JAPANESE JOURNAL OF
TROPICAL MEDICINE AND HYGIENE

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第４号
内

著
末梢血単核球と多核白血球によるインターロイキン８の産生と熱帯熱マラリア原虫の発育阻害作用
大屋ゆりジュリエッタ，松岡 裕之，向田 直史，笠原忠，石井明……………………３５１

Plasmodium yoelii 17XL 感染 ICR マウスでのジョウザンアジサイ葉水煮沸抽出液の抗マラリア活性の
季節的相違，特にフェブリフジン・イソフェブリフジン含有量に関して
石井明，藤井香，酒井美香子，飯干 昌美，宮瀬 敏男，寺田
護…………………３６１
短Original article
Ohya, J-Y., Matsuoka, H., Mukaida, N., Kasahara, T., and Ishii, A.
Growth inhibition of Plasmodium falciparum and interleukin-8 production by peripheral bloodmononuclear and polymorphonuclear cells in vitro ……………………………………………………３５１
Ohnishi, K., Kato, Y., and Iseki, M.
An imported Japanese case of cyclosporiasis ……………………………………………………………３５７
Ishih, A., Fujii, K., Sakai, M., Iiboshi M., Miyase, T., and Terada, M.
Seasonal differences in antimalarial activity of hot-water extract of Dichroa febrifuga leaves against
Plasmodium yoelii 17XL in ICR mice, with reference to febrifugine and isofebrifugine content ………３６１
Short Communication
Iwanaga, Y.
Comparative studies on the development of larval Angiostrongylus cantonensis in Puerto Rican andBrazilian Biomphalarla snails. …………………………………………………………………………３６５

and

会報・記録
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２年度（平成１４年）日本熱帯医学会役員名簿（２００
２年１２月１日現在）………………………………３７１
投稿規定…………………………………………………………………………………………………………３７５
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Medicine報
プエルトリコ産及びブラジル産 Biomphalaria 貝体内に於ける広東住血線虫幼虫の発育の比較
岩永
襄…………………………………………………………………………………………………３６５

Journal of Tropical

日本人輸入サイクロスポーラ症の１例
大西 健児，加藤 康幸，井関 基弘…………………………………………………………………３５７

DECEMBER ２０
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CONTENTS

容
Japanese

原

VOL．
３
０ No．
４

平成１
４年１
２月

Hygiene３
０巻総目次

Vol. 30 No. 4

日 本 熱 帯 医 学 会

日本熱帯医学会表紙!−" ＭＫ 中島 芦塚

DECEMBER２
０
０
２

日熱医会誌

Jpn. J. Trop.
Med. Hyg.

JAPANESE SOCIETY OF TROPICAL MEDICINE

背は２mm
（決定）

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Jpn. J. Trop. Med. Hyg., Vol.３
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GROWTH INHIBITION OF PLASMODIUM FALCIPARUM AND
INTERLEUKIN-8 PRODUCTION BY PERIPHERAL BLOOD
MONONUCLEAR AND POLYMORPHONUCLEAR CELLS IN VITROJULIETA YURI OHYA１, HIROYUKI MATSUOKA１, NAOFUMI MUKAIDA２,TADASI KASAHARA３ and AKIRA ISHII１
Accepted November, 19, 2002

Abstract: Peripheral blood mononuclear and polymorphonuclear cells have been demonstrated to kill and inhibit
malaria parasite proliferation in vitro, but most of the reports required activation of the cells by cytokines or presence of immune sera or opsonins. In our study, peripheral blood leukocytes from non-immune donor efficiently inhibited Plasmodium falciparum growth, depending on the effecter/target ratio. Moreover, these cells produced alarge amount of interlenkin-8 (IL-8) under stimulation with infected erythrocytes or supernatant of the Plasmodium
falciparum culture. IL-8 secretion in the culture supernatant of polymorphonuclear cells was noted from 6-9 hr ofstimulation with the parasites, with a substantial increase over 24 hours of culture. Attempt to elucidate whether IL8 was involved in malaria suppression was done, and the result suggested that IL-8 was not directly involved in theanti-malarial activity of the mononuclear and polymorphonuclear cells. The other mechanism besides IL-8 maywork for inhibiting parasite growth in the culture system of malaria parasites associated with leukocytes.
Key words: cytokine, interleukin-8, malaria, mononuclear cell, Plasmodium falciparum, polymorphonuclear cell

INTRODUCTION
Anti-malarial activity of peripheral blood mononuclear
cells (MNC) and polymorphonuclear cells (PMN) havebeen demonstrated (Vernes, 1980; Celada et al., 1983;
Nnalue and Friedman 1988; Bouharoun-Tayoun et al.,
1990; Kumaratilake et al., 1997), but in most of the reports,
presence of opsonising agents (Nnalue and Friedman 1988),
antibodies (Celada et al., 1983; Bouharoun-Tayoun et al.,1990) or complements (Kumaratilake et al., 1997) were required. There are considerable evidences showing involvement of cytokines such as tumor necrosis factor α (TNFα),interferon-gamma (IFNγ), interleukin lα (IL-Iα), and othersin the immunity and pathophysiology of malaria (Ockenhouse et al., 1984; Kossodo and Grau, 1993; Kwiatkowski,1995). The involvement of interleukin-8 (IL-8）and its
ligand in Plasmodium vivax infection has been suggested(Horuk, 1994), and this chemokine has been detected in theserum of patients infected with Plasmodium falciparum (P.
f.) (Friedland et al., 1993; Looareesuwan et al., 1999), but
the roles of IL-8 in falciparum malaria are not clear.
We performed in vitro experiments to study the antimalarial activity of the non-immune peripheral blood leukocytes. Moreover, we studied the IL-8 production of periph１

eral blood MNC and PMN induced by P.f., and tried to elucidate the role of this cytokine in immunity to malaria.

MATERIALS AND METHODS
Malaria parasites
Plasmodiun falciparum FCR-3 strain has been maintained in our laboratory in a continuous culture according toprevious described methods (Trager and Jensen, 1976), except for the serum supplemented to the medium. The parasites were sowed in red blood cells (RBC) at an initial hematocrit of 5% in RPMI 1640 medium (Sigma Chemical
Co, St. Louis, MO, USA) supplemented with l0% heatinactivated horse serum (Cosmo Bio Co., Tokyo, Japan).
Substitution of the human sera with horse sera greatly facilitate the malaria culture, because this permitted the use of
all types of human erythrocytes, without checking for compatibility.
Separation of MNC and purification of monocytes
50 ml of peripheral venous blood was collected fromhealthy non-immune donors in a heparinized syringe. MNC
were separated by Ficoll/Urografin density gradient centrifugation, and suspended in a plastic dish with RPMI 1640

Department of Medical Zoology, Jichi Medical School, Kawachi-gun, Minamikawachi-machi, Tochigi 329-0498, Japan
Cancer Research Institute, Kanazawa University, Takara-machi, Kanazawa 920-0934, Japan
３
Kyoritsu College of Pharmacy, Minato-ku, Tokyo l05-8512, Japan
２

