JAPANESE JOURNAL OF
TROPICAL MEDICINE AND HYGIENE

第２
９巻

第４号
内

著
蠕虫卵の定量的検出のためのナイロンメッシュ濾過法（英文）真喜屋 清 ……………………………………………………………………………………………３３５‐３４２
メフロキン，クロロキン，ピリメサミン耐性熱帯熱マラリア原虫に対するミノサイクリンのin vitro 効果（英文）林清華，片倉賢，大上 美穂，狩野 繁之，鈴木
守 ……………………………３４３‐３４８
タイで採集されたブユ（ハエ目：ブユ科）の１新種 Simulium (Simulium) weji sp. nov.
について（英文）高岡 宏行 ……………………………………………………………………………………………３４９‐３５４
Nippostrongylus brasiliensis 感染ラットにおける小腸からの寄生虫抗原吸収の機構（英文）山田稔，内川 隆一，松田 信治，有薗 直樹 ……………………………………………３５５‐３５９
マウスにおける Nippostrongylus brasiliensis の分泌・排泄抗原持続注入による IgE 抗体産生の誘導（英文）
山田稔，手越 達也，内川 隆一，松田 信治，西田稔，有薗 直樹 ……………３６１‐３６４
熱帯熱マラリア原虫の培養および薬剤感受性試験の方法としての各アネロパック!
に関する検討（英文）春木 宏介，小林富美恵，藤野 隆志，松井 利博，辻守康 ……………………………３６５‐３７０
Daengsvang らの報告したウェステルマン肺吸虫に関する形態学的再検討（英文）杉山広，柴原 壽行，Punsin Ketudat，Sodsri Thaithong，川島健治郎 ……………………３７１‐３７４
マレーシア産およびフィリピン産ウェステルマン肺吸虫脱嚢メタセルカリアの体表微細構造（英文）杉山広，Karen Lai，C.K. Ow-Yang，Bayani L. Blas，川島健治郎 …………………………３７５‐３７８

会報・記録
２
０
０
１（平成１
３）年度日本熱帯医学会役員名簿………………………………………………………………３７９
日本熱帯医学会雑誌編集委員名簿 ………………………………………………………………………３８０‐３８１
投稿規定 ……………………………………………………………………………………………………３８２‐３８３
日本医学会だより …………………………………………………………………………………………３８４‐３８５

DECEMBER ２０
０
１

CONTENTS

容
Japanese Journal of Tropical Medicine and Hygiene Vol. 29 No. ４

原

VOL．
２
９ No．
４

平成１
３年１
２月

Original article
Makiya, K.
Nylon Mesh Filtration Technique for Quantitative Detection of Helminth Eggs ……………………３３５‐３４２Lin, Q., Katakura, K., Oue, M., Kano, S. and Suzuki, M.
Effects of Minocycline against Mefloquine-, Chloroquine- and Pyrimethamine-resistantPlasmodium falciparum in vitro ……………………………………………………………………３４３‐３４８
Takaoka, H.
Simulium (Simulium) weji sp. nov. (Diptera: Simuliidae) from Thailand……………………………３４９‐３５４
Yamada, M., Uchikawa, R., Matsuda, S. and Arizono, N.
Mechanisms of Parasite Antigen Uptake from the Intestine of Rats Infected with the Nematode
Nippostrongylus brasiliensis …………………………………………………………………………３５５‐３５９
Yamada, M., Tegoshi, T., Uchikawa, R., Matsuda, S., Nishida, M. and Arizono, N.
Induction of IgE Antibody Response in Mice by Continuous Administration of
Excretory/secretory Products of the Nematode Nippostrongylus brasiliensis ………………………３６１‐３６４
Haruki, K., Kobayashi, F., Fujino, T., Matsui, T. and Tsuji, M.
Evaluation of AnaeroPack! (AnP) Type as Tools of Plasmodium falciparum cultivation
and Drug Sensitivity Tests ……………………………………………………………………………３６５‐３７０
Sugiyama, H., Shibahara, T., Ketudat, P., Thaithong, S. and Kawashima, K.
Morphological Re-examination of Paragonimus westermani Described by Daengsvang andOthers in 1964 ………………………………………………………………………………………３７１‐３７４Sugiyama, H., Lai, K., Ow-Yang, C.K., Blas, B.L. and Kawashima, K.
Surface Ultrastructure of Newly Excysted Metacercariae of Paragonimus westermani from
Malaysia and the Philippines …………………………………………………………………………３７５‐３７８

２
９巻総目次

日 本 熱 帯 医 学 会

日本熱帯医学会表紙!−" ＭＫ 稲 森木 芦塚

December２
０
０
１

日熱医会誌

Jpn. J. Trop.
Med. Hyg.

JAPANESE SOCIETY OF TROPICAL MEDICINE

背は３mm

３
３
５

Jpn. J. Trop. Med. Hyg., Vol.２
９, No．
４,２
０
０１, pp.３
３
５
‐
３
４
２

NYLON MESH FILTRATION TECHNIQUE
FOR QUANTITATIVE DETECTION OFHELMINTH EGGSKIYOSHI MAKIYA
Received April 17, 2001/Accepted September 14, 2001

Abstract: Description was given on the material and procedure of the nylon mesh filtration technique for quantitative detection of helminth eggs in the feces, and comparison was made between the filtration and the Kato-Katz
techniques on the clearness and preservation of detected eggs and the efficiency of detection. In the filtration technique, fecal matter is filtered through nylon mesh to concentrate the eggs and the eggs are sealed in the mounting
medium by melting the mesh fibers. This technique can detect all the helminth eggs including hook worm andsmall Clonorchis eggs that are difficult to be recognized by the Kato-Katz technique. The rate of coincidence ofthe positive and negative slides of Schistosoma eggs between the two techniques was 88.7%, being statisticallyhigh. The filtration technique can detect 2.4 times number of eggs that are collected by the Kato-Katz technique,and the sealed eggs are clear enough to identify and can be preserved for months. This filtration technique, simple
and suitable for field use, can contribute to find the helminth infections and to evaluate the control work even in developing countries.
Key words: Nylon mesh filtration technique, Kato-Katz technique, Helminth egg, Schistosoma japonicum, Ascaris
lumbricoides, China

INTRODUCTION
The widely used techniques for the detection ofhelminth eggs are classified into 1) direct smear, 2) flotationand 3) sedimentation techniques (Garcia and Ash, 1979;
Ash and Orihel, 1987).
The direct smear technique (WHO, 1994a) is a simple
procedure that prepares the fecal specimens by mixing asmall amount (about 2 mg) of fecal material with a drop ofphysiological saline or tap water. This technique is easy touse, but is not good for the quantitative detection ofhelminth eggs. More fecal material (60-70 mg) can be processed in the original Kato thick smear technique (Kato andMiura, 1954; Zaman and Keong, 1982), in which a cellophane strip soaked in glycerol and malachite green is placedon a sample of feces and the preparation is left until theglycerol has cleared the fecal material to enable helminth
eggs to be seen. The Kato-Katz technique (WHO, 1994c),
one of modifications of the original Kato thick smear technique, is widely used particularly in schistosomiasis control
work. The feces are first sieved through the screen to remove large fecal particles and fill the hole of plastic template to measure amount of feces (20-50 mg). This fecal
matter is covered with the cellophane strip soaked in glyc-

erol‐malachite green solution and is spread evenly by pressing against another slide glass.
Two types of concentration techniques (flotation andsedimentation) were developed to separate helminth eggs
from excess fecal debris through differences in specificgravity. The flotation technique (Ash and Orihel, 1987)
concentrates certain helminth eggs using a liquid with ahigh specific gravity such as saturated sodium chloride(Cheesbrouth, 1987), or zinc sulphate solutions (Ash andOrihel, 1987; Zaman and Keong, 1982). Feces are emulsified in the solution and the suspension is left undisturbed
for the eggs to float to the surface, where they can be collected with an inverted cover glass. This technique is usefulfor light eggs such as hookworm and fertile Ascaris eggs
but not good for heavy eggs like large trematode, sometapeworm and infertile Ascaris eggs.
The sedimentation technique is a procedure for concentrating helminth eggs by sedimenting the eggs under
their own weight or by centrifuging. The formalin detergent technique (Cheesbrough, 1987) is a simple sedimentation technique, which uses solution of low specific gravity
for sedimenting helminth eggs without centrifuge or facilities. Unfortunately, however, this technique is time‐consuming, taking one hour for heavy eggs or overnight for

Department of Parasitology and Tropical Public Health, University of Occupational and Environmental Health, 1-1 Iseiga-oka,
Yahatanishi-ku, Kitakyushu 807-8555, Japan３
３
６

lighter eggs to sediment. Formalin (formol) ether technique(WHO, 1994b) is recommended as the best overall technique for concentrating helminth eggs, in which fecal debris
and fat are separated by ether after centrifuging the fecal
suspension. This technique, however, is suitable for laboratory work with good facilities rather than for field workwithout electric power.
Among the above‐mentioned techniques, the Kato‐
Katz technique is used for field work to detect mainly schistosome eggs, but the specimens become not so clear enoughto detect thin‐shelled hookworm and small trematode eggs.
Moreover, the specimens are messy and smell bad particularly while examining under the microscope. The nylon
mesh filtration technique was devised to improve these defects and to detect easily and quantitatively all the helminth
eggs in the feces. The present paper describes the procedures of this nylon mesh filtration technique (abbreviated“filtration technique” hereinafter) and evaluates the technique based on the comparison with the Kato‐Katz technique.

MATERIALS AND METHODS
A. Description of the filtration technique
1) Materials and reagents
1. Nylon mesh disk (prepared by punching nylon meshsheet, N-NO. 380T, NBC Kogyo, Tokyo, Japan using
leather punch): diameter 25 mm, square opening size
32 µm (length of the diagonal: 45 µm) for Sch. japonicum eggs.
2. Membrane filter adapter: for 25 mm membrane filter(Swinex W/O Filter, Cat. No. SX0002500, Millipore
Co., Bedford, MA 01730, USA).
3. Plastic syringe: 10 ml (Terumo, Cat. No. SS-10S2138,
without needle; reusable after washing and lubricating
inside with detergent).
4. Applicator stick: 70 mm long×8 mm wide×3 mm
thick polypropylene spatura with sharp edges (ordinary
wooden matchstick or tooth pick can be used).
5. Organdy screen cloth: 12×12 cm (approximately 100
mesh, hole size 160-200 µm, Spark Tufter, Dandic No.
3023, Uno Co., Japan).
6. Plastic template: 24 mm long×15 mm wide×3 mm
thick polypropylene template with a hole of 8 mm diameter bored at 1/3 distance from the template edge
{an average of 206 mg feces (N=73, SD=29 mg) canbe delivered when the hole is filled completely}.
7. Microscope slide glass: 75×25 mm.
8. Microscope cover slip: 32×24 mm Cut piece (50×24mm) of OPP (oriented polypropylene, Sekisei Co. Ltd.
Cat. No. AZ-575L, Tokyo, Japan) is more convenient

than the ordinary microscope cover glass.
9. Roll film case: ordinary marketed size of 5 cm high, 3
cm in diameter (volume ca. 35 ml ).
10. Forceps.
11. Chloral hydrate solution (prepared by adding 35 g ofchloral hydrate, 2 ml of glycerol and 0.20 ml of saturated eosin solution to 5 ml of distilled water andmixing well).
12. 0.5% Tween 80 solution.
13. Used newspaper.
2) Procedure
1. Place a small mound of feces and a plastic template on
used newspaper.
2. Collect some amount of feces with an applicator stick
and fill the template hole completely. Remove excess
feces from the edge of the hole with the applicator
stick (Fig. 1-a).
3. Pour about 10 ml of 0.5% Tween 80 solution or water
into a film case (one fourth of the film case volume
measures about 10 ml ).
4. Put the template with feces in the film case (Fig. 1-b)
and close the cap of film case tightly.
5. Shake vigorously the film case lengthwise (Fig. 1-c) sothat material is mixed with 0.5% Tween 80 solution orwater to become uniform emulsion (Twenty or moretimes of shaking is necessary for ordinary fecal samples. If the fecal matter is too viscous to mix, mash itwith an spplicator stick).
6. Open the film case and take the template out of thecase.
7. Cover the film case with a piece of Organdy screen sothat the center of the screen comes to the mouth of thefilm case (Fig. 1-d).
8. Push the Organdy screen with a syringe up to the bottom of the film case so that tip of the syringe can reachevery corner of the bottom (Fig. 1-e).
9. Shake the film case sideways and suck up quickly all
the emulsion with the syringe. It is easy to suck up all
the emulsion by slanting and rotating the film caseslowly while sucking so that the debris do not stuff
mouth of the syringe (Fig. 1-f).
10. Place a piece of nylon mesh disk on the membranefilter adapter and close adapter cover tightly (Do not
use the gasket so that the excess air can leak) (Fig. 1g).
11. Connect the syringe to the adapter and filter the fecal
emulsion through the nylon mesh disk (Fig. 1-h).
12. If time is enough, pour about 10 ml of tap water into
the film case and suck up all the fecal emulsion again
in the same manner as the step 9.
13. Pass air through the mesh disk to drive out excess

３
３
７

a

f

b

g

c

h

d

i

e

j

a The hole of a template is filled with feces with an applicator.
b The template is put in a film case with feces.
c Fecal material is separated and mixed to become emulsion by shaking the film case.
d The film case is covered with Organdy screen.
e The Organdy screen is pushed by a syringe up to the bottom of the film case.
f The fecal emulsion is sucked through the Organdy screen.
g A nylon mesh disk is put on the membrane filter adapter.
h The fecal emulsion is filtered through the nylon mesh.
i Mounting medium is dropped onto the mesh disk.
j Nylon mesh disk is covered with a cover slip.
Figure 1 Procedure of the nylon mesh filtration technique.

３
３
８

water from the disk.
14. Open the membrane filter adapter and shift the nylon
mesh disk onto a slide glass by using forceps.
15. Drop enough amount {10 drops with Pasteur pipette(=ca. 0.4 ml ) or more} of the chloral hydrate solution
onto the mesh disk (Fig. 1-i) and spread the solution
together with debris all over the mesh disk.
16. Cover the nylon mesh disk with a cover slip carefully
so that the mesh disk comes to the center of the slide
glass (Fig. 1-j).
3) Comments and options
Membrane filter adapter, plastic syringe, plastic template, applicator stick and roll film case can be reused after
washing completely to avoid contamination. When necessary to disinfect the fecal materials, formalin is added to
0.5% Tween 80 solution or water to reach 1-5% formalinconcentration.
It takes about 4 minutes on average to prepare one
slide by beginners using this technique. In order to minimize time, the step 12 in the procedure can be skipped. The
nylon mesh disk begins to melt soon and disappears under
the cover slip after the specimen is prepared. The helminth
eggs become visible clearly after nylon fiber disappears in a
few minutes. Even if the nylon mesh cannot melt completely, the helminth eggs can be detected and identified byincreasing the light intensity and by forcusing appropriately.
The specimen can be preserved for months under room temperature.
The specimens should be kept on the horizontal surface for about one day to dry the mounting medium sufficiently. Delicate care is necessary not to allow the excess
medium flow out from the slide glass especially when thespecimens are carried on the rough road. Nylon mesh withdifferent opening size (20, 25, 32, 37, 42, 48, 53, 59 µm) isavailable commercially for targeted egg sizes of helminths.
Some weak or premature eggs of Ascaris lumbricoides
may slightly be distorted in the chloral hydrate solution, butthere is no practical problem for detecting and identifying
the eggs. In order to avoid the distortion, another chloral
hydrate solution (a mixture of 8 g of chloral hydrate, 5 mlof glycerol, 10 ml of m-cresol, 10 drops of saturated eosin
solution and 5 ml of distilled water) can be used. Chloral
hydrate solution has slightly acid odor and attention shouldbe paid to dry the mounting medium sufficiently before examination.
B. Comparison between the filtration and the Kato-Katz
techniques
A comparison was designed between the filtration and
the Kato‐Katz techniques by examining the fecal specimens
taken from the same fecal samples. This field experiment

was carried out in November 1999 in Gaofeng Village, an
endemic area of schistosomiasis japonica in Ruichang City,
Jiangxi Province, People’s Republic of China. One set ofthe some 100 fecal slides prepared by the filtration technique was brought to Japan and helminth eggs were identified and counted carefully under a microscope. Another setof the 100 slides prepared by the Kato‐Katz technique were
examined by the microscopists of the Jiangxi Provincial Institute of Parasitic Diseases (JPIPD). Two sets of the fielddata from the different techniques were analyzed statistically using VisualStat, Statistica 5j (StatSoft Inc.).

RESULTS
1. Comparison of the clearness and preservation ofhelminth eggs in the specimen between the filtrationand the Kato‐Katz techniques
Photographs 1-a and 1-b are Sch. japonicum eggs detected by the filtration and the Kato-Katz techniques, respectively. As can be seen in the photographs, Sch. japonicum egg is much clearer in the slide made by the filtration
than that by the Kato-Katz techniques. Anterior part andoutline of a miracidium are visible within the egg shell in
the former specimen.
Photographs 2-a and 2-b are Asc. lumbricoides eggs
detected by the filtration technique and the Kato-Katz technique, respectively. Asc. lumbricoides eggs can easily be
differentiated from Sch. japonicum eggs in the filtration
preparation by clear egg shell stained with eosin.
Photograph 3-a and 3-b are Trichuris trichiura eggs
detected by the filtration techniques and the Kato-Katz, respectively. The egg can be seen more clearly by the former
than the latter technique.
Photograph 4 is a hookworm (Ancylostoma sp.) egg
detected by the filtration technique. This kind of thinshelled and transparent egg is easily missed or unrecognizable in the Kato-Katz specimen. Photograph 5 is a Clonorchis sinensis egg detected by the filtration technique. Small
eggs like this trematode egg are very difficult to find out by
the Kato‐Katz technique but easily recognized by the filtration technique.
The helminth eggs in the filtration technique are sealedwith nylon coat which is melted by chloral hydrate or m‐cresol in the above‐mentioned mounting mediums. Thesenylon‐laminated eggs can be preserved for longer time thanthose in the Kato‐Katz technique. Most of the eggs were
preserved for months and clear enough to identify.
2. Comparison of the efficiency to detect helminth eggs
between the filtration and the Kato‐Katz techniques
It was elucidated by the filtration technique that 36(37.1%) of 97 slides were positive for one species, 4 slides

３
３
９

Photo. 1-a

Photo. 1-b

Photo. 2-a

Photo. 2-b

Photo. 3-a

Photo. 3-b

Photo. 4

Photo. 5

Photo. 1-a Schistosoma japonicum egg detected by the filtration technique.
Photo. 1-b Sch. japonicum egg detected by the Kato-Katz technique.
Photo. 2-a Ascaris lumbricoides egg detected by the filtration technique.
Photo. 2-b Asc. lumbricoides eggs detected by the Kato-Katz technique.
Photo. 3-a Trichuris trichiura egg detected by filtration technique.
Photo. 3-b Tri. trichiura egg detected by the Kato-Katz technique.
Photo. 4 Hookworm (Ancylostoma sp.) egg detected by the filtration technique.
Photo. 5 Clonorchis sinensis egg detected by the filtration technique.

３４０(4.1%) for two species and 3 slides (3.1%) for 3 species ofhelminth eggs. Among these, 33 slides (34.0%) were positive for Sch. japonicum, 7 (7.2%) for Asc. lumbricoides, 3(3.1%) for Trichostrongylus sp. and one each (4.1% in total) for Trichuris trichiura, Ancylostoma sp., Clonorchis
sinensis and Taenia sp., respectively. The infection ratewith Sch. japonicum was higher by the filtration than by
the Kato-Katz techniques, being 34.0% (33/97) against
32.0% (31/97). No Ancylostoma, Trichostrongylus andClonorchis eggs were detected by the Kato-Katz technique.
Among all the 97 slides examined for egg of Sch. japonicum, 27 slides were positive and 59 negative in bothtechniques, 7 were positive by the filtration but negative by
the Kato-Katz techniques and 4 were negative by the filtration but positive by the Kato-Katz techniques. As a results,
the rate of coincidence between the two techniques was calculated as 88.7% {(27+59)/97×100}, and the McNemar
test showed that this rate was significantly high level of coincidence (observed χ２＝0.3636, p=0.5465).
Figure 2 shows the relationship of number of eggs per
slide between the Kato-Katz (x) and the filtration (y) techniques. As shown by a high value of coefficient of correlation (r=0.979, p<0.01) based on sufficient number of the fecal samples (n=97), a definite correlation is expected between the two techniques. It is especially noteworthy thatthe regression line of y on x, y=2.406x-0.789, lies far higher
than a line, y=x, along which the number of eggs are expected to be equivalent to each other between the two techniques. It means that the filtration technique is far sensitivein the sense of collecting more eggs than the Kato‐Katz
technique.
It is possible to convert the number of eggs per sample
from the Kato‐Katz to the filtration techniques using theabove regression equation. For example, a number of 239.8(95% confidence interval: 229.8‐249.8) eggs were expectedby the filtration technique against 100 eggs by the Kato‐
Katz technique from the identical fecal sample (Fig. 2)

Figure 2

Relationship of number of Schistosoma eggs
per slide between the Kato-Katz (x) and thefiltration techniques (y).

