Japanese

第3巻

Joumalof

Tropicaland第2号

Hygiene

昭和50年9月15日

内原

Medicine

容著

エチオピア産淡水貝類の研究，特に人寄生住血吸虫の中間宿主貝の同定，分布および生態（英文）

・板垣

博，鈴木

了司，伊藤

洋一，原

隆昭，TEFERRA WoNDE

107−134

・国吉真英

135−159・宮田彬

161−200

昭和48年，49年度役員会記録・……一…・一………一…一… 一…………一・・…………・

201−204

沖縄に於ける鉤虫症の疫学的研究（英文）

寄生原虫類の凍結保存（英文）

会

報

投稿規定

日熱医会誌
Jap．J．T．M．H．

日

本熱帯医学会Jap J Trop Med Hyg Vol 3 No 2 1975, pp. 107‑134

107

STUDY ON THE ETHIOPIAN FRESHWATER MOLLUSCS,ESPECIALLY ON IDENTIFICATION, DISTRIBUTION

AND ECOLOGY OF VECTOR SNAILS OF
HUMAN SCHISTOSOMIASISHIROSHI ITAGAK11, NORIJI SUZUK12, YOICHI IT02

TAKAAKI HARA3 AND TEFERRA WONDE4
Received for publication 1 7 February 1975

Abstract: Many surveys were carried out in Ethiopia from January 1969 to January1971 to study freshwater molluscs, especially the intermediate and potential hostsnails of Schistosoma mansoni and S. haematobium, to collect their ecological data, andto clarify the distribution of the snails in the country. The gastropods collected consistedof two orders, the Prosobranchia and Pulmonata. The former order contained threefamilies (Thiaridae, Viviparidae and Valvatidae) and the latter four families (Planorbidae,

Physidae, Lymnaeidae and Ancylidae) . The pelecypods contained four families : theUnionidae, Mutelidae, Corbiculidae and Sphaeriidae. Biomphalaria pfelfferi rueppellii
and Bulinus (Physopsis) abyssinicus are the most important hosts ofS. mansoni and S. haematobium

respectively. The freshwater snail species could be grouped into two distibution patterns,
one of which is ubiquitous and the other sporadic. B. pfetfferi rueppellii and Bulinus sericinus

belong to the former pattern and Biomphalaria sudanica and the members of the subgenus
Physopsis to the latter. Pictorial keys were prepared for field workers of schistosomiasis

to identify freshwater molluscs in Ethiopia. Habitats of bulinid and biomphalarian snails

were ecologically surveyed in connection with the epidemiology of human schistosomiasis.

Rain falls and nutritional conditions of habitat appear to influence the abundance anddistribution of freshwater snails more seriously than do temperature and pH, but water
current affects the distribution frequently. Slight pollution of water brought about by
immigration of people with their domestic animals under reclamation plans of land often
results in abundance of snails in the waters and subsequently new endemic foci of schis‑tosomiasis.

Two species of human schistosomes are endemic in Ethiopia: Schistosoma mansom
and S. haematobium. Some species of freshwater snails of the genera Biomphalaria
and Bulinus are known as the intermediate hosts of these blood flukes in various
parts of Africa. But, it has been insufliciently examined in Ethiopia what speciesof Bulinus and Biomphalaria act as the intermediate hosts and how wide they are
1 Dept. of Parasitology, Azabu Veterinary College, Fuchinobe, Sagamihara 229, Japan. 2 Dept.
of Parasitology, National Institute of Health, Kamiosaki, Shinagawa, Tokyo, Japan. 3 Tokyo
Association of Health Service, c/o Hokenkaikan, Ichigaya‑Sadohara, Shinjuku, Tokyo, Japan. 4
Dept. of Medical Zoology, the Imperial Central Laboratory and Research Institute, P. O. B. 1242,
Addis Ababa, Ethiopia.

1 08

distributed in the country. Ayad (1956), Brown (1964, 1965), Burch (1965) andothers conducted surveys to collect the molluscan intermediate hosts and to know
their distribution in Ethiopia, which, however, did not sufiiciently cover broad range
enough to elucidate the state of the case.

During the stay in Ethiopia surveys were many times undertaken to know the
distribution and habitat conditions of the intermediate and potential snail hostsof the human schistosomes in this area. In surveys and experiments on medical
and veterinary important flukes such as the schistosome, common liver fluke andparamphistome, it is frequently a troublesome task for parasitologists, physicians andveterinarians to identify intermediate host snails, because they have few charac‑
teristics available to discriminate each other. Meskal ( 1967) prepared an identiflca‑tion key of Ethiopian freshwater gastropods which had been known in the country,
but the key was not pictorial. Hence an attempt was made to prepare the pictorial
keys of Ethiopian freshwater molluscs for field work.

MATERIALS AND METHODS
The surveys were carried out through the country from January 1 969 to January
1971.
Addi s Abab a

Akakie. Debre zei t)

Tessenei Asmara Massawa tS'
Adi u9ri SagaO iti. Decamere

A N Adua

SU D?

IP Mo j o

'1 ! c .,; Nazareth
Koka

/,

e Makale '4)<
E G E M E R) .. T I G R E ;･.'4
Gondar ..;**,, ̲̲̲̲̲̲.‑‑‑' ¥ 'Wonji
ft::1 :i:i:L't: '; i･:.::.,'1";r. Z

"* '

way

ASel I a

L TanaBahar, bar:‑･1'.
'., W O L L O
L. Haik.̲̲̲:̲̲..;‑‑.̲( essie ' Batl

. .‑.(‑‑‑‑‑‑‑!, G O J J A j .," ¥

*･.̲
Debr
'
,. ̲ Markos
Debre S 'I ‑'n .･ iGewanl**‑‑"
H O J i re Dawa
, '‑ Erer.' AddiS /, 'Ieso,, Babile

WOLLEGA ･ , : ,' Awash Harrar
Nekempt ̲･‑･'Ambo Ababa ..
.Guderge d
1 e. f .･‑*. '. Koka･*'*. a. z,F:Wonji
a re thIL U B A,, B
O,Adami/ TuluR,Asel I ‑.
a .. HL.,A R R AR
.･'
L

/ ‑‑
Bonga"', ･' '‑!‑* ‑‑ Jimma.' Shashamane' K A F F A ̲.Awasa̲ Yir9"alem { E T/' J: ･ j L.Abaya ,*I "H IOPIAA L E},ArbaG E M Mlnch
U ,/ ,'‑1B .'‑'.

SIDAMO?

fy

vY‑' G O F,̲.A .j

o
,i?

t.

KENYA
Fig. lEthiopia. Boundaries of provinces indicated by broken lines ; routes
followed in making the present collection indicated by solid lines.

109

The collecting loci of molluscs amounted to 336 in number, which are distributed
principally along the main all‑weather roads (Fig. I ) . All the molluscan specimens
collected were identified and ecological data of the habitats were recorded in regard

to pH and water temperature, associated plants and animals, pollution of water,and so on. pH was principally obtained by measurement with pH indicator papers.
The snails collected were identified by shell characteristics alone, and Brown( 1 965) was principally followed in the present study to identify and classify Ethiopian

gastropods.

RESULTS
1 . Freshwater molluscs collected
The present survey revealed the occurrence of the following freshwater molluscs
in Ethiopia including gastropod's and pelecypods. The gastropoda consisted of twoorders of the Prosobranchia and Pulmonata. The former order contained threefamilies : Thiaridae, Viviparidae and Valvatidae and the latter did the four families

of Planorbidae, Physidae, Lymnaeidae and Ancylidae. The family Planorbidae
contained at least six genera of Bulinus. Biomphalaria. Anisus. Gyraulus. Segmentorbis
and Armiger, and the genera Bulinus and Biomphalaria include the hosts of the humanschistosomes. The Pelecypoda consisted of the following genera of Pisidium. Sphaeriumand Etheria, together with at least one unnamed species of the family Unionidae andtwo species of Corbiculidae (Table I ) .

TABLE lEthiopian freshwater molluscs col‑lected in the present survey

Class Gastropoda
Subclass Prosobranchia

Order Mesogastropoda
Family Viviparidae
Genus Bellamya
B, unicolor

Family Valvatidae
Genus Valvata
V. sp.

Family Thiaridae
Genus Melanoides
M, tuberculata

Genus Cleopatra
C. sp.

Subclass Pulmonata

Order Basommatophora
Family Lymnaeidae
Genus Lymnaea
L, natalensis

L, truncatula

1 10

Family Planorbidae
Genus Bulinus
B. (Bulinus) sericinus

B. (B.)forskalii
B. (B.) sp.
B. (Physopsis) africanus ovoideus

B. (P.) ugandae
B. (P.) abyssinicus

Genus Biomphalaria
B. pfelfferi rueppellii

B. sudanica

Genus Anisus
A. natalensis
A. coretus

Genus Gyraulus
G. castulatus

Genus Segmentorbis
S. angustus

Genus Armiger
A. crista

Family Physidae
Genus Physa
P. sp.

Family Ancylidae
Genus Burnupia
B. caffra

Genus Ferrissia
F. clessineana

Genus Ancylus
A. sp.

Class Pelecypoda

Family Unionidae
An unnamed species
Family Mutelidae
Genus Etheria
E. elliptica

Family Corbiculidae

Two unnamed species
Family Sphaeriidae
Genus Sphaerium
S. sp.

Genus Pisidium
P. sp.

lll

TABLE 2PhylumClass

Classification of Ethiopian freshwater mollusca

Subclass

Family Subfamily

Genus‑Unionidae‑Pelecypoda (Bivalves)

Mutelidae

Etheria

Corbiculidae

Corbicula ( ?)‑Sphaeriidae

sphaerium. Pisidium‑Thiaridae

Mollusca‑‑Prosobranchia ‑Melanoides, Cleopatra

Viviparidae

Bellamya. Lanistes

Pilidae (Ampullaridae)

Ampullaria I )Valvatidae

Valvata‑Amnicolidae‑Buliminae

Ga b ia(Bythininae)‑Gastropoda‑‑Ancylidae

Ancylus. Burnupia,
Ferrissia‑Bulininae‑ Pulmonata

Bulinus

Planorbidae‑Planorbinae

Anisus. Gyraulus,
Lentorbis. Segmentorbis,

Biomphalaria. Armiger

Lymnaeidae‑Physidae

LymnaeaPhysa

1 Empty shells of Ampullaria were collected by Brown ( 1 965)

2. The intermediate and suspected snail hosts of the human schistosomes and theirdistribution in Ethiopia

The genera of Bulinus and Biomphalaria contain the species incriminated as theintermediary hosts of Schistosoma haematobium and S. mansoni respectively.
Two species of Biomphalaria were collected : B. pfelfferi rueppellii and B. sudanica.
The former species is the most important snail host of S. mansoni in Ethiopia, while
the latter is of very little importance as the host. S. haematobium, on the other hand,
has been reported to be transmitted by Bulinus snails in various parts of the African

continent. The genus Bulinus is usually composed of two subgenera, Bulinus andPhysopsis. At least six species of the subgenus Bulinus have been reported from
Ethiopia: B. (B.) sericinus. B. (B.) forskalii. B. (B.) scalaris. B. (B.) hexaploidus. B. (B.)

octoploidus and B. (B.) sp., and the subgenus Physopsis includes three species in Ethio‑
pia: B. (P.) abyssinicus. B. (P.) africanus ovoideus and B. (P.) ugandae. Of these species,
B. (P.) abyssinicus is the only confirmed snail host of S. haematobium in the country.
The freshwater snails have different distribution patterns in Ethiopia according

to the species. Speaking roughly, some species have a ubiquitous distribution
throughout the country and some are sporadic in distribution. Bulinus sericinus andBiomphalaria pfelfferi rueppellii, for example, belong to the former pattern of distribution,

112

whereas the members of the subgenus Physopsis, the forshalii group complex of thesubgenus Bulinus and Biomphalaria sudanica to the latter pattern. Bulinus sericinus
was mainly distributed in the areas around Addis Ababa, along the Asmara road from
Addis Ababa to Lake Haik, and around Lake Tana and Harer (Fig. 2), while Biom‑r'¥¥ '' ‑/' / '‑)

¥,

¥.

¥.

'
L'
l¥
( ' ‑.,. ¥ ''¥ Cli:

,1

. ̲.¥

(

'/; '
.̲.//LI 'fj

L' t '.l'v'

h' ¥, J'/"‑ ' ‑ ' ‑ :/ ¥,

'l'

,,

8 . /
'/ '

i /'
. ¥L' ¥ f'l .

. ":' l'1 '. . ¥ i/ /"+‑

.,
r

I

r' ‑'

' .

7 ' '
)

'. '¥l'' ¥.

"‑'/'¥.
¥' .¥'¥,¥,

Fig. 2 Distribution of Bulinus (Bulinus) sericinus in Ethiopia.

phalaria pfeifferi rueppellii in the areas along the all‑weather roads except the areas

between Lake Haik and Makale, from Gondar to the border between the Tigre andBegemder provinces, between Debre Markos and Lake Tana, and Nazareth toMieso (Fig. 3). The areas without any species of freshwater snails have no bodies
of water suitable for the snails to inhabit. Biomphalaria sudanica was limited in dis‑tribution to some of the Great Rift Valley lakes area (the lakes of Ziway, Awasa and
Abaya) and to a separated focus at the Artuma Bilu river (a tributary of the Awash
river) about the midst between Debre Sina and Dessie (Fig. 4). Bulinus (P.) africanus
ovoideus was collected at some foci in the north‑east part of the country, principally

along the main road from Bahar Dar to Asmara and in the Little Gibe river on theroad from Addis Ababa to Jimma (Fig. 5). Bulinus (P.) ugandae and B. (P.) abys‑sinicus have a sporadic distribution ; the former species was found in Beda Buna (aswamp near Jimma) and Lake Abaya and the latter at Gewani and Ofonofei in theLower Awash Valley (Fig. 5). Bulinus (B.) sp. described by Brown ( 1965) was also
found restrictedly in the Great Rift Valley lakes area (Fig. 6) and B. (B.) forskalii

113

f '

¥ 'i

. .J',
"/ ¥.
'¥/
.

.

,.

¥. L.

' L'

¥)

‑

, (.

. ¥ '

̲ l'

' i i: ¥‑‑'‑¥¥''¥(.
'l̲/¥.7' ' f '¥ 'e L̲.le /' t'/'

.1 '

/ .1
e' e

. J

¥./'/'‑' 'r

'

/

¥,

e . / '
' ¥

. 1'
l:e

/' ‑' '

/L' ¥.f , r ¥:', , ¥'i)̲̲ "̲f 'fl .

/¥. '¥ .

' ¥¥ '¥ ¥.¥.
'¥‑'/
. '¥

r'‑'1

¥.

¥.

Fig. 3Distribution of Biomphalaria
pfelfferi rueppellii in Ethiopia.

.f '¥¥

J*'¥/ "J'

¥, ¥.

¥,

,

' '

¥,

,
r
"' . .

, .., )¥.¥.

. ¥ '

'i{ .

1, ¥
,¥ ' ' j

,¥

¥

¥'

(

. ̲̲ . / /' 'f ' '

'!

h¥'/'

/

J

/

' /

. / '

¥.J"/ '‑'

/ ¥¥' ' /'

f'‑' /' ' ' ¥ ii i
f .1:1'(I'

r' ‑'i' ¥7' f ¥ '.

'‑‑ "̲/ '

J

/

'¥

¥ . ¥ .

¥.

¥. ¥¥. ../' ' ¥

. ¥.

¥.

.¥.

¥,

Fig. 4

Distribution of Biomphalaria
sudanica in Ethiopia.

114

r'

̲l V

.

¥

'

¥

l¥

¥ '

(

̲l

¥.

L(1. '

¥ 'l
(

'f '

/! ̲.̲ / //'

.¥

.¥,

i

1'‑"

.)A

.l

/

vl
¥

'

¥. J l'

'/ ¥.

./. '

/ ¥¥

. ,(:' ' l'
l'

l‑

¥
/ ' =

:

f'/. '

lhb.

.7'

"/"
l'

.

¥

!.

'¥. .

¥

¥ l'

r,‑･J

¥.,

¥.

.¥.

¥ ¥.

¥.

Fig. 5Distribution of Bulinus(Physopsis) abyssinicus (trian‑gle), B. (P.) ugandae (square)and B. (P.) africanus ovoideus(circle) .

.f ' t

'Y'

' ̲/'
¥/.

,
, ¥,

.

¥

･¥̲
(

.¥

‑.,

¥.

.l

.¥¥..

*

. .̲./

r/'

l¥

.

l ],

Y‑¥.

r

f

;

¥'̲"

/

"I

/

,

:/ ¥.

¥' J l'

'/. '

/

'l' t
/

/･

̲.

.

/'J '

J

! e(1
r'‑'J

¥

'r'
‑‑ '̲/'

/

.

It
l'
.

.¥ .

¥.
,¥.

/

.¥

¥

¥. ¥.

¥.

Fig. 6¥.

Distribution of Bulinus(Bulinus) forskalii (circle)

and B. (B.) sp. (triangle).

115

was also sporadic in distribution (Fig. 6). No snails were found in the area between

Nazareth and Awash where no permanent bodies of water exist except for a few
alkaline lakes.

3. Environmental factors in snail habitats and their correlation with prevalenceof schistosomiasis

Water temperature of habitat ranged from 9.5 to 3 1 .5 C with the maximum
frequency near 1 5 and 22 C in both ofthe cases ofBulinus and Biomphalaria, and higher

temperatures may be more suitable for Biomphalaria than for Bulinus (Figs. 7, 8).
Bulinus, in general, has a tendency to tolerate the wider range of pH than Biomphalaria.

] Bul.( .) spp.

10

1 Bul( ･)forskalii

5l:‑ii=:=== ===ii

i･‑oFig. 7

9.5 21.5 315'C

Histogram showing range of water temperatures of habitats
of Bulinus snails and frequency (number of localities).

10

1 Biom. E:feiff,rC 2 0

I Biom. sudanica
5.:･:･:･,. ‑

.1'.b

l "'

･･ll

O

Fig. 8It5 26.5 31.5PC

Histogram showing temperature range of habitats
of Biomphalaria snails and frequency.

1 16that is, pH range was from 5.3 to 9.5 in the case of Bulinus and from 5.4 to 7.8
Biomphalaria, with the maximum frequency in pH ranges of 5.0 to 6.2 and of 6.6 to
7.8 respectively. Consequently hydrogen ion concentration appears rarely to bea limiting factor of the distribution (Figs. 9, lO).

mro

1 Bulinus(B̲.) spp.

] Bul.(B.) forskalii[] Empty shell
5

o
5.0

Fig. 97.0

1 0.6 (PH)Showing pH range of habitats of Bulinus spp. and frequency.

10,,,

Biom. feiff.rLy2aO ,1 Biom'sudaniCa,,,

L] Empty shell,,,, tl"* ,,,5"I' ,,,..,... .., l･･*･e‑OFig. lO...
,.t‑

5.0 7.0 1 0.6 ( pH)Histogram showing pH range of habitats of Biomphalaria spp.

Two main factors responsible for marked change in abundance of snails will
be temperature and rainfall. The rainfall of Ethiopia is seasonal and varies indifferent parts of the country. The year can be divided into rainy and dry seasons.
The rainy season usually consists of the '1ittle rains' from April to June and big rainsfrom July to September. Freshwater pulmonate snails can breed in temporary
waters too and snail populations rapidly increase after the start of rainy season.

Another factor for snails to breed is nutritional. The ability and willingnessof the snail hosts to eat almost any organic material offered to them are largely res‑

117

ponsible for the correlation between human pollution and number of freshwatersnails ; in other words, mild pollution by the human has an enriching effect on thesnails. The sites inhabited by a lot ofBulinus or Biomphalaria snails were often polluted

slightly with domestic wastes, excreta of domestic animals and man, and so on ; andwaters containing much organic materials were frequently accompanied with alarge quantity of floating, emersed or submersed vegetation. For example, into a

farm along the Kella river near Shashamane many people have immigrated with
their domestic animals to result in pollution of the streams where Biomphalaria snails

crowded on muddy silt. Another example was observed in the Wonji Sugar Planta‑tion where irrigation ditches had been constructed and many people, some of whomwere infected with schistosomes, had migrated, so schistosomiasis masoni had comeendemic and Biomphalaria snails were abundantly found in the ditches.
Another factor influencing snail inhabitation is water current. Bulinus. Biom‑phalaria. Lymnaea and Physa species were unusually collected from large and rapid

streams such as the Hedie river near Guder, 143 km from Addis Ababa on the roadto Ambo, the Gibe river and the Gojeb river on the Jimma road and the Awash riverin the Awash National Game Reservoir, in most of which substratum was sandy or
rocky and had no or scarce vegetation. The reason for this appears that the snails
can not tolerate a high rate of flow and fast stream may wash away muddy silt whichcontains much oforganic materials important as the food ofbulinid and biomphalariansnails. Consequently, stream gradient seems to have an important influence on the
distribution of those intermediate host snails.

Aquatic animals found with the snails do not always have direct ecologicalcorrelation with them, but sometimes they may be fair indicators of habitats suitable
for the snails. Some species of molluscs were frequently collected with bulinines orbiomphalarians. A high degree of association between Bulinus species and such small

planorbids as Anisus and Gyraulus and a very low degree of association between
Bulinus and Biomphalaria pfelfferi rueppellii make it possible to suggest that Bulinus

has ecological requirements similar to the small planorbids and different from B.
pfelfferi rueppellii. Biomphalaria sudanica, on the contrary, was frequently collectedwith Bulinus in habitats where luxriant aquatic vegetation was present. B. pfelfferi
rueppellii appears to inhabit sites with much muddy silt containing organic materials
and was often found associated with a small 'bivalve, Sphaerium sp.
Aquatic plants provide good surface for freshwater snails to feed on and lay eggs.
Some species of the plants were observed to have a high degree of association withcertain species of freshwater snail; the submersed vegetation such as Potamogeton
crispus, the water weed Elodea sp., Chara sp., Hydrilla‑1ike sp. and fllamentous algae,

the floating plants such as Potamogeton sp., Nymphaea and Lemna‑like sp., Pygonumsp., Cradamine‑Iike sp., and Commelina‑1ike sp. were seen associated with Bulinus,
Biomphalaria and Lymnaea. Bulinus forskalii was frequently collected on the under‑sides of floating leaves of Nymphaea in Lake Awasa. A few sites with luxuriant sub‑mersed vegetation and clean water were observed inhabited by many species of fresh‑water molluscs, for example, the Chacha river about 1 1 O km from Addis Ababa on
the road to Asmara and a small body of water I OO km from the capital on the road.
4. Keys of Ethiopian freshwater molluscs

118

Freshwater molluscs, in general, are diflicult to identify, because they havegenerally not so discriminative features as sea shells. For this reason it will be anecessary but troublesome job for parasitologists, physicians and veterinarians to

identify the intermediary and potential host molluscs of human and veterinary
important parasites, hence the keys of the host snails, especially the pictorial are usefulfor the purpose. Pictorial keys were prepared for the identification of important

Charactertstics

Shel I

F

,!･:
., .

*(:

･i'=

: I

I],

Radialsculpture

'I r
l
I

:

t

;. .

I Ii ! f /li.

( Dextral )(Sinistral )Patell i‑form ShilcFsha ed

Terreted

UPPer sider , , , :=: "Apex

He i 9ht

L

SutureC,'

Spiral scupture

U)

Under side

Aperture

Discoidal

Growth line

1‑

,:
a]

Nodu[esa,::

Wl ND[ NG

5
:
:b
1'

o
ao

1,

･

OF OPERCULUM

,

L

f

<
Width

Concent ri c

Pauci‑s iral b'lulti‑s iralAm

Cardinal Lateral
toothtooth
Ambo' Impressions "'addue't.or aJ:td prdiractor

muscles'

Posteriorend=,. , , terior

end

::4.z

(Right valve
Fig. I lShowing shell characteristics available for identification of molluscan species.

Ethiopian freshwater molluscs. The identification keys were principally basedon shell characteristics for the purpose of field work and the freshwater molluscs
reported from Ethiopia were delt with in the present keys (Figs. 1 1‑15, Tables 3‑6).