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supplemented with 5% fetal calf serum. Two hours after incubation at 37 °C, non-adherent cells were removed fromthe plate by washing with phosphate buffered saline and adherent cells (mainly monocytes) were detached from theplate with a rubber policeman and resuspended in the same
medium of MNC.
Separation of PMN
Once the MNC removed, PMN (about 95% neutrophils) were separated from RBC by sedimentation in 1.25%
gelatin. Mingled erythrocytes were hemolysed in lysing reagent (Ortho Diagnostic Systems, New Jersey, NJ, USA)and PMN were resuspended in the same medium of MNC.
P.f. growth inhibition assay
Growth inhibition assay was done by a radiometricmethod, described by Kumaratilake et al. (1997). Briefly,
the experiments were conducted in 96 well flat-bottomed
microplates (Becton Dickinson Co., Franklin Lakes, NJ,
USA). The cells (MNC or PMN) were subsequently diluted
and placed to the wells in 100 µl aliquots, where the parasites (1x105 parasites in 5x107 RBC in 100 µl of medium)
were added 2 hours later. Following 48 hours incubation at
37°
C in a CO2 incubator containing a gas mixture of 5%
CO2 in air, each well was pulsed with l µCi of [3H] hypoxanthine and harvested 24 hours later on a glass fiber sheet.
The disks on the sheet were air dried, placed in a 96 wellpolyethylene plate with 100 µl of toluene scintillant andcounted by a microplate scintillation counter (TopCount,
Packard Instrument Co., Meriden, CT, USA). Each dilution
was made in triplicate and the average was calculated.
Growth inhibition rate was calculated using the formula asfollows:
scintillation count of experimental well
Growth inhibition(%)=100−
x100
scintillation count of P.f. alone well
Microscopic studies
Interaction of the phagocytic cells with parasites wasobserved by preparing cytocentrifuged smears of the culture.
Parasites were cultured together with PMN or MNC andsmears were made 48 hours later. MNC were detached fromthe culture plate with a rubber policeman before cytocentrifugation. The smears were air dried and stained with Giemsa for microscopic study at 1000x magnification.
Stimulation of the PMN and MNC with parasites andparasite-derived products
P. faciparum induced IL-8 secretion by the PMN and
MNC was investigated by measuring IL-8 in the supernatant of the culture. PMN or MNC (1x105 cells in 100 µl of
medium) were cultured with viable parasitized erythrocytes(1x105 parasites in 5x107 RBC in 100 µl of medium), andthe supernatant was collected by 24 hr. For positive control,
PMN (1x105 cells in 100 µl of medium) were stimulatedwith human recombinant TNFα (20 ng in 100 µl of medium), which was kindly provided by Dainihon Pharmaceutical Co. (Osaka, Japan).
To investigate the fraction responsible for IL-8
induction, PMN (1x105 cells in 100 µl of medium) were
stimulated respectively with intact infected erythrocytes (1x105 parasites in 5x107 RBC in 100 µl of medium), 100 µl of
supernatant of the P.f. culture, 100 µl of hemolysate of theinfected erythrocytes (originally 1x105 parasites in 5x107
RBC), and partially purified 1x105 parasites obtained byhemolysing and washing out the RBC ghost. As negative
control, uninfected 5x107 RBC in 100 µl of medium wereadded to the cell culture.
IL-8 assay
IL-8 in the supernatant was measured by an enzymelinked immunosorbent assay (ELISA) method as described
elsewhere (Ko et al., 1992).
Neutralization of IL-8 activity
To study the role of IL-8 on parasite suppression by
PMN, the cells were pretreated, 2 hours before addition of
the parasites, with a mouse anti-human IL-8 monoclonal
antibody WS-4 (Ko et al., 1992) at a concentration of 50 µg/ml. For the control groups, we used monoclonal antibodies
for IL-1α (Kasahara et al., 1987) and an unspecific IgG.
After 3 days culture, proliferation of the parasite was assessed by incorporation of [3H] hypoxanthine.

RESULTSP. f. growth inhibition
Inhibitory effect of the MNC or PMN on P.f. proliferation depended upon the effecter to target ratio (E/T) (Fig. 1a). 1x105 MNC almost inhibited the parasite proliferation(parasite number at the start was 1x105/well), and to get
50% growth inhibition of the parasites, we needed 0.2x105
cells, meaning the E/T for IC50 was about 0.2. PMN also efficiently inhibited parasite growth with the E/T for IC50 of
0.7 (Fig. 1b). Monocytes (adherent cells) were then separated from non-adherent cells (mainly lymphocytes) and respectively cultured with the parasites. The E/T of the monocytes for IC50 was 0.9 (Fig. 2). Non-adherent cells alone
showed no or minimal inhibitory effect on parasite growth.
This suggests that both monocytes and lymphocytes are required to inhibit P.f. growth.

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Figure 2 Effect of the monocytes (black bars) and nonadherent mononuclear cells (dotted bars) for P.f.
growth inhibition.

Figure l In vitro inhibitory effect of normal donors (a)MNC and (b) PMN on P.f. growth. The starting
parasite number was 1x105 /well. Results are calculated as percent inhibition in relation to the [3H]
hypoxanthine uptake of the P.f. alone, and presented as the mean ± standard error from 5 isolated experiments.

Morphological studies
Morphologically, 2 days after P.f. stimulation, themonocytes greatly increased in size, and most of them contained a large number of engulfed parasites. PMN also actively phagocytosed parasites, but the number of engulfed
parasites per cell was not noticeable as did the monocytes.
IL-8 production of the PMN and MNC induced by P.f.
A remarkable amount of IL-8 was detected in the supernatant of P.f. stimulated MNC. After 24 hours culture, IL-8 level in the supernatant of P.f. stimulated culture was 9
times higher than that of unstimulated one (Fig. 3). Although less prominent than the MNC, P.f. stimulated PMN
also secreted a considerable amount of IL-8 (Fig. 4). The IL-8 was detectable in the supernatant after 6-9 hours of
stimulation, increasing progressively with time. 24 hours
later, IL-8 concentration in the supernatant of P.f. stimulated
PMN was 80 times higher than that of negative control.
TNFα at a concentration of l µg/ml also induced IL-8

Figure 3 IL-8 detection in the supernatant of the P.f. stimulated MNC (circles). Squares represent the valueof IL-8 in the supernatant of MNC cultured with
uninfected RBC.

production in PMN but no stronger than P.f. parasites did.
Experiments were also designed to determine the fraction responsible for IL-8 induction. Among the fractions
tested, P.f. infected erythrocytes and supernatant of the P.f.
culture were the most potent stimulants for IL-8 productionin the PMN cells. Hemolysate of the infected erythrocytes,
partially purified parasites, and uninfected RBC had little or
no stimulative effect on IL-8 production (Table 1).

３
５４

Figure 4 Time course of IL-8 secretion in1x105 PMN
stimulated with 1x105 parasites (circles). Squares
represent IL-8 detected in the PMN cultured with
uninfected RBC, and triangles represent IL-8 secretion after TNF-α (100 ng/ml) stimulation.
Table 1 Concentration of IL-8 (ng/ml) in the culture supernatant of PMN stimulated with infected RBC or P.f.
products

PMN + medium
PMN +uninfected RBC
PMN +infected RBC
PMN + supernatantof P.f. culture
PMN + hemolysate
of infected RBC
PMN + partiallypurified parasites

18 hr

Period of calture
24 hr
32 hr

42 hr

5.1

6.6

8.3

12.1

5.4

6.0

5.1

6.7

140

610

1100

4500

140

370

1700

2200

16

68

130

250

34

100

160

490

P.f. proliferation after neutralization of the IL-8
Treatment of the PMN or MNC with anti-IL-8 or antiIL-1α monoclonal antibodies did not influence the inhibitory effect of these cells on parasite growth (data notshown), suggesting that IL-8 and IL-1α are not directly involved in suppression of the parasite by these cells.

