also
tested in certain cell lines. The procedures were essentially the same as those de‑scribed by Yoshino et al. ( 1 966) except that calf serum and agar in the overlay were
replaced by heat‑inactivated fetal calf serum and 20/0 Sephadex G‑200 (Schneider,1973), respectively. Infected cultures were observed for plaques for 8 days.
d) Hemadsorption‑negative (HAD‑) plaque formation. This was done as described by
Makino and Mifune (1975) for dengue virus assay.
e) Persistent i fection. To examine if the percentage of fluorescent antigen‑positive
cells increased during subcultures, the cultures infected with virus at a MOI of
0.1 FFUjcell were refed with growth medium and subcultured 4 times at an inter‑val of 5 to 7 days with a split ratio of I to 4. At the time of subculture, the per‑centage of fluorescent antigen‑positive cells in a culture was determined as described
before. The cultures which contained no cells with cytoplasmic fluorescent antigens
at the 2nd and the 4th subculture levels were considered to be "cured"f) Virus yield. Monolayers of susceptible cells in culture flasks were infected withvirus at a MOI of 0.1 FFUjcell and incubated for 4 days. The cultures were frozen
and thawed 3 times and the supernatant was assayed for infectivity after low speed
centrifugation. Infectivity of the virus was assayed in CER cells by the fluorescentfocus formation method using 4‑chamber Lab‑Tek chamber slides as described bySmith et al. (1977).

RESULTSOf seven mouse‑derived cell lines tested, neuroblastoma cells were most sensitive
to all strains of rabies virus tested. By fluorescent antibody staining, almost all of

the Nl8 cells contained large, widely spread fluorescent antigens by day 4 after
infection even with a field rabies strain (1088), and persistent infection could bereadily established. In contrast, SR‑CDF1‑DBT cells and Ehrlich ascites tumor
cells were sensitive only to tbc tissue culture‑passaged, atte. nuated HEP‑Flury strai.n

204of rabies virus.

Susceptibility of other cell lines derived from theto be very low (Table 2).
Table 2

mouse was foundSusceptibility of mouse‑derived cell lines to rabies virus as determined by fluo‑

rescent antibody staining, CPE induction and ability to support virus growthduring subculture
Virus strain

CVS

Cell
1 088

L

FA')

0‑2 b )

(M)')

(mouse brain‑passaged)

HEP‑Flury

0‑2 (M)

0‑2 (M)

CPEd)
Persistent infection

cure

cure

20‑30 (M)

2 0‑30

Persistent infection

cure

cure

L 5178 YFA
CPE

l 0‑20

NeuroblastomaFA
CPE

95‑ I OO (L)')

FA
CPE

L 1210(N‑ 1 8)Persistent infection

SR‑CDF I ‑DBT
(glioma)

Mastocytoma( P‑8 1 5)

FA
CPE
FA
CPE

Ehrlich ascites

FA

tumor

* CPE

(M)

1 0‑20cure(M)

1 5‑35

(M)

cure

(M)

95‑100 (L)l 0‑20(M)

95‑lOO (L)
:1:

yes

N.D.g)
0‑0. l

(M)

N.D.

yes

yes

0‑2 (M)80‑90 (1)f)

0‑0. 1(M)

0‑2 (M)

0‑0. 1(M)

80‑95 (L)a) Fluorescent antibody staining.

b) Per cent of fluorescent antigen containing cells on day 4 after infection in 3 experiments.
c)

Minute, pinpoint like antigen.

d) Cytopathic effect.
e)

Large, widely spread antigen masses in the cells.

f )Intermediate antigen.

g)

Not done.

Cell lines derived from other animals appeared, in general, to be more permissrve
to rabies virus infection than were the mouse‑derived cells, and it was difficult tocompare the susceptibility t0' rabies virus among 'these cell lines. CER and SYM
cells, however, had the capacity to replicate the I 088 strain to almost the same degree

as the flxed rabies strains as determined by fluorescent antibody staining, and theinfection of CER cells could be'successfully maintained in serial subcultures (Table 3) .