119Fresh‑water Molluscs(With 2 sheus) '(With t shell)

̲̲, ;̲

1 'l:¥ ":' )"]' t:(With operculum) (Without operculum)f (ConcentriC(Shield‑shaped)
ANCY LI DAE

operculum )̲(SPircrl operculum){1Jl tl }j¥rJ1 (3 ('@c(Multi‑'spjral ) spjral ) 'S (Slrustral)(Sinist[
LL,i) (Dextral)L̲ (DiScoidal)
LII 'I1!
1‑( Dextral )

rcli)

tes
Beilam a
Lanistes
Beilaml¥I.,':':' (r' p(small: (Very smalD (SPire not (Shell hi9hly turreted)

ertL'resoriJ1,hi9h) ' '̲
Valvata!

epertL,reBulinu forskali‑group(Sheil thicker: (Shell hi, hly large)
surface srnooth) turrete '4surface
Cleo atra granulQr)
GabbiaMelanoides(‑(Apex not (Apex pointed)(Aperture (Apert.

poi nted, 'Ph SCLlar, ge.

sma I I :spire

spire .
retatively

small )

Buli n us

L mnceanatQlensis(Suture shallow:

upper angle oftarge)

L̲.

trunca‑tu la(Suture rather deep:
the an9le obtuse )aperture acute)･ (f･ ) africanus

ovoideus･(̲P.) yggng Ldae,
(Columel[Qtruncate )B:(Ph so is),,
olumel lcl nottruncate )･(Buli nus) tru ncatus

groupFig. l 2 Pictorial key to identification of Ethiopian freshwater molluscs.

1 20

PLANORBI DAE

$( :Q

C J

(Shell hei9ht over 3mm)
(Biom halQria)(Uppersurface flat )ehell hei9ht lessthan 3mm)(The s urface convex )all: , :i : ( )li(smal[er: Whales smallerin number: upper sur‑face rounded or Qngular )Bl OM PHA LAR f A
PFE[FFERI RUPPEL Ll l

( Larger: Whorls 9reaterin number: upper surface(W. horls smQll (Whorl sflat )

in numbergrectt i n

cmd rapjdly
increasingnLmber:in size )i ncresi ng)

GYRAULUS

AN I SUSSIOMPHALARIA SUDANICA

slowly(Shell withaJt(Shell With
interna[lamellae )lamellae )SEGM ENTORBI S LENTORBI S

Fig. 1 3 Pictorial key to Ethiopian Planorbidae.

LIMPETSBIVALVES･O
J(Basal shell shQPe (Basal shell Shapewith strai9ht lateral circular or irre9ular)
Si des ))(small :Growth li nes

not prominent)(Hiddle or lar9e :the lines prominent)

FERR I SSI Ar bi̲

‑ / rl ; i(( t( I::;:SPHAERI I DAE

j

; :.

,,:l

rLl

i'

i

:

(APex with fine (APex with radial(Ambo sub‑ (Ambo

CShell tri‑ (Ambo sublateral)

Pisidium shaeriUm

coaBIOULIDAE UNIONIDAE

terminql ) c' entra:1)

tricLngutar)

radial striation) rows of Pit )ANCYLUS BURNUPIA

Fig' 14 Pictorial key to Ethiopian
Ancylidae (limpets)'

Figl 151'lj' 1 ' ;j‑ ':'L .‑ /(Large:Su rface corse,
like oysters)
Etheria lli tica

Pictorial key to Ethiopian

Pelecypoda (bivalves).

: :'̲‑ 1 ̲ )']':':': : ¥ :t

121

TABLE 3

Key to the genera of Ethiopian freshwater mollusca{ Shell one in number and spiral or discoidal or shield‑shaped

2 (Gastropoda)(Fig. 12)1

Shell two in number and dish‑like . . . .

14 (Pelecypoda)(Fig. 15)

Shell usually thick and has a lid (=0perculum) ; (radula has 7 teeth
2 m a transverse row; dioecious) 1) . . . . . .

3 (Prosobranchia)

Shell usually thin and has no lid; (radula more than 7 teeth m
the row ; hermaphrodite) . . . . . . . . . . . . .

3 {Operculum concentric; shell large, dextral or sinistral
Operculum spiral ; shell middle or small, dextral . , . . .

9 (Pulmonata)4
6

Shell very large (up to lOcm in height), often nearly globose,smooth in surface; dextral or sinistral . . . . . . . . . . . . . . .

5 (Pilidae)

4 Shell smaller (less than 4 cm), broad conical; dextral (rrght
Bel I amyatentacle of male acts as copulatory organ) . . . . . . . . . . .

(Viviparidae)Pila

5{ Shell dextral .

6

Shell sinistral . .

Lanistes

Operculum multispiral ; shell small in size . . . . . .

Valvata(Valvatidae)

Operculum paucispiral; shell small or middle (less than 2 cm in
7

J Shell turreted, surface tuberculate or not; whorls more than 4 in

8 (Thiaridae)

Gabia

Shell turreted, surface smooth; whorls less than 4 . . . . . . . . . . . . . . .

(Bithyniidae)

Melanoides

J Shell tuberculate on surface ; (mantle edge fringed) . . . . . . . . .

8 IShell surface often smooth or with few spiral ribs ; (mantle edge
Cleopatra

Ancylidae

Shell cap‑ or shield‑shaped(see the key)

9

(Fig. 14)
Shell spiral . . . . . .

lO{ Shell turreted . . . .
lO Shell discoidal . . . .

llPlanorbidae(see the key)
(Fig. 13){ Shell dextral . . . .
l I Shell sinistral . . . .

Lymnaea13

122
Shell rather small (7‑12 mm) with more slowly increasing whorls;
spire as high as aperture; columella not obliquely folded . . . . . . . .

L. tuncatula

12 Shell large (up to 25 mm), with rapidly increasing whorls ; aper‑

ture large, more than two times as high as spire; columella ob‑
L. natalensis

Apex pointed; sutures shallow; columella twisted; (mantle withfringed edge; proboscis with pointed side‑corners; no pseudo‑
13

branch)Phy*a (Physidae)

Apex blunt, sutures deep; columella not twisted; (mantle withsmooth edge; proboscis with rounded side‑corners; with pseudo‑branch)Bulinus(see the key)
(Fig. 13)

Shell large or middle in size and thick; concentric growth lines(Fig. 15)

Shell small and thin; concentric growth lines not prominent . . . . . 16 (Sphaeriidae)(Fig. 15)

15 Shell sub‑triangular in outline; middle in size
Shell blade‑shaped or oval in contour; Iarge . . . . . . . .

Corbiculidae

Unionidae

Beak (=ambo) central or subcentral and protruded; shell oval, 2l 6 cardinal teeth in the left valve and none in the right . . . . . . . . . . . .

Sphaerium

Beak sub‑terminal and not protruded; shell inequilateral, an‑terior end of valves longer; 2 cardinal teeth in each valve . . . . . . .

Pisid iuml Features in parentheses are those of others than the shell, or supplementary

TABLE 4 Key to the genera of common Ethiopian Ancylidael) (Limpet snails) (Fig. 14)
Shell relatively
almost ovalnarrow;or broadly
oval,
or ratherin basal
basal shape;
shape .Iateral. . . .
{shellwider
in front
than angularbehind in2

lsides of shell more or less straight . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Ferrissia

Shell almost oval or broadly oval in basal shape ; apex depressed and
2 provided with fine radial striations2) . . . . . . . .

A ncylus

Shell variable in basal shape, but frequently with rather angular out‑
line; apex with radial rows of pits2) . . . . . . . . .

Burnupia

1 Some authors have divided the lipet snails into 2 families, Ancylidae and Ferrissiidae;
then the genus Ancylus belongs to the former and Ferrissia and Burnupia to the latter

2 Radial striations and rows of pits can be easily seen by removing the periostracum(epidermal horny layer of shell) by submerging shells in a sodium hypochloride solution

123

TABLE 5

Key to the genera of Ethiopian Planorbinae (Fig. 13)

JLarger species with a shell more than 3 mm in height (in thickness)l l(except in very young specimens) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Small species, Iess than 2 mm high . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2 (Biomphalaria)

3

Shell size, more than 20 mm in full grown specimens; whorls 5 1/2‑
B. sudanica

6 1 /2 in number, more closely coiled; upper side of whorls flat . . . . . .

2 Shell size, Iess than 20 mm in full grown specimens; whorls 5‑5 l/2
in number, not so closely coiled as sudanica; upper side rounded or3{

angular; or partly flat . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

B. pfelfferi rueppellii

Shell serrated in contour and minute in size (2‑3 mm)

Armiger cristal )4

Shell without serrated contour . . . . . . . . . . . . . . . . . . . . . . .

Shell flat or concave on both sides with only slightly embracing

4 Shell lens‑shaped or convex above and flat below; whorls deeply

56JShell consists of 4‑5 slowly increasing whorls . . . . . . . . . . . . . . . . . . . . .

Anisus5 IShell consists of 3‑4.5 more rapidly increasing whorls; (verge with

6{

stylet, pear‑shaped at base) . . . . . . . . . . . . . . . . . . . . . . . . . . . .' . . . . . . . .

Gyraulus

Shell without internal lamellae . . . . .

Lentorbis

Shell has 4‑9 sets of internal lamellae .

Segmentorbis

1 This species is very small and of rare occurrence in Ethiopia, which is not shown in
Fig. 13TABLE 6 Key to the species of Ethiopian Bulinus (Fig. 13)
l Shellturreted;{shellhighly
not highly
turreted;spire
spire high;not so (radular
high . . .teeth. . . .small). . . . . . . 3 (forskalii group)
2{Columella truncate in lower portion

4 (africanus group)(Subgenus Physopsis)

2
Columella not truncate . . . . . . . . . . . . .

6 (truncatus & tropicus groups)

Shell greater ; spire thicker; aperture larger ; Iower whorls
not shouldered . . . . . . . . . . . . . . . . . . . . . . . L . . . . . . . . . . . . . .

3

B. (B.) scalarisl)

Shell smaller; spire narrower; aperture smaller; the whorls
B. (B.) forskalii

Whorls fiattened near suture to form no or blunt shoulder;spiral microsculpture well developed especially on apex ;4 inner edge of aperture folded well over umbilicus, which
completely open . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

B. (P.) africanus ovoideus

Whorls shouldered ; upper angle of aperture obtuse . . . . . . . 5

Whorls shouldered near suture ; especially the upper angle ofaperture obtuse and frequently of a right‑angle; micro‑
sculpture fine, never consisting of well defined nodules;
inner edge of aperture folded narrowly over umbilicus which
5 is open . . . .

B. (P.) u,gandae

124

5 Whorls convex and shouldered ; upper angle of aperture is
obtuse, microsculpture in upper part of shell is finely cor‑rugate rather than spirally sculptured; inner edge wellfolded over umbilicus; colour of shell varying from purewhite to a light yellowish grey. . . . . . . . . . . . . . . , . . . . . . . . . .

B. (P.) abyssinicus2)

Ultimate (or body) whorl relatively large and spire larger
than in the following species ; spiral sculpture occasionallypresent; colour of shell horny brown . . . .

6

B. (B.) sericinus

Ultimate whorls large ; spiral sculpture well developed on

apex in many cases and occasionally present in ultimate
whorl also ; shell colour of living snails pale straw‑coloured or

almost white

B. (B.) sp.

l Intermediate form of these two species has been found in Ethiopia and another small
species belonging to forskalii group, B. reticulatus reticulatus, is restrictedly distributedin the Begemeder province (37 km south of Gondar) and is like those of truncatus groupin shape (Brown, 1967)

2 The distribution of this species is restricted in the Lower Awash Valley (Assaita,
Gewani, etc.)

3 Bulinus sp. from Lake Awasa was proposed by Burch ( 1 964) to belong to the tropicus

group

DISCUSSION
Surveys had insufficiently conducted on the distribution of human schistosomiasis

in Ethiopia. Broadly speaking, Schistosoma mansoni infection was found mainly inthe highland areas, but has also been reported to be endemic in the Lower andUpper Awash Valley (Lemma, 1 969). Namely, this infection was recorded from
Asmara, Decamere, Maaraba, Saganeiti and Adi Ugri in Eritrea, Bahar Dar in the
Gojjam province (Ayad, 1956), Harrar in the Harrar province (Ayad, 1956) , Tes‑senei in Eritrea (Nutrition Survey, 1 959), Gorgora north of Lake Tana in the Begem‑
der province, Adua in the Tigre province (Duncan, 1970), the regions along the Gojeb

river near Bonga (Oliver and Buzo, 1 964) and around the lakes of Awasa, Langano

and Ziway (Oliver and Buzo, 1964; Ito et al., 1973), and the Akaki and Adami
Tulu districts (Ito et al., 1973). On the other hand, urinary schistosomiasis ismainly restricted to the warm and arid lowlands such as the Middle and Lower
Awash Valley and the endemic foci were reported in the Gewani and Awash districts(Russell, 1958; Lo, 1970a, b), though immigrated patients have been detected in
Addis Ababa, Massawa, Tessenei and other regions.
Bulinines act as the intermediary hosts of Schistosoma haematobium and Biomphalaria

snails are those of S. mansoni. Experimental infections of Biomphalaria and Bulinus
snails with S. mansoni and S. haematobium respectively have yielded divergent results.
This appears to be caused by the difference in susceptibility of the different species,

1 25

subspecies or local populations of the snails to the different strains of the schistosomes.

The genus Bulinus is usually divided into two subgenera Bulinus and Physopsis.

though another subgenus Pyrgophysa may be added to the above two. By the way,four species groups of Bulinus were proposed by Mandahl‑Barth ( 1 958) instead of thesubgenus system and he considered the 4‑group system was the better classiflcation

method in view of parasite‑host relationship. The four groups are the africanus,
tropicus, truncatus and forskalii ; and the first group belongs to the subgenus Physopsis

and the rest to the subgenus Bulinus. If the subgenus Pyrgophysa is considered to bevalid, the fourth group belongs to it. The africanus group, confined in distribution
to the Ethiopian geographical region (south of the Sahara), contains important hostsof the schistosomes with terminalspined eggs including S. haematobium in the African
continent. The truncatus group involves the host species of human and cattle schis‑tosomes. On the other hand, the tropics group has not been known to be the hosts
for the schistosomes and the forskalii group contains only two species implicated as
the snail host of the urinary schistosome in the African continent (Mandahl‑Barth,l 958; Wright, 1 966), hence these two groups are generally of little importance as thevector snails of the human schistosomes. All the four groups will occur in Ethiopia,
if the species "sericinus" is considered to belong to the truncatus grouPb y means of

the features in the radula, mantle pigmentation, reproductive maturity and pro‑portion ofeuphallic specimens as stated by Brown (1965) and Wu and Burch (1973),
and a Bulinus species from Lake Awasa, tentatively considered by Brown ( 1 965) amember of the truncatus group, rather belongs to the tropicus group by the fact thatit has haploid chromosome number (n=18) (Burch, 1964). Of the four groups
the africanus and truncatus groups will be most suspected for translTlission of humanurinary schistosomiasis. The sole incriminated host of S. haematobium in Ethiopia
is B. abyssinicus (Lo, 1970a, b), which is also responsible for the infection in Somalia.
In addition to this species B. africanus ovoideus and B. ugandae, both belonging to theafricanus group, occur in Ethiopia. The former species is responsible for transmitting
S. haematobium in Kenya (Teesdale, 1 962), while the latter is not the host of the blood

fluke in Uganda, the Lake Region ofTanzania (Cridland, 1955) and Kenya (Teesdale
and Nelson, 1 958). Hence B. africanus ovoideus may act as the host in Ethiopia too.

Bulinus sericinus had been considered to be a suspected vector of the urinary
schistosome which has been recorded from a broad range of the Ethiopian plateau
up to 2,900 m elevation (Brown, 1964a, b, 1965), but the incrimination of thisspecies will require further veriflcation. Lo (1972) failed to infect Bulinus sp. (n= 18)

from Lake Bishoftu near Debre Zeit and from Asmara with the Egyptian strain of
S. haematobium, but recently B. sericinus from its type locality (the Toquor river at
Kekerka, Ethiopia) was successfully infected with the Egyptian and lranian strains

by Wu and Burch (1973). The so‑called "Bulinus sericinus" from the Ethiopian
highlands, furthermore, has been known to include a few polyploids (Burch, 1 967a,b, 1972 ; Brown and Burch, 1967), of which an octoploid bulinine B. octoploidus was
infected with the endemic strain of the blood fluke at a rate of 70/0 (Lo, 1972).

One of the considerable reasons for the different results of infection trials isthat S. haematobium has local strains or might wrongly contain other related species.

LeRoux ( 1958) stated that the schistosome infecting the truncatus group was S.

1 26

haematobium and that from the africanus group was S. capense (Harley, 1964) , and the
10cal strains ofS. haematobium and S. capense were reported by Wright ( 1 962). Anotherreason is present in complexity of identifying host snails. Bulinus (P.) africanus.
for example, is discriminated with difficulty from B. (P.) globosus by means of shellcharacters alone, but reliably done by the characters of the copulatory organ (Wright,

1973). Identification used by Brown (1965) of the Ethiopian freshwater snails
was principally followed in the present paper and only shell characters were used
for identification. But, other characteristics must be utilized in further study, fordifferences in immuno‑cytological patterns are present between different Bulinus
populations (Burch and Lindsay, 1970).
Biomphalaria snails containing the vectors of S. mansoni are divided into four species

groups : the pfeifferi, sudanica, alexandrina and choanomphala, of which the first two

have been reported from Ethiopia (Ayad, 1956; Wright and Brown, 1962 ; Brown,l 965). B. pfelfferi and its subspecies have been confirmed as the hosts of S. mansoni(Teesdale and Nelson, 1958; LeRoux, 1961 ; Webbe, 1962a, 1965; Ito et al., 1973b).
But, field and experimental infections of B. sudanica and its subspecies with S. mansoni
revealed that this species varied in susceptibility to the local strains of the parasite(McClelland, 1956; Webbe, 1962a, 1965; Wright, 1962) and, at least in Ethiopia,
is not so much responsible for transmitting the parasite (Ito et al., 1973).
Considering the distributions of the blood fluke diseases and of the vector snail.s
and the susceptibility of the snails to the parasites, the most predominant host snails
of S. mansoni in Ethiopia will be Biomphalaria pfelfferi rueppellii and the only host of S.

haematobium incriminated by field and experimental examinations is Bulinus abys‑sinicus which is principally distributed in the Gewani district of the Lower Awash
Valley. In addition to this species of snail, B. africanus ovoideus might 'be a responsible

host in Ethiopia. Consequently it will be highly possible that schistosomiasis hae‑matobium' becomes endemic in the highlands, when the northern African or Near
Eastern urinary schistosome strain would be introduced into the highlands, as sur‑mized by Wu and Burch (1973).
ldentification keys have been prepared by previous authors of the intermediatesnail hosts of blood flukes and the related species of snail for physicians and veter‑

inarians' use, and Mandahl‑Barth (1962a) dealt with the East and Central African
freshwater snails and Meskal (1967) with the Ethiopian freshwater gastropods.
Meskal's key is not pictorial, with insufficient explanation of terms and with a few
mistakes.

Main ecological factors responsible for marked changes in abundance of fresh‑water snails will be temperature and rainfall in sub‑tropical and temperate regions,but in the tropical arid regions such as Sudan and the coastral plain of Tanzania it

is only the high temperature of summer that appears to reduce the reproduction(Malek, 1 962). The maximum temperature for Biomphalaria pfeifferi appears to beabout 32 C, but the colonies might not survive temperatures much above 28 C underfield conditions (Sturrock, 1966). The optimum temperature for rapid population
expansion of B. pfeifferi and Bulinus globosus is close to 25 C, and at other temperatures

the snail expands population much more slowly, surviving favourably at 1 9 C andn:ot at 30 C (Shiff 1964; Harrison and Shiff, 1966; Sturrock, 1966). Biomphalaria

1 27

and Bulinus snails will be able to tolerate the climatic condition in Ethiopia except
very high altitudes of the plateau, but the low temperature might inhibit the develop‑
ment of the intramolluscan stages of blood flukes to limit the distribution of schisto‑

somiasis in the country. The data on prepatent periods obtained from experimental
infection with S. haematobium conducted in outdoor aquaria in South Africa showedthat transmission in natural habitats in this area was curtained during the four

coldest months of the year when the mean maximum temperature is about 20 C orless (Pitchford and Visser, 1 965). Monthly mean temperatures in Addis Ababa
during 1959 and 1967 ranged from 13.6 to 16.4 C with an average of 13.0 C; andmonthly means of maximum temperature were 1 8.9 to 23.3 C with an annual averageof 21.0 C and those of mean minimums 6.2 to l0.2 C with an average of 5.8 C (The
Geographical Observatory, Haile Sellasie I Univ.).
As to the alkalinity of habitats Watson (1958) mentioned that establishment
and multiplication of Bulinus truncatus was favoured by neutral or slightly alkaline
water, and hard water was better than soft one. In the present survey Bulinus wasfound more abundantly in slightly acid waters, whereas Biomphalaria was moreabundant in slightly alkaline waters. Bilharziasis vectors appear to tolerate a wide
range of pH, generally speaking, from 6.0 to 9.0 (Malek, 1958).
In Tanzania Bulinus nasutus rapidly increases in population after the start ofrainy season (Webbe, 1962a) and B. globosus responds to rainfall by very active

breeding in Rhodesia (Shiff, 1964). The same phenomenon was observed in
the case of B. truncatus rohlfsi in Ghana. Biomphalaria sudanica is flushed out of its
stream habitats by rains and reaches high densities only during the dry season (Webbe,
1 962a). Bulinus breeds, in general, more rapidly than Biomphalaria under favourable
conditions and bulinines were frequently found in Ethiopia at temporary waters such .
as road‑side pits from which clay had been taken for making houses.

Another important factor affecting snail distribution may be water current.
Bulinus truncatus does not occur in rapidly flowing waters ; a rate of 20 to 30 crn/sec

is approximately the maximum in which this species is found (Watson, 1950, 1958)
and the distribution of B. glabrata (=Australorbis glabrata) and B alexa d '. n rma wasalso observed to be seriously influenced by stream gradient (Harry and Curnbie, 1956;

Pimentel and White, 1959a). Most of large and permanent rivers in the Ethiopian
plateau such as the Gibe river on the Jimma road and the Awash river near Awash

and Mojo were hardly populated by Bulinus and Biomphalaria snails, probably
owing to rapid current and absence of deposited organic materials.
Changes in the distribution and abundance of vector snails have been reporte,dto be produced by reclamation and rehabilitation of lands which bring about migra‑
tions of people and domestic animals, construction of small reservoirs and irrigation
channels, dam:ming of streams and mild pollution of waters. In the Aswan provinceof Egypt the amount of S. haematobium infection increased from four‑ to 40‑fold inthree years following the substitution of the perennial type of basin irrigation (Wright,

1 973). Watson ( 1 958) reported Bulinus truncatus was abundant in waters polluted
to some extent by human excrement, but was conspicuously absent from areas heavily
polluted with animal excrement. In Ethiopia the largest dam is situated at Koka
on the Awash river near Nazareth, which was constructed to supply water to power

1 28

mills and for irrigation, and the Koka reservoir (Lake Koka) was inhabited by a great

number of Bulinus. Biomphalaria. Lymnaea and the related pulmonate snails. On
executing developing plans the endemic foci of schistosomiasis will possibly spread overthe country, as suggested ,by Ayad ( 1 956).

Some kinds of plants have been frequently observed associated with a peculiar
species of freshwater snail. A Iarge part of the association of snails and plants is said

to be brought about by the common cause of generally favourable aquatic habitats,rather than to cause and effect (Watson, 1 958) ; but it may be sure that vegetation
provides good surface for snails to feed on and to spawn. The food of freshwatersnails is algae, especially diatomes, or a combination of this and decaying vascularplants (Watson, 1958; Dazo and Moreno, 1962). A blue green alga Osc llatorua
formosa, was proved to be a very satisfactory diet for rearing Bulinus (Xavier et al..
1 968) .

The following species of plants have been reported to be associated with Bulinus
and Biomphalaria : Nymphaea, the water hyacinth, Potamogeton crispus. Potamogeton spp.,
Callitriche. Polygonum, the rush, Ranunculus. Carex, etc. The snails are not always
associated with a specific species of aquatic animals, but a certain species of mayfly
was observed as an indicator of the habitats of Biomphalaria pfetfferi and Bulinus africanusin South Africa (de Meillon et al.. 1958).

ACKNOWLEDGEMENT
The authors wish to express their sincere thanks to Drs. T. Ishizaki and S.
Asahina, the National Institute of Health in Tokyo, for the valuable suggestions
rendered during the course of this work. Thanks are also due to Drs. T. Ohse,

K. Ogata, K. Kaneko, N. Takahashi and K. Saito, the former members of theJapanese Medical Cooperation Team to Ethiopia, and Dr. C. T. Lo who was a visiting
research worker of the Institute of Pathobiology, Haile Sellasie I University in Addis

Ababa, for collecting the materials and encouragement and also to Dr. T. Aseffa,
the former director of the Imperial Central Laboratory and Research Institute in
Addis Ababa, for his interest shown during the survey.