DISCUSSION
Some authors have demonstrated that peripheral blood
monocytes can kill malaria parasites in vivo and in vitro(Vernes, 1980; Bouharoun-Tayoun et al., 1990), but monocytes required presence of immune sera (Druilhe and Khusmith, 1987; Lunel and Druilhe, 1989) or addition of cytoki-nes (Ockenhouse et al., 1984) for activation of the cells. Peripheral blood neutrophils are also reported to be effective
against P.f. in the presence of immune sera (Celada et al.,1983) or opsonins, and this activity was enhanced by TNFα, lymphotoxin and IFNγ (Kumaratilake et al., 1997).
We demonstrated that mononuclear cells and neutrophils from non-immune individuals efficiently inhibited P.f.
growth in vitro in absence of anti-malarial antibodies orcomplements. Growth inhibition was dependent on the effecter/target ratio. The mechanism how the leukocytes restrain the parasite growth is not yet clear. For monocytes
and macrophages, phagocytosis may be the major mechanism, as observed by morphology. The non-adherent mononuclear cells, mostly lymphocytes, did not have antiparasitic activity, but when they were incubated with monocytes, they strongly enhanced the ability of the monocytes
to inhibit parasite growth. This suggests the interaction between monocytes and lymphocytes by direct contact or via
cytokines. For neutrophils, the number of engulfed parasites
per cell was not conspicuous. This suggests that, besides thephagocytic activity, neutrophils may produce soluble substances that may contribute to suppress parasite growth.
Cytokines have been demonstrated to be important in
immunity and pathogenesis of malaria. IL-8 is a CXC type
chemokine responsible for activation of a variety of cells including neutrophils (Yoshimura et al., 1987), and the receptor of IL-8 has been shown to be related to Plasmodium vivax infection (Horuk, 1994). Duffy antigen on erythrocytes
has long been reported as a target molecule for vivax malaria infection (Miller et al., 1975). Interestingly, IL-8
receptor was shown to be homologous to the Duffy antigen,and the involvement of IL-8 and its receptor in infection
with vivax malaria was suspected (Horuk, 1994). Someauthors have detected IL-8 in the sera of P.f. infected patients (Friedland et al., 1993; Looareesuwan et al., 1999),
and Wahlgren et al., (1995) have detected various cytokines,
including IL-8, in the culture supernatant of P.f. stimulated
peripheral blood MNC and PMN.
Our experiments demonstrated IL-8 in the supernatantof PMN after P.f. stimulation. Since collected fraction of
PMN was neutrophils by 95% and contamination with somemononuclear cells might occur, we histochemically confirmed IL-8 expression in PMN. Intracellular IL-8 was detected from 4 hours after P.f. stimulation (data not shown).
Whether this IL-8 production resulted from a direct stimulation by parasites or an intermediated stimulation by a secondary cytokine has to be investigated.
In our study, we did not investigate the time required
for IL-8gene expression in the P.f. stimulated PMN, but theelapsing time for IL-8 secretion may suggest presence of intermediating cytokines rather than a direct stimulation by３
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the parasite. Cytokines such as TNF-α , IL-1 and IFNγ aredemonstrated to be produced in P.f. infections (Brown et al.,
1990; Wahlgren et al., 1995), and we have to consider the
possibility of an autocrine stimulation by these cytokines.
TNF-α also induced IL-8 production in the PMN (Fig.4),
but PMN responded earlier and stronger to P.f. than did to
TNF-α. Thus, TNF-α should not be responsible for the induction of IL-8.
To elucidate the involvement of IL-8 in the immunity
to falciparum malaria, we neutralized IL-8 with anti-IL-8
monoclonal antibody in the culture mixture of P.f. and PMN,
resulting no abrogation of inhibitory ability to PMN. Therefore, we conclude that IL-8 has no direct influence on P.f.
inhibitory activity of the PMN. The other mechanism besides IL-8 may work for inhibiting parasite growth in theculture system of malaria parasites associated with leukocytes.

ACKNOWLEDGEMENTS
We are grateful to Ms. C. Seki and Ms. M. Okada for
their technical assistance.

REFERENCES
１）Bouharoun-Tayoun, H., Attanath, P., Sabchareon, A.,
Chongsuphajaisiddhi, T. and Druilhe, P. (1990): Antibodies that protect humans against Plasmodium falciparum
blood stages do not on their own inhibit parasite growthand invasion in vitro, but act in cooperation with monocytes. J. Exp. Med., 172, 1633-1641
２）Brown, A.E., Webster, H.K., Teja-Isavadharm, P. and
Keeratithakul D. (1990): Macrophage activation in falciparum malaria as measured by neopterin and interferongamma. Clin. Exp. Immunol., 82, 97-101
３）Celada, A., Cruchaud, A. and Penin, L.H. (1983): Phagocytosis of Plasmodium falciparum-parasitized erythrocytes by human polymorphonuclear leukocytes. J. Parasitol., 69, 49-53
４）Druilhe, P. and Khusmith, S. (1987): Epidemiological correlation between levels of antibodies promoting merozoite phagocytosis of Plasmodiam falciparum andmalaria-immune status. Infect. Immun., 55, 888-891
５）Friedland, J.S., Ho, M., Remick, D.G., Bunnag, D., White,
N.J. and Griffin, G.E. (1993): Interleukin-8and Plasmodium falciparum malaria in Thailand. Trans. R. Soc. Trop.
Med. Hyg., 87, 54-55
６）Horuk, R. (1994): The interleukin-8-receptor family: fromchemokines to malaria. Immunol. Today, 15, 169-174
７）Kasahara, T., Mukaida, N., Shinomiya, H., Imai, M., Matsushima, K., Wakasugi, H. and Nakano, K. (1987):

Preparation and characterization of polyclonal and monoclonal antibodies against human interleukin lα (IL-lα). J.
Immunol., 138, 1804-1812８）Ko, Y.C., Mukaida, N., Panyutich, A., Voitenok, N.N.,
Matsushima, K., Kawai, T. and Kasahara, T. (1992): A
sensitive enzyme-linked immunosorbent assay for humaninterleukin-8. J. Immunol. Methods., l49, 227-235
９）Kossodo, S. and Grau, G.E. (1993): Profiles of cytokne
production in relation with susceptibility to cerebral malaria. J. Immunol., 151, 4811-4820
１０）Kumaratilake, L.M., Ferrante, A., Jaege, r T. and MorrisJones, S.D. (1997): The role of complement, antibody,and tumor necrosis factor alpha in the killing of Plasmodium falciparum by the monocytic cell line THP-1. Infect.
Immun., 65, 5342-5345
１１）Kwiatkowski, D. (1995): Malarial toxins and the regulation of parasite density. Parasitol. Today, l1, 206-212
１２）Looareesuwan, S., Sjostrom, L., Krudsood, S.,
Wilairatana, P., Porter, R.S., Hills, F. and Warrell, D.A.
(1999): Polyclonal anti-tumor necrosis factor-alpha Fabused as an ancillary treatment for severe malaria. Am. J.
Tro.p Med. Hyg. 61, 26-33
１３）Lunel, F. and Druilhe, P. (1989): Effector cells involved innonspecific and antibody-dependent mechanisms directed against Plasmodium falciparum blood stages in vitro. Infect. Immun., 57, 2043-2049
１４）Miller, L.H., Mason, S.J., Dvorak, J.A., McGinniss, M.H.
and Rothman, I.K. (1975): Erythrocyte receptors for(Plasmodium knowlesi) malaria: Duffy blood group determinants. Science, l89, 561-563
１５）Nnalue, N.A. and Friedman, M.J. (1988): Evidence for aneutrophil-mediated protective response in malaria. Parasite Inmunol., 10, 47-58
１６）Ockenhouse, C.F., Schulman, S. and Shear, H.L. (1984):
Induction of crisis form in the malaria parasite Plasmodium falciparum by γ-interferon-activated, monocytederived macrophages. J. Immunol., 133, 1601-1608
１７）Trager, W. and Jensen, J.B. (1976): Human malaria incontinuous culture. Science, 193, 673-675
１８）Vernes, A. (1980): Phagocytosis of P. falciparum parasitised erythrocytes by peripheral monocytes. Lancet, 8207,
1297-1298
１９）Wahlgren, M., Abrams, J.S., Fernandez, V. Bejarano, M.
T., Azuma, M., Torii, M., Aikawa, M. and Howard, R.J.
(1995): Adhesion of Plasmodium falciparum-infected
erythrocytes to human cells and secretion of cytolkines(IL-1β, IL-1RA, IL-6, IL-8, IL-10, TGFβ, TNFα, G-CSF,
GM-CSF). Scand. J. Immunol. 42, 626-636
２０）Yoshimura, T., Matsushima, K., Tanaka, S., Robinson, E.
A., Appella, E., Oppenheim, J.J. and Leonard, E.J.
(1987): Purification of a human monocyte-derived neutrophil chemotactic factor that has peptide sequence similarity with other host defense cytokines. Proc. Natl. Acad.
Sci. USA 84, 9233-9237

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AN IMPORTED JAPANESE CASE OF CYCLOSPORIASISKENJI OHNISHI１）, YASUYUKI KATO１）and MOTOHIRO ISEKI２）
Accepted December, 5, 2002