In like manner, the number of eggs is estimated by another regression line of x on y, x=0.398y+0.454, from thefiltration to the Kato-Katz techniques. As a result of calculation, a total of 200 eggs by the filtration technique correspond to about 80.1 (95% confidence interval: 76.8‐83.4)
eggs by the Kato-Katz technique.
The density of egg (eggs per gram: EPG) can be calculated by dividing the total number of eggs by the mean
weight of feces (200 mg) in case of the filtration technique.
The egg density is important parameter for estimating theworm burden of patients and for evaluating the morbidity
rate of a population.

DISCUSSION
Besides above-mentioned parasitological techniques
for the detection of helminth eggs, some other techniques
have been developed especially for detecting Schistosoma
eggs in feces of human or domestic animals, i.e. Bell filtration (Bell, 1963), Visser filter (Visser and Pitchford, 1972)
and DCEP (Danish Center for Experimental Parasitology)(Willingham et al., 1998) techniques. All of them, however,
have the disadvantage that more equipment is required thanthe Kato-Katz for making specimens in the field (Jordan etal., 1981; Shutte et al., 1994; Willingham et al., 1998).
According to an extensive epidemiological study(Ebrahim et al., 1997), prevalence estimates and mean egg
counts revealed the distinct superiority of the Kato-Katz
technique (three to four smears) compared with the formol
ether sedimentation. They explained this superiority by thesieving effect, in which the quantity of stool in the KatoKatz technique was measured after the removal of all fibrous and other coarse material. The paradox of low egg
count and the diagnostic capability of the formol ether technique was explained by them as follows: Eggs may be lostin the procedural steps of the sedimentation technique despite the fact that more feces is processed (1 g comparedwith 167 mg/four Kato-Katz smears) and the eggs are easier
to distinguish than on Kato‐Katz smear.
However, the following disadvantages were pointed
out for the Kato-Katz technique: 1) it is “messy”, 2) theeggs are obscured by the thickness of the preparation (Jordan et al., 1981), 3) three or four more smears are necessary
to detect light infections (Sleigh et al., 1982; Ebrahim et al.,1997). The present study introduced a new simple techniqueto improve above-mentioned defects of the Kato-Katz technique. This method can process more (some 200 mg) fecal
material than four Kato‐Katz smears and eggs are easier todetect than on the Kato‐Katz preparation.
In addition to common eggs detected in the endemic
area of Sch. japonicum in China, the filtration technique

３
４
１

was applied to some other eggs of human and domestic animals using the same or different opening size of nylon mesh.
Successful results were obtained for human helminths suchas cestodes (Diphyllobothrium latum, Taenia saginata andEchinococcus multilocularis) and trematodes (Sch. mansoni, Fasciola hepatica, Paragonimus westermani andMetagonimus yokogawai), and for helminths of domestic
animals such as nematodes (Asc. suum, Toxocara canis,Tox. cati, Tox. vulpis, Tri. suis and Ancylostoma caninum).
The filtration technique was applied to analyze the relationship between the prevalence of hepatic milk spots in
pig and the egg density of Asc. suum. As a result, a significant correlation was observed between the average egg density and the group prevalence rate of the hepatic milk spots
in pig populations. This result proved that this technique
can be used as an effective inspection method for detecting
milk spot liver in pig (Kano and Makiya, 2001).
As previously mentioned, the flotation technique is
useful for some light eggs but not good for heavy eggs likelarge trematode and some tapeworm eggs. The sedimentation technique is time-consuming or not suitable for fieldwork without electric power. The filtration technique is not
only suitable for all kinds of helminth eggs in feces but also
for urine-originated eggs like Sch. hematobium, in whichsmall aliquot (e.g., 10 ml ) of urine sample is enough to prepare a slide specimen.
The filtration technique can detect small fecal particles
other than helminth eggs, for example, immature nematodelarvae such as Strongyloides stercoralis, house dust mites
like Dermatophagoides spp. and their eggs and many kindsof pollens. Actually, a total of 9 mite eggs were detectedfrom 2 fecal samples of the same villagers in the present
study. A modification of this filtration technique was utilized for collecting house dust mites and their eggs and juveniles (Makiya, 1999).
The plastic template used in the present study is 3 mm
thick with a hole 8 mm in diameter, giving the capacity of
151 mm3. The capacity of the hole can be increased up to
290 mm3 by using template 4 mm thick with a hole 9.6 mm
in diameter. A regression line, y=1.528x+1.447 was obtained between the number of eggs collected by the bigger
hole (y) and that by the smaller hole (x) in a preliminary experiment (n=14, r=0.977, p<0.01). This means that some
1.5 times as many as Sch. japonicum eggs were collectedby the template with a bigger hole than by that with asmaller hole. As a result, it is expected that some 3.7 times(=2.406×1.528) more Schistosoma eggs can be collectedby the filtration than by the Kato-Katz techniques using thisbigger hold template.
The detected eggs are clear enough to recognize andidentify despite the fact that the fecal matter is almost dou-bled in the bigger hole. This new template is recommended
to use in order to increase the detection efficiency when theegg density of Schistosoma decreased after control measurein future.
In this technique, cut piece (50×24 mm) of OPP (oriented polypropylene) sheet is recommended instead ofcover glass to cover the fecal specimen. This cheap material is plastic and light (specific gravity ca. 0.9, weight 64.8
mg/piece against 280 mg/piece of cover glass), and suitable
for adding more mounting medium afterwards if the fecal
specimen dries. The nylon mesh disk, an important material for the filtration technique, costs only one-tenth (ca. 20
Japanese Yen or 0.16 US $/sheet) of membrane filter that isoften used for the same purpose.
It can be concluded from these facts that the filtration
technique is “all‐inclusive” and cheap technique suitablefor detecting all the helminth eggs in the field even where
good facilities are not available. Considering the background that most of helminth infections are distributed indeveloping countries with financial problem, this filtration
technique can contribute to find helminth infections andevaluate the control work in such areas.

ACKNOWLEDGMENTS
The author wishes to express his sincere gratitude to
Dr. Chan Shaoji, former Director of Jiangxi Provincial Institute of Parasitic Diseases (JPIPD) and Dr. Lin Dandan,
Department of Epidemiology, JPIPD, for their generous
welcome and kind cooperation.
The author also thanks Dr. Hidenori Murakami, Chiefof Aid for Study of Schistosomiasis in China and Asia
(ASSCA), for his kind permission to join ASSCA project inChina.
The author is also indebted to Messrs. Hideaki Ito, Tomohisa Nagata, Nobuaki Sakamoto, Shoichi Shimizu andNaoya Miyamoto, Misses Yoko Hanaoka and Yuko Tanaka,
medical students of University of Occupational and Environmental Health, and Mr. Shigeaki Kano, Kitakyushu Municipal Meat Inspection and Control Center, for their technical assistance in the field or laboratory.

REFERENCES
１）Ash, L.R. and Orihel, T.C. (1987): Examination of freshand preserved feces. In “Parasite: A guide to laboratory
procedures and identification”, 15-35, American Societyof Clinical Pathologists Press, Chicago.
２）Bell, D.R. (1963): A new method for counting S. mansoni
eggs in faeces. Bull. W.H.O., 29, 525-530
３）Cheesbrough, M. (1987): Techniques used to identify

３
４２

parasites. In “Medical laboratory manual for tropicalcountries”, 178-198, Eng. Lang. Book Soc., Butterworths.
４）Ebrahim, A., El-Morshedy, H., Omer, E., El-Daly, S. andBarakat, R. (1997): Evaluation of the Kato-Katz smear
and formol ether sedimentation techniques for quantitative diagnosis of Schistosoma masoni infection. Am. J.
Trop. Med. Hyg., 57, 706-708
５）Garcia, L.S. and Ash, L.R. (1979): Macroscopic and microscopic examination of fecal specimens. In “Diagnostic parasitology, clinical laboratory manual”, 9-18, The C.
V. Mosby Co. St. Louis.
６）Jordan, P., Bartholomew, R.K. and Peters, P.A.S. (1981):
A community study of Schistosoma mansoni egg excretion assessed by the Bell and a modified Kato technique.
Ann. Trop. Med. Parasitol., 75, 35-40
７）Kano, S. and Makiya, K. (2001): Relationship between
the prevalence of hepatic milk spots in pig and the egg
density of Ascaris suum in Kitakyusyu Meat Inspection
and Control Center (in Japanese with English summary).
J. Univ. Occupat. Enviroment. Hlth., 23, 255-262
８）Kato, K. and Miura, M. (1954): Comparative examinations (in Japanese). Parasitol., 3, 35
９）Makiya, K. (1999): Monitoring and its evaluation (inJapanese). In “Sanitary management of rats and medically important insects (Series of Environmental andSanitary Management Technology)”, 268-280, Fuji Technosystem, Tokyo.
１０）Shutte, C.H.J., Pienaar, R., Becker, P.J., Viljoen, E.,
Growther, G., Van Deventer, J.M.G. and Markus, N.B.

(1994): Observations on the techniques used in the qualitative and quantitative diagnosis of schistosomiasis. Trop.
Med. Parasitol., 3, 305-316
１１）Sleigh, A., Hoff, R., Mott, K., Barreto, M., De Paiva, T,
Pedrosa, J.S. and Sherlock, I. (1982): Comparison of filtration staining (Bell) and thick smear (Kato) for the detection and quantitation of Schistosoma masoni eggs infaeces. Trans. Roy. Soc. Trop. Med. Hyg., 76, 403-406
１２）Visser, P.S. and Pitchford, R.J. (1972): A simple apparatus for rapid recovery of helminth eggs from excreta withspecial reference to Schistosoma mansoni. South African
Med. J., 46, 1344-1346
１３）WHO (1994a): Direct fecal smears - saline and iodine wet
mount preparations. In “Bench aids for the diagnosis ofintestinal parasites”, Plate l, Geneva.
１４）WHO (1994b): Fecal concentration procedure - formalinether/ethyl acetate/gasolin. In “Bench aids for the diagnosis of intestinal parasites”, Plate 2, Geneva.
１５）WHO (1994c): Kato-Katz technique - cellophane fecal
thick smear. In “Bench aids for the diagnosis of intestinal parasites”, Plate 3, Geneva.
１６）Willingham, A.L., Johansen, M.V. and Barnes, E.H.
(1998): A new technic for counting Schistosoma japonicum eggs in pig feces. Southeast Asian J. Trop. Med.
Public Health, 29, 128-130１７）Zaman, V. and Keong, L.A. (1982): Basic diagnostic techniques. In “Handbook of Medical Parasitology”, 195215, AIDS Health Science Press, New York.

３
４
３

Jpn. J. Trop. Med. Hyg., Vol.２
９, No．
４,２
０
０１, pp.３
４
３
‐
３
４
８

EFFECTS OF MINOCYCLINE AGAINST MEFLOQUINE-,
CHLOROQUINE- AND PYRIMETHAMINERESISTANT PLASMODIUM FALCIPARUM IN VITROQINGHUA LIN1, KEN KATAKURA1, MIHO OUE1,SHIGEYUKI KANO2 AND MAMORU SUZUKI1
Received May 8, 2001/Accepted November 14, 2001

Abstract: Tetracyclines are used for prophylaxis of malaria and treatment of drug-resistant falciparum malaria because of their safe drug action. We re-evaluated effects of three tetracyclines against drug-resistant Plasmodium
falciparum in vitro. Minocycline was approximately 4 times and twice more potent in inhibiting the in vitro
growth of falciparum parasites than tetracycline and doxycycline, respectively. Compared with doxycycline, significant inhibitory effects of minocycline to chloroquine, pyrimethamine and mefloquine resistant P. falciparumstrains were affirmed by the present in vitro study. By electron microscopy a number of electron dense vesicles
with a single membrane bound were observed in the cytoplasm of minocycline-treated parasites, although no distinct structural alternations of mitochondria was noted. Minocycline may be a better therapeutic drug than doxycycline which is widely accepted as the standard antimalarial tetracycline.
Key words: Plasmodium falciparum, Drug-resistance, Electron microscopy, Tetracyline, Minocycline,
Chloroquine, Mefloquine, Pyrimethamine

INTRODUCTION
Chemotherapy is the primary defense against malaria.
Therefore, the spread of drug resistant Plasmodium falciparum is a world-wide threat. Development of new antimalarials is one of the major goals of malaria research. However,development and deployment of a new drug is extremely
expensive, causing discouragement for pharmaceuticalcompanies to search for new therapeutic agents. This leads
to re-evaluation of antimalarial activities of any drugs whichhave already been accepted for clinical use in patients withvarious infectious diseases other than malaria. Antibiotics
are one of the major sources for such trials. Amongst commonly used antibiotics, a group of tetracyclines has been
most widely used for treatment of malaria (Puri and Dutta,
1982). The effect of chlortetracycline against a malaria
parasite was first reported by Coatney (Coatney et al.,1949). Since then, tetracyclines have been tried in malaria
treatment (Pang et al., 1987; Looareesuwan et al., 1992;
Rieckman et al., 1971). The conventional tetracycline is a
short acting agent (plasma half-life: 8.5 hr), while doxycy-

cline and minocycline have relatively long half-lives(plasma half-life: 18-22 hr and 12-16 hr, respectively (Dollery, 1999), permitting better selection of these tetracyclines
for malaria treatment than tetracycline. Doxycycline in particular, is accepted as an antimalarial for chemotherapy aswell as chemoprophylaxis (Colwell et al., 1972; Shanks etal., 1992). The present study focused on the potency of theantimalarial activity of minocycline to cultured drugresistant P. falciparum parasites in comparison with tetracycline and doxycycline. We show here the first report that
minocycline was effective against mefloquine-resistant falciparum parasites in vitro. The presumed molecular mechanisms of tetracyclines and the reason for the highest potency shown by minocycline are discussed.

MATERIALS AND METHODSParasites
Two established strains and two recent clinical isolatesof P. falciparum were used in the present study (Table 1).
SGE-1 is a drug sensitive strain of Gambian origin, donated1 Department of Parasitology, Gunma University School of Medicine, Maebashi 371-8511, Japan
2 Research Institute, International Medical Center of Japan, Tokyo 162-8655, Japan
Corresponding author: Mamoru Suzuki, Department of Parasitology, Gunma University School of Medicine, 3-39-22 Showa-machi,
Maebashi 371‐8511, Japan
Tel: 027‐220‐8020 Fax: 027‐220‐8025
E‐mail: suzuki@med.gunma‐u.ac.jp

３
４
４

by Dr. P. Ambroise‐Thomas of the University of the Grenoble in 1979, and has been maintained by in vitro culture alternating occasional freezing in liquid nitrogen in our laboratory. Regardless of long-term cultivation, the parasite has
maintained its virulence, causing fatal sickness in Aotus
monkeys. K-1 is a chloroquine-resistant strain isolated in
Thailand, which was donated by the London School of Hygiene and Tropical Medicine in 1984. One isolate, MZG, isa chloroquine-sensitive but mefloquine-resistant parasite
isolated from a Japanese patient who developed falciparum
malaria after returning from Mozambique in 1998. Anotherisolate NGG is a moderate chloroquine-resistant and highly
pyrimethamine-resistant parasite obtained from a Japanesepatient who also developed falciparum malaria after visiting
Nigeria in 1997.
Cultivation of P. falciparum parasites and in vitro drug susceptibility test
Culture of falciparum parasites was carried out according to a modified method of Trager and Jensen (Trager andJensen, 1976), using RPMI 1640 (Nissui PharmaceuticalCo., Ltd., Japan) medium with 10% human serum {RPMI
1640 (+)} and type O human red blood cells. The drug susceptibility test was performed by a semi-micro method, described previously (Bras and Deloron, 1983; Inaba et al.,2001). Parasites were synchronized by D-sorbitol treatment(Lambros and Vanderberg, 1979), and parasitized erythrocytes with a 0.15-0.3% infection rate were adjusted to a 5%
packed cell volume in RPMI 1640 (+) at the start of incubation. The test was done using 24-well plates (Falcon, 3047,
Becton Dickinson, NJ, USA) and each well contained 500µl of parasitized erythrocyte suspension and 20 µl of drug
solution containing the respective concentrations of eachdrug. Final drug concentrations of tetracyclines were 0,0.03, 0.1, 0.3, 1, 3, 10, 30, 100 and 300 µg/ml . The incubator was kept at 37°
C continuously with a gas flow mixture
composed of 5% O2, 5% CO2 and 90% N2. The medium
was changed every day and the cultivation was continued
for up to 4 days (Divo et al., 1985; Geary and Jensen, 1983;

Inaba et al., 2001). The effects of drugs on the growth ofthe parasites were expressed by the 50% inhibitory concentrations (IC50), which were calculated by computerized probit analysis.
Drugs used and the sources were as follows: tetracycline hydrochloride and minocycline hydrochloride from
Lederle Ltd., Japan; doxycycline from Sigma Chemical Co.,
USA; chloroquine sulfate from Winthrop Stearns Inc., Manila, Philippines; mefloquine from Roche Diagnostics,
Switzerland; pyrimethamine from Wako Pure Chemical Industries, Ltd., Japan.
Electron microscopy
Erythrocytes infected with SGE-1 parasites weretreated with tetracycline at 1.0 µg/ml and minocycline at
0.3 µg/ml , approximately at the IC50 values described in the
text. Blood samples were taken every 24 hr after drug exposure and fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) at 4°
C for 2 hr (Kawai et al., 1996).
The specimens were postfixed in 1% osmium tetroxide foranother 2 hr, dehydrated in a graded alcohol series, treatedwith propylene oxide, and embedded in Epon 812. The resultant blocks were cut with a Porter Blum (Newton, CT)
MT-2 ultramicrotome with a Diatome (Bienne, Switzerland)diamond knife. Thin sectioned specimens were mounted on
200-mesh copper grids, stained with uranyl acetate plus
lead citrate. The prepared specimens were observed using atransmission electron microscope (Hitachi, H-800, Tokyo,
Japan).

RESULTS
Drug resistant profiles of P. falciparum parasites used in
the present study
Drug susceptibilities of three P. falciparum parasites
were examined by a semi-micro in vitro drug susceptibility
test, in comparison with SGE-1 as a standard strain (Table1). The IC50 of K-1 for chloroquine was 4.4-fold higher
than that of the SGE-1 strain. The K-1 strain also showed

Table 1 Drug susceptibility of P. falciparum isolates
Strain
SGE-1
K-1
MZG
NGG

Chloroquine (µM)
0.086±0.002
0.378±0.019
0.040±0.001
0.22±0.017

IC＊50
Mefloquine (nM)
13.97±2.6412.78±3.85248.3±50.17
4.88±0.89

Pyrimethamine (nM)69.1±7.93
126±10.86
75.8±8.52
2,774±109.4* Values are mean±SD for three experiments
SGE-1 was used as a standard P. falciparum strain which showed susceptibilities to chloroquine, mefloquine and pyrimethamine. K-1, MZG and NGG strains showed selective resistance to chloroquine, mefloquine and pyrimethamine respectively.