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1) Ayad, N. (1956) : Bilharziasis survey in British Somaliland, Eritrea, Ethiopia, Somalia, the Sudan

and Yemen, Bull. Wld Hlth Org., 14, 1‑1 1 72) Brown, D. S. (1964a) : The distribution of intermediate hosts of Schistosoma in Ethiopia, Ethiop.
3)Med. J., 2, 250‑259(1964b) : Observation on the distribution and ecology of freshwater gastropod mol‑lusca in Ethiopia, Contr. Facul. Sci. Haile Sellasie I Univ. Ser. C (Zool.), No. 5‑6, 9‑40

4)(1965) : Freshwater gastropod mollusca from Ethiopia, Bull. Brit. Mus. (Natl. Hist.)
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5)(1967) : Records of Planorbidae new for Ethiopia (Gastropoda, Pulmonata), Arch.

Moll., 96 (3!6), 181‑185
6) Brown, D. S. and Burch, J. B. ( 1967) : Distribution of cytologically different populations of the1 29

genus Bulinus in Ethiopia, Malacologia, 6, 189‑198

7) Burch, J. B. ( 1964) : Cytological studies of Planorbidae. I. The African subgenus Bulinuss.s., Malacologia, l, 387‑400

8) (1967a) : Chromosomes of intermediate hosts of human bilharziasis. Malacologia, 5,
127‑1369) ( 1967b) : Some species of the genus Bulinus in Ethiopia, possible intermediate hosts ofschistosomiasis haematobium, Ethiop. Med. J., 5, 245‑257lO) (1972) : Names for two polyploid species of African Bulinus. Malacol. Rev., 5, 7‑8l l) Burch, J. B. and Lindsay, G. K. ( 1970) : An immuno‑cytological study of Bulinus s.s., Malacol.
Rev., 5, 7‑812) Cridland, C. C. ( 1955) : The experimental infection ofseveral species of African freshwater snails
with Schistosoma mansoni and Schistosoma haematobium. J. Trop. Med. Hyg., 58, 1‑1 1

13) Dazo, B. C. and Moreno, R. G. (1962) : Studies on the food and feeding habits of Oncomelania
quadrasi, the snail intermediate host of Schistosoma japonicum in the Philippines, Trans. Amer. Mi‑

crosc. Soc., 81, 341‑347

14) de Meillon, B., Frank, G. H., and Allanson, B. R. ( 1958) : Some aspects of snail ecology in South

Africa, Bull. Wld Hlth Org., 18, 771‑783
15) Duncan, A. (1970) : A brief description of schistosorniasis in Ethiopia, OAU Symp. Schisto‑
somiasis, Addis Ababa, 1970, Document, CS/ 15 (1)16) Harrison, A. D. and Shiff, C. J. (1966) : Factors influencing the distribution of some species ofaquatic snails, S. Afr. J. Sci., 62, 253‑258l 7) Harry, H. W. and Cubie, B. G. (1956) : Stream gradient as a criterion of lotic habitats suitablefor Australorbis glabratus in Puerto Rico, Amer. J. Trop. Med. Hyg., 5, 92 1‑928
18) Ito, Y., Itagaki, H. and Teferra, W. (1973) : Studies on the susceptibility of Biomphalaria pfelfferi
rueppellii and B. sudanica to Schistosoma mansoni in Ethiopia, Jap. J. Trop. Med. Hyg., I ( I ), 1‑519) Ito, Y., Tada. I., Itagaki, H., Iwamoto, I., Teferra, W. and Aseffa, T. (1973) : Epidemiologicalsurvey of schistosomiasis in the Rift Valley Lake area, Ethiopia. Jap. J. Parasit., 22 (1, suppl.),1 7‑1 820) Lemma, A. ( 1969) : Bilharziasis in Awash Valley. I. An epidemiologic l study with special
emphasis on its possible future ecologic and public health importance, Ethiop. Med. J., 7, 147‑
l 76

2 1 ) LeRoux, P. L. (1958) : The validity of Schistosoma capense amended as a species, Trans. R. Soc.

Trop. Med. Hyg., 52, 1222) (1961): Some problems in bilharziasis in Africa and the adjoining countries, J.
Helminth., Leiper Suppl., I 1 7‑126

23) Lo, C. T. (1970a) : Snail intermediate host of Schistosoma haematobium in Ethiopia, OAU Symp.
Schistosomiasis, Addis Ababa 1970, Document, CS!30 (1)24) ( 1 970b) : Experimental exposure of Bulinus spp. to miracidia of Schistosoma haematobium

from Ethiopra OAU Symp Schistosomrasls Addis Ababa 1970, Document, CSj29 (1)25) (1972) : Compatibilrty and host parasrte relationship between speaes of the genus
Bulinus and an Egyptian strain of Schistosoma haematobium. Malacologia, I I (2), 225‑280

26) Malek, E. A. (1958) : Factors conditioning the habitat of bilharziasis intermediate hosts of thefamily Planorbidae, Bull. Wld Hlth Org., 18, 785‑818

27) (1962) : Bilharziasis control in pump schemes near Khartoum, Sudan and an elevationof the efficacy of chemical and mechanical barriers, Bull. Wld Hlth Org., 27, 41‑58

28) Mandahl‑Barth, G. ( 1958) : Intermediate hosts ofSchistosoma : African Biomphalaria and Bulinus.

Wld Hlth Org. Mongr. Ser., No. 37, Geneva, 8929) (1962) : Key to the identificatron of East and Central Afrrcan freshwater snails of
medical and veterinary importance, Bull. Wld Hlth Org., 27, 135‑1501 3030) (1965) : The species of the genus Bulinus mtermediate hosts of Schlstosoma Bull Wld
Hlth Org., 33, 33‑44
3 l) McClelland, W. F. J. (1956) : Studies on snail vectors of schistosomiasis in Kenya, J. Trop.

Med. Hyg., 59, 229‑24232) Meskal, F. H. ( 1967) : A preliminary identification key to the freshwater gastropod molluscs ofEthiopia with some conunents of the medical signiflcance of a few species, Ethiop. Med. J., 5 (4),

227‑235
33) Nutrition survey, Ethiopia ( 1959) : A report by the Inter‑departmental Committee on Nutrition

for National Defense, Department of Defense, Washington D. C.
34) Olivier, L. J. and Buzo, Z. J. ( 1964) t Bilharzia in Ethiopia. Report on a visit to Ethiopia by

the Inter‑regional Bilharziasis Advisory Team, 4r2 1 Feb. 1964. WHO unpublished document,
PA f 23 1

35) Pimentel, D. and White, P. C.,Jr. ( 1959) : Biological environment and habits of Australorbis
glabratus. Ecology, 40, 541‑55036) Pitchford, R. J . and Visser, P. S . ( 1 965) : Some further observations on schistosome transmission

in the Eastern Transvaal, Bull. Wld Hlth Org., 32, 83‑l0437) Russel, H. B. L. ( 1958) : The Pilot Mobile Health Team, Ethiopia, Final report. WHO
unpublished document, EMjPHAj6238) Shiff, C. J. ( 1964) : Studies on Bulinus globosus in Rhodesia. I. The influence of temperature onthe. intrinsic rate of natural increase, Ann. Trop. Med. Parasit., 58, 94‑l05

39) Sturrock, R. F. (1966) : The influence of temperature on the biology of Biomphalaria pfelfferi,an intermediate host of Schistosoma mansoni. Ann. Trop. Med. Parasit., 60, 100‑l0540) Teesdale, C. ( 1962) : Ecological observations on the molluscs of significance in the transmissionof bilharziasis in Kenya, Bull. Wld Hlth Org., 27, 759‑782
41) Teesdale, C. and Nelson, G. S. ( 1958) : Recent work on schistosomes and snails in Kenya, E.
Afr. Med. J., 35, 427‑43842) Watson, J. M. (1950) : Studies on bilharziasis in lraq. Part V. Habitat of the vector snail,Bulinus truncatus and its distribution in relation to the irrigation system, J. Roy. Fac. Med. Iraq,

14, 833‑894

43) (1958) : Ecology and distribution of Bulinus truncatus in the Middle East, with commentson the effect of some human activities in their relationship to the snail host on the incidence ofbilharziasis haematobia in the Middle East and Africa, Bull. Wld Hlth Org., 18, 833 94
44) Webbe, G. (1962) : Population studies on intermediate hosts in relation to transmission of bil‑harziasis in East Africa, Bilharziasis : Ciba Foundation Symposium, 7‑22, London45) ( 1965) : Transmission of bilharziasis. 2. Production of cercanae Bull Wld Hlth
Org., 33, 155‑16246) Wright, C. A. (1962): The significance of infra‑specific taxonomy in bilharziasis, Bilharziasis :Ciba Foundation Symposium, 103‑120, London47) (1966) : Relationships between schistosomes and their molluscan hosts in Africa, J.
Helminthol., 40 (3j4), 403‑41248) Wright, C. A. and Brown, D. S. ( 1962) : On collection of freshwater gastropod molluscs from the
Ethiopian highlands, Bull. Brit. Mus. (Natl. Hist.) Zool., 8 (6), 287‑31 l49) Wright, W. H. ( 1973) : Geographical distribution of schistosomes and their intermediate host,
Epidemiology and control of schistosomiasis, 32‑249, University Park Press, Baltimore
50) Wu, S. K. and Burch, J. B. ( 1973) : Experimental infection of Bulinus sericinus with Schistosoma
haematobium. Malacol. Rev., 7, 56

131

APPENDIx
Collecting loci

Distance

in

of

km

fヒeshwater molluscs

shows

that

f｝om

ecological observations

Addls Ababa，whentemperatures show those of water．Freshwaterses withwheresnailswithout

collected

are

were

made．

any notice，and

given

in

parenthe−abbreviations（Aニノ4n4麗5sp．，An＝z厩5郷sp．，Ar＝ノ477n歪g87sp．，B＝β㍑1乞n勝（β痂鯛sp・，Ba−B痂n郷（P伽卿5）吻5舳麗5，Bf−B％」繍（B．）伽ん幽，Bis−
Bぎ卿hα1α吻舶n楓Bpr一βぎ卿hα」α吻癖蜘ゴ躍吻811蕗，Bs一β撹1幡（β．）吻繍，
Bu＝β躍πψ歪αsp．，E＝E∫h6吻6Jlψ孟加．F＝F6漉5吻sp．，G＝（勤7側1％5sp．，Ln二功襯α6απ伽16繍，Lt二勿襯α6α」7πηo伽1α，M＝ハ4ぬηo蜘励召剛α如．PニP耀伽sp，，Ph＝P伽αsp・，S一勘hα6吻吻sp・，Se−S69規㈱渤sp．，V一％1撫sp．）． 翻幡吻6傭（Bs）may contain the related

1．Along

the

River

polyploid

species．

road

f｝om

Addis

Ordidanear

Kob風，100km，13C，pH6．8，（Ln，An，G，Bpr，Bs，V，Bu）lAbaba to Asmara via Dessie

River Chacha near Chacha，110km，18．O C，pH6、8，（Bpr，Bs，An，V，A）l A pool byRiver Baresa near Debre Berhan，2L5C，pH6．8，（Bs，An）l River Baresa near Debre
Berhan，about130km，2LO C，pH7．4，（G，An）l Lake Gorbo（＝L．Gobara）in Gorbo
Village
house

north

in

streamnear

theslaughter

Kembolcha，（Bpr）l Lake Haik，24．O C，pH8．2，（Bs，G）lof

Dessie，24．O

C，pH7．6，（Bpr，Bs，Ln）l

A

River

Tita

in

Tita

Village near Dessic，665−670km，16C，pH6．8，（BsフAn）l River Haik in Haik Vil−
1age，（Ln，Bpr）l R．iverKeralu，19．O C，pH7．6，（Bpr，Bs，Ln）l R．iver勾owa呈nWuchale
Village，23．5C，pH7、2，（Bpr）l River Silinga in Silinga Village，23．O C，pH7．0，（Bpr）l River Borchenna in Kembolcha，365km，21．O C，（Ln，Bpr）；R．iver Wolke
in Kamise Village，315km，22．5C，pH7、4，（Bs，Bpr）l RiverArtuma Bilu near Mita，

about303km，25．O

C，pH7．6コ（Bis，Ln，An）l

256km，25．5C，pH7．6，（Bpr）1、R．iver

Sibilu（aRiverSenbetetributary

of

near

Senbete，about

IR．Mugher）in

ChanchoVillage，about50km，（A）l R．iver Damotu in Chancho，about55km，（Bpr，Bs，A，G，S，V，Bu）l R．iver Alellu in Chancho，about60km，（Bpr，Bs，An，G，S）；A stream，
35−40km，20．O q pH5、6，（Lt，A）l River Ubebarha，11km，15．O C，pH5．6，（A）lA stream between Chacha and Debre Berhan，120−125km，9、5C，pH5．4，（A）lR．iver Kabigiza，115420km in Chacha Village，lLO C，pH5．4，（SフBs，An，Bs）lR．iver Chacha in Chacha Village，110km，16．O C，pH5．6，（Bpr，Bs，AnフG，V，S）lRiver Ordida in Chacha Village，100km，14．5C，pH5．4，（An，G，Ln，Bu，Bs）l A
poo1・75km・18・o c，pH5・6，（Bs）l River Jisa，70−75km，in Sheno village（Amsgaba
Saraishu），18．O C，pH5．6，（An，Bpr）l A stream near Yegudo，65−70km，in Yegudo
Village，16．O C，pH5。8，（Bs，Bpr，Lt）l A stream，65km，16、O C，pH5．6，（Bpr，Bs，An，S）l A stream in AlelfヒVi11age，60−65km，17C，pH5．8，（G）l Rivcr Kamte in
AlelfU Village，55−60km，20．O C，pH5．4，（Bpr）l New R．eservoir of Addis Ababa，30km，20、5C，pH5．6，（Bs，Bpr，An）lBorkena，（Bpr，Ln，Bu，P）l
Bs，An）lRiver Atai near

2．Along

the

Awash

road

Fall

Pk．，（M，An）lfヒom

inAwashRiver

A

streamA

stream5km fヒom Kekere，（Bpr）l

bctwcen Makale andRiverEnda−Sellasie，（Bpr，Ln，Atai Town，（G）

Addis

Ababa

to

Dire

Dawa

and

Erer

Natl．Pk．，（BuラM，Unionidae）lHirunain

HirunaHot

via

Nazareth

Sp血ginAwash

Village，365−370km，27．O

Natl．

C，pH7．6，（Bpr）1

132

一
㎜

133

心

134

エチオピァ産淡水貝類の研究，特に人寄生住血吸虫の中間宿主貝の同定，分布および生態板垣博1・鈴木了司2・伊藤洋一2

原隆昭3・テフェラウオンデ噂

1969年1月より1971年1月にわたる期間にエチオピア各地で，マンソン住血吸虫およびビルハルツ住血吸虫の中間宿主貝に重点をむいて数度にわたる採集をおこない，これらの中間宿主貝の分布および生息条件を調査した。採集された腹足類（巻貝類）は2目（前鯉目および有肺目）からなり，前鯉目には3科が含まれ，有肺目には4科が認められた。また，斧足類（二枚貝類）には4科が含まれていた（Table1）。エチオピアにお・いてはBわ御ρhαZz万α魏携万御砂ρεllゴfとB％1初鰯（Phツ50ゆ5∫5）α61y35勉∫o％5とが，それぞれマンソン住血吸虫およぴビルハルツ住血吸虫の最も重要な中間宿主である。エチオピアにお・ける淡水貝類の分布には2つの型がみられ，1つは全土に広く分布するもの（広布種）と他は散在するものである。Bo〃z．地携万耀砂ρ81薦とB祝1勉郷（Bπ1初％5）58r∫6勉％5とは前者の型に属し（Figs．2，3），研o噸hαZαプ」α3％4αηガoαやB読鰍5属のPhッ50ρ5乞5亜属に属する各種など（Figs．5，6）は後者にはいる。淡水貝の同定は困難なことが多く，住血吸虫症の研究者のためにエチオピア産淡水貝の検索表（Tables3，4，5，6）および検索図（Figs．12，13，14，15）を作成した。淡水貝の分布を左右する生態学的条件としては，降雨および生息地の栄養的条件（餌料の多少）が水温やpHなどよりも重要であり，また水流も重要な条件となることがある。地域の開発にともなう人の移住によって水域が排泄物や家庭廃水によって軽度に汚染されると中間宿主貝にとって好適な生息場所を提供することとなり，これらの地点が新しい住血吸虫症の流行地となる可能性がある。

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

3予防医学協会，東京都

4エチ

Jap. J Trop Med Hyg Vol 3 No 2 1975, pp. 135‑159

1 35

AN EPIDEMIOLOGICAL STUDY OF HOOKWORM
INFECTION IN OKINAWA ISLAND
SHIN‑EI KUNIYOSHI
Received for publication I September 1975

Abstract : This is a survey of the incidence of hookworm infection and a study of infection

process in a rural village named Haebaru, Okinawa Prefecture. Mass treatment of hook‑worm carriers was repeated for six years from 1963 to 1968, and its effect on the wholeincidence and new and re‑infections was studied extensively. In addition, the biologicalbehaviour and resistance of N. an7ericanus and A. duodenale have been investigated. Inci‑dence ofhookworm infection in this community was 32.40/0, mostly caused by N. americanus.

The incidence was highest among the farmers as judged by the age and distribution of
infected persons in regard to farming, indicating the correlation between the length offarm

work hours and the incidence of hookworm infection. The infection took place mainlyin the fields where sugar cane, white gourd melon and Chinese cabbage were planted,
according to the investigation of plant distribution, mode of fertilization and the incidenceof hookworm dermatitis. Infection was not affected by seasonal difference. The factors

contributing to the high incidence of hookworm infection in this area must be the warm
humid climate, the unique agricultural pattern of producing sugar cane and other vege‑
tables, and improper disposal of the night soil. Six years of mass treatment of hookworm

infection resulted in the decrease of the infection rate, markedly among the aged andchildren, but insignificantly among the farmers who showed frequent new and re‑infec‑
tions. A. duodenale infection decreased markedly, far more than N. americanus infection.
The incidence of new infection was highest among the adult females, mostly infected by

N. americanus. The survival time of hookworms in distilled water was longer at low
temperature and shorter at high temperature, and the hatching rate was highest at 27 C.
A . duodenale survived longer than N. americanus at high as well as low temperature. Toxicityof fertilizers tested was strongest with calcium cyanamide, followed by ammonium sulfate,superphosphate, and potassium chloride. They affected N. americanus much more thanA. duodenale. Three tested pesticides, Dieldrin, Sumithion and Malathion demonstrated
weak toxicity on hookworms. Hookworms, N. americanus in particular, proved little
resistance to nitric acid, phosphoric acid and lactic acid in the order listed, and also toalkali such as sodium hydroxide, ammonium hydroxide and sodium carbonate. The
larvae were vulnerable to sodium nitrite, carbon disulfide and distilled water, showing nodifference due to pH.

INTRODUCTION
A number of investigators have reported in the literature their various studies
epidemiology;
biology, pathology and clinical aspects of hookworms. As the re‑on
Dept. of Parasitology, Institute for Tropical Medicine, Nagasaki University, Nagasaki, Japan.

1 36

sults of these studies, the incidence of hookworm infection declined year after year
in this country. Yet, there exist those areas where the incidence is higher than 200/0'
It was revealed that a high degree of damage has been inflicted in those humid dis‑

tricts, particularly in Kyushu where high temperature and high humidity prevailand notoriously high incidence of hookworm infection is still a major public healthproblem. Previous studies, however, were concerned merely in the study of inci‑
dence based on stool examinations or in the result of mass treatment. There havebeen few studies concerning the cause of prevalence, environmental conditions andsocio‑economical factors in order to understand the mode and behaviour of hookworm
infection. In fact, it is obvious that hookworm infection is inseparably related to theclimate, Iandscape, soil contents and other given natural environment as well as tosocio‑biological factors including the life, occupation, age, race and others. There‑
fore, the study of these various factors is mandatory in establishing an effective eradica‑tion programme against hookworm infection in rural districts. On the other hand,the night soil used as a fertilizer is the source of hookworm larvae. Concentration andvariety of the hookworm larvae in the night soil and their life cycle play importantroles in hookworm infection. The survival and resistance of hookworms in natureand in vivo have been studied by many scholars who agree in weaker resistance of
hookworms as compared with roundworms. Because of complex natural condit.ions,each community has its own individual environment. An extremely unique environ‑ment is found in Okinawa Island, which is located in the southern end ofJapaneseIslands and exposed to a subtropical climate, consequently being affected seriously
by hookworm infection which requires an effective eradication programme.
The author selected Haebaru village, Okinawa Prefecture, as a sample of typical
Okinawa villages with their specific environment, and studied the mode of hookworm
infection there, including the place and time of hookworm infection, since 1963.
Mass treatment of hookworm infection was repeated for six years from 1963 to 1968,
and the change of infection rate, new cases of infection as well as reinfection andmany other consequences were investigated annually. The investigation also included
a series of experiments to test hatching and survival of hookworms in vivo and toevaluate their resistance to pesticides and other chemical agents.

GROUND OF SURVEY
The ground of this survey, Haebaru village, is located in the South of Okinawa
Island as shown in Fig. I , consisted of 1 2 small communities including Yamakawa

community which was most thoroughly investigated. The total population of thevillage was 9,858, with 1,863 families. The size of the village is 8,91 1 km2, boundedby Shuri‑nishihara village in the north, by Yonabaru town in the east, and by Naha

City and Tomigusuku village in the west. It has thrived as an old farming village
with its numerous roads crossing all over. Regular buslines run on the four national
highways running through the village, Route 44, 46, 5 and 40, indicating a convenient
10cation for transportation. Another advantage is its close distance to Naha City,
the largest city in Okinawa. Therefore, the increase of industrial plants and theirworkers has been noted in the recent years, adding suburban features to the village.

137

Of 1,395 farmer families, which consist 740/0 of total families, only 278, or 200/0'
are engaged in full‑time farming. The rest of farmers have other occupations such as
government officials, business flrm employees, construction laborers and military
institution workers, beside off‑time farming. The sugar cane fields consist 790/0

of all farms, representing the unique Okinawa agriculture. The small communityof Yamakawa, where most of our studies were made, has a population of 695 and 1 20
families, with a large number of full‑time farmers. The landscape is flat and the soil
is rich in this community which is ideal for agriculture with its good supply of water.

Vegetable production has increased lately, owing to modernized farming technique,making many farmers prosperous.

Okinawa Island),./O ¥ ‑,br N

O f
EAST cHINA
SEA

ll
Ll¥;‑I ,,
,'
I

l

yld
/

;i O

J 11
･1
t , Q;OalL

NAHA
CITY I ,' oO

PACIFICOCEAN

l '!tc) d
HAEBARU VILLAGF.

Fig. I A map of Okinawa Island showmg the areas of the survey

METHOD OF SURVEY
1 . Stools of all school children and other inhabitants were examined for hook‑worms in I O communities of the Southern districts of Okinawa Island for 7 years
from 1 960 to 1 967, applying three methods of test, the thin layer smear, egg concentra‑tion, and cultivation.

2. In the community of Yarnakawa, stools were examined for parasite eggs on
6 1 5 of 695 inhabitants, using the thin layer smear (double cover glass 1 8 x 1 8 mm)
and magnesium sulfate centrifugal floatation technique, and the species of hookworms
were differentiated using the filter paper cultivation method.

3. Every family in the community was interviewed to investigate on the sizeof farms, process of farming, disposal of the night soil, history of hookworm dermatitis
in relation to the place and season ofinfection, and other informations helpful to learn

1 38

the contributing factors of hookworm infection.
4. For six consecutive years from 1963 to 1968, except the year of 1965, massstool examinations were conducted annually, and bepheniurn hydroxynaphthoate
and I ‑bromo‑naphthol were administered to the hookworm‑egg positive individuals.
A. duodenale was treated with bephenium hydroxynaphthoate, 5.0 g per day for adults
and 2.5 g per day for children; and N. americanus was treated with I ‑bromo‑naphthol,
6.0 g for adults, 4.5 g for high school children, 3.0 g for primary school children, and

2.0 g for younger children. Two drugs were administered together for mixed infec‑tion.

5. After three weeks of medication, stools were examined for negative change
and to differentiate the species of hookworms, if they still existed, applying the filter

paper cultivation method.

6. Hatching and survival of hookworms were studied under the microscope on
the hookworm‑egg containing samples cultivated on the filter paper at various tem‑
peratures ranging from 4 to 37 C.
7. After cultivating 10 days at 27 C, the infective stage larvae (F‑larvae) were
dropped in the solutions containing various chemical and medical agents, and theirsurvival time under 27 C incubation was studied. When the survival was question‑able, negative response to pin prick was interpreted as dead. Each medium waschanged anew every three days.

