ロ■■藷

第24巻第3号平成8年9月
内
原

容

著
HTLV−1感染による培養末梢血リンパ球の遅発自発増殖現象：感染細胞に対する細胞性免疫応答の存在
一伊地知信二，町頭 幸一，永井 将弘，納William W．Ha11……………・・…
四6万o

光弘
143−149

ohol8彫の産生する溶血毒の溶血活性と抗原活性

一lsabel Chinen，Claudia Toma，本馬
比嘉 直美，岩永 正明一……一……

恭子
151−155

マレーシア・サバ州のブユ1新種の記載
・・高岡

タイにおけるブユ（双翅目

15H61

宏行・・

ブユ科）の1新種および新記録種について
・・高岡

宏行，斎藤

一三………

163−169

症例報告

メフロキンにより治療された卵型マラリアの2例と四日熱マラリアの1例
一木村幹男，田辺学，緒形芳久，綿矢 有佑 171−175
メニール鞭毛虫感染により引き起こされたと思われる海外旅行者下痢症の一例……………一…・…………・…森本 徳仁，是永 正敬，小松 千津，杉原 重喜
西田 政明，安岡 真理，熊澤秀雄，佐々木匡秀
橋口 義久…一…一……・…………・一…甲………… 17H80
研究ノート

長崎市内の犬糸状虫伝搬における予防薬の使用の影響
一小田力，三根真理子，末永
藤田紘一郎，加藤克知，田原（裏面に続く）敏，黒川弘幸……・…

憲次
181−1器1■

143

J pn. J. Tro p. Med. H yg ., Vol. 24, No. 3, 1996, pp. 143 149

LATE PHASE SPONTANEOUS PROLIFERATIONOF CULTURED PERIPHERAL BLOOD
LYMPHOCYTES ASSOCIATED
WITH HTLV‑ I INFECTION=

INVOLVEMENT OF CELLULAR RESPONSES
AGAlNST THE INFECTED CELLS
SHlN JI IJICH11, KOICHI MACHIGASHIRA1, MASAHIRO NAGA11

MITSUHIRO OSAMEI AND WILLIAM W. HALL2
Received June 26, 1996/Accepted July 29, 1996

Abstract: Common features of patients with human T Iymphotropic virus type I (HTLV‑ I ) ‑associatedmyelopathy/tropical spastic paraparesis (HAM/TSP) compared to asymptomatic HTLV‑ I carriers
include a genetically determined high cellular immune responsiveness to HTLV‑ I and a distributional biasof viral activation between the blood flow and central nervous systern (CNS) . It had been proposed that
increased and altered in vitro late phase spontaneous proliferation (SP) of peripheral blood lymphocytes,

which is concomitant with in vitro viral activation, is associated with the pathogenesis of HAM/TSP. To

assess whether SP might epitomize the peculiar cellular inflammation in the CNS of patients with HAM/

TSP, fractionated peripheral blood lymphocytes from HAM/TSP patients were employed to reconstitutethis phenomenon in vitro. Although CD8 + cells had no inherent responsive potential in the absence ofexogenous interleukin‑2 (IL‑2), the SP observed in CD4 + cell cultures was facilitated by the addition of
autologous CD8 + cells to the cultures. It could be shown that proliferative responses of the CD8 + cells
appeared against cultured and irradiated autologous CD4 + cells but not against purified HTLV‑ I virions.
These findings clearly demonstrate that the cellular response against the infected cells is involved and is oneof the major components of the late phase SP, and support the view that this phenomenon may represent anin vitro counterpart of the susceptibility to HAM/TSP.