３
４
５

moderate resistance to pyrimethamine. The clinical isolate,
MZG of Mozambique origin, showed a 17.8-fold higher
IC50 for mefloquine. Another clinical isolate, NGG of Nigerian origin, was highly resistant to pyrimethamine with a
40-fold higher IC50 than that of the SGE-1 strain. Thesefindings confirmed that all strains and isolates of falciparum
parasites used in the present study had maintained their stable phenotypes in terms of original drug sensitivities to eachantimalarial drug.
Comparison of effects of three tetracyclines on the growthof P. falciparum parasites in vitro
We compared the inhibitory effects of tetracycline,
doxycycline and minocycline on the standard SGE-1 strain
by determining the IC50 values at 24, 48, 72 and 96 hr after
drug exposure. The antimalarial activities of all three tetracyclines were enhanced when drug exposure was increased
from 24 to 96 hr (Table 2), as reported previously (Divo etal., 1985; Geary and Jensen, 1983). However, the inhibitory effect of minocycline was the most potent at each time

point examined (Table 2). The IC50 value for minocycline at
24 hr was 3.90 µg/ml , while those for doxycycline and tetracyline were 10.11 and 17.20 µg/ml , respectively. At 96
hr the IC50 for minocycline (0.24 µg/ml ) was 2.1-fold lessthan that for doxycycline (0.50 µg/ml ) and 5.1-fold less fortetracycline (1.23 µg/ml ). Overall, the mean IC50 value atdifferent time points for minocycline was approximately 4fold and 2-fold lower than that for tetracycline and doxycycline.
The efficacy of minocycline was further studied using
drug-resistant parasites in comparison with tetracycline.
Growth inhibition of parasites was assayed in the presenceof tetracycline at 1.0 µg/ml or minocycline at 0.3 µg/ml ,approximating the IC50 values, according to the above findings. All falciparum parasites, such as SGE-1, chloroquineresistant K-1, mefloquine-resistant MZG and pyrimethamineresistant NGG, showed substantially similar susceptibilities
to both tetracycline and minocycline (Table 3). The inhibitory effects of these antibiotics were notable at 72 hr with
20-30% growth inhibition. Eventually, the effect reached toTable 2 Comparison of effects of three tetracyclines on the growth of P. falciparum
Drug
Tetracycline(n=5)
Doxycycline(n=2)
Minocycline(n=5)

IC50 (µg/ml ) at time points
48 hr
72 hr

24 hr

96 hr

17.2±1.52

4.36±0.91

2.26±0.38

1.23±0.24

10.11±1.84

2.45±0.14

1.10±0.01

0.50±0.13

3.90±1.60

2.00±0.25

0.85±0.22

0.24±0.01

By in vitro comparative study on tetracyclines on SGE-1 strain, it is remarked that minocycline showed
4.4 times higher anti-malarial activity than that shown by doxycycline at 24 hr test. It is notable that minocycline shows inhibitory effect to the parasites at the early stage of the test.

Table 3 Inhibitory effects of tetracycline and minocycline on the growth of drugresistant P. falciparum% Inhibition of growth at time points†
48 hr
72 hr
96 hr

Drug*

strain

MC

SGE-1
K-1
MZG
NGG

0
-12
0
7

13
16
14
15

31
28
33
30

68
72
69
65

TC

SGE-1
K-1
MZG
NGG

-9
-6
4
-20

26
2
10
2

31
31
30
17

44
48
40
43

24 hr

＊ TC, tetracycline at 1.0 µg/ml ; MC, minocycline at 0.30 µg/ml .
† Inhibitory effect was determined by comparison of the parasite growth of untreated
and drug-treated parasites in triplicate experiments.
By 24 hr test with low dose tetracyclines, no effect was remarked. Percent growth inhibitory values were equally elevated at high levels at 96 hr test both in the standard strain
and resistant strains. Still, MC effect> TC effect is also noted.

３４
６

the maximum at 96 hr, resulting in 40-48% inhibition by
tetracycline and 65-72% by minocycline.
Ultrastructural changes of parasites treated with tetracyclines
The structural alterations of parasites caused by treatment with tetracycline at 1.0 µg/ml and minocycline at 0.3µg/ml were examined at 24, 48, 72 and 96 hr after treatment. Distinct differences in the structural changes were
not observed between parasites treated with tetracycline andminocycline so far as the present study concerns. Enlargement of the perinuclear cisterna space was noted in thespecimen at 24 hr of tetracycline treatment, compared withuntreated parasites (Fig. 1A, B and C). A dilatation of cisternae of endoplasmic reticulum was also noted at 24 hr and48 hr (data not shown). In parasites exposed to tetracyclines for 72 hr, a number of electron dense vesicles with asingle membrane bound were observed and the cytoplasmic
structure was largely disintegrated (Fig. 1D). However, dis-tinct structural alterations of the organelles, such as mitochondria and plastids, were not noted in the present study.

DISCUSSION
The present study showed that in a group of tetracyclines, minocycline was the most potent for suppressing the
growth of cultured P. falciparum. The IC50 values of tetracyline, doxycycline and minocycline for the P. falciparum
SGE-1 strain were 1.23, 0.50 and 0.24 µg/ml , respectively.
Acute systemic toxicity studies showed that the LD50 valueof these drugs, when orally administered, was 2,000-3,000,
1,700 and 1,900-3,600 mg/kg of body weight, respectively(Dollery, 1999; personal communications from Lederle Japan and Pfizer Japan). When simply comparing the ratio of
the LD50 versus the IC50, the ratio of minocycline was estimated to be 7,900‐15,000, whereas the ratio of tetracycline
was 1,600‐2,400. This finding indicates that minocycline
appears to be 3.3‐9.4‐fold more potent than tetracycline.

Figure l Electron micrograph of P. falciparum treated with tetracyclines. An early trophozoite (A) and schizont (B) are shown as acontrol. Early trophozoites were treated with tetracyclines for 24 hr (C) and 72 hr (D). Dilatations of perinuclear cisterna
space are indicated by arrows. Electron dense vesicles are indicated by arrow heads. N, nucleus; M, mitochondrion; P, pigment. Bar=0.2 µm

３４７The index of minocycline also exceeds that of doxycyclinewith a value of 3,400.
Minocycline eventually showed killing activity for
mefloquine-resistant P. falciparum parasites as well as
chloroquine- and pyrimethamine-resistant parasites, although
this antibiotics required a longer time for an appearance of
the inhibitory effect than other antimalarial drugs. As far as
we know, this is the first report on the effect of minocycline
against mefloquine-resistant P. falciparum in vitro. Thisfinding suggests that minocycline should be further studiedin clinical cases as an excellent candidate for chemotherapyof drug-resistant malaria, even though there were someclinical trials for chloroquine-resistant malaria using thisantibiotics in the early 1970’s (Colwell et al., 1972; Willerson et al., 1972). Tetracycline has already been used totreat drug-resistant falciparum malaria, generally in combination with quinine (Looareesuwan et al., 1992; Puri andDutta, 1982; Rieckman et al., 1971; Watt et al., 1992). Our
recent experiments showed that a combination of minocycline with artemether and/or chloroquine resulted in an excellent therapeutic effect without showing recrudescence inmice infected with P. berghei and chloroquine-resistant P.
chabaudi (manuscript in preparation).
The rapid anti-parasite action of minocycline in vitro
study has also been shown in the present study (Table 2).
The inhibitory action shown at the early stage of drug exposure is a key point to evaluate antimalarials. All these results
support the use of minocycline as a better tetracycline than
doxycycline for the treatment of chloroquine-, pyrimethamineand mefloquine-resistant falciparum malaria, respectively.
The highly inhibitory effects of minocycline on parasite growth shown in the present study may be attributable
to the high lipophilicity of minocycline (Tomas, 1989).
Lipophilic features of this antibiotics may permit the highpermeability through the plasma membrane, mitochondrial
membrane and even plastid membrane. The minocycline
may be more efficiently transported from the serum to theparasites in the erythrocytes than the other tetracyclines because of its lipophilic feature. Mitamura et al. (2000)
showed that serum albumin (SA) derived fatty acid species
in the neutral lipid fraction in the serum plays an crucialrole in the parasite growth at the erythrocytic stage. It appears that SA derived fatty acid passes through the erythrocytic membrane by some mechanism. The finding will suggest a probable role of lipid as the carrier for lipophilic minocycline to pass through erythrocytic memberane andeventually reach to the parasites.
The ultrastructural alterations, such as the dilatation ofperinuclear space and appearance of electron dense single
membrane‐bound vesicles were marked in falciparum parasites treated with tetracyclines in the present study. The

electron dense vesicles were also observed in late trophozoites and schizonts of P. falciparum taken from Aotus
monkeys treated with pyronaridine (Kawai et al., 1996). It
was notable that these alterations in the parasites occurred
before no change came out in mitochondria, which is considered as the target of teracyclines. However, it is difficult,at present, to explain whether the structural changes resulted from the specific action mechanism of tetracyclines.
Further investigations into the morphologic changes focusing on the parasite mitochondria and plastids will be of particular importance in future studies.

ACKNOWLEDGMENT
This study was supported, in part, by a grant for Research on Emerging and Re‐emerging Infectious Diseases(H12‐Shinkou‐17), Ministry of Health and Welfare, andGrant‐in‐Aid for Scientific Research (A) (11307004) from
the Ministry of Education, Science, Sports and Culture, Japan.

REFERENCES
１）Bras, J.L. and Deloron, P. (1983): In vitro study of drug
sensitivity of Plasmodium falciparum: Evaluation of a
new semi-micro test. Am. J. Trop. Med. Hyg., 32, 447451
２）Coatney, G.R., Greenberg, J., Cooper, W.C. and Trembley,
H.L. (1949): Effect of chlortetracycline against Plasmodium gallinaceum. Proc. Soc. Exp. Biol. Med., 72, 586587
３）Colwell, E.J., Hickman, R.J., Intraprasert, L.R. andTirabutan, C. (1972): Minocycline and tetracycline treatment of acute falciparum malaria in Thailand. Am. J.
Trop. Med. Hyg., 21, 144-149
４）Divo, A.A., Geary, T.G. and Jensen, J.B. (1985): Oxygen
and time-dependent effects of antibiotics and selected mitochondrial inhibitors on Plasmodium falciparum in culture. Antimicrob. Agents Chemother., 27, 21-27
５）Dollery, C. (1999): Therapeutic drugs. Churchill Livingstone., London.
６）Geary, T.G. and Jensen, J.B. (1983): Effects of anitibiotics
on Plasmodium falciparum in vitro. Am. J. Trop. Med.
Hyg., 32, 221-225
７）Inaba, H., Ohmae, H., Kano, S., Faarado, L., Boaz, L.,
Leafasia, J. and Suzuki, M. (2001): Variation of incubation time in an in vitro drug susceptibility test of Plasmodium falciparum isolates studied in the Solomon Islands.
Parasitol. Int., 50, 9-13
８）Kawai, S., Kano, S., Chang, C. and Suzuki, M. (1996):
The effects of pyronaridine on the morphology of Plasmodium falciparum in Aotus trivirgatus. Am. J. Trop.
Med. Hyg., 55, 223-229
９）Lambros, C. and Vanderberg, J.P. (1979): Synchronization

３
４
８

of Plasmodium falciparum erythrocytic stages in culture.
J. Parasitol., 65, 418-420１０）Looareesuwan, S., Wilariratana, P., Vanijanonta, S., Kyle,
D. and Webster, K. (1992): Efficacy of quininetetracycline for acute uncomplicated falciparum malaria
in Thailand. Lancet, 339, 369
１１）Mitamura, T., Hanada, K., Ko-Mitamura, E.P., Nishijima,
M. and Horii, T. (2000): Serum factors governing intraerythrocytic development and cell cycle progression of
Plasmodium falciparum. Parasitol. Int., 49, 219-229
１２）Pang, L.W., Limsomwong, N., Boudreau, E.F. andSingharaj, P. (1987): Doxycycline prophylaxis for falciparum malaria. Lancet, i, 1161-1164
１３）Puri, S.K. and Dutta, G.P. (1982): Antibiotics in the chemotherapy of malaria. Prog. Drug Res., 26, 197-205
１４）Rieckman, K.H., Powell, R.D., McNamara, J.V., Willerson, D.J., Kass, L., Frischer, H. and Carson, P.E. (1971):
Effect of tetracycline against chloroquine-resistant andchloroquine-sensitive Plasmodium falciparum. Am. J.
Trop. Med. Hyg., 20, 811-815
１５）Shanks, G.D., Edstein, M.D., Suriyamongkol, V., Tim-saad, S. and Webster, H.K. (1992): Malaria chemoprophylaxis using proguanil/dapsone combinations on theThai-Cambodian border. Am. J. Trop. Med. Hyg., 46,
643-648
１６）Thomas, D.E. (1989): Tetracyclines as antiparasitic
agents: Lipophilic derivatives are highly active against
Giardia lamblia in vitro. Antimicrob. Agents Chemother., 33, 2144-2145
１７）Trager, W. and Jensen, J.B. (1976): Human malaria parasites in continuous culture. Science, 193, 673-675
１８）Watt, G., Loesuttivibool, L., Shanks, G.D., Boudreau, E.
F., Brown, A.E., Pavanand, K., Webster, H.K. and Wechgritaya, S. (1992): Quinine with tetracycline for the treatment of drug-resistant falciparum malaria in Thailand.
Am. J. Trop. Med. Hyg., 47, 108-111
１９）Willerson, D. Jr., Rieckman, K.H., Carson, P.E. andFischer, H. (1972): Effect of minocycline against
chloroquine-resistant falciparum malaria. Am. J. Trop.
Med. Hyg., 21, 857-863

３４９Jpn. J. Trop. Med. Hyg., Vol.２
９, No．
４,２
０
０１, pp.３
４
９
‐
３
５
４

SIMULIUM (SIMULIUM) WEJI SP. NOV.
(DIPTERA: SIMULIIDAE)
FROM THAILAND
HIROYUKI TAKAOKA
Received August 7, 2001/Accepted November 30, 2001

Abstract: Simulium weji sp. nov. is described based on the reared females, reared males, pupae and mature larvae
collected from Tharn Thong Waterfall in Lampang Province, Thailand. This new species is assigned to the tuberosum species-group within the subgenus Simulium s. str., and seems to be closely related to S. rufibasis Brunetti andfour other allied species, reported from India and Thailand, by having a pair of hair clusters on the ventral surfaceof the seventh abdominal segment of the female, which are, though, much longer and fewer in this new species.
This new species seems to be also related to S. nigrifacies Datta from India and S. puliense Takaoka from Taiwan,by having the similar arrangement of six short pupal gill filaments, but is easily differentiated from these known
species by the small cone-shaped tubercles on the head and thoracic integuments.
key words: Black fly, Simuliidae, Simulium, Thailand, New species

Recent investigations on simuliid fauna of Thailand
have increased the total number of black-fly species up to
40 (Takaoka and Saito, 1996; Takaoka and Adler, 1997;
Takaoka and Kuvangkadilok, 1999; Kuvangkadilok andTakaoka, 2000). During our recent surveys of black flies innorthern Thailand we found one more new species belonging to the tuberosum species-group within the subgenus
Simulium (Simulium), defined by Takaoka and Davies(1996).
This new species is here described based on the reared
females, reared males, pupae and mature larvae.
Holotype and most paratype specimens will be deposited at the Natural History Museum (BMNH), London,
U. K.
Simulium (Simulium) weji sp. nov.
DESCRIPTION. Female. Body length ca. 2.3 mm. Head.
Narrower than width of thorax. Frons black, shiny, widelybare except several dark stout hairs along each lateral margin and a few hairs near antennal base; frontal ratio 1.2-1.4:
1.0:1.2; frons-head ratio 1.0:3.7. Fronto-ocular area (Fig.
1) moderately developed, with round lateral tip. Clypeus
black, shiny, widely bare medially except dark stout hairs
along each lateral margin and several dark stout hairs nearlower margin. Proboscis 0.51-0.55× as long as clypeus.
Antenna composed of 2+9 segments, light to dark brown

except scape, pedicel and base of 1st flagellar segment yellow. Maxillary palp brownish black, composed of 5 segments with proportional lengths of 3rd, 4th and 5th segments 1.0:1.3:2.7; 3rd segment (Figs. 2 and 3) of moderate
size; sensory vesicle medium in size, ellipsoidal, with rugged surface, ca. 0.3× length of 3rd segment, with medium
round opening. Maxillary lacinia with 15 or 16 inner and
18 outer teeth. Mandible with ca. 26 inner and 12 outerteeth. Cibarium (Fig. 5) with several minute processes.
Thorax. Scutum blackish brown, shiny, not patterned,
moderately covered with recumbent dark-brown pubescence, interspersed with long upstanding dark hairs on prescutellar area; scutum thinly grey-pruinose when illuminatedin certain angle of light. Scutellum brownish black, withlong and short dark hairs. Postscutellum brownish black,shiny, without hairs. Pleural membrane bare. Katepisternum longer than deep, and bare. Legs. Foreleg: coxa whitish yellow; trochanter and femur dark yellow; tibia lightbrown except apical 1/5 dark brown, and outer surface ofbasal 4/5 pale white and silvery iridescent in certain angleof light; tarsus entirely brownish black; basitarsus, 2nd and3rd tarsal segments dilated (basitarsus ca. 4.9× as long as
its greatest width), with distinct dorsal crest of medium‐long hairs. Midleg: coxa brownish black; trochanter lightbrown with base whitish yellow; femur light brown withapical cap dark brown; tibia light brown except base somewhat yellowish narrowly, with white basal sheen on poste-

Department of Infectious Disease Control, Oita Medical University, Hasama, Oita 879‐5593, Japan３
５
０

rior surface in certain angle of light; tarsus light brown except basal 4/5 of basitarsus, basal 1/3 or 1/2 of 2nd tarsal
segment and base of 3rd tarsal segment whitish yellow, and5th tarsal segment brownish black. Hind leg (Fig. 6): coxa
medium brown; trochanter whitish yellow; femur medium
brown with base whitish yellow and with apical cap darkbrown; tibia dark brown to brownish black except basewhitish yellow, and with large white sheen on posterior surface in certain angle of light; tarsus dark brown except basal
2/3 of basitarsus and basal 1/2 of 2nd tarsal segment whitish
yellow; basitarsus nearly parallel-sided, ca. 5.8× as longas its greatest width, and ca. 0.79× and ca. 0.64× as wide
as hind tibia and femur, respectively; calcipala short, nearly
as long as wide, and ca. 0.4× as wide as greatest width ofbasitarsus; pedisulcus distinct. All tarsal claws simple,
without subbasal or basal tooth. Wing. Length 1.7-1.9mm; costa with spinules and hairs; subcosta haired; basal
section of vein R bare; hair tuft at base of stem vein darkbrown; basal cell absent. Abdomen. Basal scale brownish
black with fringe of long hairs; dorsal surface of 2nd segment pale yellow on anterior 1/2 or 2/3 with a pair of large
dorsolateral whitish iridescent spots broadly connected toeach other in middle; dorsal surface of posterior 1/2 or 1/3
of 2nd segment and those of other segments dark brown andwith dark hairs; tergites 3, 4 and 5 small and dull, tergites 68 large and shiny. Ventral surface of abdominal segment 7
with a pair of submedian clusters of 6 or 7 very long stout
hairs (their length range 90-140 µm) on the posterior cornerof weakly-developed sternal plates (Fig. 8). Genitalia (Figs.
9 and 10). Sternite 8 well sclerotized, bare medially butwith 8-13 short and long stout hairs as well as a few short
slender hairs laterally on each side; anterior gonapophysis
triangular in shape, membranous, covered with 5 or 6 shorthairs as well as numerous microsetae; inner border slightly
sinuous, narrowly sclerotized, folded dorsally. Genital forkof inverted-Y form, with well sclerotized stem; arms slender,
each with strongly sclerotized apical bulge having a distinct
projection directed anteriorly. Paraproct in ventral viewnearly as long as wide, with no distinct concavity on ventral
surface along anterior margin; anteromedial surface of paraproct strongly sclerotized, and with 5 or 6 short sensilla;
paraproct covered with ca. 20 short stout hairs on ventral
and lateral surfaces. Cercus in lateral view rounded posteriorly, ca. 0.5× as long as wide, covered with many shorthairs. Spermatheca nearly ovoid, well sclerotized with nodefinite reticulate pattern, with minute internal setae; tube
and small area around tubal base unsclerotized; accessary
tubes subequal in diameter to each other and to major one.
Male. Body length 1.9-2.1 mm. Head. Width slightly wider
than thorax. Upper eye consisting of large facets in 17 hori-zontal rows and in 16 vertical columns. Clypeus black,white pruinose, and silvery iridescent when illuminated,widely bare except dark stout hairs along each lateral margin and a few dark stout hairs near lower margin. Antennacomposed of 2+9 segments, light to dark brown except 1st,or 1st and 2nd, or 1st to 3rd flagellar segments, as well as
scape and pedicel, yellow when viewed ventrally; 1st flagellar segment somewhat elongated, ca. 1.7× as long as 2ndflagellomere. Maxillary palp composed of 5 segments, with
its proportional length from 3rd to 5th segments 1.0:1.3:2.6;
3rd segment (Fig. 4) of normal size; sensory vesicle small,
ellipsoidal, with small opening. Thorax. Scutum black,dull, with white-pruinose shiny pattern composed of an anterior pair of triangular spots with rounded apex (a part ofwhich disappears in certain angles of light) on shoulders,each extending posteriorly forming a broad band along eachlateral margin up to base of wing, and a large transverse
spot entirely covering prescutellar area, which is narrowly
contiguous to each lateral band; these pruinose areas silvery
iridescent when illuminated in certain angles of light;
scutum uniformly covered with dark-brown recumbent pubescences, interspersed with long upright hairs on prescutellar area. Scutellum brownish black, shiny, with several
upright dark hairs. Postscutellum brownish black, shiny,
without hairs. Pleural membrane and katepisternum as infemale. Legs. Foreleg: coxa whitish yellow; trochanter andfemur light brown; tibia medium brown except apical 1/5
dark brown, and outer surface of basal 4/5 narrowly pale
white and silvery iridescent in certain angle of light; tarsus
entirely brownish black; basitarsus, 2nd and 3rd tarsal segments dilated (basitarsus ca. 4.5× as long as its greatest
width), with distinct dorsal crest of medium-long hairs.
Midleg: coxa brownish black; trochanter medium brownwith base whitish yellow; femur medium brown with apicalcap dark brown; tibia medium brown except base narrowly
white; tarsus light brown except basal 4/5 or 5/6 of basitarsus, basal 1/3 of 2nd tarsal segment and base of 3rd tarsal
segment whitish yellow, and 5th tarsal segment brownish
black. Hind leg: coxa dark brown; trochanter whitish yellow; femur dark brown with base whitish yellow and withapical cap brownish black; tibia dark brown to brownish
black except base whitish yellow; tarsus (Fig. 7) dark brown
except basal 3/5 of basitarsus and basal 1/2 of 2nd tarsal
segment whitish yellow; basitarsus (Fig. 7) much enlarged,
ca. 3.4× as long as its greatest width at apical l/4, and ca.
0.93× as wide as greatest width of hind tibia which is subequal to that of hind femur. Calcipala small, nearly as longas wide, ca. 0.24× as wide as greatest width of basitarsus.
Pedisulcus well marked. Wing. Length 1.5-1.6 mm; otherfeatures as in female except subcosta bare. Abdomen. Basal scale blackish with fringe of long dark hairs. Dorsal