RESULTS
I. Incidence of Parasite Infection in the Southern District of Okinawa Island

Every year from 1960 to 1967, stool examinations were carried out on 10,531
inhabitants, 5,062 males and 5,469 females, of the 10 communities in the Southern
District of Okinawa Island, including school children as well as general public.
Tables I and 2 are the results of this survey based on three testing methods, thin layer

smear, egg concentration and cultivation. The highest incidence of parasite‑egg
positive carriers was found in Tomigusuku, 39.50/0 in 1961‑1962, followed by Osato,
38.20/0 in 1961, Kochinda, 33.30/0 in 1961, Tokashiki, 32.lo/o in 1960, and Zamami,
28.20/0 in 1960. The city of Naha showed the lowest incidence of 8.70/0 in 1961 and1967.

Among the identified parasite eggs, the hookworm eggs were found most frequent‑ly, in I ,757 or 1 6.7 percent. The incidences of other eggs were ; Ascaris lumbricoides 3 1 5(3.00/0) ' Trichuris trichiura 248 (2.40/0) ' Strongyloides stercoralis 93 (0.90/0) ' Rhabditis hominis

54 (0.50/0)' Hymenolepis nana 6 (0.10/0) and others 130 (12.30/0)' showing very low

incidences of roundworms and whipworms.

II. Mode of Hookworm Infection
In Yamakawa, Haebaru village, stool examinations have been carried out on
615 inhabitants, nearly all residents, using three above‑mentioned techniques. As
shown in Table 3, there were found 276 parasite‑egg carriers (35.1 o/o)' including ten
arnong the children younger than 6 years (8.20/0)' 35 in the 7 to 12 years age group(30.00/0)' 19 in the 13 to 15 years age group (23.80/0)' and 152 in the group older than

16 years (51.00/0)' confirming 'the high incidence among the adults. The sexual

139

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TABLE 2

Number and percentage of hookworm egg carriers

No. ofEgg carriers

Location
examined subject

N. americanus

A . duodenale

Both

Unidentified

Naha City

56

ItomanKochinda
Gushikami

1 15

3 ( 2.6)

89 (77.4)

3 ( 2.6)318

32 (l0.1)

245 (77.0)

19 ( 6.0)

52

3 ( 5.8)

39 (75.0)

Tamagusuku

36

3 ( 8.3)

33 (91.7)Osato

1 ( 1.9)45 (83.3)

27 ( 8.2)

235 (71.2)

12 ( 3.6)

33

Tokashiki

54
330
47

9 (19.8)

24 (51.1)

6 (12.8)

8

Zamami48

12 (25.0)

27 (56.3)

2 ( 4.2)

7

Tomigusuku

701

88 (12.6)486 (69.4)

38 ( 5.4)

89

Total

l,757

178 (l0.1)

l,269 (72.2)

80 ( 4.6)

207

Male

l ,OO 1116 (11.6)

699 (69.8)

48 ( 4.8)

62 ( 8.3)

570 (75.4)

32 ( 4.2)

Haebaru

Female756

10

46 (82.6)

20
22
10
8

( ):"/'

ratio was I 10 males (37.70/0) versus 106 females (32.40/0)' slightly higher among malepopulation. As to the species ofparasites, hookworm eggs were found in 199 (32.40/0) 'roundworm eggs in 3 (0.50/0)' whipworm eggs in 1 1 (1.80/0)' strongyloides eggs in
4 (0.70/0)' and others in one (0.20/0)' confirming the fact that majority are hookworm‑
egg carriers.

TABLE 3Age and sex distribution of parasite egg carriers at Yamakawa in l 963
No. of persons

Age group

examined

positive

Hookworm Roundworm Whipworm

O‑ 6
7‑12

122

lO ( 8.2)

9 ( 7.4)

l 15

35 (30.0)28 (24.3)

1 3‑ 1 5

15Strongyl.

Others1

1

80

19 (23.8)

18 (22.5)

l 6‑

298

152 (51.0)

144 (48.3)

2

4

3

Total

615

216 (35.1)

199 (32.4)

3 (0.5)

11 (1.8)

4 (0.7)l (0.2)Male292

l 10 (37.7)l04 (35.6)

2

lO

Female323

l06 (32.8)

95 (29.4)

l

1

4

1

l

( ):olo

1 . Variety of hookworms and the state of infection
Table 4 shows the result of filter paper cultivation of stools among 1 99 hookworm
carriers. Ankylostoma duodenale was found in 8 (4.00/0)' Necator americanus in 146

141

TABLE 4 Age and sex distribution of hookworm egg carriers at Yamakawa in 1 963
No. of persons

Age group

Unidentified

positive

examinedA . duodenale

Mixed infection

N. americanus495 (55.6)

7‑1228

18 (64.3)l (3.6)l 3‑ 1 5

18

12 (66.7)

1 (5.6)

5

l 6‑

l 44

8 (5.4)lll (77.1)

10 (6.9)

15

Total

1 99

8 (4.0)146 (73.4)

12 (6.0)

33

Male

1 04

5 (4.8)73 (70.2)

6 (5.8)20

95

3 (3.2)73 (76.8)

6 (6.3)13

O‑6

Female9( ):o/o(73.4010)' and both were found in 12 (6.00/0)' The ratio between A. duodenale andN. americanus was 20 versus 1 58, N. americanus outnumbering A. duodenale. Slight
sex affinity was found between A. duodenale and the male population and also between
N. americanus and the female population. Age distribution of hookworm‑egg carriers
is shown in Figs. 2 and 3. The incidence is lowest, 7.40/0 among the children younger
( )

80
( )
a)

J

60

60

t‑･ A.

50e‑1) N americanus

Ee

H
F

O
,

U
O

40

40

t

30

F:

H

20

20

lO

o 7 13 20 30 40 50 60

7

13

l

l

l

6

12

19

o

I i I I I I I I
6 12 19 29 39 49 59

Fig. 2

̲̲'p‑ J‑ .̲J‑‑‑e‑‑ '

20 30

40

29 39

49

l l

l

50 60

l l
59

Age groupAge groupAge distribution curve of hookworm
infection rate at Yamakawa in 1963.

Age distribution curve of A . d uod enale

Fig. 3and N. americanus infection rates at

Yamakawa in 1963.

than age 6, but it increases with the age, reaching the maximum 6 1 . I o/o among theforties, declining thereafter gradually. There was found no significant difference ofage distribution between the two species, the peak being in the forties with N. americanus(56.00/0) and in the fifties with A, duodenale (7.80/0)' In younger generations, the age

142

distribution curve is evenly low with A. duodenale, zero among infants, 0.80/0 among
primary school children, I .30/0 among high school children, and 5.90/0 among thetwenties, but it shows a sharp increase with N. americanus. 4.10/0' 16.50/0' 16.30/0
and 40.60/0 respectively.

As shown in Table 5, occupations of total 120 families were divided into 88 full‑time farming families (483 members), 14 part‑time farming families (78 members),
and 1 8 non‑farming families (54 members), with their hookworm infection rates of
33.90/0' 29.50/0' and 22.20/0 respectively, the rate being higher among the farming
families.

TABLE 5 Occupations and hookworm infection rates at Yamakawa in 1 963
No. of persons

Occupation

FarmerNon‑farmer

Total

No. offamilies

examined

positive ( '/* )Main job

88

48 3

164 (33.9)

Side job

1478

23 (29.5)

Office worker

3

Carpenter
Dressmaker
Shopkeeper

354

12 (22.2)

Driver1

Teacher4

Others2615

199 (32.4)

4120

2. Farm activities and hookworm infection(a) Farm work and its time in regard to hookworm infection rate : This small
community has 27.2 hectares of sugar cane fields and 29.4 hectares ofvegetable fields.
The average size of the field owned by a family is 22.6 ares of sugar cane field and24.5 ares of vegetable field. There is no rice field in this area. All families wereclassified into four groups according to the size of farm per person, below 4.0, 4.1 to
7.0, 7.1 to l0.0 and over l0.1 ares. The corresponding hookworm infection rates inthese groups were 22.20/0' 28.60/0' 27.60/0 and 37.70/0 respectively, with a proportionalcorrelation of higher incidence of hookworm infection to larger size of farm per family

member. The farmer families have higher infection rates than non‑farmer families,

indicating a close relationship between the farm work and hookworm infection.
Duration of farm activities was also classified into five groups, full‑time, half‑time,intermittent, harvest time only, and none. Hookworm infection rates of first threegroups were 71 .80/0' 60.30/0 and 40.00/0' indicating higher infection rates in the longer

farming time groups. When the first four groups were compared with the non‑farming Group V, the incidence of the former is much higher, 58. I o/o' than the latter,
1 7.30/0' as shown in Table 6.
(b) Night soil and its disposal : The disposal of the night soil was also inves‑

143

tigated on 102 farming families. In 66 families (64.70/0)' the night soil is still a fer‑
tilizer, and one‑third of the night soil is carried to the fleld directly from the cesspool.

Storage time of the indirectly manured night soil is less than a week in the half of them.
This means that most of the night soil is raw when it is manured into the field, suggest‑
ing high opportunities for hookworm‑eggs to infest the field.
TABLE 6 Comparison of hookworm infection rate between
farmers and the others
No. of persons

examined

positive ( o/o )

227
388

132 (58.1)

Farmers
Others67 (17.3)(c) Farm work and hookworm dermatitis : In the last five years 25 cases of"Koemake" dermatitis with erythema and quaddel were found and studied. A11
patients were farmers and 16 of them (64.00/0) were full‑time farmers. As shown in
Table 7, infection occurred in all seasons, 5 in February‑March, 10 in April‑May, 5

in July‑August, and 5 in Septembel November. The places of infection were, 9in the sugar cane field, 6 in the white gourd melon fleld, 4 in the Chinese cabbage
field, 2 in the sweet potato field, one in the green pepper and celery field and anotherone in the bitter cucumber and snake gourd fleld. There are seasonal prevelences,
the Chinese cabbage field in February‑March, the sugar cane and white gourd melon
fields in April‑May, the sugar cane fleld in July‑August, and the Chinese cabbage
and cabbage flelds in September‑November. The sugar cane fleld seems to be thefavorite place of infection throughout a year because of its long growth time. There
are three peaks of manure fertilization in a year, the spring, the summer and the
TABLE 7

Season and farm field in relation to "Koemake" dermatitis at Yamakawa(1963 to 1967)

Feb.

Season

I

Apr.
l

Mar.
Sugar cane

Jul.

May.

Sept.

Total

f

l

Aug.

6

3

4

l

Nov.
9

White gourd melon
Chinese cabbage

1

Fields of

Sweet potato

1

infection

Cabbage2

2

Green pepper

1

l

2

Total

l

5

4
2

1

and celery

Bitter cucumber
and snake gourd

6
2

1

lO

5

5

25

1 44

autumn. And hookworm dermatitis occurred most frequently in April and May
when the night soil was added most often, particularly to the sugar cane and white
gourd melon fields. Yet, dermatitis is not infrequent in other months. February
and March are the months to plant spring vegetables, July and August for summer

sugar cane, and September to November for autumn vegetables. These findings
indicate the importance of sugar cane and white gourd melon flelds as the place of

hookworm infection in Okinawa.
3. Change of hookworm infection rate following mass treatment
Mass treatment of hookworm infection was attempted annually for six years from1963 to 1 968 in a model community ofYamakawa, and the subsequent change ofinfec‑
tion rate was evaluated. Table 8 shows the number of the tested inhabitants, inci‑
dence of hookworm‑egg carriers, the number of treated carriers, their rate of negativechange, the number of positive remainders, and the species of hookworm discovered.
As the result of mass treatment project, the incidence of hookworm infection de‑creased from 32.40/0 in 1963 to 16.IO/o in 1964, 12.70/0 in 1966, 15.20/0 in 1967, and15.20/0 in 1968. There were 199 hookworm‑egg carriers in 1963, and 1 14 remained
positive after the first treatment, but they decreased to 31 in 1964, 41 in 1966, 48 inl 967 and 39 in 1 968, approximately one‑fifth of the initial number. The numbers
pf A. duodenale infections in the respective years were 3, 2, O, O, O, and those of N.
americanus infections were 94, 25, 37, 47 and 36, and those of mixed infections were5, 2, 3, O and I . In the final year, Ieft were almost only cases of N. americanus.
As shown in Fig. 4, the eggs disappeared from feces after mass treatment, more sig‑
nificantly among the aged and children than among the other adults, though the latter
had a higher rate of infection.

TABLE 8Yearly change of hookworm infection rate under repeated mass treatmentof hookworm carriers at Yamakawa
l 963

l 964

l 966

1967

l 968

examined

615

413

40 l

416

394

positive

1 99

66

51

63

51

(16.1)(12.7)(15.2)(15.2)

Year

No. ofpersons(o/o)(32.4)

treated

1 64

63

41

53

45

85

35

lO

15

12

turned to ilegative(5 1 .8)(55.6)(24.4)(28.3)(26.7)

remained positive ( I )

79

28

31

38

33

untreated (2)

35

3

10

lO

6

1 14

31

41

48

39

A. duodenale

3

2

o

o

N. americanus

94

25

37

47

o
36

2

3

O

(o/o)

(1 ) + (2)Mixed infection

Unidentified

12

2

Remarks : Stool examination and mass treatment were not made in 1965

2

145
( )

60

Q‑O 1963
so･‑･ 1967

t‑1 1968,h‑4 196440

O,

t‑･ 1966

CU
F1
F

O

30

.l

+J

U
O
LH

'//
l ;/

F:

H

;i t

t

r ¥N

IJ

20
l

lO

,r J ̲ ll',

lo 7I 13I 20I 80I 40
I 50
I l60
6 12 19 29 39 49 s9
Age group

Yearly change of age distribution curve of infection

Fig. 4rates with hookworm eggs at Yamakawa.

Teble 9 shows a follow‑up study made for 6 years on these 352 patients to evaluate

the trend of hookworm infection, particularly of new and reinfection. The subjectsof the study included 1 70 males and 1 82 females. Hookworm‑eggs were found inl 1 9 of them, 60 males and 59 females, before the treatment. No eggs were found in
the remaining 233, 1 10 males and 123 females. As the results of the treatment, 14
TABLE 9 Six‑year follow‑up of hookworm carriers at Yamakawa under repeated mass
treatment

1 963 1 964

1 966

1967

+
+
+

+
+

+
+

+
+
+
+
+

+
+

+
+
+
Total

+

+
+

l 968Male
Female Total
+

4
44

82

4

6

lO

8

4

12

O
O

l

2

1

3

2

6

8

1 05

lll

216

l 70

182

352

lO

38

+

+
+
+

Remarks

14

Reinfection

1

4

N w infection

1 46

TABLE I O Yearly change of hookworm infection rate under repeated mass treatment,in relation to age, sex and species

No. of persons

Age group

positive in

examined
l 963

1 964

1 966

1967

1 968

O‑ 6

61

4 ( 6.6)

2 ( 3.3)

o

7‑1 5

67

15 (22.4)

3 ( 4.5)o

1 6‑ 1 834

9 (26.5)

3 ( 8.8)

2 ( 5.9)2 ( 5.9) I ( 2.9)

l 90

92 (48.8)

49 (25.8)

39 (20.5)45 (23.7) 44 (23.2)

[l]

[8]

l 9‑

[7] [1]

Total

352

120 (34.1) 57 (16.1) 41 (11.6) 47 (13.4) 45 (12.8)

Male

1 70

59 (34 7) 32 (18 8) 21 (12 4) 20 (ll 8) 22 (12.9)

Femalel 82[ I J [8] [7] [ I J[3]

61 (33.5) 25 (13.7) 20 ( .O) 27 (14.8) 23 (12.6)[ I J [5]A. duodenale

15

N. americanus 1 20

lO1

Unidentified

[2]

4
54

12

[5] [ I J

4

2

2

41

47

45

[8]

[7]

[1]

O

o

O

[l]

( ) : o/o' [ I : New mfection( 10 males and 4 females) remained as positive carriers, 82 (38 males and 44 females)changed from positive to negative, I I (4 males and 7 females) changed from positive
to negative and again to positive, but 1 2 (8 males and 4 females) from positive to nega‑tive, to positive again, and to negative at last, Nine others (2 males and 7 females)changed from negative to positive and again to negative, and 8 (3 males and 5 females)from negative to positive, whereas the rest, 2 1 6 ( 105 males and I I I females), remained

negative. This means that there were 1 7 (5 males and 1 2 females) new infection casesand 27 (14 males and 13 females) reinfection cases during 6 years. Yearly distribu‑
tion of these infection cases according to age, sex and species of hookworms is tabulated
in Table 10. New infection cases were found one in 1964, 8 in 1966, 7 in 1967, andone in 1 968. They were all adults, mostly carriers of N. americanus. It seems extreme‑

ly difEicult to eradicate hookworms by mass medical treatment alone in the endemic

community.
III. Resistance of Eggs and Larvae
1 . Influence of temperature to maturation : Hookworm‑egg containing stools
were cultivated in the filter paper at various temperatures from 4 to 37 C at every fifth

grade. The results are shown in Table I I . The larvae were born at the temperature
ranging from 18 to 37 C. The rate of hatching varied depending on the species ofhookworms. The rate with N. americanus was 3.50/0 at 18 C, 8.80/0 at 22 C, 31.00/0
at 27 C, 32.80/0 at 32 C, and 0.30/0 at 37 C. With A. duodenale, the rate was 4.00/0'

1 47

8.70/0' 73.30/0' 90.60/0 and 58.00/0 respectively, a higher maturation rate than N.
americanus at any given temperature, with a significant difference at high temperature.

There was found no difference in the speed of larva maturation, changing to theinfective‑stage larvae (F‑1arvae) in 1 1 days at 27 C arid in 8 days at 32 and 37 C.

2. Temperature and survival time : The survival time of the larvae after hatch‑ing was studied at the temperature from 18 C to 37 C, as shown in Table 12. The
average survival time at various temperatures was as follows : N. americanus survived
66 days at 18 C, 69 days at 22 C, 44 days at 27 C, 27 days at 32 C and 13 days at
37 C. A. duodenale survived 74 days, 73 days, 63 days, 45 days and 27 days at therespective temperatures. They survived longer at the lower temperature, regardlessthe species of hookworms. The longest survival of N. americanus at the respective
temperatures were 75 days, 75 days, 85 days, 33 days and 16 days, and those of A.
duodenale were 80 days, 82 days, 70 days, 6 1 days and 39 days. At the higher tem‑
perature hookworms died at the stage of R‑larvae.
IV. Resistance of the Infective‑stage Larvae (F‑Larvae) to Various Chemical Solu‑
tions

The survival time of F‑larvae kept in the various concentrations of chemicalfertilizers, pesticides, acid and alkali, and ovicides was as shown in Table 13. All
experiments were conducted at 27 C.
l . Survival time in the distilled water : The survival time of hookworms in the

distilled water was studied at the temperature range of 1 8 to 37 C. The averagesurvival time of A. duodenale larvae was 86 days at 1 8 C, 88 days at 22 C, 69 days at

27 C, 46 days at 32 C, and 12 days at 37 C, and that of N. americanus larvae was
74 days, 79 days, 47 days, 1 7 days and 6 days at the respective temperatures, showing

the longer survival at the higher temperature, markedly with A. duodenale. Fig. 5
shows the death curve at the different temperatures. The death curve of F‑1arvae
was analyzed to compare the destiny of N. americanus with A. duodenale. When thehorizontal plane is log X and the vertical plane is probit M, then the death curve
takes a shape as illustrated in Fig. 6. Y=4.18 x ‑ 1.55 in A. duodenale, suggestinga different death curve due to species. The following study of resistance of the two
species of hookworm larvae was conducted at 27 C.
2. Survival time of larvae in the diluted fertilizers : The average survival timeof A. duodenale larvae in the diluted superphosphate was 1 8 days in 200 X dilution,
1 9 days in 400 >< , 29 days in 800 x , and 27 days in I ,600 x . The average survival
time of N. americanus larvae was 1 2 days, 1 3 days, 1 5 days and 1 5 days respectively,
significantly shorter than the survival time in the distilled water. The longest survival
time of N. americanus was shorter than that of A. duodenale.

The average survival time of A. duodenale in calcium cyanamide solution was
9 days in 200 x , 12 days in 400 x , 13 days in 800 x and 1 7 days in 1,600 x dilutions,whereas that of N. americanus was 3 days, 4 days, 7 days and 1 1 days respectively,
much shorter than the former owing to its poorer resistance. In ammonium sulfate
solution, A. duodenale larvae survived 14 days in 200 X dilution, 1 7 days in 400 >< ,19 days in 800 X , and 23 days in 1,600 x on the average, whereas N. americanus
larvae survived 1 3 days, 14 days, 1 6 days and 1 5 days respectively, suggesting theinhibiting effect of the agent to hookworms, especially to N. americanus. In potassium︵O．︒oめ︶お寸︵O．OO︶の黛︵︒o．︒りト︶O︒oめ︵卜．︒o︶O創寸

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1 50

TABLE 1 3

Survrval time on infective‑stage larvae in various chemical solutions

Chemical agent

Survival of larvae (days)

DilutionA . duodenale

Superphosphate:)

Calcium Cyanamide

'

200 x

14‑23 (17.6)

l0‑13 (ll.8)

400 x
800 x

12‑25 (18.6)

12‑16 (13.1)

18‑33 (29.1)

14‑19 (15.3)

1 ,600 x

18‑30 (27.4)

14‑20 (15.1)

200 x
400 x
800 x

8‑10 ( 9.2)

3 ( 3.0)

8‑14 (1 1.5)

3‑ 6 ( 3.8)

8‑18 (13.2}

6‑ 9 ( 7.2)

l ,600 x

9‑24 (16.9)

10‑11 (l0.8)

200 x
400 x
800 x

11‑21 (14.2)

12‑15 (13.2)

12‑22 (17.2)

13‑15 (14.1)

12‑29 (18.5)

14‑22 (15.9)

l ,600 x

9‑26 (23.4)

14‑21 (15.1)

200 x
400 x
800 x

29‑43 (36.3)

22‑28 (25.1)

28‑45 (38.4)

27‑36 (32.7)

33‑55 (45.0)

l ,600 x

39‑51 (40.7)

27 L8 (39.4)
40‑52 (46.7)

27‑39 (32.7)

l 7‑28 (20.0)

29‑52 (40.6)

19‑27 (21.5)

29‑55 (41.6)
28‑53 (42.9)

20 9 (29.1)

L1;ia'

C
L'
a,

::
(:)

Ammonium Sulfate

Potassium Chloride

Dieldrin

1 OO ppm
lO
l

O. 1
G'

1,
,

Sumithionl OO ppm
lO

Malathion

19‑24 (22.0)

13‑19 (15.8)

14‑32 (21.1)l

21‑37 (30.5)

18‑33 (23.9)

O. l

19 2 (38.2)

2C 38 (26.8)

l OO ppm
10
1

O. 1

Nitric Acid!*

Phosphoric Acid

23‑28 (24.9)

16‑19 (17.1)

27‑48 (41.2)

19‑53 (27.6)

28‑45 (37.7)
34‑51 (45.7)

20‑52 (38.5)

22 9 (39.5)

200 x

16‑20 (17.2)

2‑ 3 ( 2.5)

400 x
800 x

16‑24 (21.2)

5‑11 ( 7.5)

23‑31 (28.6)

l0‑18 (13.3)

l ,600 x

27‑43 (34.9)

1 7‑30 (20.9)

200 x

22‑36 (27.4)

5‑12 ( 7.7)

400 x

16‑38 (29.1)

12‑16 (13.9)800 x
l ,600 x

Lactic Acid

19‑33 (23.9)

2C 35 (30.4)

"'q,

A

N. americanus

16 1 (31.6)

15‑22 (17.6)

2

18‑49 (31.0)

50 (35.2)

200 x

23‑38 (31.1)

15‑22 (17.3)

400 x

23 LI (32.9)

18‑31 (20.8)

800 ><

26 3 (36.5)

18‑38 (23.5)

l ,600 x

3 46 (44.9)

22‑55 (38.3)

151

Ammonium

Hydroxide

200x
400×

22−30（27．7）

12−13（12．6）

20−32（28、8）

12〜14（13．1）

800x29−35（31．0）

13−16（13．9）

26−36（3Ll）

13−22（15。1）

1ン600×国当＜

SodiumHydroxide

200×15−31（24．6）

400×29−40（35．7）

14−16（14．7）

800×

36一生4（42、7）

1奮32（16．6）

30−51

25−49（39．8）

1，600x

Sodium

SodiumCarbonate

Nitriteo鷹o哨＞○

CarbonDisulflde

200×31−49（40．7）2543（33．2）

400×
800×

30−50（42．4）

25−45（40．6）

39−51（50．2）

25−49（39．0）

1，600×44−55（52．1）

31−47（39．1）

1，000x17−40（29．0）

22−30（24．4）

2，000×29−44（37．2）

22−44（30．4）

4，000×24−42（35、5）23−39（28．2）

8，000×2346（32。8）2241（27．1）

16，000×

23−50（39、3）

22−47

water

27−40（34．7）

20−29（24．5）

400×15−41 （31．1）

20−33（25。6）

800×

17−41

20−43（28．8）（31．1）

1，600×18−49（35．0）

20−42（27．9）

3，200×19−42

20−43（26．5）

4680

22C
27C
32C
37C

pH

Saline solution（29．6）

200×

18C
Distilled（45．3）

3−4（3．7）（34、9）（86．1）（73．8）（88．1）（79．3）（68，6）（47．2）（45．5）（17．4）

（1L8）

（6．2）

6−33（29。0）6−14（7．7）

23−34（29．4）

8−16（10．0）

26−33（27、6）

13−18（1生8）25−33（28．9）

15−30（25．9）

1．3％

16−32（20．8）

12−25（17．4）

1．1

14−34（20．8）

16−29（20．7）

0．9

18−33（23．0）

18−29（23．7）

0．7

16−35（27、1）

19−33 （26．5）

0．5

17−37（31．6）

20−34（24．7）（ ）：Average

ch1・rides・luti・n，オ・4㍑・伽」61arvaesurvived36daysin200×diluti・n，38days圭n
400×，45days in800×，and41days in1，600×，whereasハτ．α膨r初肱51arvae survived
25days，33days，39days and4・7days respectively．In summary，the surviva，l timeof hookworm larvae was shortest in calcium cyanamide solution，and prolonged insuperphosphate and potassium chloride solutions，demonstrating the strongest para−
siticidal action ofcalcium cyanamide．ノV．α膨7加鰍51arvae proved poorer resistance

152
(

If

,

,
e

: 80

e

HEti 60

,

,

20

,

/

8

l

el,

,
,

37 C

,

40

; :

H
,d

h 60

N. amerieanus )

32 C

18 C
,

4

e‑testx

P
M : mortality rate,,e

Pr> o . 05

l.5 l'g
2.0
2.5
p : ine batio period

,

e

e

2

,

e

o

,

e

,

40

20

4‑
d:)

H
l

t ,'
..
,
,
80

o:

lOO

80

60

lOO

:h

Y‑5 . 35 X, ‑4.48(A,duodenale)

6

,

,

20

Y‑4. 18 >( ‑1 .55(N,americanus)

o

e

e
,

/

Y

,

,

,
,
,

(

( )

,
,

,
,
,

H

o 40

18 C

32 c

37 c

( )

lOO

:

A'duodenale )Fig. 6.