Abstract: A stool examination revealed oocysts of Cyclospora cayetanensis in a 54-year-old Japanese man who
had recently returned from the Phillipines. He suffered from watery diarrhea for about two weeks prior to visiting
our hospital. An oral dose of 1,600 mg sulfamethoxazole and 320 mg trimethoprim was administered daily for ninedays, and his diarrhea had disappeared by the fifth day of medication. Most Japanese physicians and laboratory
technicians are unfamiliar with this protozoan disease, and need to be reminded of its existence when they encounter patients with diarrhea who have recently returned from a cyclosporiasis endemic area.
Key words: Cyclospora cayetanensis, diarrhea, Japanese

INTRODUCTION
The protozoan Cyclospora cayetanensis is recognized
as causing diarrhea (Herwaldt, 2000), and has mainly been
identified in residents and travelers of North, Central, andSouth America, the Caribbean Islands, Eastern Europe,
Pakistan, India, Nepal, Southeast Asia, the Solomon Islands,
Papua New Guinea, Morocco, and South Africa (Wurtz,
1994; Soave, 1996). To our knowledge, the first Japanesecase of cyclosporiasis upon returning from Thailand andCambodia was reported in 1996 (Iseki, 1996), since then,few Japanese patients with cyclosporiasis have been reported (Iseki et al., 1998; Oki et al., 2001; Masuda et al.,
2002; Ohnishi et al., 2002; Shiota et al., 2002; Yamaura etal., 2002). This, perhaps, is due to the fact that cyclosporiasis is unfamiliar to Japanese physicians. Here we report thecase of a Japanese patient with cyclosporiasis who had recently returned from the Philippines. The purpose of this report is to emphasize the importance of taking into accountcyclosporiasis in the differential diagnosis of diarrhea, especially when related to countries in tropical and subtropicalareas.

CASE REPORTA 54-year-old Japanese man stayed in the Philippinesfrom April 11 to June 30, 2002. He was admitted to a hospital in Mandaluyong City, Philippines on June 21, 2002, because of watery diarrhea and fever lasting for 2 days. He
１）

was treated with metronidazole and ciprofloxacin, but thewatery diarrhea did not improve. He was discharged onJune 25, 2002, and visited our hospital on July 2 because of
the diarrhea. His medical history was unremarkable except
for a fatty liver (bright liver had been identified by ultrasonography in another hospital in Japan), and he had been
well before visiting the Philippines. When we saw him on
July 2, he was afebrile, alert, and without palpable necklymph nodes. Auscultation of the heart and lungs was also
unremarkable, and bowel sounds were mildly hyperactive.
Laboratory data on July 2, 2002, showed normal hemogramand blood chemistry, except for slightly increased whiteblood cell counts of 9,200/mm３, AST 47 IU/l, ALT 64 IU/l,
ALP 432 IU/l, and C-reactive protein 0.5 mg/dl. Serum
HIV antibody was not checked. No pathogenic bacteria
were cultured from the stool. Microscopic stool examination, using a formalin-ether concentration technique forhelminth ova and protozoan, revealed a lot of spherical organisms that were 8-10 µm in diameter and contained numerous globules enclosed within a wall (Fig.1). The organisms appeared as bright-blue circles under ultraviolet
epifluorescence microscopy (Fig.2). When unpreserved organisms isolated from the stool specimen were incubated in
2.5% potassium dichromate for 7 days at 27°
C, a cluster ofglobules inside the organism was transformed into two ellipsoidal structures (Fig.3). On the basis of these characteristics, the organisms were identified as oocysts of C. cayetanensis. Our patient was treated with a daily oral dose of
1,600 mg sulfamethoxazole and 320 mg trimethoprim for

Department of Infectious Diseases, Tokyo Metropolitan Bokutoh General Hospital
Department of Parasitology, Graduate School of Medical Science, Kanazawa University
Correspondence to: Kenji Ohnishi, M.D., Department of Infectious Diseases, Tokyo Metropolitan Bokutoh General Hospital, 4-23-15
Kohtohbashi, Sumida City, Tokyo 130-8575, Japan

２）

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８

Table 1: Reported Japanese cases of imported cyclosporiasis

Fig. 1: Immature oocyst of Cyclospora cayetanensis
isolated from the patient’s stool specimen; bar
=8 µm

Case

Age(years)

Sex

Visited countries

References

1

22

M

Thailand and Cambodia

4, 7

2

50

M

Myanmer, Singapore,
Thailand, and Cambodia

7

3

26

M

Thailand

5

4

70

M

Southeast Asia

9

5

46

M

Indonesia

7

6

36

M

Thailand and Cambodia

7

7

35

F

Hong Kong and Indonesia

10

8

36

M

Philippines

8

9

53

M

Mexico13

Present case

54

M

Philippines

DISCUSSION

Fig. 2: Autofluorescence of the oocyst wall; bar=8 µm.
(Ultraviolet epifluorescence microscopy using a
365-nm excitation filter)

Fig. 3: Mature oocyst with two sporocysts; bar=5 µm.
(Nomarski interference-contrast microscopy)nine days, and his diarrhea disappeared by the fifth day after starting the medication.

Infections with C. cayetanensis can be detected in 0.07
-2.8% of European diarrheal patients returning from developing countries (Gascon, et al., 1995; Clarke and McIntyre,
1996; Jelinek, et al., 1997). Many Japanese people have
traveled to areas where cyclosporiasis is endemic, but to ourknowledge, only nine other cases of Japanese travelers infected with C. cayetanensis upon returning from thesecountries have been reported (Table 1)(Iseki, 1996; Iseki etal., 1998; Oki et al., 2001; Masuda et al., 2002; Ohnishi etal., 2002; Shiota et al., 2002; Yamaura et al., 2002). These
facts indicate that some Japanese patients with diarrhea
upon returning from developing countries may, infact, be
infected with C. cayetanensis, and some of them with cyclosporiasis may be overlooked. This oversight may be dueto the fact that most Japanese physicians and laboratory
technicians are unfamiliar with this protozoan disease. Ourcase indicates that C. cayetanensis is one of the causative
organisms associated with diarrhea, especially when no improvement is observed, in spite of the administration of antibacterial agents and metoronidazole, as is similar to ourcase. Most cases with cyclosporiasis resolve themselves
spontaneously in immunocompetent hosts, but cyclosporiasis is one of the diarrheal diseases which physicians should
be aware of, since cyclosporiasis is fatal in immunocompromised patients. C. cayetanensis is probably indigenous
to Japan, since a Japanese patient with cyclosporiasis who
had no history of traveling to other countries was found in2001 (Shiota et al., 2002). Therefore, we have to be awareof the fact that people have a chance of contracting cyclosporiasis even in Japan.