３
５１

Figures 1-20. Morphological features of S. weji sp. nov. 1, female fronto-ocular area; 2-4, 3rd segment of maxillary palp with sensory vesicle (2 and 3, female; 4, male); 5, female cibarium; 6, femur, tibia, basitarsus and 2nd tarsal segment of female
hind leg; 7, basitarsus and 2nd tarsal segment of male hind leg; 8, median portion of the ventral surface of female abdominal segment 7 showing weakly-sclerotized sternal plates and hair clusters; 9, sternite 8, anterior gonapophyses,
genital fork, right paraproct, right cercus (left paraproct and cercus omitted) and spermatheca of female genitalia insitu (ventral view); 10, right paraproct and cercus (lateral view); 11, coxites, styles and ventral plate of male genitalia
in situ (ventral view); 12, right style showing a spiny basal protuberance (end view); 13, ventral plate and mediansclerite (lateral view); 14, ventral plate (end view); 15, left paramere and aedeagal membrane (end view); 16, and 17,
10th abdominal segment and cercus (16, lateral view; 17, end view); 18, pupal gill filaments (left side, lateral view);
19, basal portion of pupal gill filaments; 20, apical portion of larval mandible. Scale bars: 0.2 mm for fig. 18; 0.1
mm for figs. 6, 7 and 19; 0.05 mm for fig. 1; 0.03 mm for figs. 2-5, 8-17; 0.02 mm for fig. 20.

３
５
２

surface of 2nd segment mostly dark yellow except narrowportion along posterior margin medium brown, those ofother segments brownish black and with dark hairs; segments 2, 5, 6 and 7 each with a pair of silvery iridescentspots dorsolaterally, those on segment 2 connected broadly
to each other in middle, those on other segments connected
narrowly to each other anteriorly. Genitalia (Figs. 11-17).
Coxite in ventral view nearly quadrate; style elongate, ca.
3.2× as long as its greatest width near middle, ca. 1.6× aslong as coxite, curved inward from middle to apex, andwith a terminal spine; style with basal protuberance produced dorsomedially, bearing numerous spinules on its surface. Ventral plate with base shorter than wide, rounded laterally, concave medially on posterior margin, having aventrally-produced process which is setose on anterior andposterior surfaces, and with posterolateral margins dentate
though becoming indefinite apically; basal arms of ventral
plate diverging from each other. Median sclerite moderately sclerotized, plate-like, widening apically, withrounded apex. Parameres broad basally, each with several
hooks apically. Aedeagal membrane moderately setose.
Abdominal segment 10 with 6 or 7 short hairs on each posterolateral corner (4 or 5 ventrally and 2 or 3 laterally).
Cerci rounded, each with 7-9 short hairs.

Photographs A-E. Cocoon and larva of S. weji sp. nov. A, cocoon and pupal exuvia thickly covered
with deposits (probably calcium carbonate); B and C, mature larva (B, dorsal
view; C, lateral view); D, larval head capsule (dorsal view) showing a negative headspot pattern on cephalic apotome; E, larvalhead capsule (ventral view) showing a triangular postgenal cleft. Scale bars: 1.0mm for photos. A, B and C; 0.2 mm forphotos. D and E.

Pupa. Body length (excluding gill filaments) 2.0-2.5 mm.
Head. Integument dark yellow to light brown, densely andellaborately covered with small cone-like tubercles, andwith 3 pairs of simple trichomes; 2 frontal trichomes oneach side slender and short, 0.4-0.6× as long as facial
medium-long trichome, and moderately separated fromeach other. Thorax. Integument dark yellow to lightbrown, densely and ellaborately covered with small conelike tubercles on anterior 1/2, and moderately or sparselywith smaller cone-like tubercles on posterior 1/2; pit-likecuticular organ absent; thorax with 8 pairs of simple
medium-long trichomes (2 anterodorsally, 2 anterolaterally,
1 posterolaterally and 3 ventrolaterally). Gill (Figs. 18 and19) with 6 thread-like filaments arranged in 3 pairs; upper
pair very shortly stalked but middle and lower pairs almost
sessile; filaments of dorsal and middle pairs arising side byside, while those of ventral pair vertically; filaments decreasing in length and diameter from dorsal to ventral withouter filament of dorsal pair longest and thickest of all (1.51.7 mm long), and lower filament of ventral pair shortest(0.5-0.9 mm long); all filaments pale yellow to medium
brown, gradually tapered toward apex, with marked annularridges and furrows (though ridges becoming indistinct apically), densely covered with minute tubercles. Abdomen.
Tergum 1 with 1 medium-long simple slender hair on eachside. Tergum 2 on each side with 1 medium-long simple

３
５
３

hair and 4 short simple spinous setae. Terga 3 and 4 eachwith 4 hooked spines along posterior margin on each side.
Terga 5-7 without spine-combs though tergum 7 with atransverse row of comb-like groups of minute spines; terga
8 and 9 each with a transverse row of distinct spine-combs
on each side. Tergum 9 without terminal hooks. Sternum 4
with 1 simple hook and 1 simple seta submedially on eachside. Sternum 5 with a pair of bifid hooks submedially oneach side. Sterna 6 and 7 each with a pair of bifid inner andsimple (or bifid) outer hooks, widely spaced on each side.
Grapnel-like hooklets absent. Cocoon (Photo. A). Simple,
slipper-shaped, tightly woven, not extending ventrolaterally;
2.5-3.0 mm long × 0.9-1.1 mm wide.
Mature larva. Body length 4.8-5.0 mm. Body color darkgreenish on thoracic segment 1 and abdominal segments 58, and light brown or light green on the rest (Photos. B andC), or almost entirely dark greenish. Cephalic apotome(Photo. D) medium brown with pale areas medially andposteriorly. Lateral and ventral surfaces of head capsuledark brown with negative spots (Photo. E). Antenna composed of 3 segments and apical sensillum, much longer thanstem of labral fan; length ratio of segments (from base totip) 1.0:1.0:0.6. Labral fan with ca. 42 main rays. Mandible (Fig. 20) with mandibular serrations composed of 1 medium tooth and 1 small one, without supernumerary serrations; comb-teeth decreasing in length from 1st to 3rd. Hypostomal teeth 9 in number, median tooth and each corner
tooth longer than others; lateral margins moderately serrate
apically; 5 or 6 hypostomal bristles diverging posteriorly
from lateral border on each side. Postgenal cleft (Photo. E)
deep, triangular, pointed anteriorly, ca. 3.1× as long as
postgenal bridge. Thoracic cuticle almost bare. Abdominal
cuticle bare except last segment moderately covered withshort colorless setae on each side of anal sclerite. Rectal
papilla of 3 lobes, each with 14-16 finger-like secondary
lobules. Anal sclerite X-shaped, with broadened base, posterior arms ca. 1.4× as long as anterior ones. Last abdominal segment slightly bulged laterally but lacking ventral papillae. Posterior circlet with ca. 86 rows of hooklets
with up to 17 hooklets per row.
TYPE SPECIMENS. Holotype female, slide mounted together with pupal exuvia, Tharn Thong Waterfall, northern
Lampang Province, near the border to Lamphun Province,
northern Thailand. 15. VI. 2001, by H. Takaoka, W.
Choochote, and T. Suntaravitun. Paratypes 5 females, 7
males, 3 pupae, 6 pupal exuviae and 4 mature larvae, samedata as holotype.
ECOLOGICAL NOTES. The pupae and larvae of S. weji

were found to be attached to trailing grasses and stones in amoderately-flowing stream 1.0-3.0 m wide, exposed to thesun. Most of the pupae and their cocoons of this species, aswell as stones in the streambed and submerged trailing
grasses, were covered with a thick layer of calcareous sediments (Photo. A). Altitude was 580 m. Water temperature
was 23°
C. No other black-fly species was found in thesame stream.
ETYMOLOGY. The species name weji is given after Mr.
Wej Choochote, Associate professor, Department of Parasitology, Faculty of Medicine, Chiang Mai University, Chiang
Mai 50200, Thailand, for his great contribution in the research field of Parasitology and Medical Entomology as
well as his kind cooperation during the present survey.
REMARKS. This new species is assigned to the tuberosum
species-group within the subgenus Simulium Latreille s. str.
by the shape of the genitalia of both sexes and the pupal gillwith six filaments per side (Takaoka and Davies, 1996), andseems to be closely related to S. rufibasis Brunetti, S. ramosum Puri, and S. fasciatum Puri, all originally describedfrom India (Brunetti, 1911; Puri, 1932), and also to two unnamed species (S. sp. D and S. sp. E), reported only fromadult female specimens collected together with S. rufibasis
from Thailand (Takaoka and Suzuki, 1984) by having a pairof hair clusters on the ventral surface of the seventh abdominal segment of the female, which are, though, much
longer and fewer in this new species (Fig. 8). The female ofS. weji is however distinguished from those of all the five
known species by the dark hind tibiae leaving only a small
pale area basally (Fig. 6) (cf. the hind tibiae of the otherspecies are dark on apical 3/5 to 1/2 and pale on the basal
2/5 to 1/2). The pupa of S. weji is easily separated fromthose of S. rufibasis and S. fasciatum by the almost sessile
gill filaments (cf. gill filaments are stalked in the latter two
species), and from S. ramosum by the simple trichomes on
the head and thorax (cf. trichomes are branched in the latter).
This new species seems to be also related to S. nigrifacies Datta from India (Datta, 1974) and S. puliense Takaoka
from Taiwan (Takaoka, 1979) by having the similar arrangement of the short pupal gill filaments. The female ofthe latter two species are still unknown. However, the maleof these two species are easily separated by the absence of apair of silvery iridescent spots dorsolaterally on the fifth abdominal segment. In the pupa, there is a slight difference:
the tubercles on the head and thoracic integuments are coneshaped in this new species while those are disc-like in S.
nigrifacies or round in S. puliense.
This new species is distinct from S. brevipar and S.

３
５
４

tani, both originally described from Peninsular Malaysia(Takaoka and Davies, 1995) and later recorded from Thailand (Takaoka and Saito, 1996), by having the hair clusters
on the ventral surface of the seventh abdominal segment in
the female, the ventral plate rounded laterally (Fig. 11) inthe male, and the cone-shaped tubercles on the head andthoracic integuments in the pupa.

ACKNOWLEDGEMENTS
I am grateful to Associate Professor Wej Choochote,and Mr. Trenayut Suntaravitun, Department of Parasitology,
Chiang Mai University, Chiang Mai, Thailand, for theirkind help and arrangement of collections of black flies innorthern Thailand. Thanks are due to Dr. Yasushi Otsuka,
Department of Infectious Disease Control, Oita Medical
Unversity, who kindly took photographs of a cocoon and alarva needed for this paper. This work was financially supported by the Grant-in-Aid of Japan Society for Promotionof Science (the fiscal year 2001).

REFERENCES
１）Brunetti, E. (1911): New Oriental Nematocera. Rec. Indian Mus., 4, 259-316
２）Datta, M. (1974): Some black flies (Diptera: Simuliidae)of the subgenus Simulium Latreille (s. str.) from the Darjeeling area (India). Oriental Insects, 8, 15-27３）Kuvangkadilok, C. and Takaoka, H. (2000): Taxonomic
notes on Simuliidae (Diptera) from Thailand: Descriptionof a new species and new distributional records of nine
known species. Jpn. J. Trop. Med. Hyg., 28, 167-175
４）Puri, I.M. (1932): Studies on Indian Simuliidae. Part II.
Descriptions of males, females and pupae of Simulium
rufibasis Brunetti, its variety fasciatum nov. var. and of
three new species from the Himalayas. Indian J. Med.
Res., 19, 899-915
５）Takaoka, H. (1979): The black flies of Taiwan (Diptera:
Simuliidae). Pacific Insects, 20, 365-403
６）Takaoka, H. and Adler, P.H. (1997): A new subgenus,
Simulium (Daviesellum), and a new species, S. (D.)
courtneyi (Diptera: Simuliidae) from Thailand and Peninsular Malaysia. Jpn. J. Trop. Med. Hyg., 25, 17-27
７）Takaoka, H. and Davies, D.M. (1995): The black flies(Diptera: Simuliidae) of West Malaysia. viii + 175p.,
Kyushu University Press, Fukuoka８）Takaoka, H. and Davies, D.M. (1996): The black flies(Diptera: Simuliidae) of Java, Indonesia. viii + 81p.,
Bishop Mus. Bul. Entomol., 6, Bishop Museum Press,
Hawaii.
９）Takaoka, H. and Kuvangkadilok, C. (1999): Four new
species of black flies (Diptera: Simuliidae) from Thailand.
Jpn. J. Trop. Med. Hyg., 27, 497-509
１０）Takaoka, H. and Saito, K. (1996): A new species and newrecords of black flies (Diptera: Simuliidae) from Thailand. Jpn. J. Trop. Med. Hyg., 24, 163-169
１１）Takaoka, H. and Suzuki, H. (1984): The blackflies (Diptera: Simuliidae) from Thailand. Jpn. J. Sanit. Zool., 35,
7-45

３
５
５

Jpn. J. Trop. Med. Hyg., Vol.２
９, No．
４,２
０
０１, pp.３
５
５
‐
３
５
９

MECHANISMS OF PARASITE ANTIGEN UPTAKEFROM THE INTESTINE OF RATS INFECTED
WITH THE NEMATODE NIPPOSTRONGYLUS
BRASILIENSISMINORU YAMADA１, RYUICHI UCHIKAWA１, SHINJI MATSUDA２
AND NAOKI ARIZONO１
Received August 15, 2001/Accepted November 12, 2001

Abstract: The mechanisms of antigen-uptake at the host mucosal barrier during infection with lumen-dwelling intestinal parasites have not been well elucidated. In this study, we investigated immunolocalization of parasite excretory/secretory (ES) products in the small intestinal epithelium of rats infected with Nippostrongylus brasiliensis.
Immunoelectronmicroscopy revealed intense ES immunoreactivity in the absorptive cell cytoplasm in association
with lysosome-associated membrane glycoprotein (LAMP-1), and in the paracellular spaces. These results indicated that nematode antigen could be absorbed by two pathways: absorption by endocytosis and transport to lysosomes, and absorption through the paracellular pathway, suggesting altered mucosal barrier function.
Key word: Nippostrongylus brasiliensis, Rat, Intestine, Parasite antigen, Uptake

INTRODUCTION
Infection of the lumen-dwelling intestinal nematode
Nippostrongylus brasiliensis induces various immune responses such as IgE antibody production and mastocytosis
which are associated with the generation of Th2 cells (Matsuda et al., 1995). Along with immune reactions, N. brasiliensis infestation also induces pathological alterations in the
jejunal epithelium, such as partial villus atrophy, increases
in epithelial cell turnover and decreases in expression ofbruch border enzymes (Hyoh et al., 1999; Perdue et al.,1989). Pathophysiological studies have also revealedchanges in intestinal permeability after infection: decreasein glucose absorption rate, increased lactulose/mannitol ratio (Nolla et al., 1985; Cobden et al., 1979) and proteinleakage into the intestinal lumen which results in hypoalbuminemia (Nawa, 1979; Lunn et al., 1986). Inductions of
immunological responses and pathological alterations require absorption of parasite-derived molecules across intestinal epithelium, which normally have a barrier function toprevent absorption of potentially harmful materials from theintestinal lumen: uptake of macromolecules by normal absorptive cells is limited and paracellular channels are sealedby tight junctions (Anderson and Van, 1995). In this context, it is interesting to know the route of absorption of N.
1
2

brasiliensis-derived molecules through the intestinal epithelium of infected animals. In the present study, we examined
ultrastructural localization of excretory/secretory (ES) product of N. brasiliensis in the rat intestinal epithelium. ES
product of N. brasiliensis comprise biologically active
molecules such as acetylcholinesterase, cysteine proteinase,an interferon-γ suppressive factor and major allergens whichevoke marked IgE antibody response in the host (Yamada etal., 1991; Kamata et al., 1995; Nakazawa et al., 1995;
Uchikawa et al., 2000).

MATERIALS AND METHODS
Male Spragus-Dawley rats were purchased from SLC
Inc. (Shizuoka, Japan). Animals at 8 weeks of age were infected with 2,000 N. brasiliensis L3 larvae by subcutaneous
injection. ES product form adult N. brasiliensis worms
were prepared as described (Yamada et al., 1991). Polyclonal antibody against ES product was raised in Japanesewhite rabbits by injecting 10 µg ES with Freund’s complete
adjuvant 3 times every 4 weeks, and the IgG fraction was
prepared using a protein-G column (Amersham Pharmacia
Biotech, Buckinghamshire, UK). Immunoblotting analyses
showed that this antibody recognized multiple molecules ofES product from 17-220 kDa, but not of normal intestinal

Department of Medical Zoology, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji, Kyoto 602-8566, Japan
Department of Parasitology, Akita University School of Medicine, Hondo-1-1-1, Akita 010-8543, Japan３
５
６

lysates of mice and rats (data not shown).
Preliminary immunohistochemical studies using
paraffin-embedded tissue sections of the proximal jejunum
showed that there was intense ES immunoreactivity in thevillus epithelium 7 and 10 days p.i., while not in uninfectedanimals or those 3, 14 and 21 days p.i., suggesting that absorption of ES product is detectable during the period whena large numbers of adult worms are parasitized in the lumen.
Therefore, immunoelectronmicroscopic studies were carriedout 7 days p.i. using 4 rats.
For immunoelectron microscopy, segments of the jejunum approximately 20 cm distal to the pyloric ring were removed and fixed in a solution containing 0.5% glutaraldehyde, 4% paraformaldehyde and 0.1 M phosphate buffer,
pH 7.4, for 1 hr at 4°
C, and embedded in LR White resin(London Resin Co., Surrey, UK) in a position so that villus
structures were cut longitudinally. Ultra-thin sections were
cut and incubated with 5% (w/v) non-fat dry milk, followed
by incubation with anti-ES rabbit IgG (50 µg/ml ) overnightat 4°
C. For double immunostaining of ES and lysosome associated membrane glycoprotein-1 (LAMP-1), sections
were further incubated with anti-LAMP-1 goat IgG (2 µg/
ml : Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 2
hr at room temperature. After washing, sections were incubated with 15 nm gold particles-conjugated goat anti-rabbit
IgG (diluted to 1:20) or 5 nm gold particles-conjugated rabbit anti-goat IgG (diluted to 1:20) (BB International,
Golden gate, UK) for detections of ES and LAMP-1 for 1
hr at room temperature, respectively, followed by staining
with 2% uranyl acetate, and observed under a Hitachi H7000 electron microscope.