Cummulative mortality of infective
stage larvae in distilled water at 27 C.

e,20
40 60 80 roo
Incubation period (days)
Fig. 5

Survival time of infective stage larvae indistilled water at various temperatures.

than A. duodenale.

3. Survival time of hookworm larvae in the pesticides : The survival time ofhookworm larvae was tested flrst in the Dieldrin solution. A. duodenale larvae sur‑

vived 33 days in 100 ppm of Dieldrin, 41 days in 10 ppm, 42 days in one ppm and43 days in 0.1 ppm, whereas N. americanus larvae survived 20 days, 22 days, 24 daysand 29 days respectively. In the Sumithion solution, the survival time ofA. duodenale

larvae was 22 days in 100 ppm, 30 days in 10 ppm, 31 days in one ppm and 38 daysin 0.1 ppm, whereas that of N. americanus larvae was 1 6 days, 2 1 days, 24 days and
27 days respectively, the shortest survival among the pesticides tested. In theMalathion solution, A. duodenale larvae survived 25 days in 100 ppm, 41 days in
10 ppm, 38 days in one ppm and 46 days in 0.1 ppm on the average, whereas N.
americanus larvae survived 1 7 days, 28 days, 40 days and 39 days respectively. Thus,
Sumithion is the most potent pesticide followed by Dieldrin and Malathion.
4. Survival time of larvae in acid solutions : The survival time of A. duodenale
larvae in the nitric acid solution was 1 7 days in 200 x dilution, 21 days in 400 >< ,29 days in 800 x , and 35 days in I ,600 x , whereas that of N. americanus larvae was
3 days, 8 days, 1 3 days and 2 1 days respectively. In the phosphoric acid solution
A. duodenale larvae survived 27 days in 200 x dilution, 29 days in 400 x , 32 daysin 800 x , and 35 days in I ,600 >< , whereas N. americanus larvae survived 8 days, 1 4days, 1 8 days and 3 1 days respectively, similar to the survival in the nitric acidsolution. In the lactic acid solution, A. duodenale larvae survived 3 1 days in 200 x
dilution, 33 days in 400 x , 37 days in 800 x and ‑45 days in 1,600 X , whereas N.
americanus larvae survived 1 7 days, 2 1 days, 24 days and 38 days respectively. There‑

153

fore, the pesticidal action is highest with nitric acid and lowest with lactic acidamong the three acid solutions tested.

5. Survival time of hookworm larvae in alkali solutions : A. duodenale larvae
survived in the ammonium hydroxide solution, 28 days in 200 x dilution, 29 daysin 400 >< , 3 1 days in 800 x , and 3 1 days in I ,600 x , whereas N. americanus larvae sur‑

vived 1 3 days, 1 3 days, 14 days, and 1 5 days respectively, revealing no significant
difference due to dilution, but significantly short survival of N. americanus larvae.

In the sodium hydroxide solution, A, duodenale larvae survived 25 days in 200 x
dilution, 36 days in 400 x , 43 days in 800 x , and 45 days in 1,600 x , whereas N.
americanus larvae survived 4 days, 1 5 days, 1 7 days and 40 days respectively, obviously

less resistant than A. duodenale larvae. The average survival time of A. duodenale
in the sodium carbonate solution was, 41 days in 200 x dilution, 42 days in 400 >< ,50 days in 800 x , and 52 days in I ,600 x , whereas N. americanus larvae survived 33
days, 41 days, 39 days, and 39 days respectively. The parasitocidal action of sodium
carbonate was weaker than other two alkali with little difference due to dilution.
Therefore, the order of larva‑cidal potency of the three alkali is : sodium hydroxide,ammonium hydroxide and sodium carbonate.
6. Survival time of hookworm larvae in ovicides : The average survival timeof A. duodenale larvae in the sodium nitrite solution was 29 days in 1,000 x dilution,
37 days in 2,000 x , 36 days in 4,000 x , 33 days in 8,000 x and 39 days in 16,000 x ,whereas N. americanus larvae survived 24 days, 30 days, 28 days, 27 days, and 30 days
respectively, shorter than the controls, with no significant difference due to concentra‑tion. In the carbon disulfide solution, the survival time of A. duodenale larvae was
35 days in 200 >< dilution, 31 days in 400 x , 31 days in 800 >< , 35 days in 1,600 x
and 35 days in 3,200 x , whereas N. americanus larvae survived 25 days, 26 days, 29days, 28 days and 27 days respectively, resembling to the result with sodium nitrite.

7. Survival time of hookworm larvae and pH of culture media : The averagesurvival time of A. duodenale larvae varied depending on pH of the media: 29 daysin pH 4, 29 days in pH 6, 28 days in pH 8, and 29 days in pH 10, whereas N. american‑
us larvae survived 8 days, I O days, 1 5 days and 26 days respectively, showing an insighi‑

ficant difference due to pH.
8. Survival time of hookworm larvae in various dilutions of saline water : Five
different concentrations of saline water were used to test the survival time of the lar‑vae. The average survival time ofA. duodenale larvae was 2 1 days in I .30/0 Saline solu‑
tion, 21 days in 1.lo/o' 23 days in 0.90/0' 27 days in 0.70/0' and 32 days in 0.50/0'
whereas that of N. americanus was 1 7 days, 2 1 days, 24 days, 27 days, and 25 days
respectively, Ionger survival in lower concentration.

DISCUSSION
Extensive epidemiological surveys of hookworm infection have been carried out
by various scholars in many areas ofJapan, and their reports have been published one
after another. According to these reports, the incidence of hookworm infection is
generally high in every prefecture in Kyushu which is known for its subtropical en‑
vironment. Higher than 500/0 of infection rate was found in several communities.

1 54

In Okinawa, however, the study concerning hookworm infection had been rare before
the War. The only report to be mentioned was an article entitled as "A Survey ofParasites in Okinawa and the Result of Eradication Programme" by the Health Cen‑ter, Okinawa Prefecture. After the War, however, a number of investigations oflarger scales have been launched and reported by many authors including Kuniyoshi(1945, 1947, 1949, 1962), Major Hunter 111 (1949), Shiroma (1956, 1960), Sasa etal. (1958), Sato et al. (1958), Kuniyoshi et al. (1959), Heishiki (1961), Katamine et al.

(1962), Kawashima et al. (1963), Yamamoto et al. (1964), Miyahara et al. (1964),
Kato (1967, 1968), and Tada et al. (1968). A11 these reports confirmed the muchhigher incidence of hookworm infection in Okinawa in comparison with the otherparts ofJapan, even higher than the rate of 28.60/0 reported by the Health Center,
Okinawa Prefecture, in 1938. This high incidence is due to mass stool examinations
frequently conducted after the War on school children and adult inhabitants and also
due to improved technique of egg collection and cultivation. The result of our mass
survey in 10 communities of the Southern districts of Okinawa Island, 10,531 indi‑viduals in total, showed various incidences of hookworm infection from 3.0 to 64.00/0'
16.70/0 on the average.

Considering the fact that majority of our subjects were primary and secondary
school children, the real incidence of the general population must be much higher,

although the infection rates of the roundworm and whipworm were as low as 3.0
and 2.4 percent.
A11 former reports stated the dorninance of N. americanus among hookworms foundin Okinawa (Sasa et al., 1958; Kuniyoshi, 1959; Kinjo et al., 1959; Heishiki, 1961 ;Yamamoto et al., 1964; Kuniyoshi et al., 1963; and Tada et al.. 1968). Our present
survey also confirmed the predominance of N. americanus. There is no doubt that A.
duodenale is uncommon in Okinawa. It is noteworthy that N. americanus prevalence ismore or less limited to the Southern sections of Japan, including Okinawa Prefec‑
ture, the Southern half of Miyazaki Prefecture (Shinkado, 1959), and a district inKagoshima Prefecture, although it has been believed that N. americanus was foundmore in the South than in the North, and more in the lower lands than in the higher
altitudes.

After confirming the prevalence of N. americanus in Okinawa, the author at‑
tempted to study the mode of hookworm infection in its relation to farming, the placeand season of infection in particular, in a community called Yamakawa, Haebaru
village. The incidence of hookworm infection in Yamakawa was 32.40/0' mostly due
to N. americanus, representing a typical farm community in Okinawa. The incidence
was 7.40/0 among the infant, considerably high for the age, 24.30/0 among the school
children, and 48.30/0 among the adults, showing gradual increase as the age advanced.
A steep increase curve was found at the age range of twenties, i.e. after finishing schooland starting as farmers. The highest peak comes at the age of forties, 61.1 percent.
The full‑time farming families showed higher incidence than the part‑time farming
families, 33.9 versus 22.20/0' suggesting a close relation of infection to farm activ‑
ities. No difference of incidence was found between male and female population, as
expected from the fact that both men and women share the sarne farm labors.
The farmers consist 31. I o/o of the whole population, but their incidence of hook‑

155

worm infection was 59.00/0' strikingly higher than the rest of the population, 1 7.3
percent. The incidence is higher among the full‑time farmers than part‑time farmers.
The larger the size of farm, the higher was the incidence of hookworm infection.
All the figures indicate that hookworm infection is related to farm work, to frequency
and duration of contact with farms.
Treatment of the night soil was investigated on I 02 farm houses, and it was foundthat the night soil was used as a fertilizer in 64.70/0 of these farm houses, and the raw
night soil was poured into the field directly in one‑third of the cases. Even when thenight soil was stored before the use, halfof the stored manure was used within one weekof storage. There exist the alarming opportunities of heavy contamination of the
fields with fresh hookworm‑eggs.

Next come the place and season of hookworm infection in this community.
Hookworm dermatitis due to percutaneous infection occurred in 23 farmers, of whom
1 6 were full‑time farmers. There were 10 cases of hookworm dermatitis in April‑May, but only five in February‑March, as in July‑August and September‑November.
Infection seems to occur throughout the year. The peak of hookworm infection wasin May and early September at Saitama Prefecture according to Komiya et al. ( 1 953),
in AprillJune at Kagawa Prefecture according to Yoshida ( 1 956), in April to August
except June at a village in the suburb of Tokyo according to Suzuki (1959), and in

MaylJune and September‑October at Nagasaki Prefecture according to Sakaguchi( 1 961) . It is obvious that the season of infection varies depending upon the agricul‑
tural practice, the kind of products and the different climates. The sugar cane field
had the highest chance of infection, followed by white gourd melon, Chinese cabbage,

green pepper and celery, bitter cucumber and snake gourd fields. The factors con‑tributing to the high incidence of hookworm infection in this district are, therefore,

humid warm climate, the type of agriculture producing sugar cane and vegetables,
and improper treatment of the night soil.

The most effective method to control hookworm infection in the endemic areas
is to conduct a mass treatment continuously. There have been, however, only a few re‑ports ofrepeated mass treatment in the literature; Matsubayashi et al. (1952), Komiya

et al. (1953). Isoda (1958) and Uchida (1958). That is why the author continued amass treatment programme annualy from 1 963 to 1 969 at Yamakawa, a community
in the Southern district of Okinawa Island, and studied the change of infection trend,

incidence of new cases in regard to age and sex, and comparison of two hookworm
species. As stated already, the incidence of hookworm infection prior to the mass
treatment programme was 32.40/0' mostly caused by N. americanus. The incidence
dropped to 16.1 o/o in 1964, one year after the first mass treatment, but no further de‑crease followed in spite of repeated community‑wide anti‑hookworm medical treat‑ment, owing probably to the egg‑positive carriers who failed to take medicine and also

due to new infections. Therefore, a follow‑up study was made on 352 cases whichwere examined annually for 6 years. Negative change followed medical treatment
among the young and the aged who had relatively lower incidence of carriers,
more often among A. duodenale carriers than among N. americanus carriers. The
newly infected individuals during the programme were all adults, as were the 327
reinfected individuals. Concerning the sex, there were more fernales among the1 56

newly infected cases. In 1 968, as the result of 6‑year‑10ng mass treatment programme,
hookworm infection was found only among the adults of age 20 to 59, and A. duodenale
has virtually disappeared. It should be emphasized that a full‑scale control program‑me is necessary to eliminate hookworms in an endemic area, although there is no
doubt that a mass treatment with anthelmintics is an effective means to prevent anderadicate hookworm infection. It would be ideal if the hookworm larvae could bekilled in the soil in the endemic areas.
In the past, the resistance of hookworm‑eggs and larvae has been studied by vari‑

ous scholars including Inatome (1932), Sasada (1934), Sano (1952), Kobayashi et al.

(1955)) Kutsumi et al. (1955), Yasuda (1957) and Kozai (1960, 1961). All theauthors agreed in the low resistance of N. americanus. The present author also
investigated the resistance of hookworm larvae to various chemical fertilizers, pest‑
icides and other agents, with particular consideration to the practical concentrations
applicable to field sprays.
The control survival tirne of the hookworm larvae in the distilled water was short‑
est at 37 C, but it was prolonged at 32 C, 27 C, and 18 C progressively, demonstrat‑

ing the significant influence of temperature. Of the two species of hookworms,
A. duodenale larvae have stronger resistance and survived longer at any temperaturethan N. americanus larvae .

Both species did not survive long in the fertilizers. Calcium cyanamide proved

to be the most potent larva‑cide, followed by ammonium sulfate and potassium
chloride. Calcium cyanamide of I ,600 x dilution can kill A. duodenale larvae in 1 7days, and N. americanus larvae in only I 0.8 days on the average. This means thatthe ordinary concentration of fertilizers is potent enough to eliminate hookworm lar‑vae in the flelds, most effectively with calcium cyanamide.
The pesticides can also destroy the hookworm larvae, but none of them was aspotent as calcium cyanamide, although only Sumithion, Dieldrin and Malathion
were tested among the pesticides.
Among the acid chemical agents, nitric acid had the most powerful action to kill
the two species of hookworms, followed by phosphoric acid and lactic acid. Among

alkali, sodium hydroxide was most pQwerful, followed by ammonium hydroxide andsodium carbonate, with a significantly strong effect on N. americanus. However, thedifference due to concentration was not significant. Both sodium nitrite and carbon
disulfide seemed to possess larva‑cidal action as well as ovicidal action, because thesurvival of larvae were shorter in these ovicides than the control. The survival time
was prolonged in lower concentrations, although N. americanus had a weaker resistanceto the ovicides. It can be assumed that spray of chemical fertilizers and pesticides

is a most effective means to eliminate hookworm larvae. Therefore, a dramatic
eradication can be achieved by using chemical fertilizers in the place of the night soil

which is the major source of hookworm infection.

ACKNOWLEDGMENT
The author is deeply indebted to Professor Daisuke Katamine and Dr. Yuji
Sakaguchi who have offered invaluable advices and have read this manuscript to the157details. He also wishes to express his appreciation of the assistance generously givenby the officials of Haebaru village, all residents of Yamakawa community, Mr. Seikichi

Shiroma, Mr. Yoshiyasu Heishiki, and late Mr. Naozo Uehara, Ryukyu Public
Health Laboratory, and Mrs. Matsuko Henzan, Naha Health Center, and Mr. Isamu
Yaka, the former administrator of the Parasite Prevention Association of Okinawa.

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1) Heishiki, Y. ( 1961) : Recent results on intestinal parasite surveys, Ryukyu Eisei Kenkyusho
Ho, (2), 61‑71

2) Inatome, T. (1932) : Experimental study on poisonous effect of calcium cyanamide to human
hookworm larvae in soil, Kei lgaku, 12 (2), 159‑187
3) Ishizaki, T. and Komiya, Y. (1966) : Treatinent of hookworm infection. Progress of Medical

Parasitology in Japan. Vol. 3, 1 15‑182, Meguro Parasitological Museum, Tokyo
4) Isoda, Y. ( 1958) : Studies on parasite control in a farm village in Nagano Prefecture. Ist

Report. Hookworm control, Teishin lgaku, 10 (3), 197‑208
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Parasit., 8 (3), 394

6) Kobayashi, A., Yasuda, I. and Saito, T. ( 1955) : Ovicidal effects of several chemicals on hook‑worm ova in vitro. Jap. J. Parasit., 4 (3), 308‑3 1 17) Komiya, Y. ( 1957): Hookworm and hookworm disease, Sekibundo Shuppan and Co. Ltd.,

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2 (2), 157‑163

9) Komiya, Y. and Yasuraoka, K. (1966) : The biology of hookworms. Progress of Medical
Parasitology in Japan, Vol. 3, 1‑114, Meguro Parasitological Museum. Tokyo
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J. Parasit., 9 (2), 202‑2101 l) Kozai, I. (1960) : Re‑evaluation of sodium nitrite as the ovicide used in the nightsoil (2). Jap.
J. Parasit., 9 (5), 519‑528

12) Kozai, I. (1960) : Re‑evaluation of sodium nitrite as the ovicide used in nightsoil (3), Jap.
J. Parasit., 9 (5), 529‑540

13) Kozai, I. (1962) : Re‑estimation of sodium nitrite as an ovicide used in nightsoil. IV. On thefield trial with Na‑nitrite‑Ca‑superphosphate mixture and the effect of its application upon ascarisand hookworm infection among people in treated rural areas of Saitama Prefecture. Jap. J.
Parasit., I I (5), 400‑410

14) Kuniyoshi, S. (1959) : A report on parasitic surveys in Ryukyus by Colonel Hunter 111. Ryukyu

Health Laboratory, Naha

15) Kuniyoshi, S., Nakachi, K., Heishiki, Y., Shiroma, S. and Uehara, N. ( 1963) : On the resultsof human parasite surveys at Kushi‑mura, Okinawa Main Island, Ryukyu Eisei Kenkyusho Ho,(4), 5‑1 116) Kutsumi, H. and Ote, H. (1955) : Ovicidal effects of carbon disulphate and Neo‑Gikuron on
the ascaris and hookworm ova, Jap. J. Parasit., 4 (1), 5‑1 l17) Matsubayashi, K., Takada, I. and Yamada, H. (1952) : Field experiment on mass treatment forparasite carriers ( I ) , Annual Report of the Kanagawa Prefecture Public Health Laboratory,
195118) Matsubayashi, K. and Yamada; Y. (1952) : Field experiment fQr decrease of hookworm infec‑

158tion rate among inhabitants by mass treatment of carriers. Proceedings on the Japanese Societyof Parasitology (21), 122‑12319) Matsusaki, G. (1966) : Hookworm disease and prevention. Progress of Medical Parasitology
in Japan, Vol. 3, 183‑282, Meguro Parasitological Museum, Tokyo
20) Miyagawa, Y. (1956) : The newest clinical parasitology, Chiigai lgakusha, Tokyo2 1 ) Mizuno, T. (1975) : The exploration of hookworm ‑ their biology and control ‑, Kikuya
Shobo, Tokyo
22) Sakaguchi, Y. (1961): Studies on ancylostomiasis in the farm district. I . Epidemiologicalstudy on the mode ofhookworm mfection m a farm village of Nagasaki Prefecture Endem Dis.

Bull. Nagasaki, 3 (2), 1 14L129 '23) Sakaguchi, Y. ( 1963) : Studies on ancylostomiasis in the farm district. 2. Seasonal observa‑
tions ofsurvival ofhookworm ova in the privy pits and out‑side soil‑reservoirs, Endem. Dis. Bull.

Nagasaki, 5 (1), 32‑37
24) Sano, T. (1952) : On the poisonous effect of some pesticides to the eggs and larvae of parasites.

2. Resistance test of human hookworm eggs to some pesticides, Okayama lgakkai Zasshi,
64 (6), Il40‑1 143

25) Sasa, M. (1957) : New practical stool examination technique for parasite eggs by smear‑, floata‑tion‑ and cultivation‑methods, Dept. of Parasitology, Institute for Infectious Diseases, Universityof Tokyo26) Sasa, M., Teruya, H., Ikemiya, Y., Kuniyoshi, M. and Kaneshiro, S. (1958) : On the incidenceof parasitic diseases in farm villages of Okinawa, Nihon Ishikai Zasshi, 39 (9), 600‑60427) Sasada, T. ( 1934) : On the influence of various medicaments upon the mature larvae of Ancylos‑

toma. Necator and Trichostrongylus. Kei6 Igaku, 14 (9), 1331‑1631

28) Sato, H., Fukushima, H., Toyama, H., Nonaka, T., Teruya, H., Kuniyoshi, S. and Shiroma, M.
(1958) : A study on parasitic helminth and filariasis bancrofti in Okinawa, Med. J. Kagoshima
Univ., 10 (4), I 148‑1 160

29) Shinkado, O. ( 1959) : Studies on ancylostomiasis in Southern Kyushu (A‑2). Studies on theinfection with hookworm in rural districts, Med. J. Kagoshima Univ., 1 1 (1), 351‑369
30) Shiroma, H. ( 1956) : Results of stool examination among elementary school and junior highschool pupils, and ofmass treatment by l‑bromonaphthol (2) (Warmin) in Okinawa Main Island,
Shinyaku to Rinsho, 5 ( 12), 1041‑l044
31) Suzuki, N. (1959) : Epidemiological studies on hookworm in a village ofTokyo Prefecture, Jap.
J. Parasit., 8 (1), 50‑56

32) Uchida, A. ( 1958) : Studies on preventive and eradication ofhookworm and ascaris in rural area.