REFERENCES
１）Clarke, S. C. and McIntyre, M. (1996): The incidence of３５９Cyclospora cayetanensis in stool samples submitted to a
district general hospital. Epidemiol. Infect., 117, 189-193
２）Gascon, J., Corachan, M., Bombi, J. A., Valls, M. E. and
Bordes J. M. (1995): Cyclospora in patients with traveller’s diarrhea. Scand. J. Infect. Dis., 27, 511-514
３）Herwaldt, B. L. (2000): Cyclospora cayetanensis: a review,
focusing on the outbreaks of cyclosporiasis in the 1990s.
Clin. Infect. Dis., 31, 1040-1057
４）Iseki, M. (1996): The first human case of Cyclospora infection in Japan. I. A. S. R.,17, 241-242 (in Japanese)
５）Iseki, M., Kimata, I., Nakamura, S., Matsumoto, Y.,
Kuroki, T. and Koezuka T. (1998): A case of traveler’s diarrhea due to Cyclospora cayetanensis. J. J. A. Inf. D., 72,
1138 (in Japanese)
６）Jelinek, T., Lotze, M., Eichenlaub, S., Loescher, T. and
Nothdurft, H. D. (1997): Prevalence of infection with
Cryptosporidium parvum and Cyclospora cayetanensis
among international travelers. Gut, 41, 801-804
７）Masuda, G., Ajisawa, A., Imamura, A., Negishi, M. andIseki, M. (2002): Cyclosporiasis: four case reports with areview of the literature. J. J. A. Inf. D.,76, 416-424 (inJapanese with English Abstract)８）Ohnishi, K., Kato, Y., and Iseki, M. (2002): An imported
case of Cyclospora cayetanensis in Japan. J. J. A. Inf. D.,
76, 118-120
９）Oki, K., Fujiue, Y., Watanabe, Y., Shimizu, S. and Moro,
K. (2001): A case of traveler’s diarrhea with Cyclospora
cayetanensis. J. Jpn. Soc. Clin. Microbiol., 11, 130 (inJapanese)１０）Shiota, T., Yamada, M., Wakabayashi, N. and Funakoshi,
H. (2002): Cyclospora cayetanensis: first domestic infection and an imported infection in Japan. Program and Abstracts of the 71st Annual Meeting of the Japanese Societyof Parasitology, 158 (in Japanese)
１１）Soave, R. (1996): Cyclospora: an overview. Clin. Infect.
Dis., 23, 429-437
１２）Wurtz, R. (1994): Cyclospora: a newly identified intestinal pathogen of humans. Clin. Infect. Dis., 18, 620-623
１３）Yamaura, H., Kikuchi, K., Itoda, I., Yasunami, T., Totsuka,
K., Kobayashi, M., Takasaki, K., Isoda, N., Shimada, K.,
Miyazawa, M. and Kobayakawa, T. (2002): A case of cyclosporiasis detected after returning from Mexico. Program and Abstract of the 13th Congress of the Japan
Clinical Parasitology Meeting, 51 (in Japanese)３
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Seasonal differences in antimalarial activity of hot-water extract of Dichroa
febrifuga leaves against Plasmodium yoelii 17XL in ICR mice, with reference tofebrifugine and isofebrifugine content
Akira Ishih１＊, Kaori Fujii１, Mikako Sakai１, Masami Iiboshi２,
Toshio Miyase２, and Mamoru Terada１
Accepted November, 15, 2002

Abstract: The antimalarial activity of the hot-water extract of leaves and roots of Dichroa febrifuga was evaluatedagainst Plasmodium yoelii 17XL in ICR mice. Untreated control mice died with a gradual body weight loss and increase of parasitemia by day 9 after infection. The hot-water extract of leaves collected in June showed an antimalarial activity, and furthermore the possible adverse side effect was also observed. All mice given orally the extractof leaves (0.1 g/ml) collected in June died by day 9 without parasite multiplication. The mice given the lower concentration (0.025 g/ml) of the same leaf extract showed low parasitemia levels during administration. Following atransient increase of malaria parasites in the bloodstream, no parasites could be detected by a microscopic examination, and all mice survived during the experiment. On the other hand, the extract of leaves (0.1 g/ml) collected in
December showed no activity. The extract of roots (0.1 g/ml) of D. febrifuga collected in December, however, had
an antimalarial activity, and three mice out of four survived during the experiment. The leaves collected in June
contained about 30 times as much febrifugine and isofebrifugine mixture as those in December.
Key words: Plasmodium yoelii 17XL,Dichroa febrifuga, febrifugine, antimalarial activity

INTRODUCTION
Malaria is one of the most important tropical diseases
in the world. World-wide emergence of chloroquineresistant Plasmodium falciparum has stimulated the development of new effective treatments against malaria (Payne,
1987; Winstanley, 2000). Of several strategies on malaria
control it is a new interest to investigate the folk medicine
exhibiting a potent antimalarial activity. Isolation of artemisinin from leafy portions of Artemisia annua L. in 1972
has led to further trial for identifying other medicinal compounds of potential as antimalarial agents from natural
products (Iwu et al., 1986; Ajaiyeoba et al., 1999; Isaka etal., 1999; Rahman et al., 1999; Boonlaksiri et al., 2000).
In our continuation of investigation of plants having
antimalarial activity, the hot-water extract of leaves of Hydrangea macrophylla Seringe var. Otaksa Makino showeda high in vivo antimalarial activity against the rodent malaria parasite P. yoelii 17XL though the extract of roots had
no activity (Sakai et al., 2000; Ishih et al., 2001). Furthermore the fraction including the febrifugine and isofebrifugine mixture having an antimalarial activity was isolated
from the leaves of this plant. These compounds had already１２

been isolated from the roots of Dichroa febrifuga Lour.
(Koepfli et al., 1947) and from the leaves of Hydrangea
umbellata Rehd. Et Wils. (Ablondi et al., 1952). It is therefore of interest to clarify the distribution and/or content ofthese principles in the different parts of the plant. This paper
deals with a preliminary in vivo antimalarial evaluation of
the hot-water extract of leaves of D. febrifuga collecting in
June or in December using ICR mice infected with P. yoelii
17XL.
MATERIALS AND METHODS
Animals and parasites
All animal experiments were performed according to
the Guidelines for Animal Experimentation, Hamamatsu
University School of Medicine. Outbred male ICR mice,8weeks old, purchased from SLC Inc. (Hamamatsu, Japan),were used. For experiments, the blood stage parasites of
Plasmodium yoelii 17XL from frozen stock were injected
into two mice. The mouse showing 10-15% of parasitemia
was bled under ether anaesthesia to collect P. yoelii 17XLparasitized blood. Experimental mice were infected intraperitoneally with1x105 parasitized erythrocytes. Infected mice

Department of Parasitology, Hamamatsu University School of Medicine, Hamamatsu 431-3192, Japan
Department of Pharmacognosy, School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka 422-8526, Japan３
６
２

Fig.1. Body weight profile of each mouse in response to treatment with the hot-water extract of roots or leaves of D.
febrifuga. P. yoelii 17XL-infected mice were orally
given each extract at 75 µl/10 g body weight, twice aday, for 5 consecutive days from day 3 to day 7 after inoculation intraperitoneally with 105 parasitized erythrocytes. Control group (A, n=5) of mice received anequivalent volume of distilled water. Each symbol represents an individual mouse. Extract of leaves collected
in December (B, n=5;0.1 g/ml), extract of roots collected in December (C, n=4;0.1 g/ml) and extract ofleaves collected in June (D, n=5;0.1 g/ml and E, n=5;0.025 g/ml).

Fig.2. Parasitemia profile of each mouse in response to treatment with the hot-water extracts of roots or leaves of D.
febrifuga. P. yoelii 17XL-infected mice were orally
given each extract at 75 µl /10 g body weight, twice aday, for 5 consecutive days from day 3 to day 7 after inoculation intraperitoneally with 105 parasitized erythrocytes. Control group (A, n=5) of mice received anequivalent volume of distilled water. Each symbol represents an individual mouse. Extract of leaves collected
in December (B, n=5;0.1 g/ml), extract of roots collected in December (C, n=4;0.1 g/ml) and extract ofleaves collected in June (D, n=5;0.1 g/ml and E, n=5;0.025 g/ml).

treated with extracts were monitored for % parasitemia anddays of survival relative to control mice up to 60 days postinfection.
Preparation of the hot-water extract of leaves and roots of
D. febrifuga
Leaves (before fallen, dark green and hard) and rootsof D. febrifuga were collected from the Botanical Garden,
University of Shizuoka in December, 1999, and leaves (infull bloom, green and soft) of the same plant were also collected in June, 2000. Five g of air-dried leaves or roots ofthe plant put in a herbal bag (Tokiwa Industry, Ehime, Japan) was added with 500 ml of distilled water and boiled.
When the fluid volume came to about 250 ml, leaves orroots were taken out and the extract was finally concentrated to 50 ml. Each hot-water extract (equivalent to the 0.1
g of dried material /ml extract) was stored at 4°
C until use.
Quantitative analysis of febrifugine and isofebrifugine mixture

Dried leaves or roots (1 g) of D. febrifuga collected in
December, 1999 and leaves in June, 2000 were extractedwith 50 ml of water under boiling for1hr and the extract
was filtered and the residue was washed with 20 ml of boiling water. The combined filtrate was acidified with hydrochloric acid (1 ml) and extracted with chloroform (10 ml)
three times. To the water layer, 40% of potassium hydroxide
aq. was added until the solution became to alkali. The alkaline solution was extracted with chloroform (10 ml) threetimes. The chloroform layer was concentrated to dryness after washing with water. The residual chloroform extract was
analysed by liquid chromatography (HPLC) comparing thepeak area. HPLC condition: Column, Develosil ODS - 74.6mm x 25 cm (Nomura Chemical, Aichi, Japan); Solvent, 20
mM NaH２PO４- 20 mM H３PO４ (pH 2.1) / CH３CN (9:1);
Flow rate 1.0 ml/min; UV, 276 nm; Retention times; 7.4
min, 9.2min.
In vivo antimalarial activity of the hot-water extract of３
６３

leaves collected in June and of leaves or roots collected in
December
To evaluate antimalarial activity of hot-water extract,
infected ICR mice were divided into four to five per groupfor activity assay of each extract and untreated control.
From3days after injection of parasitized erythrocytes, mice
were orally given each hot-water extract at 75 µl/10 g bodyweight in the treated group and distilled water in the control
one, respectively, twice a day for5consecutive days.