RESULTS AND DISCUSSION
In uninfected animals, ES-specific antibody showed
negligible levels of non-specific binding to absorptive cell
cytoplasm, and moderate levels of nonspecific binding tomicrovilli (Fig. 1a). Seven days p.i., intense immunoreactivity was found on microvilli, paracellular spaces and thecytoplasm of villus absorptive cells, in which some immunoreactivity was localized to vesicular structures (Fig.
1b). Low level ES immunoreactivity was also found on thecellular membrane along the basal lamina (Fig. 1c). In a
few absorptive cells, there was markedly dense immunoreactivity in the lysosome-like organelle (Fig. 1d). To determine whether ES product is transported to absorptive celllysosomes, double immunostaining of ES and LAMP-1 wascarried out. As shown in Fig. 2a, b, at least some of ES immunoreactivity showed co-localization with LAMP-1.
These localizations of immunoreactivity were not observedin all villus absorptive cells, but in patchy distributions withfrequencies higher in the upper part of villi. Crypt lining
cells did not reveal ES immunoreactivity, while M cells
over Peyer’s patches showed similar distribution of ES immunoreactivity to that of villus absorptive cells (data notshown). Using nonimmune rabbit or goat IgG, there was nogold particle deposition in paracellular spaces or in the cytoplasm of absorptive cells.
It has been presumed that only M cells over Peyer’s
patches are specialized for the uptake of macromolecules
and antigen presentation to T cells (Mayer, 1997). However,the present studies showed that parasite-derived molecules
could be transported not only through paracellular spaces
but also absorbed in the villus epithelial cells, indicatingthat the normal barrier functions of intestinal epithelium are
disrupted after infection of lumen-dwelling nematodes. The
mechanism of the antigen uptake through paracellular channels is not clear from the present study. It is possible thatnematode infection altered expression of adhesion molecules of villus epithelial cells. In fact, we reported previously that expression of E-cadherin, a major epithelial celladhesion molecule, was significantly reduced at the tip ofvilli after N. brasiliensis infection (Hyoh et al., 1999). Recently, it was reported that a cysteine proteinase allergen
Der pl is transported through the paracellular pathway of
respiratory epithelium possibly mediated by the breakdownof tight junctions by the allergen’s own proteolytic activity(Wan et al., 1999). In this respect, it is interesting that N.
brasiliensis ES product also contains a cysteine protease(Kamata et al., 1995). Alternatively, increase of paracellular permeability might have been triggered indirectly by signals from absorptive cells, as suggested by reports that thesolute permeation across tight junctions is not static and canbe reversibly regulated by intracellular events with a common effector mechanism probably tied to the cytoskeleton(Madara, 1998).
The present results also showed that the ES productwas absorbed in the villus epithelial cells, and at least some
ES immunoreactivity was co-localized with LAMP-1, suggesting that ES product was internalized by endocytosis andtransported into lysosomes. These results are consistent
with the report that at least some species of macromolecules
can be transported across the normal mature gut by a pinocytotic processes (Walker and Isselbacher, 1974). Since villus epithelial cells express MHC class II antigens and its expression was enhanced after N. brasiliensis infection(Mayrhofer and Spargo, 1990; Masson and Perdue, 1990),
it is possible that some of the processed antigens are directly presented from intestinal absorptive cells to lymphocytes.
Taken together, the present studies showed that N.
brasiliensis antigen is partly internalized in intestinal ab-３５７

Figure 1 ES immunoelectronmicroscopy of rat small intestinal villus epithelium from uninfected rats (a) or animals 7 days after N.
brasiliensis infection (b-d). a: nonspecific gold-particle deposition is noted on microvilli, while deposition in the absorptive cell cytoplasm and paracellular spaces are negligible. ×20,000. b: Intense ES immunoreactivity is noted on
microvilli, paracellular spaces (arrows), and in the cytoplasmic vesicular structures (arrowheads). ×20,000. c: ES immunoreactivity is observed on the basal cell membrane (arrows) and in a small vesicular structures (arrowheads) of absorptive cells. ×21,360. d : Intense immunoreactivity is noted in lysosome-like structures (arrows). ×34,860.

３
５
８

Figure 2 ES (large gold particles) and LAMP-1 (small gold particles) double immunostaining of rat small intestinal villus epithelium from rats 7 days after N. brasiliensis infection. ES and LAMP-1 (arrowheads) are occasionally co-localized inmembrane-bound structures in the cytoplasm of absorptive cells. a: ×72,000. b: × 134,000.

３５９sorptive cells and partly transported through the paracellularpathway, probably in association with the disruption of barrier function. The uptake of parasite antigen through the intestinal epithelium would have primary importance for the
induction of immune reactions and development of intestinal pathology.

ACKNOWLEDGEMENTS
This study was supported by grants from the Ministryof Education, Culture, Sports, Science and Technology, Japan.

REFERENCES
１）Anderson, J.M. and Van, I.C. (1995): Tight junctions andthe molecular basis for regulation of paracellular permeability. Am. J. Physiol., 269, g467-475
２）Cobden, I., Rothwell, J. and Axon, A.T. (1979): Intestinal
permeability in rats infected by Nippostrongylus brasiliensis. Gut, 20, 716-721
３）Hyoh, Y., Nishida, M., Tegoshi, T., Yamada, M.,
Uchikawa, R., Matsuda, S. and Arizono, N. (1999): Enhancement of apoptosis with loss of cellular adherence inthe villus epithelium of the small intestine after infection
with the nematode Nippostrongylus brasiliensis in rats.
Parasitology, 119, 199-207
４）Kamata, I., Yamada, M., Uchikawa, R., Matsuda, S. andArizono, N. (1995): Cysteine protease of the nematode
Nippostrongylus brasiliensis preferentially evokes an IgE/
IgG1 antibody response in rats. Clin. Exp. Immunol.,102, 71-77
５）Lunn, P.G., Northrop, C.A., Behrens, R.H., Martin, J. and
Wainwright, M. (1986): Protein losing enteropathy associated with Nippostrongylus brasiliensis infestation andits impact on albumin homoeostasis in rats fed two levelsof dietary protein. Clin. Sci., 70, 469-475
６）Madara, J.L. (1998): Regulation of the movement of solutes across tight junctions. Ann. Rev. Physiol., 60, 143159
７）Masson, S.D. and Perdue, M.H. (1990): Changes in distribution of Ia antigen on epithelium of the jejunum and ileum in rats infected with Nippostrongylus brasiliensis.

Clin Immunol. Immunopathol., 57, 83-95
８）Matsuda, S., Uchikawa, R., Yamada, M. and Arizono, N.
(1995): Cytokine mRNA expression profiles in rats infected with the intestinal nematode Nippostrongylus
brasiliensis. Infect. Immun., 63, 4653-4660
９）Mayer, L. (1997): Local and systemic regulation of mucosal immunity. Aliment. Pharmacol. Ther., 3, 81-85
１０）Mayrhofer, G. and Spargo, L.D. (1990): Distribution ofclass II major histocompatibility antigens in enterocytesof the rat jejunum and their association with organelles ofthe endocytic pathway. Immunology, 70, 11-19
１１）Nakazawa, M., Yamada, M., Uchikawa, R. and Arizono,
N. (1995): Immunocytochemical localization of secretory
acetylcholinesterase of the parasitic nematode Nippostrongylus brasiliensis. Cell. Tissus Res., 280, 59-64
１２）Nawa, Y. (1979): Increased permeability of gut mucosa inrats infected with Nippostrongylus brasiliensis. Int. J.
Parasitol., 9, 251-255
１３）Nolla, H., Bristol, J.R. and Mayberry, L.F. (1985): Nippostrongylus brasiliensis: malabsorption in experimentally infected rats. Exp. Parasitol., 59, 180-184
１４）Perdue, M.H., Ramage, J.K., Burget, D., Marshall, J. and
Masson, S. (1989): Intestinal mucosal injury is associatedwith mast cell activation and leukotriene generation during Nippostrongylus-induced inflammation in the rat.
Dig. Dis. Sci., 34, 724-731
１５）Uchikawa, R., Matsuda, S. and Arizono, N. (2000): Suppression of gamma interferon transcription and production by nematode excretory-secretory antigen during
polyclonal stimulation of rat lymph node T cell. Infect.
Immun., 68, 6233-6239
１６）Walker, W.A. and Isselbacher, K.J. (1974): Uptake andtransport of macromolecules by the intestine. Possible
role in clinical disorders. Gastroenterology, 67, 531-550
１７）Wan, H., Winton, H.L., Soeller, C., Tovey, E.R., Gruenert,
D.C., Thompson, P.J., Stewart, G.A., Taylor, G.W., Garrod, D.R., Cannell, M.B. and Robinson, C. (1999): Der plfacilitates transepithelial allergen delivery by disruptionof tight junctions. J. Clin. Invest., 104, 123-133
１８）Yamada, M., Nakazawa, M., Matsumoto, Y. and Arizono,
N. (1991): IgE antibody production in rats against multiple components of excretory-secretory products of thenematode Nippostrongylus brasiliensis. Immunology, 72,
104-108

３
６
１

Jpn. J. Trop. Med. Hyg., Vol.２
９, No．
４,２
０
０１, pp.３
６
１
‐
３
６
４

INDUCTION OF IGE ANTIBODY RESPONSEIN MICE BY CONTINUOUS ADMINISTRATION OF
EXCRETORY/SECRETORY PRODUCTS OF
THE NEMATODE NIPPOSTRONGYLUS BRASILIENSISMINORU YAMADA１, TATSUYA TEGOSHI１, RYUICHI UCHIKAWA１,
SHINJI MATSUDA２, MINORU NISHIDA１ AND NAOKI ARIZONO１
Received August 15, 2001/Accepted November 20, 2001

Abstract: IgE-inducing potential of excretory/secretory products (ES) of the nematode Nippostrongylus brasiliensis was examined in mice immunized with ES without use of adjuvant. Repeated i.p. injections or continuous i.p.
or s.c. infusion of ES induced significant increases in total serum IgE. Continuous i.p. or s.c. infusion of ES also
induced ES-specific IgE antibody, while repeated i.p. injections did not. IL-4 production was increased by ex vivo
stimulation with ES of mesenteric lymph node cells from repeatedly immunized mice. On the other hand, continuous intraduodenal infusion of ES induced neither total nor specific IgE responses. These results suggest that continuously secreted ES from the nematode are relevant for the up-regulation of IL-4 and IgE responses, although additional factor(s) may be necessary for the uptake of ES across the intestinal mucosal barrier.
Key words: Nippostrongylus brasiliensis, IgE, Mice, Continuous administration, Excretory/secretory products

INTRODUCTION
Previously, we showed that excretory/secretory products (ES) of N. brasiliensis contain several major antigensof 16 to 130 kDa including some functional molecules suchas acetylcholinesterase as well as cysteine protease, that are
produced in and secreted from the subventral gland of theworm (Burt and Ogilvie, 1975; Kamata et al., 1995;
Nakazawa et al., 1995; Yamada et al., 1991; Yamada et al.,1993). Intraperitoneal administration of a single large doseof ES induced a small but significant increase in the level of
total serum IgE, but not ES-specific IgE in naive rats
(Uchikawa et al., 1993). The aim of the present study was
to examine whether repeated or continuous ES administration without use of adjuvant evokes IgE antibody production in high-IgE responder BALB/c mice.

MATERIALS AND METHODS
Male BALB/c mice, 8 weeks old, were purchased fromJapan SLC Inc. (Hamamatsu, Japan). The strain of N.
brasiliensis used has been maintained in our laboratory by
serial passage in rats. ES was prepared from N. brasiliensis
as described previously (Yamada et al., 1991).

In one experiment, ES was dissolved in PBS (10 or
100 µg/ml ) and was injected i.p. at various time intervals.
A group of mice was inoculated i.p. with adult N. brasiliensis worms which had been collected from the small intestine of a rat 7 days p.i. In another experiment, a total doseof 320 µg ES was continuously delivered by mini-osmotic
pumps (Alzet model 2001, Alza Corp., Palo Alto, CA) at
the rate of 1 µl /hr for 7 days. For i.p. administration, miniosmotic pumps were implanted into the peritoneal cavity
and for s.c. administration into the dorsal flank. For intraduodenal administration, mini-pumps were implanted
subcutaneously into the dorsal flank tissue and a draining
catheter attached to the pump was passed through the subcutaneous tissue and introduced into the peritoneal cavity.
The tip of the catheter was inserted into the duodenal lumen
through a small incision and the catheter was secured to theduodenal wall with Histoacrylblue (B.Braun Melsüngen AG,
Melsüngen, Germany). All skin incisions were closed withsuture silk.
For measurements of total serum IgE, IgG1 or IgG2aby ELISA, the wells of 96-well plates were coated with rat
mAb against mouse IgE (10 µg/ml : LO-ME-2; EIU Inc.,
Belgium), IgG1 (2 µg/ml : LO-MG1-2, Zymed Laboratories
Inc., San Francisco, USA), or IgG2a (2 µg/ml : LO-MG2a-1 Department of Medical Zoology, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji, Kyoto 602-8566, Japan
2 Department of Parasitology, Akita University School of Medicine, Hondo 1-1-1, Akita 010-8543, Japan３
６
２

3; Zymed Laboratories Inc.) dissolved in carbonate buffer,
pH 9.6, overnight at 4°
C. After washing, the wells were incubated with bovine milk protein (Block Ace; Dainihon
Pharm. Co., Osaka, Japan), followed by incubation with
serially diluted test sera for 2hr. The plates were then incubated with goat anti-mouse IgE serum (Bethyl Laboratories
Inc., Montgomery, USA), followed by incubation withaffinity-purified biotin-conjugated rabbit IgG against goat
IgG (MBL Inc., Nagoya, Japan) or for detection of IgG1 or
IgG2a, the plates were incubated with biotin-conjugatedanti-mouse IgG1 or IgG2a rat monoclonal antibodies(Zymed Laboratories Inc.). After subsequent incubation
with alka-line phosphatase-labeled streptavidin (Boehringer
Mannheim Biochemica, Mannheim, Germany), 10 mM 4nitrophenylphosphate dissolved in 10 mM diethanolamine
buffer (pH 9.8) containing 0.5 mM MgCl２ was added as a
substrate. The OD value at 405 nm was measured with amicroplate reader. Standard curves were generated by serial
dilutions of purified IgE protein (mouse anti-DNP IgE monoclonal antibody; Yamasa Shoyu Co., Tokyo, Japan), mouse
IgG1 myeloma protein, or mouse IgG2a myeloma protein(Zymed Laboratories Inc.) and amounts of IgE, IgG1 andIgG2a in test sera are expressed in µg/ml .
For detection of ES-specific IgE, IgG1 or IgG2a by
ELISA, the wells were coated with ES (10 µg/ml ) dissolved in carbonate buffer, pH 9.6, overnight at 4°
C. EScoated plates were further incubated with bovine milk protein, then with test sera (1:10 dilution) for 2 hr. The plates
were further incubated with biotin-conjugated rat mAb
against mouse IgE (PharMingen, San Diego, USA), IgG1
(Zymed Laboratories Inc.) or IgG2a (PharMingen) followed
by incubation with horseradish peroxidase-labeled streptavidin (Amersham, Bucks, U.K.).
Reaction with ophenylenediamine was carried out, and the OD value at 492
nm was determined.
For measurement of IL-4 production, mesentericlymph nodes (MLN) were removed from mice, and single
cells were prepared and cultured with ES as described previously (Tegoshi et al., 1997). Concentrations of IL-4 inculture supernatant were determined using ELISA kits (Endogen Inc., Cambridge, USA) according to the manufacturer‘s instructions.
Statistical analysis was performed using Mann-Whitney Utest.

RESULTS AND DISCUSSION
To determine whether antigens released from adultworms have the potential to induce IgE antibody responses,
living adult worms were transplanted into the peritoneal
cavity of naive mice. These mice developed significant in-

Table 1 IgE antibody responses after repeated ES administration or transplantation of adult N. brasiliensis
worms into the peritoneal cavity of mice
Group
A
B
C
D
E
F

Treatment

Total quantity Total IgEof ES (µg)(µg/ml )0.12±0.04
PBS
0.25±0.1320
10 µg ES0.52±0.5050
10 µg ES0.23±0.07
200
100 µg ES
2.53±1.31**
500
100 µg ES
Adult worm
1.79±0.61*

ES-specificIgE (O.D.)
0.094±0.023
0.080±0.031
0.079±0.023
0.059±0.009
0.079±0.017
0.300±0.120*

A: PBS was injected i.p. five times every three days. B, C: 10 µg of
ES was injected i.p. twice at 1 week interval (B), or five times every
three days (C). D, E: 100 µg of ES was injected i.p. twice at 1 week
interval (D), or five times every three days (E). F: Adult worms (500)
were surgically transplanted into the peritoneal cavity. Sera were obtained 14 days after the first injection of ES or surgical transplantationof adult worms. Data shown are means±S.D. of 4-6 mice. Significantly different from the values for group A: ＊p<0.05, ＊＊p<0.01(Mann-Whitney U-test)

creases of total and specific IgE levels (Table 1, Group F).
Most worms had penetrated mainly to the omentum andperipancreatic tissue, by day 7. They were still alive on day
7, and a few had survived by day 14 (Arizono et al., 1994).
It was assumed that these transplanted worms potentiatedIgE production. When mice received repeated i.p. injections of 10 µg ES (Table 1, Group B, C), no IgE response
was observed, while administration of 100 µg of ES 5 times
every 3 days induced significant increases in total serum
IgE (Table 1, Group E). Although specific IgE was not detectable in these mice, ex vivo stimulation of MLN cellswith ES induced significant IL-4 response (56.3±23.5
pg/ml in mice administered with 100 µg ES 5 times every 3days i.p. vs <5 pg/ml in mice injected with PBS 5 times
every 3 days i.p.).
Next, we examined IgE responses in mice after continuous infusion of ES in the peritoneal cavity, subcutaneous tissue or duodenum using implanted mini-osmotic
pumps. Two weeks after implantation of the pumps, i.p.
and s.c. routes of administration induced significant increases in the levels of total and specific IgE (Table 2). Total and specific IgG1 levels were also increased significantly following i.p. and s.c. administration (Figure 1A, B).
On the other hand, IgG2a levels did not change significantly(data not shown). With intraduodenal administration, neither IgE (Table 2), nor IgG1 (Figure 1A, B) or IgG2a (datanot shown) responses developed.
The reason for the failure of specific IgE response byrepeated i.p. administration of ES, despite ES-specific IgE
response developed by continuous i.p. or s.c. administration,
is not clear. It is possible that repeated exposure of ES antigen with short intervals might rather suppressed the specific３６３IgE production.
Previously, we showed that a single i.p injection oflarge dose of ES to rats induced a low level increase in total
serum IgE without production of specific IgE antibodies(Uchikawa at al., 1993). The present results in mice
showed that although IgE response did not occur with 10µg of ES every three days or with 100 µg of ES twice at 1
week interval, administration of 100 µg of ES every threedays induced significant increases in total serum IgE. Further, continuous i.p. or s.c. infusion of ES induced significant increases in both total and ES-specific IgE comparable
to those observed following implantation of living adultworms into the peritoneal cavity. These results suggest thatthe critical amount of ES for induction of the IgE responsemust be over 320 µg, whereas less than 200 µg will not beeffective. We previously reported that ES is produced at
levels of >200 µg by 1,000 adult N. brasiliensis worms per
Table 2 IgE antibody responses after continuous ES administration into the duodenal lumen, peritoneal
cavity or subcutaneous tissue of mice
PBS
intraduodenal
intraperitoneal
subcutaneous

Total IgE (µg/ml )ES-specific IgE (O.D.)