Part I . The field experiment ofthe hookworm control in a village ofGumma Prefecture, Nihon
Eiseigaku Zasshi, 13 (4), 499‑509

33) Yamamoto, T., Kuniyoshi, S., Shiroma, S. and Heishiki, Y. ( 1964) : Epidemiological surveyof hookworm infection among the school children in Okinawa Island, Proc. Jap. Soc. Trop.
Med., 5 (1), 92‑9334) Yanagisawa, R. (1966) : The epidemiology of hookworm disease. Progress of Medical Para‑sitology in Japan, Vol. 3, 283‑366, Meguro Parasitological Museum, Tokyo35) Yasuda, I. (1957) : The effect ofvarious chemicals on the larvae of hookworms. ( I ) The in vitro
test of ovicides of ascaris, Jap. J. Parasit., 6 (1), 75‑86

36) Yasuda, I. (1957) : The effect of various chemicals on the larvae of hookworms. (2) The
in vitro test of anthelmintics and insecticides etc., Jap. J. Parasit., 6 (5), 479‑490

37) Yasuda, I. (1957) : The effect of various chemicals on the larvae of the hookworm. (3) Chemi‑
cal control of the larvae of the canine hookworms in the soil, Jap. J. Parasit., 6 (6), 509‑514

159

沖縄に於ける鉤虫症の疫学的研究
国

吉

真

英

沖縄南部の1農村で鉤虫の感染状況と感染成立の動態要因を観察，6年間を通じて集団駆虫を行って，感染率の推移を追究した。又くをo碗orα勉θrづcαn鰯とノ4noヅ05の規α4％04επαZ6両種鉤虫の生態と抵抗性にっいて実験的観察を行った。対象となった南風原村の住民の鉤虫感染率は32．4勉で大部分は亙
α窺θrfoαη粥である。感染率は農耕従事者に画然と高く，しかも農耕作業時間と密接な関係がある。鉤虫皮膚炎の発生からみて最も主要な感染の場所は沖縄特有の甘蕪畑で，冬瓜畑，白菜畑がこれに次ぎ，1月の最寒期を除いて年間を通じて感染が成立しているものと思われる。また農業の形態と尿尿の高率な使用が鉤虫蔓延を助長している。6力年の集団治療により著明な感染率の低下がみられるが，農業専

従者には毎年，新旧両感染がくりかえされている。しかも成人女性に多く，大部分は亙α椛8r加n粥である。両種鉤虫感染幼虫の蒸留水中での生存期間は高温で短く，低温で長いが，いずれの温度でもA．
4麗04εηαZθで長い。虫卵艀化率は27C前後で最も高い。各種化学肥料（石灰窒素，硫酸アンモニア，過燐酸石灰，塩化カリ），農薬として用いられる各種殺虫剤（スミチオン，デルドリン，マラサイオン）
は一定濃度以上で両種鉤虫感染幼虫の生存に対して影響を与えるが，いずれも瓦α規碗0απ鰯で著明である。又，色々の濃度の酸，アルカリ，（硝酸，リン酸，乳酸，アンモニア，炭酸ソーダ，亜硝酸ソー

ダ，二硫化炭素）や殺卵剤，食塩水の殺虫作用にっいても観察されたが，常に瓦α〃3θr加η螂がA．
伽04θπα距に比較して抵抗力が弱いことが証明された。

長崎大学熱帯医学研究所寄生虫学部門

Jap. J Trop Med Hyg Vol 3 No 2 1975, pp. 161‑200

161

CRYO‑PRESERVATION OF THE PARASITIC PROTOZOAAKIRA MIYATA
Received for publication I September 1975

Abstract: In the present paper, about 200 Iiteratures on the cryo‑preservation of theparasitic protozoa have been surveyed, and the following problems have been discussed :cooling rate, storage periods at various temperatures, effects of cryo‑protective substancesin relation to equilibration time or temperatures, and biological properties before andafter freezing. This paper is composed of three main chapters, and at first, the historyof cryo‑preservation is reviewed in details. In the second chapter, other literatures, whichwere not cited in the first chapter, are introduced under each genera or species of the
protozoa. In the last chapter, various factors on cryo‑preservation mentioned above are
discussed by using the author's data and other papers in which various interesting prob‑
lems were described. The following conclusions have been obtained in this study: Before
preservation at the lowest storage temperature, it appears preferable that samples are
pre‑cooled slowly at the rate of I C per minute until the temperature falls to ‑25 to‑ 30 C. The cooling rate might be obtained by the cooling samples for 60 to 90 minutes
at ‑25 to ‑ 30 C freezer. For storage, however, Iower temperatures as low as possible are

better for prolonged storage of the samples. Many workers recommended preservationof the samples in liquid nitrogen or in its vapor, but the storage in a dry ice cabinet or amechanical freezer is also adequate, if the samples are used within several weeks or at least

several months. Cryo‑protective substances such as glycerol or DMSO are highly effective

to keep higher survival rate of the protozoa in frozen state. Most workers recommended
to use 100/0 glycerol or 5 to 7.50/0 DMSO for this purpose. For the use ofglycerol, at least

30 to 60 minutes of equilibration at temperatures as high as 37 C, is necessary to produce

satisfactory results, because at 25 C or lower temperatures, the cryo‑protective action
ofglycerol becomes insufficient. In DMSO, however, samples should be cooled as soon as

possible after adding the substance into protozoan suspension. Prolonged equilibration

with DMSO is apparently toxic to trichomonad. Many workers pointed out that biologi‑
cal properties of the protozoa such as infectivity, virulence, antigenicity, and drug re‑sistance, were not changed by prolonged period of preservation at the low temperature.
These aspects are greatly advantageous for cryo‑preservation. By adopting the cryo‑preservation technique, furthermore, we save expenses for the maintenance throughanimal passages or in vitro culture, and thus we can store much more protozoan species
or strains in the laboratory. Finally, the author proposed to build the cryo‑preservation
center of protozoan strains, because in this country we have no adequate center to deposit
our strains. Even if nobody use a certain strain for experiment at the moment, yet we canpreserve them for the future need. Other laboratories also have many strains, andsome of them may be used frequently, but others are not. If all strains be collected
in one center, and if the center supplies each strain in case of need, we could save a lot of

expense to preserve strains in each laboratory.

Dept. of Epidemiology, Institute for Tropical Medicine, Nagasaki University.
This work was supported in part by a grant for the scientific research from the Ministry of Educationin 1975.

162

Before the advent of cryo‑preservation technique, various strains of the parasitic
protozoa had been preserved by animal inoculation or by cultivation in laboratories.
For instance, maintenance of Toxoplasma gondii (RH strain) or Trypanosoma gambiense
necessitates storage in mice every 3 to 4 days, and Trichomonas vaginalis requires to sub‑culture every 48 hours. In our laboratory, we preserve the following parasites byanimal passage : Plasmodium gallinaceum. P. berghei. Trypanosoma gambiense, and two
strains of Toxoplasma gondii. Solely to preserve those parasites, 640 mice and 1 20
chickens are necessary in every year, costing us about 1 1 3,200 yen (374 dollars) for the

animals. Moreover, we have several strains of Entamoeba. Leishmania, and Trichomonasin culture media. At a moderate estimation the total expense for time, culture mater‑
ials, cages or feeds for the animals, might cost more than 300,000 yen (1,000 dollars)a year, which is equivalent about 100/0 of the total budget per year for our laboratory(6 researchers) .

Since the cryo‑preservation was adopted widely in various laboratories, it has

become possible that not only we could save money but also we could store morevarious species and strains of the parasitic protozoa in a frozen state, and even someof "type specimens" preserved by other workers can be re‑examined in a living state.
Furthermore, in many strains, it was often reported that pathogenicity and biologicalproperties of parasite changed gradually during the long period of animal passage or

sub‑culture. In cryo‑preservation, however, such properties of each strain can bepreserved without apparent loss or change.

Lumsden and Hardy (1965) and Lumsden (1972) proposed the terms stabilate
and stabilation for population of organisms stored in frozen state. According tothem the term stabilate means a population of organisms viably preserved "on a uniqueoccasion", and stabilation is the process of preparing a stabilate. In cryo‑preservation,organisms arrest "the continuous reproduction of a population" and thus "avoid thecontinuous selection" without change of its biological properties such as infectivity,
drug sensitivity, and antigenicity. The term strain which means organismal popula‑tions maintained by serial passage is distinguished from stabilate. A strain mightchange during a period of serial passage in animals or in culture media. It is agreat advantage to adopt cryo‑preservation for workers.

During past 30 years, more than 200 papers concerning cryo‑preservation of
the parasitic protozoa at various temperatures ( ‑ 70 to ‑ 190 C) were published andmost of them were reviewed by Muhlpfordt (1960), A. U. Smith (1961), Diamond( 1964), and Dalgliesh ( 1 972). Even today, however, there are still many problems

to be solved. The present paper will introduce and discuss the problems in moredetails from a historical view point.

I. HISTORICAL REVIEW
According to A. U. Smith (1961), from the time of Antony van Leeuwenhoek(1676‑1677), who first observed cryo‑resistance of the protozoa, to the beginningof this century, there are several reports concerning the life and death of the protozoa

after freezing and thawing. On the parasitic protozoa, Laveran and Mesnil ( 1 904)
showed that trypanosomes survived and retained virulence after exposure for several

163

minutes to liquid air at ‑ 191 C. For example, Trypanosoma lez )isi survived after
75 minutes at that temperature, and still could infect rats. Gaylord (1908) reported
that Trypanosoma gambiense survived even after freezing in liquid air for 20 minutes,

but could not survive after about 40 minutes, and De Jong (1922) also observedthat Trypanosoma equiperdum survived for 2 1 days in liquid air ( ‑ 1 9 1 C).
After these pioneer's trials, Coggeshall ( 1 939) succeeded to preserve Plasmodium

knowlesi and P. inui in a ‑ 76 C dry ice box for 70 days, and the parasites were still
infective to fresh hosts. His attempt was the first important step in cryo‑preservationof the parasitic protozoa. Since his report, many workers have attempted to store
their materials at ‑ 70 to ‑ 1 90 C, and approximately 200 papers have been reportedon cryo‑preservation of various protozoa.
1 ) Coggeshall's technique of cryo‑preservation and its modiflcations
Sorne early trials of cryo‑preservation of pathogenic microorganisms such as
spirochetes, Treponema spp. or filtrable viruses at ‑ 75 C were carried out by several

workers including T. B. Turner (1938) during the ten years from 1930 to 1940.
These studies have encouraged protozoologists, and Coggeshall ( 1 939) wrote a paperon preservation of malaria parasites. He used the following freezing and thawing
technique. One ml of the blood containing Plasmodium knowlesi or P. inui was placedin screw‑capped celluloid tube of 2 cm in diameter. With whirling motion eachtube was immersed in a mixture of solid C02 and alcohol. Then, as. soon as thematerial is frozen, it was rapidly transfered to a low temperature storage cabine.t,

which was maintained at temperatures between ‑72 and ‑80 C by solid C02'
For testing, the tube was thawed as rapidly as possible in a water bath at 37.5 C.

Monkeys were inoculated with I ml of the infected blood stored at ‑76 C for 70
days, and both malaria parasites were able to infect to the monkeys. According to
Horsfall (1940), Coggeshall's unpublished studies proved that P. knowlesi had remain‑
ed infectious for 140 days, P. inui for 1 5 1 days, P. cathemerium for 35 days, Trypanosoma

brucei for 1 35 days, T. duttoni for 34 days, and T. equiperdum for 62 days. Furthermore,

Horsfall described an ideal dry ice cabinet for preservation of microorganisms,
and the cabinet had been used by Coggeshall ( 1939) before Horsfall's report published.
Modifications of Coggeshall's technique were adopted by Archetii (1941) to pre‑serve Plasmodium gallinaceum for 50 days at ‑ 75 C, and Manwell andJeffery ( 1 942),

and Manwell (1943) who kept avian malaria parasites of 10 different species in theinfected blood at ‑55 to ‑78 C for a maximum period of 212 days. Manwell andEdgett (1943) described some modifications of cooling method in which they used
the tubes with thinner wall varied between 0.018 and 0.030 inches in thickness toincrease the rate of cooling and thawing in the storage of Plasmodium lophurae. The

tubes were rotating during cooling. According to their paper, the superiorityof the thinner tubes was also a logical inference from the fact that Toxoplasma gondii(may be RH strain) was found to be still viable after freezing and thawing in suchtubes, whereas it had never been possible to preserve this species in the thicker tubes(about double thickness of the thinner tube) previously used. They concluded thatthe temperature for freezing was not so important as the temperature of storage,
and it was possible to preserve P. Iophurae in viable condition after freezing at atemperature as high as ‑ I O C. For storage, Iower temperatures were better because

1 64

P. Iophurae survived for 244 days at ‑75 C. According to Manwell et al. (1945),
T, gondii was kept frozen only a few minutes in a mixture of dry ice and ethyl alcoholusing the thinner tube, but long period of preservation was not reported. F. Wolfson( 1 945) also preserved P. Iophurae. P. cathemerium, and P. relictum for period varying from

2 days to one year at ‑75 C. Saunders and Scott (1947) were the first to preserve

the human malaria parasite, Plasmodium vivax. About 2 to 3 ml of the citrated
human blood infected with P. vivax were put into small vials and the blood was rapidly

frozen at ‑75 C, then stored in dry ice box at ‑50 C. The parasites survived up
to 16 days or longer and, in a case, up to 37 days, and the blood was capable totransmit malaria and induce fever in the patients who have been treated for neuro‑syphilis by this means. Boyd ( 1949) also reported that Plasmodiumfalciparum survived

for 156 days at ‑72 to ‑80 C.
Weinman and McAllister ( 1 947) have studied to preserve many species of thepathogenic protozoa at ‑ 70 C. They reported that better results were obtained
when the material was first frozen at ‑ 15 C and then stored at ‑70 C than when
it was immediately brought to a temperature of ‑ 70 C. There were survivors withboth procedures, but in the case of Trypanosoma rhodesiense or T. cruzi, the number ofsurvivors using the former procedure increased 2 to I O times of the latter. This
modifled cooling technique is the first proof that slow cooling is less distructive than

rapid cooling of the protozoa. According to their paper, following results were
included ; Trypanosoma gambiense survived for 561 days at ‑ 70 C, T. rhodesiense for595 days, T. cruzi for 653 days, T. Iewisi for 531 days, Leishmania tropica for 794 days,
L. donovani for 276 days, Pentatrichomonas hominis for 407 days, and Plasmodium lophuraefor 235 days, but Trichomonas vaginalis. Chilomastix mesnili. Balantidium coli. Tetrahymena
geleii (non parasitic ciliate) , and Entamoeba histolytica did not survive under the same
condition. In the case of Trichomonas vaginalis, only 2 out of I O samples frozen at‑ 1 5 C and stored at ‑ 70 C contained living organisms when thawed. Toxoplasma
gondii also could survive after freezing, but could not be stored. Later, Weinman( 1 958) reported that T. rhodesiense survived for 8 years at ‑ 70 C, and the parasite
was still infectious to fresh host. Levaditi ( 1 952) also preserved Trypanosoma congo‑
lense for I OO days and Plasmodium berghei for 1 5 days at ‑ 70 C. The malaria parasite

was usually stored as the infected host blood in a frozen state. Only Brumpt andBao Van Ty ( 1 945) recorded unsuccessful experiment in which blood parasites
and sporozoites of Plasmodium gallinaceum were rapidly frozen to ‑25 C, and stored

at the temperature, but no viable malaria parasite was observed when thawed.
The freezing and storing temperature might have been inadequate. The successof preservation of malaria sporozoites owes to Jeffery and Rendtroff ( 1 955), whoreported that viable human malaria sporozoites were obtained after cryo‑preservation
at ‑ 70 C. The longest survival records of each malaria parasite at this temperature
were as follows : Plasmodium vivax for 375 days, P. falciparum for 1 80 days, and P.
ovale for 70 days. In 1 957, Jeffery again recorded longer survival of sporozoites ofP. ovale for 997 days under the same condition described above.
The attempts of cryo‑preservation, before the discovery of cryo‑protective actionof glycerol to living cells by Polge et al. ( 1 949), were discussed already. As a rule,
Plasmodium spp., Trypanosoma spp., and Leishmania spp. are the easiest parasites to

165

be stored in frozen state without any cryo‑protectant. The successful preservation
for prolonged period seems to depend upon constant maintenance of temperature of
the storage cabinet. In early days, most workers used dry ice cabinet to preserve
their materials, because mechanical freezers were not available, and the temperature
within the freezers sometimes fluctuated often. Even now, many workers (Lumsden,
1 972, and others) recommended to use chemical refrigeration (dry ice or liquid nitro‑gen) for cryo‑preservation of the protozoa rather than mechanical refrigeration.
2) Discovery of cryo‑protective effect of glycerol
In 1938, T. B. Turner described that viruses placed in glycerol and maintainedat refrigerator temperature will remain virulent for a number of weeks. In this case,l cerol have been used only as suspended media of the material. The effect ofgyglycerol as a cryo‑protectant was not recognized until Polge et al. ( 1949) found thatglycerol had a protective action to the bull semen in cryo‑preservation. Stimulatedby their finding, many workers tried to use glycerol in cryo‑preservation of theparasitic protozoa (Fulton and A. U. Smith, 1953, may be the flrst). At that time,
one of the most important problems was how to avoid contamination of Tritrichomonas
foetus to the bull semen, because artificial insemination may spread trichomonad in‑
fection among cattles. Since the bull semen can be stored safely at ‑ 79 C in thepresence of glycerol (Polge and Lovelock, 1952), the possibility was examined byworkers if T. foetus also can survive with spermatozoa after freezing and thawing
procedure. Joyner ( 1 954) answered that T. foetus did not survive at some concentra‑
tions of glycerol tolerated by bovine spermatozoa: they could be eliminated from
infected semen by overnight storage at 5 C in 290/0 glycerol, or preferably, by freezing
to ‑ 79 C in the presence of I Oo/o glycerol (cited from Shorb, 1 964) . Soon McWade

and Williams ( 1 954) reported that T. foetus could survive in prepared milk semen
extender in the presence of 7.50/0 glycerol at ‑ 79 C for 1 25 days. Leidl and Mahrla( 1954) also pointed out that the trichomonads were tolerated in the horse serum
broth with 100/0 glycerol frozen at ‑99 C. Joyner and Bennett (1956) again con‑firmed that T. foetus failed to survive freezing and thawing in the presence of I Oo/oglycerol (incubation for overnight at 5 C) in egg yolk citrate diluent under the same

condition which was adequate for sperm survival. They believed that T. foetus
were particularly sensitive to the toxic effects of glycerol when suspended in othermedia such as egg yolk phosphate or milk.
Prior to Joyner's and other papers, McEntegart ( 1 954) reported that the mostsatisfactory condition of cryo‑preservation of trichomonads was found to be storage

at ‑79 C following slow cooling recommended by Fulton and A. U. Smith (1953),
the trichomonads being suspended in medium containing 100/0 glycerol. The "slowcooling" was carried out by the "slow addition" of powdered solid C02 mto alcoholbath in which samples in ampoule were immersed, and from O to ‑ 1 5 C cooling wasin the rate of I C per minute, below ‑ 1 5 C in the rate of 4 C per minute. According
to McEntegart, Trichomonas gallinae was easily preserved and survived for 6 months

or more even without glycerol, and T. vaginalis remained viable for 4 months but
died out after 6 months. Pentatrichomonas hominis in the presence of I Oo/o glycerol
survived for 6 months, but with 50/0 glycerol did not survive beyond 4 months, andTritrichomonas foetus could not survive under any of the conditions tested. More

166

recently McEntegart ( 1 959) has reported that the survival period of T. vaginalis
at ‑ 79 C in the presence of 5 o/o glycerol was prolonged to 26 months, and after
rapid thawing, a few sluggishly motile trichomonads were still observed micro‑
scopically.

Levine and his co‑workers studied intensely on cryo‑preservation of T. foetus.
and their first paper appeared in 1954. The paper was only a short abstract (Levine
and Marquardt, 1954), but soon they published its detail in the next year (Levine

and Marquardt, 1955). According to their paper, they adopted slow coolingmethod ( I C per minute) similar to McEntegart ( 1 954) , and T. foetus survived morethan 128 days at ‑76 C in the presence of 1.0 to 1.1 M glycerol (9 to 100/0)' They
also tested the effect of several compounds related to glycerol such as ethylene glycol,

1, 2, 3, 4‑butanetetrol, 1, 2‑propanediol, and others, and concluded that while
glycerol was the most effective compound studied, several related compounds also
protected the protozoa against the injurious effects of freezing. In the next experi‑ment, Levine et al. (1958) wanted to determine some of the factors which affect theprotective action of glycerol both against the immediate effects of freezing and against

the subsequent slow deterioration during the storage in a ‑20 C freezer in thepresence of I .O M glycerol. They thought that if T. foetus died of slow metabolism
during prolonged preservation at this temperature, it might be possible to preventthe death by adding of the antimetabolites such as malonic acid, sodium fluoride andsodium iodoacetate. For this purpose, their selected concentration of the anti‑
metabolites failed to lower the metabolism of trichomonads at 37 C, but when themedium frozen, remaining liquid changes to a high concentration of these anti‑
metabolites sufiicient to prevent slow metabolism. However, they found that theaddition of antimetabolites did not improve the survival, they also experimented
the addition of lechitin to the medium in the hope of decreasing the brittlenessof the cell membrane, but survival rate did not increase. Increase of the sodium
chloride concentration of the medium decreased survival rate of T. foetus. They
also examined optimal concentration of glycerol in freezing at ‑21 C, and reported
that it was I .5 M. According to the author's experience (Miyata, 1973a) at ‑20 C,
the medium similar to the one used by Levine et al. sometimes did not freeze in thepresence of 100/0 glycerol (about I .O M), and the medium maintained a super‑cooled
state, in which trichomonad still survived. It was not clear whether the medium
of Levine et al. froze or not, and also even if the medium already froze, question
remained whether trichomonad froze or not. Salt concentration was examined by
the author (Miyata, 1973a), and somewhat different result was obtained from Levine
et al. : in T. vaginalis it might be said the survival rates in salt concentration of 0.9
to I .50/0 were higher than those in lower concentrations.

In other experiment, Levine et al. (1959) froze T. foetus at ‑21 C in the presenceof I .O M glycerol at different phases of the growth curve, and they concluded thatwhen the trichomonad froze during initial and logarithmic phases, they did notsurvive, and optimum survival came after at the peak of the growth curve.

In the papers written by Fitzgerald and Levine (1957 and 1961), the term
equilibration was used, and they also checked the temperature fluctuation inside
the tubes of frozen medium themselves with thermocouples. When equilibration

1 67with glycerol was carried out at 24 C, survival after freezing and thawing was better
following rapid equilibration (glycerol was added all at once, and one hour equilibra‑tion) than following slow equilibration (1/6 of flnal amount of glycerol was added

each hour for 6 hours, the equilibration time, 7 hours), but survival of T. foetus
was extremely poor following either rapid or slow equilibration at 4 C, and glycerol
might become toxic at this temperature They also reported that b ff
age medium to pH 6.4 to 7.1 with glycylglycine increased survival upon freezing,but triethanoamine had no signiflcant effect.

' u ering the stor‑

Levine et al. ( 1 962) reported that T. foetus survived much longer in the extended

storage at ‑95 C than at ‑28 C, because at ‑28 C trichomonads die off slowly,
whereas survivors remained constant at ‑95 C for 128 to 256 days. The survival
was much better in the original Diamond's medium (1957) in which trichomonads
had been grown than when they were frozen in a physiological salt solution or in

the fresh Diamond's medium. They thought that presumably some product or
products of the trichomonad's metabolism have an additional protective action
which supplements that of glycerol.
3) Discovery of the effect of DMSO as a cryo‑protectant

In 1959, Lovelock and Bishop found that dimethyl sulfoxide (DMSO) had aprotective action similar to glycerol to living cells in cryo‑preservation. This flnding

also stimulated many protozoologists. Walker and Ashwood‑Smith ( 1 961) showedthat 5 o/o DMSO and I Oo/o glycerol were equally effective for cryo‑preservation of
T ypanosoma rhodesiense and T. congolense at ‑79 C, and they concluded that the lower
toxicity of DMSO both to the host and to the parasite, as compared with glycerol incryo‑preservation. Collins and Jeffery (1963) also compared 50/0 DMSO and 100/0
glycerol in low‑temperature preservation of blood stage of Plasmodium berghei and P.
gallinaceum at ‑ 70 C, and they reported that both substances were effective to thosemalaria parasites, and although I Oo/o glycerol gave rise to superior survivals, the lower

toxicity of DMSO to the parasites made it a useful alternative for preservation.
4) Preservation in liquid nitrogen
Although the possibility of preservation of the living protozoa in liquid nitrogenwas suggested by early workers, the use of liquid nitrogen for dry ice or mechanicalrefrigeration is relatively new. The temperature in the dry ice cabinet or mechanicalfreezers was maintained from ‑70 to ‑95 C at the lowest, and at such relatively
high temperatures, the cryo‑preservation of the protozoa is not permanent but only
for several months or a few years. The cause of death of the protozoa during preser‑
vation period is thought that ice crystal can form and grow in the protozoan cells.