Vero and BSC‑1 cells were sensitive only to fixed rabies strains (CVS and HEP‑Flury
strains) by fluorescent antibody staining but produced lower yields of the virus as205
Table 3

Susceptibility of non‑mouse‑derived cell lines to rabies virus as determined byfluorescent antibody staining, CPE induction, plaque and HAD‑ plaque for‑mation and ability to support virus growth during subculture
Virus strain

CVS

Cell
1 088

FA )

Vero

(M)*)

5‑ I Ob)CPE
PlaquePersistent infection

BSC‑l

FA
CPE

cure
5‑ I O(M)

HAD‑ plaqueBHK‑2 1FA
CPE
Persistent infection

CER

FA
CPE

SYM (Human
CEFf )

FA
CPE
FA
CPE

80‑90 (1)d)

80‑90 (1)

+
+

+

yes

yes

50‑75 (1)50‑75 (1)++
95‑100 (L)

20‑25 (1)
yes

yes

yes

50‑100 (1)95‑100 (L)95‑lOO (L)

+

+
+
+

+

yes

yes

yes

65‑75 (1)95‑lOO (L)

95‑100 (L)HAD‑ plaqueneuroblastoma)

HEP‑Flury

95‑ I OO (L)')

PlaquePersistent infection(mouse brain‑passaged):ll

30‑50 (1)

95‑100 (L)N.D.g)Plaque+a) Fluorescent antibody staining.
b) Per cent of fluorescent antigen containing cells on day 4 after infection in 3 experiments.
*)

Minute, pinpoint like antigen.

d) Intermediate antigen.
e) Large, widely spread antigen masses in the cells.
f )Primary chick embryo fibroblast cells.

g)

Not done.

compared with those of CER and BHK‑21 cells (Table 4). BSC‑1 cells were shown
to be useful for the infectivity assay of fixed strains of rabies virus. Vero cells and

CER cells permitted plaque formation by the CVS strain of rabies virus.
Virus yields from a limited number of susceptible cell lines are shown in Tabl 4.
In general, tissue culture‑passaged rabies virus grew to higher titers than did mousebrain‑passaged rabies virus. Among these cell lines, murine neuroblastoma cells
and CER cells supported replication of tissue dulture‑passaged CVS rabies to thehighest titers. The highest yields of HEP‑Flury virus were' obtained in C,ER cells

and CEF cells, '206
Table 4

Rabies virus yields froin susceptible cell lines*)

Virus strain

CVS

Cell

CVS

(mouse brain‑passaged)neuroblastoma‑passaged)(plaque‑purified murine HEP‑Flury

Murine neuroblastoma (N‑18)

5.3b)

8.0

Vero

3.6

6.4

5.8

BSC‑ 1

3.2

N.D.')

5.0

BHK‑2 lSYM (Human neuroblastoma)

5.0

7.0

6.3

5.5

7.2

N.D.

CER

5.7

7.7

7.3

CEFd)

N.D.

N.D.

7.3

5.9

a) Monolayers in culture flasks were infected with virus at an MOI of 0.1 FFU/cell and incu‑

bated at 37 C for 4 days. The cultures were frozen and thawed 3 times and supernatantfluid was assayed for infectivity after low speed centrifugation.

b) Loglo FFU/ml.
c) Not done.
d) Primary chick embryo fibroblast cells.

DISCUSSION

Murine neuroblastoma cells, which were derived from an A strain mouse andwhich express some neuronal characteristics in culture, were demonstrated to havethe greatest susceptibility to rabies of the mouse‑derived cell lines tested in this study.
Since these cplls permit expression of antigen on the surface after infection with both

fixed and street rabies virus, they have been recently chosen as target cells by us(Mifune and Tignor, in press) and others (Wiktor et al., 1977) in the in vitro studiesof iminunological responses of rabies in mice. In addition, these cells have potential
for isolacion of field rabies strains (Smith et al., 1978) and provide a system in which

to examine the interaction between field rabies strains and neuronal cells.
The susceptibility of L cells and mastocytoma cells derived from C3H mouse and

DBA mouse, respectively, was shown to be very low. The high susceptibility of CER
cells to rabies virus including field strains was first described by Smith et al. (1977).

In this study, further and comparative observations were made with some other celllines regarding their susceptibility to the virus. The results showed that, in addition

to CER and BHK‑21 cells, SYM cells derived from a human neuroblastoma arehighly susceptible to both fixed and field rabies strains and might be a useful cell line

for the immunological studies of human rabies which might require a histocompatible
system in the assays.