0.13±0.02
0.45±0.44
1.30±0.41*

0.020±0.006
0.034±0.012
0.199±0.054*
0.132±0.063*

1.10±0.29*

A total of 320 µg ES was administered by implanted mini-osmotic
pumps at a rate of 1 µl /hr for 7 days. Control animals were administered PBS i.p. by mini-osmotic pumps. Sera were obtained 14 days
after implantation of the mini-osmotic pumps. Data shown are means±S.D. of 4 mice. Significantly different from the values for PBS
control: ＊p<0.05 (Mann-Whitney U-test)

Figure 1 Serum levels of total IgG1 (A) and ES-specific IgG1
(B) antibody 2 weeks after continuous administration of a total of 320 µg ES into the duodenum (i.d.),subcutaneous tissue (s.c.), or peritoneal cavity (i.p.)by Alzet mini-osmotic pumps at the rate of 1 µl /hr
for 7 days. Control animals were infused with PBS
intraperitoneally for 7 days. The bar on the top ofeach column represents SE. Significantly different
from the values for PBS control: ＊p<0.05.

day in vitro (Yamada et al ., 1991). These results are consistent with the observation that levels of total serum IgE after N. brasiliensis infection in rats were positively correlated with the numbers of infecting worms, although thenematode-specific IgE antibody response was rather suppressed at very high worm burden (Yamada et al., 1992).
In contrast to i.p. or s.c. immunization, intraduodenal
infusion of ES did not induce IgE, IgG1 or IgG2a responses.
This result is consistent with the previous report that oral
administration of ES in rats did not generate IgE responses(Uchikawa et al., 1993). It is possible that ES product introduced into the duodenum was subjected to digestion andwas degradaded before absorption from the intestine. Alternatively, absorption of ES across intact intestinal barrier
might require additional factors.
Taken together, the present results show that ES continuously secreted from nematodes is responsible for triggering IgE antibody responses.

ACKNOWLEDGMENTS
This work was supported by grants from the Shimizu
Immunology Foundation and the Ministry of Education,
Science, Sports and Culture, Japan (08670285).

REFERENCES
１）Arizono, N., Yamada, M., Tegoshi, T., Okada, M., Uchikawa,
R. and Matsuda, S. (1994): Mucosal mast cell proliferation following normal and heterotopic infections of thenematode Nippostrongylus brasiliensis in rats. APMIS,
102, 589-596
２）Burt, J.S. and Ogilvie, B.M. (1975): In vitro maintenanceof nematode parasites assayed by acetylcholinesterase
and allergen secretion. Exp. Parasitol., 38, 75-82
３）Kamata, I., Yamada, M., Uchikawa, R., Matsuda, S. andArizono, N. (1995): Cysteine protease of the nematode
Nippostrongylus brasiliensis preferentially evokes an IgE/IgG1
antibody response in rats. Cli. Exp. Immunol., 102, 71-77
４）Nakazawa, M., Yamada, M., Uchikawa, R. and Arizono,
N. (1995): Immunocytochemical localization of secretory
acetylcholinesterase of the parasitic nematode Nippostrongylus brasiliensis. Cell and Tissue Res., 280, 59-64
５）Tegoshi, T., Okada, M., Nishida, M. and Arizono, N. (1997):
Early increase of gut intraepithelial mast cell precursors
following Strongyloides venezuelensis infection in mice.
Parasitology, 114, 181-187
６）Uchikawa, R,, Yamada, M., Matsuda, S. and Arizono, N.
(1993): IgE antibody responses induced by transplantation of the nematode Nippostrongylus brasiliensis in rats:
a possible role of nematode excretory-secretory productin IgE production. Immunology, 80, 541-545
７）Yamada, M., Nakazawa, M., Matsumoto, Y. and Arizono,

３
６４

N. (1991): IgE antibody production in rats against multiple components of excretory-secretory products of thenematode Nippostrongylus brasiliensis. Immunology, 72,
104-108
８）Yamada, M., Nakazawa, M., Kamata, I. and Arizono, N.
(1992): Low-level infection with the nematode Nippostrongylus brasiliensis induces significant and sustained

specific and non-specific IgE antibody responses in rats.
Immunology, 75, 36-40
９）Yamada, M., Nakazawa, M. and Arizono, N. (1993): IgE
and IgG2a antibody responses are induced by different
antigen groups of the nematode Nippostrongylus brasiliensis in rats. Immunology, 78, 298-302

３
６５

Jpn. J. Trop. Med. Hyg., Vol.２
９, No．
４,２
０
０１, pp.３
６
５
‐
３
７
０

EVALUATION OF ANAEROPACK! (AnP) TYPE
AS TOOLS OF PLASMODIUM FALCIPARUM
CULTIVATION AND DRUG SENSITIVITY TESTSKOSUKE HARUKI, FUMIE KOBAYASHI, TAKASHI FUJINO,TOSHIHIRO MATSUI AND MORIYASU TSUJI
Received October 12, 2000/ Accepted October 23, 2001

Abstract: The AnaeroPack (AnP) system (Mitsui Gas Co., Japan) was produced for the purpose of culturing anaerobic bacteria. The idea using AnP Campylo! for cultivation of Plasmodium falciparum and its successful results were firstly reported by Haruki et al. (1997). In the present study, characterisation of 3 products of AnPs, i.e.,
AnP CO２! (at a gas phase of 5% CO２ and 15% O２), AnP Campylo! (10% CO２ and 10% O２) and AnP Plas! (5%CO２ and 5% O２), in an AnaeroPack Box Jar! (22×16×10.5 cm) was studied comparing with conditions in a gas
incubator or gas chamber (4% CO２ and 3% O２) and in a candle jar (5% CO２ and 15% O２). The growth curves ofmalaria parasites showed no difference among these four conditions by incubation for 48 hr except for AnP Plas.
The observed IC５０ levels of chloroquine were varied much by the difference of AnPs, gas phases, the pressure in a
Box Jar and free space inside. For stabilisation of IC５０ testing, free space in a Box Jar should be kept equal andminimal by placing the maximal 5 test plates by adding dummy plates. By the stable results of IC５０, gas phases canbe divided into two groups. The one involves the gas incubator, AnP Plas! and AnP Campylo!, and the other does
the candle jar and AnP CO２!. Results of chloroquine sensitivity test in the candle jar group showed more drug sensitive than in the gas incubator group. These two groups should be used properly by the purpose of field experiments. The AnP! system is proven to be a practical tool for malaria culture in the field for occupying only a small
space in an incubator.
Key words: Malaria, Plasmodium falciparum, in vitro culture, AnaeroPack!, Microanaerophilic condition, Chloroquine sensitivity

INTRODUCTION
Since discovery of cultivation method of P. falciparumby Trager and Jensen (1976), it has been possible to survey
the drug sensitivity using a microtest method which wasrecommended by Rieckmann et al. (1978) and WHO(Bruce-Chwatt et al., 1986). On the other hand, the problem of drug resistance becomes a big obstacle for prevention and treatment of falciparum malaria world widely. It is
important to survey the drug sensitivity in malarious areas
and to document the extent and distribution of resistance inthe world. Therefore, the discrepancy among drug sensitivity assay methods is not only scientific issues but also clinical and practical problems. In addition, the assay system isrequired to be less laborious and of accuracy, because thesetests will be performed usually under unsatisfied facilities
and low economical conditions in malaria infested areas.
To produce a stable gas phase in the field studies as in

the gas incubator that is approriate for malaria cultivationand drug sensitivity test, an idea of using AnaeroPack(AnP) Campylo! for cultivation of P. falciparum was firstly
reported by Haruki et al. (1997a) at the 9th Malaria meetingof British Society of Parasitology, Liverpool held in 1997,
and next in Japan by Haruki et al. (1997b) in terms ofgrowth efficiency and drug sensitivity test of P. falciparumin comparison with ordinary systems in the gas incubatorand in a candle jar. Following the above reports, a few
works have been performed using AnP system (TaylorRobinson, 1998; Onda et al., 1999; Lin et al., 1999; Mizuno et al., 2000). AnP systems is simpler and easier tohandle than the technique with a candle jar. In the present
study, the other two products, AnP CO２! and AnP Plas!
were added to AnP Campylo!, and their characterisation
was studied to clarify how and what purpose individual AnP
types should be used properly. The benefit of using AnP
system in the AnP Box Jar! was also studied from parasi-

Department of Infectious Diseases, Kyorin University School of Medicine, Mitaka, Tokyo 185-0021, Japan
Correspondence: Dr. K. Haruki, Department of Infectious Diseases, Kyorin University School of Medicene, Mitaka, Tokyo 181-8611, Japan;
phone +81-(0)422-47-5511 extention 3467, facsimile +81-(0)422-44-4603, e-mail QWJ04544@nifty.ne.jp

３
６
６

tological points of view in comparison with systems in agas incubator and in a candle jar.

MATERIALS AND METHODS
1) Malaria parasites and cultivation procedures
P. falciparum K1, (chloroquire resistant strain) originating from Thailand and HB3, (chloroquine sensitive
strain) originating from Hondulus were used (supplied incourtesy of Dr. S.A., Ward of University of Liverpool) for
the growth study. K1 is regarded as very useful strain because it shows rather highly different values of IC５０ thanother sensitive strains where it was used for chloroquine
sensitivity assays in different gas conditions. After retrieval
from cryopreservation, the parasites were maintained according to a modifird method described by Trager andJensen (1976). RPMI 1640 (Sigma) was used for parasite
growth supplemented with 2 mg L-Glutamine, 1.0 g NaHCO３, 12.5 ml of 1M HEPES (Dojindo), 0.02 mg gentamycin (Sigma) in 500 ml , and 50 ml of human AB serum
were added (Western Tokyo Red Cross Blood Centre,
Tachikawa, Tokyo). A haematocrit of 5% was maintainedwith washed human blood (type O Rh+) cells supplied also
from Tokyo Red Cross Blood Centre. Before incubation at
37°
C, the completed culture was gassed using a special gas
mixture (Setagaya Sanso) composed of 3% O２ 4% CO２ and
93% N２. Cultures were maintained on a daily basis by observing blood films to assess parasitaemia and replenishing
the medium to feed. Parasite culture were subcultured regularly usually after parasitaemia reached 5%.
2) Parasite Growth
Parasite growth was observed by culturing in 96 wellplate (Falcon 3072) in different gas compositions, 1) Gas
chamber flown with special gas mixture containing 3% O２,
4% CO２ and 93% N２ (Setagaya Sanso), 2) AnP Plas! (5%CO２ and 5% O２), 3) AnP Campylo! (10% CO２ and 10% O２),
4) AnP CO２! (15% O２ and 5% CO２), in an AnP Box-type
Jar! (the size: 22×16×10.5 cm, weight: 500 g, Fig 1.) and5) Candle Jar (15% O２ and 5% CO２). Infected erythrocytes
were diluted to 1% parasitaemia with fresh red blood cells.
Samples were then diluted with RPMI medium in 5% haematcrit, 100 µl each were transferred into wells of six rows
and ten column of the plates. The plates were placed in different gas conditions and incubated at 37°
C. Every 24 hr,
the samples were taken from six wells, fixed with methanol,
then stained with 10% Giemsa for 30 min to count parasitaemia with a light microscope. The culture media werechanged when the samples were taken. New sachets wereplaced in the jars to maintain gas condition after sampling.
Growth curves were also observed by placing the varied

Figure 1 AneroPack sachets (from right to left: AneroPack
Campylo!, CO２ and Plas!) and specific jar (AneroPack Kakugata jar!) with five plates.

number of the plates in a jar.
3) Chloroquine (CQ) sensitivity assay
CQ (Sigma) in 50% methanol (1 mM) was diluted
with RPMI medium in serial two hold dilutions at concentrations of 0, 15.6, 31.3, 62.5, 125, 250, 500, 1,000, 2,000
and 4,000 nM. Fifty (50) µl of RPMI containing CQ wereplaced into wells. Infected erythrocytes diluted into 1%
parasitaemia with fresh red blood cells were diluted with
RPMI into 10% haematocrit, and 50 µl of sample wereadded into each well to adjust the final haematcrit at 5%and final concentrations of CQ at 0 (×2), 7.8, 15.6, 31.3,
62.5, 125, 250, 500, 1,000 and 2,000 nM. The plates wereincubated for 36 hr under different conditions as describedabove. Culture samples were taken from the wells for Giemsa stain to count the number mature and immature schizonts in infected cells. To determine Percent maturation,
the number of schizonts was counted and divided by the
number of schizonts in control. The date were analysed using Grafit! programme (Enrithacus Software, Kent, UK) to
determine the 50% inhibition concentration (IC５０).
4) Gas concentration measurement
Gas concentrations in a jar were measured using Oxygen Analyser LC-700 (Toray Engineering Co., Ltd. Japan)
for O２ and Infrared Gas Analyser UR-025S (Komyo Rikagaku Kogyo KK, Japan) for CO２ 30 min, 1, 2, 4, 18, 24, 48,
72, 96, 120 and 144 hr after start of incubation.
5) Gas pressure measurement in a jar
The pressure in the Box Jar was measured by placing
different number of plates in it. The barometer (Rhythm
Altimeter, Japan) was read 30, 60, 120 min and 24 hr after

３
６
７

incubation.

required until gas composition becomes stable in the Jar, if
the free space is wider. To examine this, the growth rates
were compared by placing 1 or 5 plates in a Jar at different
gas phases by 2 types of AnP with control plates in a gas incubator and in a candle jar. Using AnP Plas, growth rates
were affected by different free space in a Box Jar. The culture in a Jar occupied by five plates shows better growththan by a single plate with AnP Plas (p=0.99) (Table 2).
The results indicated narrower free space in a Jar provided better growth and stable results, therefore, for stabilisation of cultivation in a Jar, 5 plates should be always
placed by adding dummy plates besides examining plates
up to a total of 5.
Difference of concentrations of CO２ and O２ in a Box
Jar was measured by placing 1 or 5 plates in a Jar after 24
hr incubation (Table 3).
Of course, there is no difference of growth by the different number of plates in the gas incubator or in a big can-RESULTS
Difference of growth rates of P. falciparum K1 strain
was examined among different gas phases such as in a gas
incubator, by 3 products of AnP and in a candle jar. The
cultivation started at 1% parasitaemia, and development ofparasites was followed by percent parasitaemia after 24 and48 hr. The result of growth rates thus observed is shown in
Table 1, but there was no remarkable difference of growth
rates in different AnPs except for AnP plas (Table 1). AnP
plas shows less growth with single plate (p=0.003) but notwith five plates.
As it was noticed that the growth rates showed somedifference by the different number of culture plates placedin a Box Jar, growth rates were suspected to be affected by
the free space inside of the Jar, since more time would be

Table 1 Growth of Plasmodium falciparum K1 strain expressed by % parasitaemia at different
gas conditions placing 1 culture plate in an AnaeroPack box jar and starting cultivation
at 1% parasitaemia
Hours forcultivation
jar
0

Gas

Plas

Campylo

CO２

Candle1.0±0.2%

1.0±0.2%

1.0±0.2%

1.0±0.2%

1.0±0.2%24

1.2±0.2%

1.2±0.4%

1.2±0.5%

1.2±0.3%

1.3±0.4%48

5.3±0.2%

4.2±0.3%

4.8±0.8%

4.5±0.5%

4.5±0.6%

Gas; Gas incubator, Plas, Campylo, CO２; varied types of AnaeroPack Mean±SD by two plates observed by triplicate wells (two times examination by using one plate).

Table 2 Growth by % parasitaemia with different number of plates in an AnaeroPack box jar at different gas conditions
No. of platein a box jar
One

Gas

Plas

Campylo

Candle Jar

5.3±0.2%

4.2±0.3%

4.0±0.8%

4.5±0.6%Five

4.9±0.3%

7.3±1.2%

4.8±0.4%

4.5±0.5%

Mean±SD by two plates observed by triplicate wells (two times examination by using one
plate after 48 hr incubation changing media and sachet).

Table 3 Percent (%) concentration of CO２ and O２ in abox jar with 1 or 5 plates after 24 hr incubation
Concentration
CO２ with
1 plates (%)
CO２ with
5 plates (%)
O２ with
1 plate (%)
O２ with
5 plates (%)

Gas

Plas Campylo Candle Jar

4.0

5.3

6.0

4.0

4.0

4.5

6.4

6.0

5.0

6.4

12.7

16.0

5.0±0.7

4.8

11.2

14.0±2.0

Mean by double experiments.

３
６
８

Figure 2 Gas concentration with different number of plates
using AneroPack Campylo. There are four conditions, without plate, with single, three or five plates
in an AnaeroPack box jar. AnaeroPack sachets in a
Jar were renewed every 24 hr at the time of sampling. Open figures indicate O２ concentrations andclosed figures indicate CO２. The shapes of figuresindicate the number of the plates as follows: circles
with single plate, square with three plates, triangles
with five plates and inverted triangles without plate.

dle jar as they are not enclosed in a small space. Thechange of free space in a Jar also modified gas concentrations a little in AnP methods. The concentrations of CO２
are not changed much in AnP methods as in gas incubatorand candle jar with some variations. The concentrations of
O２ however, does not go down to 10% with AnP Campylo
with a single plate but far better with 5 plates, whilst AnP
Plas with 5 plates approached to the gas composition as in agas incubator after 24 hr.
The time course of changes in gas composition in a jar
was observed using AnP Campylo when none, 1, 3, or 5

plates were placed in a Box Jar. It took about 24 hr until
gas composition became stable in a jar, and concentrationsof CO２ and O２ were modified and became more stable when
5 plates were placed (Fig 2).
Generally, high altitude with low atmospheric pressure
hinders the growth of P. falciparum in the culture (Wernsdorfer and Payne, 1988). It is feasible to speculate that thechemical reaction of AnPs may interfere the internal pressure of a Box Jar. AnPs absorb oxygen and produce carbon
dioxide in making a different ratio of gas composition finally. Therefore, the internal pressure of the Jar is one ofthe determinants to affect the culture when different AnPs
are used. In this respect, the pressure difference inside of
the Jar from the outside was measured, and it showed negative with AnP Plas and further decreased from -14 to -20
Hecto Pascal (HP) with more plates, whereas AnP Campylo
showed slightly positive. By the results with AnP Plas, thepressure was changed as an equivalent altitude of about
150 m above sea lebel (Table 4). The change was minor
and can be ignored for the parasite culture.
It is clear that the AnPs are a good tool for the parasite
culture by these experiments. It is considerable that the difference of gas concentrations and pressures affect the results of IC５０ of CQ. For determining reliable IC５０, the preferable gas concentrations are 3% O２ and 4% CO２ as in a gas
phase incubator. It is important to compare these AnP types
by evaluating the observed IC５０ figures for its reliability andimmutability. In terms of CQ sensitivity assay, differenceof IC５０ of K1 was obvious in different gas phases. IC５０ of
K1 in a gas incubator was 217±16 nM whilst those by
AnPs Plas, Campylo (no significant difference), CO２ and in
Candle Jar (significant difference between ordinary gas
method. p=0.007) were 269±15 nM, 235±38 nM, 149±
18 nM and 137±23 nM, respectively (Table 5).

Table 4 Difference of atmospheric pressure inside of abox jar from outside when placing 1 or 5 plates
in a Jar after 24 hr incubation
Number of plates

Plas

Campylo

1 plate

-14HP

+2HP

5 plates

-20HP

+4HP

Single experiment. HP=Hecto Pascal.

Table 5 IC５０ of K1 strain at different gas conditions and with 1 or 5 plates in a box jar
IC５０ (nM)with 1 plate
IC５０ (nM)with 5 plates

Gas

Plas

Compylo

CO２

Candle Jar

217±16269±15235±38149±18138±23220±22202±36217±44ND

122±34

Mean±SD by two plates observed by triplicate wells.