Key words: Human T Iymphotropic virus type I (HTLV‑ I ) ; HTLV‑ I ‑associated myelopathy/tropicalspastic paraparesis (HAM/TSP) ; peripheral blood lymphocyte; spontaneous proliferation

INTRODUCTION

Biberfeld, 1983; Gavosto et al., 1959; Epstein and Bre‑cher, 1965; Rinaldo Jr et al., 1977) , in volunteers immun‑

Spontaneous proliferation (SP) of peripheral bloodized with various bacterial or viral antigens (Crowthermononuclear cells (PBMCs) is characterized by anincrease in thymidine incorporation into the cells underculture conditions which are devoid of mitogenic stimuli.

et al., 1969), in human immunodeficiency virus type 1(HIV‑ I ) ‑infected individuals (Lane and Fauci, 1985;
deShazo et al., 1989), and in patients with multiple

SP observed in the PBMCS cultured less than 3 days

sclerosis (Brinkman et al., 1984) . In contrast, Iate phasereflects the state of in vivo lymphocyte activation (Lane

SP has been reported in patients with a number of acuteviral infections (measles, varicella, Epstein‑Barr virus,

SP, which is apparent on day 3 and peaks on days 5‑7,
is one of the hallmarks of hurnan T Iyrnphotropic virus(HTLV) infection. This late phase SP had originallybeen described and attributed to an increase in large

and cytomegalovirus) (Griffin et al., 1989; Arneborn andblastoid lymphocytes in cultures of PBMCS derived

and Fauci, 1985; Griffin et al., 1989). This early phase

1

2

Third Department of Internal Medicine, Faculty of Medicine, Kagoshima University, Sakuragaoka 8‑35‑1, Kagoshima 890, Japan
Laboratory of Medical Virology, The Rockefeller University, New York, NY 10021‑6399, USA

144

from HTLV‑ I ‑infected healthy donors (Yasuda et al.,1986) . In patients with HTLV‑ I ‑associated myelopath‑y/tropical spastic paraparesis (HAM/TSP), a chronic
neurological disorder characterized by signs of bilateralpyramidal tract lesions, the late phase SP was firstreferred to as "autologous proliferative response" or "spontaneous lymphoproliferative response" (Usuku ettive selection procedure employing washed polystyrene

magnetic beads coated with mouse monoclonal anti‑
bodies (mAbs) (Dynabeads; Dynal. Oslo, Norway) . The
thawed PBMCS Were incubated with either anti‑CD8 or ‑CD4 mAb, coated beads (20‑25 beads/target celD in
phosphate buffered saline (PBS) containing 2% (v/v)(Itoyama et al., 1988; Eiraku et al., 1992), and altera‑

fetal calf serum (FCS) for 30 min at 4'C with gentle
mixing. Rosetted cells vyere removed using a magnetic
particle concentrator according to the manufacturer's
instructions. The nonrosetted cells were collected andfurther purified by the second negative selection step

tions in lymphocyte subset populations in the late phaseusing the same conditions as in the first step to obtain

SP were also noted in patients with HAM/TSP compar‑
ed to asymptomatic carriers (Eiraku et al., 1992).
Lymphocytes from HTLV‑II carriers also exhibit latephase SP (Prince et al., 1990). However, the HTLV‑ II
SP is significantly lower than that observed in HTLV‑

the CD4+ T Iymphocyte and CD8+ T Iymphocyteal.,1988; Jacobson et al., 1988) . It could be demonstrated

that the late phase SP is more intense in HAM/TSP

patients than in asymptomatic HTLV‑ I carriers

enriched cell populations respectively (CD4+ cells andCD8+ cells) . The purity of the fractionated cell popula‑tions was confirmed by flow cytometric analysis where
the maxirnum contamination of cells was 1.0 %.

I infection (Wiktor et al., 1991), and this may reflectmolecular and biological differences (Prince et al.,Cell cultures.