RESULTS
Antimalarial activity of the hot-water extract of leaves orroots of D. febrifuga
As shown in Fig.1, mice in the untreated control andgroups treated with the extract of leaves (0.1 g/ml) collected
either in June or in December died from day 7 to day 9 witha gradual body weight loss. However, in the group given theroot extract (0.1 g/ml), the mouse body weight transiently
decreased, but turned to increase in several days, and three
mice out of four survived during the experiment. All mice
given the extract of leaves (0.025 g/ml) collected in June
showed a transient body weight loss but survived during theexperiment. Malaria parasites appeared from day 3 of infection in the bloodstream of the control and treated groups(Fig.2). Parasitemia of each mouse in the control and thegroup given the extract of leaves collected in December
gradually increased, and all mice died by day 9. On theother hand, mice in the group given the extract of leaves(0.1 g/ml) collected in June showed low parasitemia levels
during medication but all mice died by day 9. Autopsy ofdead mice showed that the livers became whitish wholly, indicating the hepatotoxicity by higher concentration of theleaf extract. Mice treated with the lower concentration(0.025 g/ml) of the extract of leaves collected in June
showed low parasitemia levels during medication. After atransient increase of parasitemia, malaria parasites in the
bloodstream of mice could not be detected by a microscopic
examination from day 16 to day 22, and all mice survived
during the experiment. Mice treated with the root extract
also showed low parasitemia levels during medication. On
day 10, malaria parasites increased in the bloodstream of
the treated mice and one mouse died on day 17. Then theparasitemia level of three surviving mice decreased and theparasites in the bloodstream could not be detected by a microscopic examination from day 16 to day 30.
Preparation of alkaloid fraction from leaves and roots
One g of dried leaves of D. febrifuga collected in December, 1999 and in June, 2000 afforded 0.024 and 0.718
mg of febrifugine and isofebrifugine mixture, respectively.
One g of dried roots collected in December, 1999 afforded

0.14 mg of alkaloids.

DISCUSSION
The roots of D. febrifuga have been used as a treatment for malaria in Chinese traditional remedies, and the
mixture of febrifugine and isofebrifugine having an antimalarial activity was first isolated from the roots of D. febrifuga (Koepfli et al., 1947). In the present study, the hotwater extract of the roots of D. febrifuga collected in December had a direct antimalarial effect on P. yoelii 17XL infection in ICR mice; namely, suppression of multiplicationof parasites during medication. Besides the extract of theroots, this effect was shown in the hot-water extract of theleaves of D. febrifuga collected in June, but not in December. The leaves collected in June contained the febrifugine
and isofebrifugine mixture about 5 times as much as theroots collected in December did. Furthermore, the former
contained the alkaloid mixture about 30 times as much as
the leaves in December did. Kuehl et al . (1948) reported
that the total alkaloidal contents having the antimalarial activity were 0.1 to 0.15% of the dried roots of D. febrifuga
but the yield of alkaloids from the stem and leaf material
was invariably much lower. There is no description available about a season of harvesting leaves, and if the leaves
were collected after flowering, the present results coincidewith their report. Previously, a solid extract of the leaves ofD. febrifuga (Shuu Chi) were found equally effective
against P. gallinaceum infection in chicks in doses of only
about one-fifth of the root extract (Ch’ang Shan) (Jang et al.,1946). Our results also agree with their results. It was also
reported that the root samples from India contained only
about one-tenth of the alkaloidal fraction present in the Chinese samples. The present results hence suggest that theseasonal changes in activity of hot-water extract of leaves ofD. febrifuga might be due to the movement of active compounds from leaves to other parts or the biological and/or
chemical inactivity of compounds.
Acute toxicity tests of Ch’ang Shan, the roots of D.
febrifuga, made on dogs showed a 50% lethal dose value of
20 g/kg (Jang et al., 1946). Fatal dose produced in dogs intense congestion with numerous hemorrhagic patches
throughout the whole gastrointestinal tract. Aside fromsome congestion, no specific lesions were found histologically in the liver, spleen and kidneys. Toxic deaths in mice
were observed at 10 mg/kg for febrifugine (Chien andCheng, 1970). In the present experiment, the hepatotoxicityof alkaloids is suggested by autopsy of mice given thehigher dose of the hot-water extract (0.1 g of dried material/ml extract) of leaves collected in June. Further experiments
are at present being undertaken to examine histologically３６４and pharmacologically the toxicity of febrifugine andisofebrifugine mixture in detail.
Recently, the evaluation of antimalarial activity of medicinal plants against malaria was extensively studied, andvarious active natural plant products were discovered (Iwu
et al., 1986; Ajaiyeoba et al., 1999; Rahman et al., 1999;
Boonlaksiri et al., 2000). The present study showed that themarked difference in the antimalarial activity of the hotwater extract of D. febrifuga leaves collected in between
June and December, and thus it should be very important toconsider the material conditions such as parts of plants, thegeographic conditions, and the season of harvesting, in an
ongoing effort to detect the antimalarial activity of medicinal plants and to identify novel antimalarial agents fromthem.

REFERENCES
１）Ablondi, F., Gordon, S., Morton, J. II and Williams, J.H.
(1952): An antimalarial alkaloid from hydrangea. II. Isolation. J. Org. Chem., 17, 14-18
２）Ajaiyeoba, E.O., Abalogu, U.I., Krebs, H,C. and Oduola,
A.M.J. (1999): In vivo antimalarial activities of Quassia
amara and Quassia undulata plant extracts in mice. J.
Ethnopharmacol., 67, 321-325
３）Boonlaksiri, C., Oonanant, W., Kongsaeree, P., Kittakoop,
P., Tanticharoen, M. and Thebtaranonth, Y. (2000): An
antimalarial stilbene from Artocarpus integer. Phytochemistry, 54, 415-417
４）Chien, P-L. and Cheng, C.C. (1970): Structural modification of febrifugine. Some methylenedioxy analogs. J.
Med. Chem., 13, 867-870
５）Isaka, M., Punya, J., Lertwerawat, Y., Tanticharoen, M.

and Thebtaranonth, Y. (1999): Antimalarial activity ofmacrocyclic trichothecenes isolated from the fungus Myrothecium verrucaria. J. Nat. Prod., 62, 329-331
６）Ishih, A., Ikeya, C., Yanoh, M., Takezoe, H., Miyase, T.
and Terada, M. (2001): A potent antimalarial activity ofHydrangea macrophylla var. Otaksa leaf extract against
Plasmodium yoelii 17XL in mice. Parasitol. Int., 50, 3339
７）Iwu, M.M., Obidoa, O. and Anazodo, M. (1986): Biochemical mechanism of the antimalarial activity of Azadirachta indica leaf extract. Pharmacol. Res. Commun., 18,
81-91８）Jang, C.S., Fu, F.Y., Wang, C.Y., Huang, K.C., Lu, G. andChou, T.C. (1946): Ch’ang Shan, a Chinese antimalarial
herb. Science, 103, 59９）Koepfli, J.B., Mead, J.F. and Brockman, J.A. Jr. (1947):
An alkaloid with high antimalarial activity from Dichroa
febrifuga. J. Am. Chem. Soc., 69, 1837
１０）Kuehl, F.A.Jr., Spencer, C.F. and Folkers, K. (1948): Alkaloids of Dichroa febrifuga Lour. J. Am. Chem. Soc., 70,
2091-2093
１１）Payne, D. (1987): Spread of chloroquine resistance in
Plasmodium falciparum. Parasitol. Today, 3, 241-246
１２）Rahman, N.N.N.A., Furuta, T., Kojima, S., Takane, K.
and Mohd, M.A. (1999): Antimalarial activity of extractsof Malaysian medicinal plants. J. Ethnopharmacol., 64,
249-254
１３）Sakai, M., Ishih, A., Takezoe, H., Fujii, K., Sano, M.,
Asanuma, N., Miyase, T. and Terada, M. (2000): A potent
antimalarial activity of hot-water extracts of plants belonging to the family Saxifragaceae against Plasmodium
yoelii 17XL in ICR mice [Abstract no A-36]. Jpn. J. Trop.
Med. Hyg., 29, 43-44
１４）Winstanley, P.A. (2000): Chemotherapy for falciparum
malaria: The armoury, the problems and the prospects.
Parasitol. Today, 16, 146-153