３６９

By the results observed IC５０, gas phases can be divided
into two groups. The one involves gas incubator, AnP Plas,
and AnP Campylo, and the other does the Candle Jar and
AnP CO２. The free space also affects the results of IC５０.
With both Campylo and Plas, IC５０ levels were lowered withfive plates than a single plate, indicating a little more sensitive to CQ in a narrower free space in a Jar. Only using
AnP plas and ordinary gas method showed significant difference with single plate (p=0.99) but not with five plates.
Of course, there is no difference in ordinary gas methods by
the different number of plates.

DISCUSSION
Since establishment of in vitro culture of P. falciparum(Trager and Jensen, 1976), drug sensitivity assay has been
introduced by WHO (Bruce-Chwatt et al., 1986). Although
CQ resistant parasite has been distributed world widely, it is
not well known quantitatively how widely and deeply it isspreading now in the world. We need to monitor the drug
sensitivity in the field widely in terms of drug choice forprevention and treatment.
WHO standard test is based on the candle jar method(Rieckmann et al., 1978). Therefore, it has been so far the
only method to culture the parasites for drug sensitivity
tests in the field. But candle jar is labour intensive and difficult to manage. Usually, gas incubator or gas chamber isused for laboratory works only in the well equipped laboratories for malaria experiments, drug sensitivity tests inclusive (Desjardins et al., 1979). Initially, Trager and Jensen(1976) suggested that the culture of the parasite was good at
low O２ (5%) and high CO２ (7%). They also suggested thatthe candle jar condition is also possible to culture the parasite in the field alternatively, because of a lack of equipment
in developing countries. But the other hand, Desjardins etal. (1979) established a drug sensitivity test using radioactive hypoxanthine in laboratory at gas concentrations of 5%
O２ and 5% CO２. This method nowadays becomes a standard laboratory method for drug sensitivity test world
widely. Although the gas condition affects on the results of
CQ sensitivity test, 2 different gas conditions are used for
drug sensitivity test at moment. That is why there is a discrepancy of the results between field and laboratory CQ
sensitivity tests. The authors tried to characterise AnP system in terms of parasite growth of malaria and CQ sensitivity test for considering a stable method other than candle jar,and eliminating a discrepancy of the results in the laboratory and field.
We reported for the first time that AnP Campylo was
producing appropriate gas phase for malaria cultivation(Haruki et al., 1997a, b, 1998). Soon after, Taylor-Robinson(1998) submitted a short paper regarding this method without using a specific jar regardless of gas composition andpressure in the jar.
The AnP system was originally used for anaerobic bacterial culture by producing CO２ and absorbing O２ from gas
phase to maintain a suitable condition for anaerobic bacterial culture by enclosing them in a specific AnP Box Jar.
There are three types of products, i.e., AnP CO２, AnP Campylo and AnP Plas. The suitable gas conditions of cultivating the parasite is known to be 2-5% CO２ and 3-18% O２(Jensen 1988). In fact, recent studies showed the O２ concentration above 5% also acceptable conditions using AnPCampylo (Haruki et al., 1997a, b, 1998). Onda et al. (1999)
reported use of AnP CO２ to maintain the parasite growth for
26 days.
Although all AnP systems showed fairly good results
for the growth of parasites, the number of incubated plates
or the volume of free space in a jar affected the results ofthe growth with AnP plas, because varied volume of freespace in a jar changed the gas concentration and pressure in
it by the following reasons;
AnP Plas ideally maintains 5% of both O２ and CO２Hence, a total of 15% volume of O２ is absorted by AnP Plas
from enclosed air, and it produces 5% volume of CO２ bychemical reaction. The total balance of volume will be minus 10%. This chemical reaction changed internal pressure
negatively. The other hand, AnP Campylo maintains 10%of both O２ and CO２ and balance of the gas volume maintained as minus 10% volume of O２ and plus 10% volume of
CO２ subsequently. Therefore, internal pressure also maintained as same as the original pressure. By placing moreplates in a Jar, AnP Plas absorbs a restricted amount of gas,
which was calculated and anticipated by the maker using
the box jar, if free space is narrower, and the initial volume
of total gas is smaller. Once chemical reaction starts, absorbed restricted amount of gas interferes the internal pressure more prominently than in the wider free space. Because of the equal balance of the gas volumes taken and released by AnP Campylo, internal pressure will be maintained nearly at the original level even in a wider space assame as in a narrower space. Whilst five plates stabilised
gas composition at 140 hr, single plate did not stabilised gas
composition as product data even 140 hr incubation. This
indicates that AnPs are not exactly same as laboratory
method and will be an alternative method to culture anddrug sensitivity assay. AnPs will be acceptable method in
the field but not in laboratory, because of its stability variation.
CQ sensitivity test using AnP CO２ was also studied by
Lin et al. (1999). As well as in that report, our results also
indicated that the IC５０ levels with AnP showed the results

３７０

similar to those in the candle jar, as gas conditions, by AnP
CO２ appear to be similar in the candle jar, because AnP CO２
was intentionally developed as a replacement of the candle
jar for anaerobic bacterial culture.
A Candle Jar is heavy and difficult to carry, easily brealable and occupies a large space in an incubator whereas
the AnP Box Jar is small in size and light weighing only
500 g. Hence the drug sensitivity test using AnP system in
an AnP Box Jar will be a set of equipment which is easily
portable in the malarious countries.
In the results with AnP Plas, although the pressure waschanged as an equivalent altitude of about 150 m above sea
level, the change was minor and can be ignored for theparasite culture. Proper condition of malaria cultivation using AnP Plas in consideration with drug sensitivity test
would be putting totally 5 culture including dummy plates
placed in an AnP Box Jar, if the testing plates were less than
5 (One plate occupies the space of 166 cm３, and then fiveplates occupy 830 cm３ of the space inside the jar). Because
AnP Plas reduce the pressure and AnP Campylo can notproduce the stable gas concentration with wider space. For
drug sensitivity test, if the gas phase common to the gas incubator is required, AnP Plas or AnP Campylo is useful, but
the latter will be slightly superior in keeping positive pressure inside the Jar and resulting in better parasite culture.
The different pressure may affect the result of IC５０ with especially CQ, because CQ is weak base and its accumulation
to parasite food vacuole interfered by pH of media (SAWard personal communication).
If the field CQ sensitivity test following WHO standard is attempted, AnP CO２ is recommended, since its gas
composition and the result of IC５０ are close to those available in a candle jar by which, however, the result may indicate more CQ sensitive than by other AnPs or in the gas incubator. Finally, to use this system for CQ sensitivity test
widely in practice in the field, the method should be studied
more in comparing laboratory experiment and WHO standard method, and after accumulating the field experience.

ACKNOWLEDGEMENT
The authors show their appreciation to Mitsubishi Gas
Chemical Co. Ltd., and Mr. Daisuke Sugiyama, Sugiyama
Gen Co. Ltd., Bunkyo‐ku, Tokyo, Japan for supplying materials. The blood materials are provided in courtesy of Japan Red Cross Western Tokyo Blood Centre. The authors
are also indebted to Dr. H. Tanaka, former Visiting Professor of this division for his help and review in preparation of
the manuscript.

REFERENCES
１）Bruce‐Chwatt, L.J., Black, R.H., Canfield, C.J., Clyde, D.
F., Peters, W. and Wernsdorfer, W.H., (1986): Chemotherapy of Malaria, 2nd ed., WHO Geneva.
２）Desjardins, R.E., Canfield, C.J., Haynes, J.D. and Chulay,
J.D. (1979): Quantitative assessment of antimalarial activity in vitro by a semiautomated microdilution technique. Antimicrobial Agents and Chemotherapy, 63,
710-718３）Haruki, K., Fujino, T., Yokota, N., Kobayashi, F., Matsui,
T. and Tsuji, M. (1997a): Evaluation of new drug sensitivity assay: Can AnaeroPack System be a novel tool todetermine drug sensitivity of P. falciparum? 9th Malaria
Meeting, British Society of Parasitology, Programme and
Abstracts, 58-59.
４）Haruki, K., Fujino, T., Yokota, N., Kobayashi, F., Matsui,
T. and Tsuji, M. (1997b): Evaluation of AnearoPack Series to detemine drug sensitivity of P. falciparum. Jpn, J.
Trop. Med., 25, Supplement, 87 (Japanese text)５）Haruki, K., Kobayashi, F., Fujino, T., Matsui, T. and Tsuji,
M. (1998): Evaluation of AnaeroPack Campylo andnewly developed AnaeroPack series for culture and drug
sensitivity assay of Plasmodium falciparum. 9th Int.
Cong. Parasitol., Makuhari, Japan. Parsit. Int., 47, Supplement, 379
６）Jensen, J.B. (1988): In vitro cultivation of malaria parasites, Erythrocytic stages, Malaria Vol. 1, ed. by Wernsdorfer, W.H. and MacGregor, Sir I., Churchil Livingstone,
Edinburgh, 307-347.
７）Lin, Q., Onda, T., Kano, S., Masuda, G. and Suzuki, M.
(1999): In vitro drug susceptibility test of Plasmodium
falciparum using a portable thermostat and CO２ gas. J.
Japan. Assoc. Inf. Dis., 73, 1099-1133 (Japanese text)８）Mizuno, Y., Hatabu, T., Kawazu, S., Masuda, G., Ohtomo,
H., Suzuki, M. and Kano, S. (2000): Cultivation of Plasmodium falciparum isolates under the AneroPack gas
condition in a portable themostatic incubator. Jpn. J. Trop.
Med. Hyg., 28, 383-385
９）Onda, T., Lin, O., Kano, S. and Suzuki, M. (1999): Continuous in vitro culture of Plasmodium falciparum using
microanaerophilic gas generators and portable incubator.
J. Japan. Assoc. Inf. Dis., 73, 1095-1098 (Japaniese text)
１０）Rieckmann, K.H., Sax, L.J., Campbell, G.H., and Mrema,
J.E., (1978): Drug sensitivity of Plasmodium falciparumAn in vitro microtechnique. Lancet, 7, 22-23
１１）Taylor-Robinson, A.W. (1998): Cultivation of Plasmodium
falciparum in vitro using microbiological gas sachets thatgenerate micronaerophilic conditions. Trans. Roy. Soc.
Trop. Med. Hyg., 92, 357-358
１２）Trager, W. and Jensen, J.B., (1976): Human malaria parasites in continuous culture. Science, 193, 673-675
１３）Wernsdorfer, W.H. and Payn, D. (1988): Drug sensitivity
test, malaria parasites. Malaria Vol. 2, ed. by Wernsdorfer, W.H. and MacGregor, Sir I., Churchil Livingstone,
Edinburgh. 1765-1794

３
７
１

Jpn. J. Trop. Med. Hyg., Vol.２
９, No．
４,２
０
０１, pp.３
７
１
‐
３
７
４

MORPHOLOGICAL RE-EXAMINATION OF
PARAGONIMUS WESTERMANI DESCRIBEDBY DAENGSVANG AND OTHERS IN 1964
HIROMU SUGIYAMA１，＊, TOSHIYUKI SHIBAHARA２, PUNSIN KETUDAT３,SODSRI THAITHONG４ AND KENJIRO KAWASHIMA５
Received October 12, 2001/Accepted November 26, 2001

Abstract: We examined specimens of adult Paragonimus westermani collected from Thai leopards by Daengsvang
et al. in 1964. The two flukes examined had a six-lobed ovary each and singly-spaced cuticular spines. The seminal receptacle and seminal vesicle were filled with spermatozoa. Based on morphological features, these flukes
were identified as the diploid type of P. westermani. The testis branched out into six lobes on the ovarian side andinto five on the opposite side in both specimens. Abnormal branching in the testes, that is, separation of one lobe
from the others and its direct connection to the vas deferens, which was found in adult P. westermani from the Philippines, was undetectable.
Key words: Lung fluke, Paragonimus westermani, Diploid, Morphology, Thailand

INTRODUCTION
The lung fluke remained undiscovered in Thailand until 1964, when Daengsvang et al. (1964) isolated adultParagonimus westermani from leopards captured in southern Thailand. Soon thereafter, Miyazaki and Wykoff (1965)
described a new species, P. siamensis, and Miyazaki and
Vajrasthira (1967a) reported the occurrence of P. heterotremus. Miyazaki and Vajrasthira then described a new species, P. bangkokensis (Miyazaki and Vajrasthira, 1967b), theoccurrence of P. macrorchis (Miyazaki and Vajrasthira,1967c) and the discovery of another species, P. harinasutai(Miyazaki and Vajrasthira, 1968). Thus the number of species of the genus Paragonimus identified in Thailand totals
six. From a medical viewpoint, P. heterotremus is the mostimportant because it infects humans readily and occurs
widely in various parts of Thailand (Vanijanonta et al.,1984). However, human infection with P. westermani has
not been documented in Thailand.
In 1977, Miyazaki reported that lung flukes, regarded
as P. westermani for almost a century, could be divided into
two types according to the contents of the seminal recepta-

cle of the adult specimens: one is the bisexual (or original)
type in which the seminal receptacle is filled with sperm
and the other is the parthenogenetic (or nonspermatozoan)
type in which it is occupied by egg cells and vitelline cells(Miyazaki, 1977). Miyazaki’s opinion has its base on the
results of the chromosome study of P. westermani by Sakaguchi and Tada (1976) and Terasaki (1980). They showedthat the bisexual type is diploid with karyotype 2n=22,
while the parthenogenetic type is triploid with karyotype
3n=33. Miyazaki (1978) then proposed that the parthenogenetic type should be referred to as a separate species
named P. pulmonalis. Some researchers, however, have argued against such separate specific naming for the parthenogenetic triploid form of P. westermani (Blair et al., 1997).
We were offered the opportunity to observe the specimens of adult P. westermani collected from Thai leopards
by Daengsvang et al. (1964). Re-examination of these
specimens revealed that they were diploid, as described below.

１ Department of Parasitology, National Institute of Infectious Diseases, Tokyo 162-8640, Japan
２ Laboratory Animal Research Center, Faculty of Medicine, Tottori University, Yonago 683-0826, Japan
３ Faculty of Science, Srinakharinwirot University, Bangkok, Thailand
４ Faculty of Science, Chulalongkorn University, Bangkok, Thailand
５ School of Health Sciences, Kyushu University, Fukuoka 812-8582, Japan*Corresponding author: Hiromu Sugiyama
Department of Parasitology, National Institute of Infectious Diseases, Toyama 1-23-1, Shinjuku-ku, Tokyo 162-8640, Japan
Tel: +81-3-5285-1111, Fax: +81-3-5285-1173, E-mail: hsugi@nih.go.jp

３
７
２

MATERIALS AND METHODS
Adult flukes of P. westermani were collected fromnaturally infected leopards caught in Choomporn Province
in southern Thailand (Daengsvang et al., 1964). The two
specimens examined in this study were stained with borax
carmine and mounted with Canada balsam.

RESULTS
The specimens 1 and 2 (Figs. 1 and 4) measured 13.5mm and 12.9 mm in length and 8.7 mm and 7.6 mm inwidth, respectively. Cuticular spines were originally single(Figs. 2 and 5), but some of them had split into two or moreat the terminal end. The oral sucker was located near theanterior end of the body, and was 1.32 and 1.21 mm wide in
specimens 1 and 2, respectively. The ventral sucker was
situated somewhat anteriorly to the middle of the body andwas 1.00 and 0.95 mm wide in specimens 1 and 2, respectively. The ventral sucker was slightly smaller than the oral
sucker in both flukes. The intestine wound gently and ran
posteriorly to the end of the body. The ovary, situated on
the anterolateral side of the ventral sucker, was divided into
six lobes, each of which was subdivided into short sections(Figs. 3 and 6). The seminal receptacle and seminal vesicle
were small but filled with spermatozoa. The uterus wasfilled with eggs and situated on the opposite side of theovary. The vitelline glands were widely and bilaterally distributed. The testes, situated at both sides of the posterior
part of the body, were almost as large as the ovary. The testis branched out into six lobes on the ovarian side and into
five on the opposite side in both flukes. Abnormal branching of the testes was not observed; that is, none of the lobes
were separated from the rest and directly connected to thevas deferens.

Figure 1 Mounted adult fluke specimen of Paragonimus westermani, No. 1, stained with borax carmine,showing the ovary (O) and testes (T).
Figure 2 Microphotograph of cuticular spines, showing singly-spaced arrangement.
Figure 3 Camera lucid drawing of the ovary, which was divided into six lobes. SR, seminal receptacle.

３
７３

Figure 4 Mounted adult fluke specimen, No. 2, stained with borax carmine, showing the ovary (O) andtestes (T).
Figure 5 Microphotograph of cuticular spines, showing singly-spaced arrangement.
Figure 6 Camera lucid drawing of the ovary, which was divided into six lobes. SR, seminal receptacle.

DISCUSSION
Daengsvang et al. (1964) isolated adult P. westermani
from Thai leopards, but the morphological characteristics ofthe flukes were not described other than that the features ofthe flukes were identical to the description of P. westermani
by Yamaguti (1958). In the present study, the adult flukes
collected by Daengsvang et al. (1964) were examined andwere identified as the diploid type of P. westermani on thebasis of morphological features. The flukes examinedclosely resembled P. siamensis in morphological features,
but the cuticular spines of adult P. siamensis are arranged ingroups all over the body surface (Miyazaki and Wykoff,1965), whereas the spines were singular in P. westermani.
We recently found Paragonimus metacercariae in thecrab, Siamthelphusa improvisa, from Surat Thani Province
in southern Thailand. Size and shape indicated that they
were P. westermani metacercariae. In a scanning electron

microscopic study, Kanla et al. (1998) observed excysted
metacercariae of P. westermani harvested from crabs in
Nakhon Nayok Province in central Thailand. Taken together, these findings suggest that the life cycle of P.
westermani is still maintained in Thailand with crabs functioning as the second intermediate host.
The phylogeny and/or intraspecific variation of P.
westermani have been studied using nuclear ribosomal
DNA {second internal transcribed spacer (ITS2)} and mitochondrial DNA (partial COI) sequences (Blair et al., 1997;
Iwagami et al., 2000). The results indicated that P. westermani could be partitioned into at least 2 groups (or species):
the northeastern Asian group and the southern Asian group.
The former group consisted of both diploid and triploidtypes of P. westermani from China, Korea, Japan and Taiwan and the latter consisted of P. westermani from Malaysia, the Philippines and Thailand, which were confirmed orassumed to be diploid. These two groups were also distin-

３
７
４

guished by the difference in snail host specificity. P.
westermani in the northeastern group utilized the pleurocercid snails as the first intermediate hosts, whereas those in
the southern group utilized the thiarid snails (Davis et al.,1994). Even within the groups, however, the morphologicalfeatures of the adult flukes were not universal. As Miyazaki(1981) described, adult P. westermani from the Philippines
exhibited some morphological peculiarities in the testes;
one or two lobes of one or both testes were often separated
from the rest and directly connected to the vas deferens.
This abnormal branching was observed in 83 (18.4%) of
450 adult flukes examined. Miyazaki (1978) also examinedat least 36 adult flukes of P. westermani from Malaysia but
did not mention such abnormalities in those specimens. Abnormal branching was undetectable in the testes of P.
westermani from Thailand in the present study. Morespecimens from various localities in Thailand should be investigated to confirm our findings.

ACKNOWLEDGEMENTS
This study was supported in part by research grants(Nos. 62041067 and 63043053) under the International Scientific Research Program from the Ministry of Education,
Science, Sports and Culture of Japan.