ACKNOWLEDGMENTS
A part of the present study was 'undertaken in collaborations with Drs. A. L.
Smith, G. Tignor and R. E. Shope ofYale ArbQvirus ResearQh Unit, Yale University.

207

The authors wish to express sincere thanks for their help and suggestions. The
authors also acknowledge the supply of SR‑CDF1‑DBT cells and SYM cells from
Dr. Aoyama, Institute for Medical Soience, Tokyo University and support by the
research grant No. 248169 from the Ministry of Educ.ation, Science and Culture ofJapan.

REFERENCESl) Buckley, S. M, and Tignor, G. H. ( 1975) : Plaque assay for rabies serogroup viruses in Verocells, J. Clin. Microbiol., 1, 241‑242

2) Cox, J. H., Dietzschold, B. and Schneider. L. G. ( 1977) : Rabies virus glycoprotein. II. Bio‑logical and serological characterization, Infect. Immun., 16, 754‑759
3) Diamond, L. ( 1967) : Two spontaneously transformed cell lines derived from the same hamster

embryo culture, Intern. J. Cancer, 2, 143‑152
4) Doherty, P. C., Zinkervagel. R. M. ( 1975) : H‑2 compatibility is required for T‑cell‑mediatedlysis of target cells infected with lymphocytic choriomeningitis virus, J. Exp. Med., 141, 502‑507

5) Grandien, M. ( 1977) : Evaluation of tests for rabies antibody and analysis of serum responses
after administration of three different types ofrabies vaccines, J. Clin. Microbiol.. 5, 263‑267

6) Hummeler, K., Koprowski, H. and Wiktor, T. J. ( 1967) : Structure and development of rabies
virus in tissue culture, J. Virol., l, 152‑170

7) Kawai, A. and Matsumoto, S. (,1977) : Interfering and noninterfering defective particles
generated by a rabies small plaque variant virus, Virology, 76, 60‑71
8) Kissling, R. E. ( 1958) : Growth of rabies virus in non‑nervous tissue culture, Proc. Soc. Exptl.

Biol. Med., 98, 223‑225
9) Kondo, A. ( 1965) : Growth characteristics of rabies virus in primary chick embryo cells, Vi‑rology, 27, 199‑204lO) Kumanishi, T. ( 1967) : Brain tumors induced with Rous sarcoma virus, Schmidt‑Ruppin strain.
I. Induction of brain tumors in adult mice with Rous chicken sarcoma cells, Jap. J. Exp. Med.,37, 461‑474
1 1 ) Makino, Y. and Mifune, K. ( 1975) : Sensitivity of rapid plaque assay method of dengue viruses,

Trop. Med., 16, 163‑170
12) Mifune, K. and Tignor, G. H. (in press) : Cytotoxic activity of peritoneal exudate cells frommice immunized with rabies virus, Infect. Immun.
l 3) Schneider, L. G., Horzined, M. and Novicky, R. (1971): Isolation of a hemagglutinating,immunizing, and non‑infectious subunit of the rabies virus. Arch. ges. Virusforsch. 34, 360‑370

14) Schneider, L. (1973): Cell monolayer plaque test. in Kaplan, M. M. and Koprowski, H.
Laboratory Techniques in Rabies, World Health Organization, Geneva, pp. 339‑342
l 5) Sedwick, W. D. and Wiktor, T. J. ( 1967) : Reproducible plaquing system for rabies, Iymphocytic

choriomeningitis, and other ribonucleic acid viruses in BHK‑2 1 j 1 3S agarose suspensions, J.
Virol., 1, 1224‑1226

16) Smith, A. L., Tignor, G. H., Mifune, K. and Motohashi, T. (1977): Isolation and assay ofrabies serogroup virutes in CER cells, Intervirol., 8, 92‑99
1 7) Smith, A. L., Tignor, G. H., Emmon , R. W. and Woodie, J. D. (1978) : Isolation offield rabies

virus strains in CER and murine neuroblastoma cell cultures, Intervirol., 9, 359‑361
18) Sokol, T., Kuwert, E., Wiktor, T. J., Hummeler, K. and Koprowski, H. (1968) : Purification ofrabies virus grown in tissue culture, J. Viro]., 2, 836‑849