The ice crystal, however, does not form and grow below the recrystallization pointof water ( ‑ 1 30 C). Therefore, the temperature for preservation of living cells
might be better kept below this point. Diamond et al. (1961 and 1963) reported
that use of liquid nitrogen refrigerator for the preservation of Entamoeba histolytica(monoxenic culture grown in association with a Crithidia sp.). Tritrichomonas foetus.
Trichomonas gallinae. T. vaginalis. Pentatrichomonas hominis. Trypanosoma cruzi, and T.

ranarum in the presence of 5 o/o DMSO. Before cooling, trichomonads and amoebae‑
Crithidia suspensions were allowed to equilibrate for 1 5 and 30 minutes, respectively,

at 35 C, and the trypanosomes for 1 5 minutes at room temperature, the samples

168

were then cooled by two‑step cooling technique, in which the samples were cooled
at flrst at the rate of I C per minute from O to ‑ 35 C, then they were plunged directly

into liquid nitrogen. To examine the survival, aliquots of the samples were momen‑
tarily immersed and rapidly swirled about in a 45 C water bath containing a few

drops of 250/0 Aerosol O. T. (American Cynamid Co.) which was added to improveheat transfer. According to their results, cultures of E. histolytica were obtained fromsamples stored as long as 14 months, and also viable cultures were obtained fromT. gallinae and T. vaginalis stored for 10 months and T. foetus and P. hominis stored

for 5 months. T. cruzi and T. ranarum survived up to 4 months. No difference in
numbers was found between samples thawed 24 hours after freezing and those thawed
after the longest period of storage. The result indicated the absence of gradualdeath during extended period of storage in liquid nitrogen, whereas such death wascommonly reported at dry ice temperature by many workers.
Herbert et al. ( 1968) also reported the storage of Trypanosoma brucei subgroup at‑ 196 C by using rapid cooling method, and without glycerol or DMSO they obtained
the excellent result. Reather and Seidenath (1972) successfully stored a total of 28
species of the parasitic protozoa containing I O genera, Entamoeba. Pentatrichomonas.
Trichomonas. Tritrichomonas. Crithidia. Leishmania. Trypanosoma. Plasmodium. Babesia.
and Eimeria, for 720 to 2,662 days at ‑ 196 C. Their cooling procedure is as follows:

The samples containing DMSO or glycerol are stored in plastic ampoules for 30
minutes at 4 C, then they cooled at 0.4 C per minute from 4 to ‑ 10 C, at 2 C per

minute from ‑ 10 to ‑30 C, at 5 C per minute from ‑30 to ‑60 C, and finallywhen reached ‑60 C, the samples are stored in liquid nitrogen. Their paper shows
the possibility of extremely prolonged preservation in liquid nitrogen, and this is
the longest storage record in the frozen state reported in the literature. The record,

however, does not mean the maximum and more extension may be possible in future.

I I . OTHER LITERATURES

1 ) Amoebae

In the history of cryo‑preservation of the parasitic protozoa, glycerol as a cryo‑protectant was used at first by Fulton and A. U. Smith ( 1953) for the storage of
Entamoeba histolytica at ‑ 79 C. Their success stimulated many workers. Fulton
and A. U. Smith used E. histolytica which cultured monoxenically with Bacillus
coli, and in the presence of 5 to 100/0 glycerol, the amoebae were cooled by two
step technique ; O to ‑ 15 C for 20 minutes, ‑ 15 to ‑70 C for 30 minutes, and storageat ‑ 79 C. Before cooling, the samples were allowed to equilibrate with glycerol for
30 minutes at 37 C. Thawing after 65 days of freezing, the samples were inoculated

to fresh media, and the survived amoebae multipled in the media.
Kasprzak and Rydzewski ( 1 970) reported that E. histolytica (3 strains), E. invadens.
and free‑living E. moshkovskii survived for 80 days, 30 days, and 450 days, respectively,

at ‑ 75 C in the presence of I Oo/o glycerol by slow cooling.

Gordon et al. ( 1969) using DMSO, preserved axenic E. histolytica for 5 months
at ‑ 196 C in liquid nitrogen. They cooled the samples at the rate of I C per minuteuntill temperature reached ‑ 70 C, then the samples were stored in liquid nitrogen.

169

Bosh and Frank ( 1 972) observed that E. invadens and E. histolytica could survive freez‑ing in liquid nitrogen using 100/0 DMSO. Neal et al. (1974) also reported theirsuccessful storage of 6 species of the genus Entamoeba in liquid nitrogen in the presenceof DMSO. Other papers concerning the preservation of Entamoeba spp. in liquid
nitrogen were introduced in page 1 67 of this review.

In the papers introduced above, trophozoite stage samples were used for cryo‑preservation, but Halpern and Dolkart (1954) and Chang (1955) examined thecyst of E. histolytica at about ‑ 30 C without cryo‑protectant, but their results were
unsuccessful except for several hour survival after freezing.

Molinari (1955, 1956a, and 1956b) wrote several papers on the effect of low
temperature (4, ‑20, ‑ 79, or ‑ 1 80 C) on survival of trophozoites of E. histolytica
without using cryo‑protectant.
According to the author's own experiments (Miyata, 1 973b), the cryo‑preserva‑
tion of Laredo strain of E. histolytica at ‑ 75 C did not succeed even in the presenceof glycerol or DMSO, and the main reason of this failure could be explained thatrice powders which were contained in the medium became injurious, because underfreezing condition, amoeba decreased the cell volume, and rice powders ingested
inside the cell might be destroyed by mechanical pressure to organelles such as nucleus

or cell membrane.

2) Trypanosornatids
Polge and Soltys ( 1 957) studied cryo‑preservation of trypanosomes with orwithout glycerol by two different cooling methods; (a) Quick freezing : the samples
were frozen directly in a mixture of dry ice and absolute alcohol at ‑ 79 C. (b)Two stage freezing : samples were first placed in alcohol at room temperature, thencooled slowly (2 C per minute) by adding small pieces ofdry ice untill the temperaturereached ‑20 C. After the desired temperature was obtained, the samples were
continued to cool at 5 C per minute to ‑79 C.
According to their paper, both the whole horse serum and the yolk citrate
diluent proved unsatisfactory without glycerol. In Alsever's solution (glucose 4.66
g, sodium chloride I .05 g, sodium citrate 2.0 g, and distilled water 200 ml) and sperm‑

Ringer or other diluents, but the best results (about 500/0 Survival) were obtainedwith the fastest rate of cooling without glycerol, and survived trypanosomes decreased

when the cooling rate was slower. In contrast, in the media containing glycerol,the viability after thawing was poor after quick freezing and was much better after
slow cooling. The best results in all diluents (about 800/0 Survival) were obtainedwith slow cooling and with 5 or I Oo/o glycerol in the medium.

Lymph tubes, instead of test tubes or vials, were used by Cunningham et al.
( 1 963b) for preservation of trypanosomes at ‑80 C. The trypanosome suspensions
containing 7.50/0 glycerol were distributed in "about 25 mg quantities" to the tubes,

and after sealing, they were cooled slowly. By the use of these small tubes, many
materials could be preserved in a small space.
The following authors also studied cryo‑preservation or cryo‑resistance of try‑

panosomes at various temperatures : ReuBe (1956), Molinari and Mont zin (1956aand 1956b), Molinari (1960b), Gordon (1961), Fhick (1962), Berson (1962), Cun‑ningham and Harley (1962), Cunningham et al. (1963a and 1965), Resseler et al.

1 70(1965), Mieth (1966), Lapierre and Vinh Hien (1968a and 1968b), Dar et al. (1972),
Miyata (1973b), and Filardi and Brener (1975).
Leishmania spp. were easily preserved by Foner (1963), Most et al. (1964), andHeymen and Monsour ( 1 962). Foner reported that Leptomonas culicidarum and Her‑
petomonas muscidarum survived for 1 3 months at ‑60 C in the presence of I Oo/o glycerol.

3) Trichomonads
The following papers are not cited in this review : Blackshaw and Beattie(1955), Honigberg and King (1962), Honigberg et al. (1965), Resseler et al. (1965),

Lumsden et al. (1965 and 1966), Muller (1966), Kasprzak and Rydzewski (1970),
and Miyata (1973a, 1974, 1975a, and 1975b).
4) Giardia spp.
As far as the author knows, there are only three papers containing cryo‑preserva‑

tion of Giardia spp. Bemrick (1961) reported that trophozoites of Giardia muris
could survive for 40 days in frozen state (at ‑38 C?) in the presence of glycerol.
At ‑20, ‑38 and ‑70 C, the parasites would survive at least 24 hours if glycerol
were used. Slow freezing in cotton insulated tubes (a modification of Levine andMarquardt, 1955) resulted in a higher survival rate than quick freezing, and if I o/o

Tween 80 was added into basal medium containing glycerol, the number of survived
parasites after freezing and thawing was greater than that where the Tween 80
was not present. The temperature of ‑ 38 C appeared to be the most favorable forthe survival of G, muris, and at the temperautre, optimum concentration of glycerol
was 14.2 percent. But G. muris may survive quick freezing at ‑70 C for several
months.
Meyer and Chadd (1967) preserved trophozites of Giardia spp. obtained from
culture at ‑70 C for at least 2 years in the presence of 70/0 glycerol. They com‑pared various concentrations of glycerol (1, 3, 5, 7, 9, and 150/0) and DMSO (1,3, 5, 7, and 90/0)' A11 samples were frozen slowly in four steps: room temperaturefor 15 minutes, ‑ 12 to ‑ 15 C for 30 minutes, and ‑22 C until frozen (45 to 90
minutes) ; then they were stored at ‑70 C. The parasites survived after freezing andthawing (in a 37 C water bath) at every glycerol and DMSO concentration examined,

but most viable parasites were recovered after using 70/0 DMSO. The parasite
did not survive in cultures frozen without cryo‑protectants.

The paper written by Le Corroller et al. (1970) also dealt with preservationof G. muris as cited in page 1 77 of this review.

5) Dientamoeba fragil is

According to Dwyer and Honigberg ( 1 971), Dientamoeba fragilis could survive
for 6 months after storage in liquid nitrogen by using slow‑cooling technique inthe presence of DMSO.
6) Ciliates
As far as the author knows, only one paper (Weinman and McAllister, 1947)
was published on the cryo‑preservation of Balantidium coli at ‑ 70 C with negative
result cited in page 164 of this review. After discovery of the effect of cryo‑protectants

such as glycerol or DMSO, there are still few papers on the study of low‑temperaturepreservation of ciliate.
Hwang et al. ( 1 964) reported that Tetrahymena pyrlformis could survive for 3 months

171

after freezing at ‑ 1 96 C in the presence of I Oo/o DMSO. The samples were cooledfor 20 minutes in a ‑20 C freezer (2 C per minute), then they were stored in liquid

nitrogen. The number of survived ciliate after freezing and thawing reduced toabout one fourth of that before cooling.
Wang and Marquardt ( 1 966) also preserved T. pyriformis for 1 1 2 days in a ‑95 C

deepfreezer using 100/0 DMSO, and the ciliate still survived. They also adopted
two step freezing; the samples were first cooled in a ‑27 C freezer for 20 minutes(4.5 C per minute), and those were transfered into a ‑95 C alcohol bath, thenstored in the freezer. Furthermore, they reported that Paramecium aurelia survived

for 7 days at ‑27 C or ‑196 C in the presence of 6.0 to 7.50/0 DMSO. Unfor‑
tunately the survival ciliates, however, did not multiply in the inoculated fresh
media, except for the storage for 20 minutes at ‑27 C. According to Marquardt
et al. ( 1 963), ciliates (Colpoda steinii) and amoebae ( Vahlkampfia sp.) were found fromsands or dusts collected in an ice tunnel of Greenland, and cyst of both species could

survive at the low temperature of ‑28 or ‑95 C, but trophozoites of either protozoa
did not.

C. Wolfson ( 1 935) observed Paramecium by using a cryo‑microscope which was
designed by himself. Other papers on the study of cryo‑biology of ciliate are as
follows; Efimoff (1924), Altavilla et al. (1971), Matsusaka (1971), and Tanno (1972).

7) Malaria parasites
Plasmodium spp. seem to be one of the easiest parasites for cryo‑preservation in
dry ice, mechanical freezers, or liquid nitrogen in the presence or absence of cryo‑protectants already mentioned above. The freezing methods adopted by various
workers are simillar to each other, and it is needless to repeat the details of the papers

written by following authors : Vargues ( 1 952), Molinari and Mont zin ( 1 956b),

Molinari (1960a and 1961), Molinari and Tebibzadeh (1961), Jeffery (1962 and1965), Shute and Maryon (1962), Yoeli et al. (1963), Collins et al. (1963), Vincke
et al. (1965), Jacobs (1965), Warhurst (1966), Bafort et al. (1966), Bafort (1968),

Killick‑Kendrick and Bruce‑Chawatt (1969), Booden and Geiman (1970 and 1973),
Allen (1970), Miyata (1973b), and so on.
Among them, Molinari (1961), Shute and Maryon (1962), and Bafort (1968),
reported on cryo‑preservation ofsporozoites in the salivary gland ofvector mosquitoes.
Estimation of the number of alive malaria parasites before and after f 'reezmg was

rather difficult. Warhurst ( 1 966), however, pointed out that percentage survivalof2 strains ofP. berghei after freezing at ‑78 C in 70/0 DMSO might be measured byapplication of a lineal relationship between log inoculum size and pre‑two percent
period (the period between intravenous inoculation of parasites and infection of two
percent of the red cells). The measurement of the effect of low‑temperature on the
protozoa by titration was reported by Overdulve and Antonisse ( 1 970a, 1 970b andl 970c) .

8) Toxoplasma gondii
Toxoplasma gondii was one of the difEicult species to preserve at low temperature
without cryo‑protectants. Recently, Fabio et al. ( 1 967), however, described that
T. gondii (RH strain) which was prepared in physiological saline could survive for47 days at ‑70 C. Miyata (1973b) also reported that T. gondii (RH starin) was

172easily preserved in a ‑ 75 C deepfreezer for at least 30 days without using any cryo‑protectant.
By using such cryo‑protectants, at least RH strain of the parasite is more easily

stored in dry ice, deepfreezers, or liquid nitrogen. For example. Chandler andWeinman (1956) preserved two toxoplasma strains at ‑70 C for 184 days by using
mouse peritoneal exudate for freezing in the presence of I Oo/o glycerol. Eyles et al.
( 1 956) also preserved T. gondii by slow freezing in glycerol solution for 209 days at‑ 70 C. They observed that very slow cooling using 5 to I Oo/o glycerol was the best,
but even with the optimum methods, the initial loss of viability after freezing was
great, whereas storage at be]ow ‑70 C resulted in little further loss of the viability

over the period examined. Recently, Stewart and Feldman (1955) described theuse of rollar‑tube tissue cultures of T. gondii as a source of parasites for cryo‑preserva‑

tion by two step cooling in dry ice, and after 360 day preservation, the cultures be‑came positive after one blind passage.

In liquid nitrogen, according to Franchi and Hahn (1968a and 1968b), T.
gondii survived in the presence of 100/0 glycerol after slow cooling. The samples were
kept at 4 C for 2 to 3 hours, then they were cooled at the rate of I C per minute.
After pre‑cooling, the samples were stored at ‑ 196 C. R. Smith (1973) stored safely
RH strain of T. gondii'which was obtained from tissue culture, in liquid nitrogen withthe fetal calf serum as a cryo‑protectant. He preserved samples directly in liquid

nitrogen. Before reaching this ideal method, he attempted to use DMSO or glycerol
by slow cooling at the rate of I C per minute to ‑ 70 C, then they were cooled andstored in liquid nitrogen.

A11 the authors of the papers cited above used proliferative form of T. gondii,
but following two authors showed the possibility of preservation of cysts or parasites

within cysts. Kwantes et al. (1967) preserved cyst‑cyst type strains which wereisolated from various human tissues or tissue fluid. After storage at ‑ 70 C thestrains produced cysts in mice and their virulence did not change during the pre‑servation. Roble (1965) also observed that cysts survived for 200 days in fresh milk
at ‑76 C. Miyata (1973b), however, reported a negative results on cysts ofBeverleystrain in preservation at ‑ 75 C with or without glycerol. According to this paper,even in those negative samples, a few cysts ofnormal appearance was detected micro‑
scopically without damage, but they have already lost the infectivity to mouse.
Frenkel and Dubey ( 1 973) observed that sporulated oocysts of T. gondii could
survive for 28 days at ‑20 C without using cryo‑protectants.

Gartner and Theile (1970) studied on deep‑frozen toxoplasm with electron
microscope. According to them, in the process of rapid cooling, water penetrated
into cell through the conoid by pressure differences between the outside and the insideof the cell, and after freezing the water changed to ice which destroyed the cell.
They also described that in slow cooling, dehydration of the cell was a cause of cell
damage, while mechanical destructions were diminished and the parasite could sur‑vive. Before the publication of their interesting paper, this kind of study has not beenreported by protozoologists, but the direction suggested by Gartner and Theile
would be followed by other workers in near future. Other papers on cryo‑preserva‑
tion of T. gondii were as follows; F. and A. Roger (1957), Fabio et al. (1967), Bugiar‑

1 73

dini et al. (1967), Bollinger et al. (1974), and Dumas (1974a, 1974b and 1974c).
9) Eimeria. Leucocytozoon. Babesia. Eperythrozoon. Nosema. Anaplasma, and Theileria

Cryo‑preservation of above genera was studied by following workers : Babesia
berbera by Pipano and Senft (1966), B. bigemina by Waddell (1963), Pipano andSenft (1966), and Barnett (1964) ; B. trautmani by Barnett (1964) ; B. calballi by
Frerichs et al. (1968) and Wayne et al. (1968) ; B. equi by Frerichs et al. (1968) ;B. rodhaini by Overdulve and Antonisse (1970b), and Mieth (1966) ; B. canis byReuBe (1956) and Mieth (1966) ; Leucocytozoon simondi by Kocan et al. (1967) ;Eimeria tenella by Kouwenhoven (1967), Doran and Vetterling (1968 and 1969),
Doran (1969a and 1970), and Norton and Joyner (1968) ; E. acervulina by Norton
et al. (1968) ; E. meleagrimitis by Doran and Vetterling (1968 and 1969) and Doran(1969a) ; E. adenoeides by Doran (1970) and Norton and Joyner (1968) ; E. ahsata.
E. arloinigi. E. duodenalis. E. maxima. E. phasiani, and E. stiedae by Norton and Joyner(1968) ; Eimeria spp. by Landers ( 1953) ; Eperythrozoon wenyoni by Ishihara and Minami(1968) ; Anaplasma centrale and A. marginale by Barnett (1964), Pipano and Senft(1966), and Ishihara and Minami (1968) ; Theileria sp. (2 strains) by Ishihara andMinami ( 1 968) ; Nosema cuniculi by Bedrnik and Vavra ( 1 971).

According to Doran and Vetterling (1969), and Doran (1969a), oocysts, sporo‑cysts released from oocysts, and excysted sporozoites of E. meleagrimitis and E.
tenella in media containing 70/0 DMSO were frozen at ‑80 C (1 C per minute), andstored in liquid nitrogen vapor for 4 months. After thawing, oocysts could not infectfresh hosts, although these samples appeared in an excellent condition under micro‑
scope. After 3 months, sporocysts of both species could infect, but in a less extent
than those infected by fresh oocysts. Frozen sporozoites caused infection to fresh

hosts comparable to those caused by unfrozen fresh oocysts. Those results are
somewhat similar to those of the cyst of Beverey strain of Toxoplasma gondii (Miyata,
1973b). According to the author's opinion, since water in the inside of the oocyst

is unable to pass the oocyst membrane during the freezing procedure, the water
would form ice crystals within the oocyst, and the ice becomes injurious to sporozoites

inside of the oocyst. Water penetration to the sporocyst membrane appeares fasterthan that to the oocyst membrane.
Doran ( 1 969b) studied freezing of excysted sporozoites of Eimeria spp. According
to his results, survival of sporozoites was better at the lower concentration of DMSO
before freezing, but after freezing and thawing, survival was better at the higher

concentration of DMSO. With 10 to 12.50/0 DMSO, various equilibration periods
and coo]ing rates were compared, and the best results were obtained where coolingrate from the freezing point to ‑30 C was I C per minute. In the concentrations of
2.5, 5.0 and 7.50/0 DMSO, various equilibration periods up to 2.5 hours were examin‑ed, but the survival rate of sporozoites was not improved.
l O) Other protozoa
The following authors studied on cryo‑biology of the protozoa : A. U. Smithet al. (1951), Gehenio and Luyet (1953), Sharf (1954), Altavilla et al. (1971), andKlein (1972).
Annear ( 1 956) preserved Strigomonas oncopelti for 1 2 months in dried state invacuo at 4 or 20 C. The contents of ampoules yielded heavy growths of the flagellates

1 74

after 1 2 month storage.

l I ) Helminths
Weinman and McAllister (1947) studied on the cryo‑preservation of helminthat ‑ 70 or ‑ 1 5 C. Microfilariae of Wuchereria bancrofti and Dirofilaria immitis could
survive at ‑ 70 C without cryo‑protectant, and infective larvae of Ancylostoma caninum
also survived for I 07 days at ‑ 1 5 C and 44 days at ‑ 70 C, respectively.

Recently, several papers were published on the attempts to preserve helminthlarvae at low temperatures in the presence of cryo‑protectant. Microfilariae or mos‑quito stages are easily preserved. The following authors published their studies
on cryo‑preservation or cryo‑biology of helminths including some papers concerning
free living nematodes: De Coninck (1951), J. H. Turner (1953), Gustafson (1953),

Poole (1956), Asahina (1959), Taylor (1960), Beye and Lawless (1961), Bemrick
et al. (1965), Restani (1968), Ogunba (1969), Parfitt (1971), Obiamiwe and Mac‑
donald (1971), Campbell et al. (1972 and 1973), Isenstein and Herlich (1972),
Campbell and Thomson (1973), McCall et al. (1975), and James (1975).

III. VARIOUS FACTORS ON CRYO‑PRESERVATION
Various factors involved in cryo‑preservation of the parasitic protozoa wereintroduced and discussed briefly in this chapter.

1 ) Cooling rate
Before preserving the parasitic protozoa in the frozen state, most workers cooled

the samples at a rate of about I C per minute to temperature around ‑30 C. To
obtain the cooling rate, the samples were pre‑cooled at ‑20 to ‑30 C for 30 to 90
minutes, then they were stored at ‑70 to ‑80 C or ‑ 1 70 to ‑ 190 C. The author
compared five different cooling rates between 0.25 to 5 C per minute in preservationof Trichomonas vaginalis (Miyata, 1 974) . Each group of I O samples in the presenceof 100/0 glycerol was pre‑cooled in each freezer of 5 different temperatures for 90
minutes, then the samples were stored in a ‑75 C freezer. According to the results,
the highest survival rate of T. vaginalis was obtained at the cooling rate of 0.78 C
per minute by the use of a ‑30 C freezer (Figs. I and 2). The survival rate at 5 C
per minute was about 20 percent lower than the highest survival rate.
Diamond ( 1 964) reported that 30 to 40 percent of Entamoeba invadens protectedwith 150/0 DMSO and 4.50/0 glucose have survived when cooled at I C per minute,but there were almost no survivors in the samples cooled at 8 C per minute.
The cooling and thawing process of the parasitic protozoa in cryo‑preservation canbe explained in Fig. 3 (after Miyata, 1973a). The sample is cooled at about ‑30 C
for one to two hours. The temperature of the sample drops without freezing, and thesuper‑cooled state continues for several minutes. The survival rate of the cells does
not change during super‑cooled state, but when the state is broken, the temperatureslightly goes up by liberation of the latent heat in crystallization, then the temperatureof the sample again drops slowly. The point at the beginning offreezing (F in Fig. 3)
is called the freezing point or the freezing temperature of the sample. The freezing

temperature is lower in higher concentration of the cryo‑protectant as shown in
Fig. 4. From the point to ‑ 30 C or lower, dehydration of the cells is carried out by

175
̲̲. ‑16 C freezer (cooling rate o. 25 C/min.)30‑..‑ ‑ZO C freezer (cooling rate O. z7 C/min. )‑‑‑‑' ‑25 C freezer (cooling rate O. 45 C/min. )‑‑‑ ‑3O C freezer (ccolipg rate O. 78 C/min.)‑75 C freezer (coQling rate 5 C/min. )

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The cooling curves of various concentrations ofglycerol (GLY) Qr DMSO
in a ‑25 C freezer (Miyata, 1973a).
Note : Each curve is shown from the beginning of freezing, and super‑cooled state is excluded.

formation and growth of extracellular ice crystals. Dehydration of the cells must
be thorough, because if the water remains in the cell, the water may be crystallizedlater and destroy the cell mechanically. After sufficient dehydration, the sample
must be cooled rapidly to the storage temperature. In the rapidly cooled samples
without pre‑cooling, the water in the cells can not go out, and changes to ice crystal
in the cell. This is the reason to explain why slow cooling is better than rapid cooling.
In the case ofvery slow cooling, salt concentration could be effective. Several workers
reported that some species or strains of the parasitic protozoa could survive after

freezing directly in dry ice alcohol or liquid nitrogen. In such cases, many ice
crystals may be formed within the cell, but the size of each crystal is too small to
injure the cell.