REFERENCES
１）Blair, D., Agatsuma, T., Watanobe, T., Okamoto, M. andIto, A. (1997): Geographical genetic structure within thehuman lung fluke, Paragonimus westermani, detectedfrom DNA sequences. Parasitology, 115, 411-417
２）Daengsvang, S., Papasarathorn, T. and Tongkoom, B.
(1964): Paragonimus westermani (Kerbert, 1878) in Thai
leopards. Ann. Trop. Med. Parasitol., 58, 304-306
３）Davis, G.M., Chen, C.E., Kang, Z.B. and Liu, Y.Y.
(1994): Snail hosts of Paragonimus in Asia and theAmericas. Biomed. Env. Sci., 7, 369-382
４）Iwagami, M., Ho, L.Y., Su, K., Lai, P.F., Fukushima, M.,
Nakano, M., Blair, D., Kawashima, K. and Agatsuma, T.
(2000): Molecular phylogeographic studies on Paragonimus westermani in Asia. J. Helminthol., 74, 315-322
５）Kanla, P., Yaemput, S., Arunyanart, C. and Maleewong,

W. (1998): Scanning electron microscopy of newly excysted metacercariae of Paragonimus westermani-like,
Nakhon Nayok, Thailand. Southeast Asian J. Trop. Med.
Public Health, 29, 748-750６）Miyazaki, I. (1977): A newly introduced question on
Paragonimus westermani (Kerbert, 1878). Jpn. Med. J.,
2788, 43-46
７）Miyazaki, I. (1978): Two types of the lung fluke which
has been called Paragonimus westermani (Kerbert, 1878).
Med. Bull. Fukuoka Univ., 5, 251-263
８）Miyazaki, I. (1981): Taxonomical studies on the lung
fluke occurring in the Philippines. Med. Bull. Fukuoka
Univ., 8, 153-158
９）Miyazaki, I. and Vajrasthira, S. (1967a): Occurrence ofthe lung fluke Paragonimus heterotremus Chen et Hsia,
1964, in Thailand. J. Parasitol., 53, 207
１０）Miyazaki, I. and Vajrasthira, S. (1967b): On a new lung
fluke, Paragonimus bangkokensis sp. nov. in Thailand(Trematoda: Troglotrematidae). Jpn. J. Med. Sci. Biol.,20, 243-249
１１）Miyazaki, I. and Vajrasthira, S. (1967c): Occurrence ofthe lung fluke Paragonimus macrorchis Chen, 1962 inThailand. J. Parasitol., 53, 894-895
１２）Miyazaki, I. and Vajrasthira, S. (1968): On a new lung
fluke found in Thailand, Paragonimus harinasutai sp.
nov. (Trematoda, Troglotrematidae). Ann. Trop. Med.
Parasitol., 62, 81-87
１３）Miyazaki, I. and Wykoff, D.E. (1965): On a new lung
fluke, Paragonimus siamensis n. sp. found in Thailand(Trematoda, Troglotrematidae). Jpn. J. Parasitol., 14,251-257
１４）Sakaguchi, Y. and Tada, I. (1976): Chromosomes of alung fluke, Paragonimus westermani. Chromosome Inf.
Serv., 20, 23-24
１５）Terasaki, K. (1980): Comparative studies on the karyotypes of Paragonimus westermani (s. str.) and P. pulmonalis. Jpn. J. Parasitol., 29, 239-243
１６）Vanijanonta, S., Bunnag, D. and Harinasuta, T. (1984):
Paragonimus heterotremus and other Paragonimus spp.
in Thailand: pathogenesis, clinic and treatment. Arzneim.
Forsch., 34, 1186-1188
１７）Yamaguti, S. (1958): Paragonimidae Dollfus, 1939, Yamaguti, S., (ed.), Systema Helminthum, Vol. 1, The digenetic trematodes of vertebrates. Part 1, 931-933, Interscience Publishers, New York３
７
５

Jpn. J. Trop. Med. Hyg., Vol.２
９, No．
４,２
０
０１, pp.３
７
５
‐
３
７
８

SURFACE ULTRASTRUCTURE OF NEWLY EXCYSTEDMETACERCARIAE OF PARAGONIMUS WESTERMANIFROM MALAYSIA AND THE PHILIPPINESHIROMU SUGIYAMA１，＊, KAREN LAI２, C.K.OW-YANG２,BAYANI L. BLAS３ AND KENJIRO KAWASHIMA４
Received October 12, 2001/Accepted November 24, 2001

Abstract: We studied the ultrastructure of excysted metacercariae of Paragonimus westermani from Malaysia andthe Philippines using scanning electron microscopy. The body surface of the excysted metacercariae was covered
with numerous single-pointed and thorn-like tegumentary spines. Dome-shaped papillae were evenly distributed
over the whole body and situated circularly around the oral and ventral suckers. Between six to eight and seven tonine small dome-shaped papillae were observed around the ventral sucker in P. westermani from Malaysia and thePhilippines, respectively. In general, the morphological features of the worms from Malaysia and the Philippines
were identical.
Key words: Lung fluke, Paragonimus westermani, Scanning electron microscopy, SEM, Ultrastructure, Malaysia,
the Philippines

INTRODUCTION

MATERIALS AND METHODSThe external appearance of newly excysted metacercariae has been studied on various species of the genus
Paragonimus using scanning electron microscopy (SEM;
Higo and Ishii, 1984, 1987; Sugiyama et al., 1990). The
number of papillae around the oral and ventral suckers havebeen reported to vary among species. Moreover, even
within a species, for example P. mexicanus, the number ofpapillae around the ventral sucker differed by geographicaldistribution (Aji et al., 1984; Tongu et al., 1985, 1987,
1995).
Recently, Kanla et al. (1998) studied the surface morphology of newly excysted metacercariae of P. westermani
from Thailand using SEM. They noted that the morphological features of flukes from Thailand and Japan were
nearly identical. In the present study, we used SEM to examine the surface ultrastructure of excysted metacercariaeof P. westermani from Malaysia and the Philippines and to
determine whether a geographical difference exists in morphology in P. westermani, similar to that seen in P. mexicanus.

P. westermani metacercariae were harvested from thecrabs, Parathelphusa maculata, from the Kuala Pilah area
in Negri Sembilan State of peninsular Malaysia in July,
1985, and, Sundathelphusa philippina, from the Jaro area in
Leyte Province of the Philippines in September, 1985. The
P. westermani populations occurring in these localities wereidentified as the diploid type based on the morphologicalfeatures of the adult flukes which were recovered from thecats given the metacercariae. Fifteen metacercariae eachwere available for the present study.
The methods for excystation of Paragonimus metacercariae have been previously described (Higo and Ishii,1984). Excysted metacercariae were washed in 0.1M phosphate buffer (pH 7.4) several times, fixed for 4 hr in 1%
phosphate-buffered glutaraldehyde and fixed for 2 hr in 1%
phosphate-buffered osmium tetroxide. The specimens were
dehydrated in a graded series of ethanol, substituted withisoamyl acetate and critically dried. The metacercariae
were mounted on aluminum stubs, coated with platinumpalladium and examined under a scanning electron micro-

１ Department of Parasitology, National Institute of Infectious Diseases, Tokyo 162-8640, Japan
２ Division of Parasitology, Institute for Medical Research, Kuala Lumpur, Malaysia
３ Schistosomiasis Research and Training Center, Palo, Leyte, the Philippines
４ School of Health Scinences, Kyushu University, Fukuoka 812-8582, Japan*
Corresponding author: Hiromu Sugiyama
Department of Parasitology, National Institute of Infectious Diseases, Toyama 1-23-1, Shinjuku-ku, Tokyo 162-8640, Japan
Tel: +81-3-5285-1111, Fax: +81-3-5285-1173, E-mail: hsugi@nih.go.jp

３
７
６

scope (Hitachi S-800) at an accelerating voltage of 5 kv.

RESULTSP. westermani from Malaysia
The excysted matacercariae had various shapes, although the body of most specimens was flattened dorsoventrally (Fig. 1A). The body surface was covered with numerous single-pointed and thorn-like tegumentary spines. Domeshaped papillae were evenly distributed over the wholebody.
The oral sucker had an inner diameter of about 20 µm
and was situated near the anterior end of the worm body(Fig. 1B). Around the oral sucker, three types of domeshaped papillae were observed: large papillae with a raggedsurface, small papillae with an irregular shaped pit andFigure 1 Scanning electron microscopy of newly excysted
metacercaria of P. westermani from Malaysia.
1A Whole body in ventral view. Bar=30 µm.
1B High power magnification of oral sucker region showing the papillae. Bar=10 µm.
1C High power magnification of ventral sucker region showing the papillae. OS, oral sucker; VS,ventral sucker; P, papillae. Bar=10 µm.

small papillae with a smooth surface. The small domeshaped papillae with a pit were distributed mainly on thedorsal or dorsloateral side of the sucker and numbered between 25 and 30. The small dome-shaped papillae with asmooth surface were situated on the dorsal or dorsolateralside of the sucker and numbered around 20.
The ventral sucker had an inner diameter of about 40µm and was situated somewhat anterior to the middle of theworm body (Fig. 1C). Around the ventral sucker, we observed a circular distribution of large dome-shaped papillae
internal to small dome-shaped papillae. The number oflarge papillae was fixed at six, whereas the number of small
papillae varied from six to eight.
P. westermani from the Philippines
No distinct difference was evident between P. wester-Figure 2 Scanning electron microscopy of newly excysted
metacercaria of P. westermani from the Philippines.
2A Whole body in ventral view. Bar=30 µm.
2B High power magnification of oral sucker region showing the papillae. Bar=10 µm.
2C High power magnification of ventral sucker region showing the papillae. OS, oral sucker; VS,ventral sucker; P, papillae. Bar=10 µm.

３
７７

mani from the Philippines and Malaysia either in the shapeof metacercarial bodies, in the shape and position of eachsucker or in the shape and distribution of the tegumentaryspines (Fig. 2A). Dome-shaped papillae also showed similar shape and arrangement to those in the flukes from Malaysia (Fig. 2B). However, the number of papillae aroundthe ventral sucker differed slightly. As seen in P. westermani from Malaysia, we observed six large dome-shaped
papillae in P. westermani from the Philippines. However,the small dome-shaped papillae varied in number fromseven to nine (Fig. 2C).

DISCUSSION
The number of small dome-shaped papillae around theventral sucker of newly excysted metacercariae of the genus
Paragonimus from Japan varies with the species (Higo andIshii, 1984, 1987): zero to six papillae are found in P. ohirai(zero to two in the metacercaria which has no inner cyst
wall and was originally described as P. iloktsuenensis, andtwo to six in the metacercaria which has inner cyst wall),five to 13 in P. westermani (five to 11 in the triploid type of
P. westermani and 10 to 13 in the diploid type), and 13 to
19 in P. miyazakii. Moreover, in P. mexicanus, a marked
geographical difference is observed in the number of these
papillae: 22 to 25 papillae are found in P. mexicanus from
Peru (originally referred to as P. peruvianus; Aji et al.,1984), 20 to 30 in those from Guatemala (Tongu et al.,1995), 30 to 35 in those from Mexico (Tongu et al., 1985),
and 28 to 38 in those from Costa Rica (Tongu et al., 1987).
In the present study, we found that the small dome-shaped
papillae around the ventral sucker of excysted metacercariaeof P. westermani numbered six to eight and seven to nine in
Malaysian and Philippine P. westermani, respectively. In
contrast, the papillae numbered 10 to 13 in P. westermani
metacercariae found in Thailand (Kanla et al., 1998). Although the number of the papillae varied between P. westermani from Thailand and Malaysia, and the Philippines, thevariation was small and was within the range of the observed papillae in P. westermani from Japan. Our data suggest that a geographical difference does not exist in morphology of excysted metacercariae of P. westermani andthus, the number of the papillae (5 to 13) could be used as acriterion for identifying P. westermani in the metacercarial
stage.
The phylogeny of P. westermani has been studied using ribosomal and mitochondrial DNA sequences (Blair etal., 1997; Iwagami et al., 2000). The results indicated that
the P. westermani examined could be partitioned into atleast two groups (or species): the northeastern Asian groupand the southern Asian group. The northeastern group con-sisted of both diploid and triploid P. westermani from China,
Korea, Japan, and Taiwan, and the southern group consistedof P. westermani from Malaysia, the Philippines and Thailand, which were confirmed or assumed to be diploid. Even
within each group, however, the internal morphology of theadult flukes was not universal. For instance, as Miyazaki(1981) described, adult P. westermani from the Philippines
exhibited some morphological peculiarities in the testes:
one or two lobes of one or both testes were often separated
from the rest and directly connected to the vas deferens.
This kind of abnormality was undetectable in adult P.
westermani from Malaysia (Miyazaki, 1978) and Thailand(Sugiyama et al., unpublished data). However, informationon the external morphology of adult P. westermani using
SEM is still lacking and thus, the potential existence of geographical differences in the surface ultrastructure of P.
westermani in the adult stage remains to be investigated.

ACKNOWLEDGMENTS
We express our gratitude to all the members of the Institute for Medical Research in Kuala Lumpur of Malaysia,
and Schistosomiasis Research and Training Center in Palo,
Leyte of the Philippines for collecting materials in thisstudy. We also thank Dr. Shigehisa Habe of Fukuoka University, and Dr. Takeshi Agatsuma of Kochi Medical School,
who provided encouragement throughout this study. This
study was supported in part by research grants (Nos.
60041052 and 61043047) under the International Scientific
Research Program from the Ministry of Education, Science,Sports and Culture of Japan.

REFERENCES
１）Aji, T., Oh, H., Tongu, Y., Inatomi, S., Hata, H., Kobayashi, M. Yokogawa, M., Miranda, H. and Ibáñz, N.
(1984): Ultrastructure of tegumental surface of themetacercaria of Paragonimus peruvianus. Jpn. J. Parasitol., 33,15-21
２）Blair, D., Agatsuma, T., Watanobe, T., Okamoto, M. andIto, A. (1997): Geographical genetic structure within thehuman lung fluke, Paragonimus westermani, detectedfrom DNA sequences. Parasitology, 115, 411-417
３）Higo, H. and Ishii, Y. (1984): Scanning electron microscopy of the newly excysted juveniles of Paragonimus
westermani (Kerbert, 1878) Braun, 1899 (parthenogenetic type) and P. miyazakii Kamo, Nishida, Hatsushika
and Tomimura, 1961. Jpn. J. Parasitol., 33, 421-427
４）Higo, H. and Ishii, Y. (1987): Comparative studies on surface ultrastructure of newly excysted metacercariae ofJapanese lung flukes. Parasitol. Res., 73, 541-549
５）Iwagami, M., Ho, L.Y., Su, K., Lai, P.F., Fukushima, M.,
Nakano, M., Blair, D., Kawashima, K. and Agatsuma, T.

３
７８(2000): Molecular phylogeographic studies on Paragonimus westermani in Asia. J. Helminthol., 74, 315-322
６）Kanla, P., Yaemput, S., Arunyanart, C. and Maleewong,
W. (1998): Scanning electron microscopy of newly excysted metacercariae of Paragonimus westermani-like,
Nokhon Nayok, Thailand. Southeast Asian J. Trop. Med.
Public Health, 29, 748-750
７）Miyazaki, I. (1978): Two types of the lung fluke which
has been called Paragonimus westermani (Kerbert, 1878).
Med. Bull. Fukuoka Univ., 5, 251-263
８）Miyazaki, I. (1981): Taxonomical studies on the lung
fluke occurring in the Philippines. Med. Bull. Fukuoka
Univ., 8, 153-158
９）Sugiyama, H., Horiuchi, T., Tomimura, T., Shibahara, T.,
Agatsuma, T., Habe, S., Kawashima, K., Ketudat, P. andThaithong, S. (1990): Surface ultrastructure of newly excysted metacercariae of Paragonimus heterotremus.
Southeast Asian J. Trop. Med. Public Health, 21, 109-113
１０）Tongu, Y., Aji, T., Oh, H., Ishiii, A., Yokogawa, M., Hata,
H., Ito, J. and Lamothe-Argumedo, R. (1985): Surface ultrastructure of Paragonimus mexicanus Miyazaki et Ishii,
1968. Jpn. J. Parasitol., 34, 441-447
１１）Tongu, Y., Hata, H., Orido, Y., Pinto, M.R., LamotheArgumedo, R., Yokogawa, M. and Tsuji, M. (1995): Morphological observations of Paragonimus mexicanus from
Guatemala. Jpn. J. Parasitol., 44, 365-370
１２）Tongu, Y., Iwanaga, Y., Hata, H., Tsuji, M., Yokogawa, M.,
Morera, P. and Conejo, M. (1987): Morphological features of Paragonimus metacercariae from Costa Rica. Jpn.
J. Parasitol., 36, 236-241

JAPANESE JOURNAL OF
TROPICAL MEDICINE AND HYGIENE

第２
９巻

第４号
内

著
蠕虫卵の定量的検出のためのナイロンメッシュ濾過法（英文）真喜屋 清 ……………………………………………………………………………………………３３５‐３４２
メフロキン，クロロキン，ピリメサミン耐性熱帯熱マラリア原虫に対するミノサイクリンのin vitro 効果（英文）林清華，片倉賢，大上 美穂，狩野 繁之，鈴木
守 ……………………………３４３‐３４８
タイで採集されたブユ（ハエ目：ブユ科）の１新種 Simulium (Simulium) weji sp. nov.
について（英文）高岡 宏行 ……………………………………………………………………………………………３４９‐３５４
Nippostrongylus brasiliensis 感染ラットにおける小腸からの寄生虫抗原吸収の機構（英文）山田稔，内川 隆一，松田 信治，有薗 直樹 ……………………………………………３５５‐３５９
マウスにおける Nippostrongylus brasiliensis の分泌・排泄抗原持続注入による IgE 抗体産生の誘導（英文）
山田稔，手越 達也，内川 隆一，松田 信治，西田稔，有薗 直樹 ……………３６１‐３６４
熱帯熱マラリア原虫の培養および薬剤感受性試験の方法としての各アネロパック!
に関する検討（英文）春木 宏介，小林富美恵，藤野 隆志，松井 利博，辻守康 ……………………………３６５‐３７０
Daengsvang らの報告したウェステルマン肺吸虫に関する形態学的再検討（英文）杉山広，柴原 壽行，Punsin Ketudat，Sodsri Thaithong，川島健治郎 ……………………３７１‐３７４
マレーシア産およびフィリピン産ウェステルマン肺吸虫脱嚢メタセルカリアの体表微細構造（英文）杉山広，Karen Lai，C.K. Ow-Yang，Bayani L. Blas，川島健治郎 …………………………３７５‐３７８

会報・記録
２
０
０
１（平成１
３）年度日本熱帯医学会役員名簿………………………………………………………………３７９
日本熱帯医学会雑誌編集委員名簿 ………………………………………………………………………３８０‐３８１
投稿規定 ……………………………………………………………………………………………………３８２‐３８３
日本医学会だより …………………………………………………………………………………………３８４‐３８５

DECEMBER ２０
０
１

CONTENTS

容
Japanese Journal of Tropical Medicine and Hygiene Vol. 29 No. ４

原

VOL．
２
９ No．
４

平成１
３年１
２月

Original article
Makiya, K.
Nylon Mesh Filtration Technique for Quantitative Detection of Helminth Eggs ……………………３３５‐３４２Lin, Q., Katakura, K., Oue, M., Kano, S. and Suzuki, M.
Effects of Minocycline against Mefloquine-, Chloroquine- and Pyrimethamine-resistantPlasmodium falciparum in vitro ……………………………………………………………………３４３‐３４８
Takaoka, H.
Simulium (Simulium) weji sp. nov. (Diptera: Simuliidae) from Thailand……………………………３４９‐３５４
Yamada, M., Uchikawa, R., Matsuda, S. and Arizono, N.
Mechanisms of Parasite Antigen Uptake from the Intestine of Rats Infected with the Nematode
Nippostrongylus brasiliensis …………………………………………………………………………３５５‐３５９
Yamada, M., Tegoshi, T., Uchikawa, R., Matsuda, S., Nishida, M. and Arizono, N.
Induction of IgE Antibody Response in Mice by Continuous Administration of
Excretory/secretory Products of the Nematode Nippostrongylus brasiliensis ………………………３６１‐３６４
Haruki, K., Kobayashi, F., Fujino, T., Matsui, T. and Tsuji, M.
Evaluation of AnaeroPack! (AnP) Type as Tools of Plasmodium falciparum cultivation
and Drug Sensitivity Tests ……………………………………………………………………………３６５‐３７０
Sugiyama, H., Shibahara, T., Ketudat, P., Thaithong, S. and Kawashima, K.
Morphological Re-examination of Paragonimus westermani Described by Daengsvang andOthers in 1964 ………………………………………………………………………………………３７１‐３７４Sugiyama, H., Lai, K., Ow-Yang, C.K., Blas, B.L. and Kawashima, K.
Surface Ultrastructure of Newly Excysted Metacercariae of Paragonimus westermani from
Malaysia and the Philippines …………………………………………………………………………３７５‐３７８

２
９巻総目次

日 本 熱 帯 医 学 会

日本熱帯医学会表紙!−" ＭＫ 稲 森木 芦塚

December２
０
０
１

日熱医会誌

Jpn. J. Trop.
Med. Hyg.

JAPANESE SOCIETY OF TROPICAL MEDICINE

背は３mm