19) Sokol, F., Schlumbetger, H. D., Wiktor, T.J. and Koprowski, H. ( 1969) : Biochemical ahd
biophysical studjjes on tbc nuQ eocapsid and on the RNA of rab. ies virus, Virolo y, 38, 65 l. 665

20820）wiktor，T・J・，Femandes，M、v．andKoprowski，H．（1964）：cultivationofrabiesvirus inhuman
diploid cell strain

WI−38，」．ImmunoL，93，353−36621）wiktorJ・J・，sokol，E，Kuwert，E・and Koprowski，H（1969）：1血niunogenicity
centrated

and

of

con−

purined rabies vaccine oftissue culture origin，Proc．Soc．Expt1．Biol．Med．，131，

799−805
22） Wiktor，T・J・，Doherty，P．c。and
immunity

after

exposure

ofmiceto

Koprowski，H．（1977）：In

both

live

and

inactivatedvitrorabiesevidencevirus，

ofProc．NatL

celLmediatedAcad、Sci．，

74，334−338
23）

Yoshino，K，Taniguchi，S。and Arai，K．（1966）：Plaquecells，

assay ofrabiesvirus in chickembryoArch．ges．Virusfbrsch．，18，370−373

狂犬病ウイルスに対する種々細胞株の感受性
三舟求真人1・3・牧野芳大1・万年

和明2

7株のマウス由来組織培養細胞株を含む12の細胞株と初代鶏胎児細胞について，狂犬病ウイルスに対する感受性を検討した結果，これまで報告のあったCER細胞ならびにBHK−21細胞の他に，マゥス神経芽細胞腫およびヒト神経芽細胞腫（SYM細胞）由来の細胞が，狂犬病ウイルスの固定毒ならぴに街上毒に高い感受性を示し，マウスおよびヒトにおける狂犬病の研究，特に，組織適合抗原の適合性が重要な免疫学的研究に，これらの細胞が非常に有用であることが示唆された。

ヨー長崎大学熱帯医学研究所ヴイ・ルス学部門一2農林水産省動物検疫所，長崎分室
日3二・文献請求先：大分医科大学微生物学講座（昭和55年以降）

Japan. J. Trop. Med. Hyg., Vol. 7, No. 3, 4, 1979, pp. 209 21 1

209

THE EPIDEMIOLOGY OF MEASL(RUBEOLA)GILFORD AMARH ASHITEY
Received fbr publication 7 May 1979

There are important differences between the epidermiological features of measles

in developed countries (e.g. Japan) and those in developing countries. In Japan,
for example, measles is a disease of nursery school and kindergarten children (Figtire1). It follows a 2‑4 year cycle and has a seasonal distribution (Figure 2). Its clini‑
cal couse is mild and its fatality rate is very low (0.0150/0 in 1975). This picture isvery different from that of West Africa where measles is one of the most serious andfeared diseases of childhood. The attack rate is very high and the median age of thedisease is about 18‑24 months (Figure 3). The disease occurs throughout the year
A'ith peaks during the dry months of November to May (Figure 4). The disease isoften accompanied by one or moye of the following complications : stomatitis, purulent

c.onjunctivitis, otitis media, diarrhoea and bronchopneumonia. Many children
die from measles and others are disabled through blindness or deafness. The general
fatality rate is about 2‑5 per cent, but higher rates (200/0 Or more) have been recorded
among hospital c,ases (Morley, 196: 9).
60

E 1 Male50

D:1 Female

cs 40

60
50

" 40

v

cs

o 30

* .20

o

lO

Z 10
O

o

l 2 3 4 5 6 7 8 9+Age in years , j

Figure l30* 20

Cases of Measles rtported in the
Wadayam t district (rural Japan)
1977.

‑ Age/sex distribution.

O

J. F.

M. A. M. J. J. A S O N D
MonthFigure 2

Cases of Measles reported in
Wadayama district (rural Japan)
1977. ‑ Monthly distribution.