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Short communication

COMPARATIVE STUDIES ON THE DEVELOPMENT OFLARVAL ANGIOSTRONGYLUS CANTONENSIS IN PUERTO
RICAN AND BRAZILIAN BIOMPHALARIA SNAILS.
YUZURU IWANAGA
Accepted November, 28, 2002
Since the first report of angiostrongylosis cantonensis
by Nomura and Lin (1945), Angiostrongylus cantonensis
has been recognized as a causative agent of eosinophilicmeningoencephalitis in humans in South Asian and Pacificareas (Roux et al., 1987; Purohit et al., 1991; Alibhoy et al.,
1999; Cooke-Yarborough et al., 1999; Re and Gluckman,
2001). The diagnosis of the disease is usually based onclinical manifestations and immuno-serological test.
Immuno-serological tests require a large amount of antigens.
Therefore, it is a paramount importance to establish the life
cycle of A. cantonensis in the laboratory. For this purpose,
it is necessary to maintain the intermediate hosts in thelaboratory. It is well known that various molluscan species
can serve as intermediate hosts for A. cantonensis (Lim andHyneman, 1965; Liat et al., 1965; Hori et al., 1976;
Iwanaga et al., 1983). In general, snail species used to establish the life cycle of the parasite in the laboratory is
Biomphalaria sp. that is easy to breed them. Although thelarval development of A. cantonensis in Biomphalaria
glabrata (Yousif and Lammler, 1977), Achatina fulica(Hori and Yamaguchi, 1982) and Ampullarium sp(Uchikawa et al., 1986) has been described, few detailed
observations on the location of larvae and susceptibility ofthe pigmented and albino types of Biomphalaria snails areavailable.
The present paper examines this relationship using
laboratory colonies of the pigmented and albino types ofPuerto Rican and Brazilian B. glabrata and pigmented B.
straminea collected in Brazil. Only adult snails were used.
The pigmented and albino types of Puerto Rican strains ofB. glabrata were obtained from Puerto Rico via NIH in U.S.
A. The Brazilian strains of snails were from the following
areas in Brazil: Pigmented B. glabrata from Jaboatao, Pernambuco, albino B. glabrata from Belo Horizonte and pigmented B. straminea from Sao Lourenco da Mata. The
snails have been reared in our laboratory by the modified

method of Iwanaga and Tsuji (1972). Albino rats, Rattus
norvegicus experimentally infected with A. cantonensis
served as a source of the first stage larvae (L1) for snail infection. A. cantonensis L1 were isolated from feces of infected rats by means of a Baeruman apparatus. About 200
snails each were individually exposed to 50 L1 in 10 ml
beaker for 20 hrs at 24 - 26°
C. Snails were then washed withdechlorinated tapwater and kept in 24 - 26°
C in a soil filtrated aquarium. To delineate the larval infection sites in thesnail, the snail was divided into four regions: the foot (region 1), seminal receptacle sac and vas deferens (region 2),
prostate gland and oviduct (region 3), and ovotestis and digestive gland (region 4). 10 snails each were examined 10,
15, 20, 25 and 30 days after larval exposure. Some snails
were examined with a dissecting microscope, while others
were fixed in Bouin’s solution, serially sectioned at 8 - 10
µm, and stained with hematoxylin-eosin for histological observations. Furthermore, some of the third stage larvae (L3)obtained from each snail were tested for infectivity by feeding rats on them using a stomach tube. The second stage
larvae (L2) and L3 were distinguished by morphologicalexaminations after fixation in 10% formalin; The intestineof L2 is composed of large cells filled with refractile foodgranules, whereas L3 have two well-developed chitinousrods with expanded knob-like tips near the anterior end andtheir intestinal cells are clear without food granules.
The number of L1 penetrated into pigmented B.
glabrata, Puerto Rico, ranged from 20 to 29 (avg. 26).
Similarly, those numbers were 18 to 26 (avg. 24) for pigmented B. glabrata, Brazil, 20 to 25 (avg. 22) for albino B.
glabrata, Puerto Rico, 19 to 25 (avg. 23) for albino B.
glabrata, Brazil and 7 to 11 (avg. 9) for pigmented B. straminea, Brazil. Although there was no significant difference
between pigmented and albino types of B. glabrata, pigmented B. straminea showed a markedly lower larval countthan those of other snails (p<0.0001, Student’s t-test). It

Department of Immunology and Parasitology, Division of Molecular Medical Science, Graduate School of Biomedical Sciences, Hiroshima University

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Figure 1. Distribution of the second stage larvae of Angiostrongylus cantonensis in Biomphalaria snails

may be due to differences in biochemical factors of thesnails associated with invasion of the larvae. Figs.1 and 2
show the distribution in the snail bodies of L2 and L3, respectively. L2 were found in all regions, but especially inregions1and 2 at 15 days postexposure (dpe). On 15 dpe, L
3 firstly were observed in regions 3 and 4 in pigmented B.
glabrata and B. straminea, and in regions 2, 3 and 4 in albino B. glabrata. The L3 counts increased in regions 3 and
4 in the course of time. There was a distinct difference indistribution between L2 and L3. The majority of L2 weredetected in regions1and 2 at 15 dpe, where they generally
remained without reaching to L3. Furthermore, some larvae
died and mostly remained ensheathed. Especially, manydead larvae were observed in pigmented B. straminea andthese dead larvae were readily identified as L2. Chao et al(1987) reported that L2 in Ampullarium canaliculatus werefirst observed on 5 dpe and reached the peak on day 22,
mainly harboring in the head foot. In the present study, themaximum L2 count was observed during 10-15 dpe andmost L2 were found in viscera and genital grand existing inregion 2. This finding differs slightly form their report. On
25 dpe, most L2 developed into the L3, and the majority of

Figure 2. Distribution of the third stage larvae of Angiostrongylus cantonensis in Biomphalaria snails

the L3 were detected in regions 3 and 4. These L3 became
active when transferred to 0.9% NaCl solution. In contrast,some larvae were morphologically similar to L3, but they
were killed in an artificial digestive solution. It seems likely
therefore that they were preinfective L3. The majority of L2grew to L3 within 30 dpe. At 30 dpe, total numbers of L3
obtained from 10 snails each of Puerto Rican and Brazilian
pigmented B. glabrata were 129 and 120. Similarly, 72 and63 L3 were recovered from albino Puerto Rican and Brazilian B. glabrata, respectively. Interestingly, however, pigmented B. straminea produced only 23 L3. It seems likelythat pigmented B. glabrata snails tend to harbor more L3
than albino B. glabrata and pigmented B. straminea. The
distribution pattern of L3 was quite different from that inother reports. For instance, Richards and Merritt (1967) reported that the dispersion of L3 in B. glabrata occurred
through digestive tract, whereas Yousif and Lammler (1977)
showed that most of L3 were found in headfoot, columellar
muscle and mantle collar, although it is not clear whether B.
glabrata used in their studies are pigmented or albino. In
the present study with Biomphalaria snails, most of L3
were found in the digestive gland and genital duct, especially, in the digestive gland in pigmented B. glabrata. With