Most workers recommended that the frozen samples must be thawed in a water
bath at about 40 C as soon as possible after taking out from a storage cabinet, because

recrystallization of ice can be avoided by such rapid thawing. Diamond ( 1 964)
and Dalgliesh ( 1 972) pointed out that the survival rate increased in proportion to
the warming rate.,

1772) Storage period at various temperatures
In Table I , storage periods of the parasitic protozoa (24 genera including
about 90 species) at various temperatures were summarized. The longest preserva‑
tion period of each species at different temperatures in the presence or absence of
cryo‑protectants was sought from many literatures as far as the author knows. Several
species could survive after preservation at relatively high temperatures as ‑20 to ‑ 30
C, but the storage period is rather short. According to such authors, gradual deathoccured during preservation period at such a high temperatures, and finally allorganisms died. At '‑70 to ‑90 C, survival period prolonged to months or often
years,' but at this temperature still living parasites decreased gradually with theincrease of storzige period. At the temperatures of liquid nitrogen itself or its vapor,the protozoa could survive for an extremely long period. At present, the longest
survival record so far tested is longer than 2,500 days (Raether and Seidenath, 1972),

but the period might have been extend infinitely. It was said by many workers that
before cooling and after storage for several years in liquid nitrogen, there were no
significant loss of survival number and of their infectivities to the host.

3) Use of chemical compounds for cryo‑preservation

Both glycerol and DMSO are widely used by workers as cryo‑protectants,
whereas recently, DMSO instead of glycerol was recommended by several workers(Dalgliesh, 1972; Walker and Ashwood‑Smith, 1961; Collins and Jeffery, 1963 ;Diamond, 1964; and Miyata, 1973a), because of its rapid equilibration and lower
toxicity. In addition to these two 'chemicals, some other substances were reported
having the cryo‑protective action to living cells, for example, dimethylacetamide by
Djerassi et al. (1971) and pyridine N‑oxide by Nash (1961). Dobbler (1966) review‑
ed and discussed of structure and function of cryo‑protective compounds.
As a protozoologist, O'Connel , nd his co‑wor ers ( 1 968) examined the cryo‑protective action of 83 chemicals such as alcohol, sugars, amines, and others, forpreservation of Crithidia fascilatus at ‑20 C. According to their results, the bestprotectant among the chemicals tested was glycerol at I O percent (w/v), and as agroup the alcohols were most effective, and several sugars also showed soine protective
activities. Levine and Marquardt ( 1 954) also tested several compounds related to glyc‑
erol in cryo‑preservation of Tritrichomonas foetus introduced in page 1 66 of this review.

The protective action of the serum in cryo‑preservation of the protozoa wasreported by several workers (Jeffery and Rendtroff, 1 955; Polge and Soltys, 1 957;
R. Smith, 1973; and Miyata, 1973b).
More recently, Le Corroller et al. ( 1 970) examined macromolecular, extracellularcryo‑protectants such as polysaccarides and polyvinyl pyrrolidone instead of micro‑
molecular, intracellular cryo‑protectants such as glycerol and DMSO, because thetoxicity of intracellular cryo‑protectants were pointed out by various workers. They
prepared a mixture containing 1 5 g of dextran sorbitol (Rheomacrodex) and 1 5 g
ofpolyvinyl pyrrolidone in 100 ml distilled water. Protozoan suspensions were mixed
in equal volume of the cryo‑protectant mjxture. The samples which distributed in
one to 4 ml in ampoules were cooled and stored at ‑ 70 C. Trypanosoma lewisi. T.
cruzi. T. congolense. T. brucei. T. gambiense. Tritrichomonas muris. Trichomonas tenax. T.
vaginalis. Pentatrichomonas hominis. Giardia muris. G. intestinalis. Toxoplasma gondii, and178

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185

Plasmodium berghei survived for more than 2 years at the temperature without losing
their virulence.

(a) Equilibration
In the use of cryo‑protectants, samples should be kept or incubated for several
minutes at least at temperatures higher than sub‑zero temperature before cooling.

The adequate temperature and period in the incubation was reported by someworkers (Levine et al.. 1958; Dalgliesh, 1972; Miyata, 1974, 1975a and 1975b).
They preferred the term equilibration or to equilibrate to incubation or to incubate. As

one of the authors using the term, the reason of the choice should be explained.
In the previous papers (Miyata, 1974, 1975a, and 1975b), the author showed several
figures, and in the present paper, some of them are presented in Figs. 5 and 6. In
the Fig. 5, after incubation at 25 C or 37 C for various intervals between O to 180
minutes, the samples ( Trichomonas vaginalis. 5 strains) were cooled in a ‑ 30 C freezer(cooling rate, I C per minute) in the presence ofglycerol, then the samples were storedat ‑ 75 C. The survival rate of T. vaginalis increased gradually with the prolonged
incubation period, and after several minutes, the maximum survival rate has appeared,
and no more incubation was needed. The effect of glycerol as a cryo‑protectant onsurvival is enhanced with prolonged incubation, which is necessary to equilibrate
protozoan cells to glycerol.

The temperature for equilibration is also important, as shown in Fig. 6. In
this experiment, the samples were allowed to equilibrate with I Oo/o glycerol for I OO
minutes at various temperatures, and they were stored at ‑ 75 C, after pre‑coolingin the ‑30 C freezer. The survival rate was markedly improved at higher tem‑
peratures. Incubation at O C was not effective, but even at this temperature, thesurvival rate was still very high, if compared with the samples without glycerol. The
survival rate for non‑glycerol samples was less than one percent, or sometimes nosurvivor was observed microscopically. Fitzgerald and Levine ( 1 961), and Dalgliesh( 1 972) pointed out that glycerol was toxic at around 4 C. The author's opinion, how‑ever, is quite different from them, and he believes that glycerol is not toxic at 4 C,
but at this temperature, cryo‑protective action does not work sufliciently.

These observations clearly show the importance of the temperature and theperiod for the equilibration between cells and glycerol. According to Lovelock( 1953), glycerol must penetrate into the cells, and for the penetration, time andtemperature must be important. At least, in the present time, the author is un‑
certain whether or not glycerol is really present within trichomonad cells. Therefore,
the volume of glycerol within cells must be measured at various temperatures andin various equilibration periods. According to literatures surveyed so far, there
has been no report dealing with such problems in the protozoological fleld.
The action of DMSO is very different from that of glycerol as shown in Figs. 6

and 7. These experiments were carried out by the same method mentioned above.
The good effectiveness of DMSO as a cryo‑protectant was demonstrated at lower
temperatures (Fig. 6), and the lowest survival rate of T. vaginalis was obtained fromthe samples which were incubated at 37 C. The effect of equilibration time at 25 C
or 37 C on the survival was also very different. At either temperature, survival rate
decreased with the prolonged equilibration time, and the highest survival rate was

186
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Notes for Figs. 5 to 9 :DMSO‑‑‑ at 37C
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After thawing in a 37 C water bath,cls

survival rate was examined. Theipooled survival rate of 5 strains of

Trichomonas vaginalis was shown in

the figures. The details of theseexperiments were seen in Miyata,
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J qun[bration Time (mlnute)Fig. 7The effect of the equilibration timeon trichomonad survival with 7.50/0

DMSO at 25 C or 37 C (Miyata,
1975a).

obtained in the sample without equilibration. According to Lovelock's theory(1953), the time for penetration ofDMSO into cells must be required. The author's
results, however, show that penetration of DMSO is rather injurious, and if this sub‑stance is used, the samples should be cooled as soon as possible after addition of

DMSO. In the samples without cooling, Iiving trichomonads did not change in

187

the number during equilibration period, even after 3 hour incubation. This showsthat injurious action of DMSO to cell is caused not by prolonged incubation but

by cooling and thawing after prolonged incubation. The action of DMSO andglycerol varied among the species or strains used as reported by various workers.

Meyer and Chadd (1967) also pointed out that both glycerol and DMSO proved
toxic to the parasites in cultivation at 37 C. DMSO are clearly more toxic; 3.50/0
DMSO killed more than 990/0 Giardia in 2 days while 3.50/0 glycerol killed about
500/0 of the parasites in the same period. Therefore, they recommended that theprotective agent should be diluted as quickly as possible after thawing.
(b) Optimal concentration of cryo‑protectants

The optimal concentration of glycerol or DMSO were studied by most workers
who attempted to preserve the protozoa in a frozen state. Their opinion varied,
but most ofthem recommended to use 100/0 glycerol and 5 to 7.50/0 DMSO. Accord‑
ing to the author's experiment (Miyata, 1975a and 1975b), the optimal concentra‑tion of glycerol in Trichomonas vaginalis changed with equilibration period and tem‑

perature. For example, the optimal concentration was higher at 25 C than 37 C
in equilibration for 100 minutes (Fig. 8). In the case of DMSO, the optimal con‑centration is flxed between 5 to I Oo/o at various temperatures (Fig. 9). The incuba‑tion period is important, and in higher concentration many parasites may die during
prolonged equilibration as pointed by Doran ( 1 969b).

Diamond (1964) used 50/0 DMSO, because this concentration gave him satis‑
factory results for many protozoa. By using 50/0 DMSO, Entamoeba invadens survived
by slow cooling and storage at ‑ 1 70 C, but recoveries of viable organisms were

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2.5 5.0 7.5 Io,o 12.5 15.0 17.5 20.0oFig. 8The effect of the concentration of glycerol
on trichomonad survival after 100 minutes
equilibration at 25 C or 37 C (original).

2.0' 5.0 7.5 Io.o 125 15 oconcentration of DMSo (v/v "'1r')

concentration ot Chycerol (v/v %)Fig. 9The effect of the concentration of

DMSO on trichomonad survival
after 60 minute equilibration at
25 C (original).

188

only one percent. Then, the concentration of DMSO was increased to 15 percent,and this gave rise to a maximum recovery of about 25 percent. Stimulated by
Djerassi and Roy (1963) who employed DMSO and sugar in freezing preservationof blood platelets of the rat, Diamond attempted to add 4.50/0 glucose to sample
containing 150/0 DMSO, and 45 percent ofamoebae recovered, whereas no protection
was shown with glucose alone at concentrations from 2.25 to 90/0 (w/v).
4) Maintenance of biological properties of the parasitic protozoa in frozen state
It is well known that biological properties such as virulence, antigenicity, anddrug resistance of various parasites change gradually by prolonged passage in animals
or culture media. Some workers reported that several strains have lost their patho‑
genicity to inoculated mice after prolonged storage in a frozen state. For example,
Allain ( 1 964) observed the loss of pathogenicity in one of 9 strains of Trypanosoma
cruzi stored at ‑64 C for 6 months, but other 8 strains retained their original virulencesand viabilities. Many workers, however, claimed that the properties did not change
by using cryo‑preservation.

(a) Virulence
According to Stabler et al. (1964), the highly virulent strain of Trichomonas
gallinae (Jones' Barn strain) survived for 52 weeks at ‑ 72 C by addition of I .O M
glycerol ( I hour equilibration at room temperature), and its virulence did not change
during the prolonged preservation period, but the continued in vitro cultivation at
37.5 C resulted in the gradual loss of pathogenicity of the trichomonad to domestic
pigeons.
Lindgren and lvey ( 1 964) also reported that the virulence of Trichomonas vaginalis(12 strains) did not change during a period of 8 week preservation at ‑43 C in thepresence of 100/0 glycerol, whereas the virulence decreased in after 2 to 3 months
cultivation at 37 C. Jeffries and Harris (1967) obtained the result similar to Lindgren
and lvey on T. vaginalis and Tritrichomonas foetus. Diamond et al. ( 1 965) examinedthe virulence of T. vaginalis to mice after 730 day storage at ‑ 1 70 C by using 50/0

DMSO, they found that the virulence did not change singnificantly by such long
period storage.

Weathersby and McCall ( 1 967) reported that sporozoites of Plasmodium gal‑linaceum were preserved in the vector, Aedes aegypti, for 767 days in liquid nitrogen

without apparent loss of viability or infectivity. Minter and Goedbloed (1971)
obtained live trypanosomatid parasites from the frozen tsetse flies and sandflies
kept in liquid nitrogen with no obvious loss of viability or infectivity. Filardi andBrener ( 1 975) pointed out that prolonged preservation at ‑ 1 96 C apparently did
not change the biological characteristics of different strains of Trypanosoma cruzi.

(b) Gametogenesis
Many workers observed the disappearance of gametocytes of certain malaria
parasites such as Plasmodium spp. by serial blood transferes in vertebrate hosts. By
the application of cryo‑preservation, the original nature to produce gametocytes
in various blood parasites might be maintained. This is another advantage of cryo‑preservation. Furthermore, according to Bafort et al. ( 1 966), the ability to re‑produce gametocytes in P. vinckei which had lost the power by continuous animalpassages recovered in the parasites which were kept in frozen state at ‑ 75 C.

189(c) Drug resistanceSchneider et al. (̲1968) examined the survival time and retention of antimalarial
resistance of malaria parasites (Plasmodium berghei and P. gallinaceum) in liquid nitrogenin the presence of 100/0 glycerol. The samples were cooled at I .3 C per minute for
60 minutes, then rapidly cooled in liquid nitrogen. One of the strain of P. berghei
survived for 639 days and P. gallinaceum also for 62 days. There was no remarkabledifference of antimalarial resistance between frozen samples and unfrozen ones.
Since the antimalarial resistance of the malaria parasite is reduced if the parasites
were maintained from host to host without medication, the cryo‑preservation might
be useful to keep such characters of the parasites.

(d) Antigenicity
According to Gordon et al. ( 1 969), no permanent antigen change had occured
as a result of 5 month preservation of Entamoeba histolytica at ‑ 1 96 C.

(e) Loss of kinetoplast
According to Overdulve et al. (1970c), one strain of Trypanosoma evansi lost its
kinetoplast after storage at ‑76 C and at ‑ 196 C, and it did not reappear in sub‑sequent mouse passage. This kind of observation, however, was not reported byother workers who preserved trypanosomes in a frozen state.

IV. CONCLUSION
In the introduction of this paper, the author has already pointed out the advan‑
tages of cryo‑preservation of the parasitic protozoa, and he would not repeat the
subject again. In cryo‑preservation, biological properties such as infectivity, viru‑
lence, antigenicity, drug resistance, or to produce gametocytes, might be maintained

without apparent loss or change as reported by many workers. Of course, several
undesirable results were described by some workers, for example, Ioss of pathogenicity
or, even loss of kinetoplast permanently in parasites after freezing. Even consideredthose minus results, still cryo‑preservation is a useful method to maintain our materials
because during prolonged period of animal passages or in vitro cultivation, originalnatures of parasites frequently changes in infectivity, virulence, or other properties

as pointed out by many workers. To avoid such undesirable changes, we must im‑prove our preservation technique furthermore, and there is no doubt that problems
will be solved in future. The advantages of cryo‑preservation are apparently greaterthan those in animal passage or serial cultivation, and especially the preservation
period is extremely prolonged by the use of liquid nitrogen. At present, however,the fo]10wing problems are not yet solved clearly, for instance, fate of ice crystals in
the frozen cell, optimal cooling rates, action of cryo‑protectants, effect of salt con‑centration (or other substances present in suspended media), or penetration ratesof cryo‑protectants into the cells.

Today, cryo‑preservation of the protozoa is popular all over the world, andvarious new techniques vyere proposed and discussed by many workers, but thestandard methods might be summarized as follows :( I ) For most parasitic protozoa, cryo‑protectants such as DMSO or glycerol

are useful. Several workers recommended to use DMSO (5 to 7.50/0) rather than

1 90

glycerol (about I Oo/o) because equilibration is unnecessary. Glycerol, however,seems also an useful protectant because DMSO is rather toxic than glycerol in higher
concentration and in prolonged equilibration. The equilibration with glycerolmust be carried out for 30 to 60 minutes at a higher temperature as 37 C. DMSO,
however, should be added at a lower temperature and the materials must be cooled
as soon as possible after adding the substance.

(2) Slow cooling in the rate of I C per minute is ideal in most parasites, andthis cooling rate can be obtained by cooling at ‑25 to ‑ 30 C, or by slow addition ofsmall pieces of dry ice into alcohol bath.

(3) For cryo‑preservation, the lower temperature is better, and stdrage inliquid nitrogen or its vapor is the best.

(4) Frozen materials should be thawed as rapidly as possible to avoid recry‑stallization of the ice during warming, and for that purpose, 37 to 40 C water bathis useful.

In our country, we have no center in which various protozoa strains are stored

to supply to other researchers. In one laboratory, some strains might be usedroutinely for experiments, but other many strains are not. For example, in ourlaboratory, nobody uses Trypanosoma gambiense. Entamoeba histolytica. Leishmania donovani.

and Toxoplasma gondii (2 strains) at present. Other laboratories may have somestrains out of use. If those strains were deposited certain center, the center could

supply the strains soon whenever the need for them arose. For these reason, theauthor would like to propose to build such a preservation center of protozoan strainsin this country. Ifwe could have the center, it would benefit all researchers who haveinterest in protozoology and tropical medicine.

ACKNOWLEDGEMENTS
The author wishes to express his deepest appreciation to Dr. Toshio Nakabayashi,

Professor, and to Dr. Masuhisa Tsukamoto, Associate Professor of the Department,
for their continuous encouragements and advices to this work.

LITERATURESl ) Allain, D. S. ( 1964) : Evaluation of the viability and pathogenicity ofhemoflagellates after freez‑

ing and storage, J. Parasit., 50, 604‑607

2) Allen, E. G. ( 1970) : Preservat.ion of Borrelia kansas and Plasmodium berghei. Appl. Microbiol.,20, 224‑226
3) Altavilla, J. C., Meskill, V. P., and Garner, J. G. (1971) : Survival of Colpidium campylum andEuglena gracilis following freezing, J. Protozool., 18 (suppl.), 20
4)

Annear, D. I. ( 1956) i Preservation ofStrigomonas oncopelti in the dried state, Nature, 1 78, 4135) *Archetti, I. (1941) : Conservazioni di P. g llinaceum a bassa temperatura, Riv. Parassit., 5, 247‑

2496) Asahina, E. ( 1 959) : Frost‑resistance in a nematode Aphelenchoides ritzema‑bosi. Low Tem‑
perature Science, Ser. B, 1 7, 51‑62 (in Japanese with English summary)7) Bafort, J. (1968) : The effects of low temperature preservation on the viability of the sporozoitesof Plasmodium berghei. Ann. Trop. Med. Parasit., 62, 301‑304

1918) Bafort, J., Vincke, I. H., and Timperman, G. (1966) : Observations on the gametogenesis of
Plasmodium vinckei, Nature, 2 1 1 , 439 L40

9) * Barnett, S. F. (1964) : The preservation of Babesia bigemina. Anaplasma centrale and A. marginale

by deep freezing, Vet. Rec., 76, 4 8lO) Bedrnik, P., and Vavra, J. (1971): Cryopreservation of the mammalian microsporidian
Nosema cuniculi. J. Protozool., 19 (suppl.), 46l l) Bemrick, W. J. (1961) : Effect of low temperatures on trophozoites of Giardia muris. J. Parasit.,

47, 573‑576
12) Bemrick, W. J., Buchli, B. L., and Griffiths, H. J. ( 1965): Development of Dirofilaria immitis
in Anopheles quadrimaculatus after exposure of the microfilariae to a freezing temperature, J. Para‑sit., 51; 954‑957

13)* Berson, J. P. ( 1962): Utilisation du liquide de Hanks pour la conservation de Trypanosoma
congolense par le froid, Bull. Soc. Path. Exot., 55, 804‑807

14) Beye, H. K., and Lawless, D. K. ( 1961): Viability of microfilariae of Wuchereria bancrofti
during prolonged storage at ‑ 25 C, Exptl. Parasit., I l, 319‑322

15)* Blackshaw, A. W., and Beattie, H. E. R. ( 1955): The preservation of Trichomonas foetus at‑ 79 C, Aust. Vet. J., 31, 214‑216
16) Bollinger, R. O., Musallam, N., and Stulberg, C. S. ( 1974) : Freeze preservation of tissue cul‑
ture propagated Toxoplasma gondii. J. Parasit., 60, 368‑369

l 7) Booden, T., and Geiman, Q. M. ( 1970) : Low temperature preservation of Plasmodium sp.
using dimethyl sulfoxide, J. Protozool., 1 7 (suppl.), 1 618) Booden, T., and Geiman, Q. M. ( 1973) : Plasmodiumfalciparum and P. knowlesi: Iow temperaturepreservation using dimethylsulfoxide, Exptl. Parasit., 33, 495‑498
19) Bosch, I., and Frank, W. (1972) : Beitrang zur Gefrierkonservierung pathogener Amoeben und
Trichomonaden, Ztschr. Parasitkde., 38, 303‑31220)* Boyd, N. F. ( 1949) : Malariology, Vol. 1, p. 69, W. B. Saunders Co., Philadelphia
2 1)* Brumpt, E., and Bao Van Ty ( 1945) : Essais n gatifs de conservation par le froid ( ‑25 C) desshizontes, des gam6tes et des sporozoites de Plasmodium gallinaceum. Bull. Soc. Path. Exot., 38,

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200

寄生原虫類の凍結保存
宮田彬

最近30年間に発表された原虫の凍結保存に関する論文は，200篇を越えている。そこでこの論文では，
それらのうち主な論文を紹介するとともに凍結保存が可能な原虫類の保存方法や保存期間などを総括し，さらに今後の問題点を論じた。また今までに十分な検討を加えずに用いられていた凍害保護剤にっいて，

特にグリセリンとDMSOの用い方，平衡時間などについて，著者の研究を中心に紹介した。原虫類の最適保存法及ぴこの論文の論旨は次の通りである。1）原虫は，適当な保護剤を含む溶液あるいは培地中に撹伴し，試験管またはアンプルに分注する。2）保謹剤の濃度は，グリセリンは10％前後・DMSO
は，7，5％前後が適当である。グリセリンの場合は，比較的高い温度（例えば37C）で30−60分平衡させる。高温に耐えない原虫は，25C前後で60−90分平衡させる。DMSOは，低い温度（例えばOC）で加え，平衡時間をおかず直ちに凍結する。3）凍結は2段階を用いる。すなわち，一30C前後のフリー
ザー中で約90分予備凍結し（この時冷却率は約1C，1分），ついで保存温度へ移す。4）保存温度としては，液体窒素のような超低温が好ましいが，一75Cでも数ヵ月程度は保存可能である。5）凍結材料は，37〜40Cの恒温槽中で急速融解し，融解後は，すみやかに動物あるいは培地へ接種する。6）原虫の種類によっては，もっと簡単に保存できる。原虫ごとに予備試験を行い，目的の保存温度に数日保存して高い生存率の得られる方法を採用するとよい。7）今後の問題点としては，保存原虫の性質（薬剤耐性，
抗原性，感染性など）の長期保存における安定性を検討することと純低温生物学的な立場から超低温下における細胞の生死のメカニズムを解明することである。前者については，多くの研究者が凍結保存による実験株の性質の変化は認められないと指摘している。8）終りに数多くの実験株を保存し，研究者に提供する低温保存センターの設置の必要性を提案した。

長崎大学熱帯医学研究所疫学部門

JA ANESE JOURNALOF
TROPICAL MEDICINE AND HYGIENE
Septenrber, 1975

Vol. 3 No. 2

CONTENTS,

Original article
ITAGAKI. H., SuzuKI, N., ITo, Y., HARA, T. AND WONDE. T.
Study on the Ethiopian Freshwater Molluscs, Especially on Identification, Distribution
and Ecology of Vector Snails of Human Schistosomiasis . . . .. . .

KUNIYOSHI, S.
An Epidemiological Study of Hookwdrm Infection in Okinawa Island ... .

107‑134

135‑159MIYATA, A.
. Cryo‑preservation of the Parasitic Protozoa . . . . . .

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

12‑4 Sakamoto‑machi, Nagasaki, 852, Japan

161‑200