The difference between measles in developed countries and that in developing
countries is due to a combination of many factors which include malnutrition, Iow
standard of living and cultural pra,qtices. In many of the developing countries,
malnutrition is very prevalent among preschool children, andeasles is severe in
the malnourished. Besides, measles itself can precipitate malnutrition in border‑line cases. In West Africa, it is customary for mothers to carry their infants on their
Department of Community Health, University of Ghana, Medical School, P.O. Box 4236, Accra,
Presently, JICA participant at' Kobe University School (jf Medicine

NOTE

210
l 50

140
l 30

EZI Male12

C:1 Female

llO
lOO
90

80

80

70

70

60

60

50

c, 50

,

V
O
u
q,
::

:

q,
(,,

40

,:s ) 40

+

30

o

20

20
::

10

lO
O

Figure 330o

1 2 Age3 in4year5 6 7 8 9+O J. F.

Cases of Measles reported in the

Figure 4

M A M J J A S. O. N. D
Month

Akosombo district (rural Ghana)
1977. ‑ Age/sex distribution.

Cases of Measles reported in the
Akosombo district (rural Ghana)
1977.

‑ Monthly distribution.

backs. This means that a child goes everywhere (markets, festivals and othergatherings) with mother. This may start as early as the second week of life. Chil‑dren are thus exposed to measles and other droplet infections very early in life andthey are, therefore, Iiable to be attacked by measles as soon as maternal antibodies
disappear at about the age of six ngonths. Furthermore, the adverse effects of thedisease are enhanced by traditional beliefs and practices. Sometimes food is with‑held or purgatives are given "to bring out the rash", because it is believed thatmeasles rash on the body is less dangerous than rash on the intestinal lining. The
child may not be bathed for days, and instead, it is plastered with local mixtures
again in an attempt to bring out the rash. Other important factors in the patho‑
genesis of measles in West Africa include the low standard of living and the lack ofadequate medical care, especially in rural areas. All the above combine to makemeasles a very serious and important disease in many developing countries. From1966 to 1972, a campaign against measles using vaccination was conducted in West
Africa as part of the worldwide smallpox eradication programme. West Africa was
declared free from smallpox in 1975, but no lasting impression was made on measles.
The prevention and control of measles are, therefore, among the urgent challengesof tropical medicine today.

ACKNOWLEDGEMENT
Mrs. E N. Blankson, Public Health Nurse, Health and Safety Division,
A̲ kosombo, provided rne with the data on Akosombo and Dr. H. Kinoshita, former

NOTE

211

Director ofthe Wadayama Health Centre，Hyogo，gavedata．To both I am extremely gratefULmeaccessto

REFERENCES
1）

Ministry ofHealth，Ghana（1978）：Unpublished data

2）

Ministry3）

o田ealthandwelfare，Japan（1977）：HealthMorley，D．（1969）：Brit．Med．J，1，297−350

services inJapan・P・9

theWadayama,JAPANESE JOURNALOF
TROPICAL MEDICINE AND HYGIENE
Vol. 7 No. 3, 4

December, 1979

CONTENTS
Original article
TsUJrrA, J., MAYUZUMI, M., TANAKA, N. AND HoRI, S.
Comparison of Physique and Body Temperature Among Subtropical Natives,
Immigrants of Subtropical Natives to Temperature Zone and Temperature
Natives (in Japanese) . . . , , .

1 53‑160

FUKUMA, T., KANBARA, H. AND NAKABAYASHI, T.
An Attempt for Short‑term Cultivation of Trypanosoma gambiense in Coexistence with

the Mou e L‑cell (at 37 C)

161‑169

CHUANG, C. K., NAKAJIMA, Y. AND AoKI, Y.
Development of Brugia pahangi Microfllariad from Jird Peritoneal Cavity in Aedes
aeg ypti

171‑189

IGARASHI, A.. OKUNo, Y. AND FUKAI, K.
Comparative Sensitivity of Focus Counting Method in the Infectivity Titration of
Dengue Viruses and in the Neutralization Test

191‑199

MIFUNE, K., MAKINO, Y. AND MANNEN, K.
Susceptibility of Various Cell Lines to Rabies Virus

20 1 ‑208

Note
ASHITEY, G. A.

The Epidemiology of Measles (Rubeola)

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

12 ' Sakamoto‑machi/ Nagasaki, 852, Japan20g 21 1