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７

Figure 3. Section of the second stage larvae in seminal receptacle sac and vas deferens region of pigmented
Biomphalaria glabrata, Puerto Rico on 10 day after exposure to the first stage larvae. Hematoxylin and eosin stain, x200

Figure 4. Section of the third stage larvae in prostate gland and oviduct region of pigmented Biomphalaria
glabrata, Puerto Rico on 20 days after exposure to the first stage larvae. Hematoxylin and eosin
stain, x200

３
６
８

regard to the distribution of L3, most of them were detectedin the mantle, foot and digestive gland of A. fulica (Horiand Yamaguchi, 1982), similarly, in the head of Ampullarium sp. (Uchikawa et al., 1986), in the body muscle of Laevicaulis alte (Noda et al., 1985), and in the kidney and intestinal regions of Oncomelania hupensis snails (Iwanaga,
1995). It seems therefore that the development and distribution of L3 differ depending on snails species. Clearly, further work needs to be done on this point. Infection rates ofBrazilian and Puerto Rican pigmented B. glabrata were62.0 and 77.1%, respectively. Likewise, corresponding albino B. glabrata snails were 51.5 and 59.3%. In contrast,
the infection rate of pigmented B. straminea was only
39.8% (data not shown). Hence, in B. glabrata, pigmented
types show a slightly higher susceptibility than the albino
types. When compared the current data with those of otherexperimental intermediate hosts (Iwanaga et al., 1983;
Iwanaga, 1995), there was a distinct difference in susceptibility among snails. There were no distributional differenceof both L2 and L3 between pigmented and albino B.
glabrata. However, numbers of L3 recovered from pigmented B. glabrata at 30 dpe were slightly higher than thatfrom albino B. glabrata. Especially, it is of interest to notethat pigmented B. straminea showed a significantly lower
susceptibity than B. glabrata sanils (p<0.001). These differences are probably due to a combination of physiologicaland biochemical differences among the snails (Newton and
Brand, 1955; Iwanaga, 1992). Host’s responses against larvae were histologically examined at 10 and 20 dpe. No hosttissue response was noted around L2 at 10 dpe (Fig.3). At
20 dpe, a few L3 were observed in region 3, and they wereattacked by a definite host reaction consisting of amoebocytes surrounded by fibroblasts (Fig.4). These results agreewith the fingings by Iwanaga (1995) for O. hupensis snails.
Mean recovery rate of adult worms from rats infected with
L3 grown in Biomphalaria snail populations tested in thisstudy were 70 to 80%. The morphological features of these
adult worms coincided with those report by Mackerras andSandars (1955). Finally, these data suggest that B. glabrata,
especially pigmented type, is a suitable intermediate host to
establish the life cycle of A. cantonensis in the laboratory.

Acknowledgement
The author is greatly indebted to Dr. Jose Valfrido deSantana, Universidade Federal de Pernambuco, Brazil for
supplying the snails and also to Prof. Masamoto Kanno, Hiroshima University, for his cooperation throughout this research.

REFERENCES
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eosinophilic meningitis. Ceylon Med. J., 44,173-174
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larvae in Ampullarium canaliculatus. Southeast Asian J.
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３）Cooke-Yarborough, C.M., Kornberg, A.J., Hogg, G.G.,
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４）Hori, E. Kano, R. and Ishigaki, Y. (1976): Experimental
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on the development of Angiostrongylus cantonensis in
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31, 265-270 (in Japanese with English abstract)６）Iwanaga, Y. (1992): Comparative studies on Brazilian species of Biomphalaria snails by immunoelectrophoresis.
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767-773８）Iwanaga, Y. and Tsuji, M. (1972): Fundamental studies onlaboratory breeding of Oncomelania hupensis nosophora.
Hiroshima J. Med. Sci., 20,１-12 (in Japanese with English abstract)９）Iwanaga, Y., Tsuji, M. and Tanaka, N. (1983): Studies onhost parasite relationship of Angiostrongylus cantonensis.
(1) The antigenic communities between Angiostrongylus
cantonensis adult worm and snails. Jpn. J. Parasitol., 32,
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１０）Liat, L.B., Kong, D.C. and Joe, L.K. (1965): Natural infection of Angiostrongylus cantonensis in Malaysian rodents and intermediate hosts, and preliminary observations on acquired resistance. Am. J. Trop. Med. Hyg., 14,
610-617１１）Lim, B.L. and Hyneman, D. (1965): Host-parasite studies
of Angiostrongylus cantonensis (Nematoda, Metastrongylidae) in Malaysian rodents: Natural infection ofrodents and molluscus in urban and rural areas of control
Malaya. Ann. Trop. Med. Parasitol., 59, 425-433
１２）Mackerras, M.J. and Sandars, D.F. (1955): The life history of the rat lung-worm, Angiostrongylus cantonensis(Chen) (Nematoda; Metastrongyliidae). Australian J.
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１３）Newton, W.L. and Brand, T. (1955): Comparative physiological studies on two geographic strains of Australorbis
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(1985): Acquisition of Angiostrongylus cantonensis in-

３６９fection in Achatina fulica under natural field conditionand localization of Angiostrongylus cantonensis thirdstage larvae in Acathina fulica and Laevicaulis alte. Jpn.
J. Parasitol., 34, 457-463 (in Japanese with English abstract)１５）Nomura, S. and Lin, P. H. (1945): First case report to human infection with Haemostrongyllus ratti Yokogawa.
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１６）Purohit, A.K., Dinakar, L., Sundaram, C. and Ratnakar, K.
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Clin. Inf. Dis., 33, e112-115

１８）Richards, C.S. and Merritt, F. (1967): Studies on Angiostrongylus cantonensis in molluscan intermediate hosts. J.
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Angiostrongylosis; an important endemic in the Marquesa island. Med. Trop., 47, 141-144
２０）Uchikawa, R., Mori, T. and Sato, A. (1986): Experimental
infection of Ampullarium sp. with Aigiostrongylus cantonensis. Jpn. J. Parasitol., 35, 369-371 (in Japanese withEnglish abstract)２１）Yousif, F. and Lammler, G. (1977): The mode of infection
with and the distribution of Angiostrongylus cantonensis
larvae in the experimental intermediate host Biomphalaria
glabrata. Z. Parasitenk., 53, 247-250

JAPANESE JOURNAL OF
TROPICAL MEDICINE AND HYGIENE

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内

著
末梢血単核球と多核白血球によるインターロイキン８の産生と熱帯熱マラリア原虫の発育阻害作用
大屋ゆりジュリエッタ，松岡 裕之，向田 直史，笠原忠，石井明……………………３５１

Plasmodium yoelii 17XL 感染 ICR マウスでのジョウザンアジサイ葉水煮沸抽出液の抗マラリア活性の
季節的相違，特にフェブリフジン・イソフェブリフジン含有量に関して
石井明，藤井香，酒井美香子，飯干 昌美，宮瀬 敏男，寺田
護…………………３６１
短Original article
Ohya, J-Y., Matsuoka, H., Mukaida, N., Kasahara, T., and Ishii, A.
Growth inhibition of Plasmodium falciparum and interleukin-8 production by peripheral bloodmononuclear and polymorphonuclear cells in vitro ……………………………………………………３５１
Ohnishi, K., Kato, Y., and Iseki, M.
An imported Japanese case of cyclosporiasis ……………………………………………………………３５７
Ishih, A., Fujii, K., Sakai, M., Iiboshi M., Miyase, T., and Terada, M.
Seasonal differences in antimalarial activity of hot-water extract of Dichroa febrifuga leaves against
Plasmodium yoelii 17XL in ICR mice, with reference to febrifugine and isofebrifugine content ………３６１
Short Communication
Iwanaga, Y.
Comparative studies on the development of larval Angiostrongylus cantonensis in Puerto Rican andBrazilian Biomphalarla snails. …………………………………………………………………………３６５

and

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Medicine報
プエルトリコ産及びブラジル産 Biomphalaria 貝体内に於ける広東住血線虫幼虫の発育の比較
岩永
襄…………………………………………………………………………………………………３６５

Journal of Tropical

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