1994) including their in vivo cellular tropism (Ijichi et

The components of the late phase SP was recon‑al., 1992) between these closely related viruses whichmight be ultimately linked to the clinical outcomes instituted using fractionated viable cells. A series ofgraded mixtures of CD4+ and CD8+ cells from a

infected individuals (Hall et al., 1994).

patient with HAM/TSP was incubated for 7 days in

In viro interactions between the virus and the cellu‑

lar immune‑responses in the late phase SP in HTLV‑ I
infections have been suggested to be important (Usuku

RPMI‑1640 supplemented with 100 U/ml penicillin, 100
pglml streptomycin (GIBCO BRL) , and 10% (v/v) FCS
in 96‑well U‑bottorn plates without any mitogenic

et al., 1988), and the HTLV‑ I activation could bedemonstrated in cultured PBMCS derived from infected

stimuli (RPMI medium) . Triplicate wells were used foreach culture condition.

individuals (Hinuma et al., 1982; Minato et al., 1988).

Responsive transformation of uninfected

However, the role of cellular responses in the HTLV‑ I
late phase SP remains poorly understood. To investigate
this we have analyzed the in vitro response of CD8 +lymphocytes was evaluated as the proliferation of
CD8+ cells responding to autologous irradiated CD4+lymphocytes to cultured and irradiated autologous
CD4+ cells from individuals with HAM/TSP.

MATERIALS AND METHODSPatients.

cells. The in vitro emergence of HTLV‑ I activation in
the precultured CD4 + cells was previously demonstrat‑

ed by detection of HTLV‑ I pl9 and p24, increase inproviral and viral RNA amount, and using assays ofreverse transcriptase activities in culture supernatants(Ijichi et al., 1995) . As HTLV‑ I antigen presenting
cells, CD4+ cells were obtained from a patient with

A total of 6 patients who met WHO diagnostic
criteria for HAM/TSP (WHO, 1989) and two HTLV‑I seronegative normal donors were studied. PBMCS

HAM/TSP and cultured for 10 days in RPMI medium
containing 2 pg/ml phytohernagglutinin (PHA‑P,

were isolated from heparinized peripheral blood samples
on density gradients using Ficoll‑Hypaque, and the cells
were viably cryopreserved in liquid nitrogen until tested
using methods previously described (Usuku et al., 1988;

2) (Genzyme, Cambridge, MA) . The concentration ofIL‑2 was maintained by half medium changes every 3
days. Viable cells were isolated by density gradientcentrifugation, washed with PBS, resuspended in RPMI
medium and irradiated (5000 rad) . The proliferative
response of autologous CD8 + cells was evaluated by

Eiraku et al., 1992; Ijichi et al., 1995). Trypan blue dye

exclusion tests demonstrated that the thawed PBMCS

Sigma, St. Louis, MO) , and 10 I.U./ml interleukin‑2 (IL‑

had a viability of more than 95%.

incubating triplicate wells containing 1.0 X 105 CD8+

Cell separations.

cells and the CD4+ cells (1.0 X 105) in 96 well plates in
the presence 10 I.U̲/ml IL‑2 for 3 to 7 days.

T cell subsets were prepared using a double‑nega‑

To compare the potential of purified virus virions

145

with that of infected cells as the stimulant for CD8 +cells, 5.0 X 10' irradiated CD4+ cells prepared as
described above (precultured for 13 days) or 8 pglml
HTLV‑ I virions (EISAI, Tokyo, Japan) were added toautologous CD8+ cells (5.0 X 104) in 5 patients with

absence of exogenous IL‑2, the CD8+ cells (3.0 X 105
cells/welD from the same patient had no proliferative

response without viable CD4 + cells. However, theincrease in [3H] TdR incorporation observed in CD4+
cell cultures was facilitated by the addition of

HAM/TSP. The cells were incubated for 5 days in

autologous CD8+ cells (2.5 x 104 to 2.0 X 105 cells/

RPMI mediurn containing 10 I.U./ml IL‑2 in triplicate

welD in a dose dependent manner, showing that thewells. To assay the mitogenic effect of HTLV‑ I
virions on normal cells, PBMCS and CD4 + cells isolated

extraordinary excess of CD8+ cell population (at 0.5 of
the CD4 + /CD8 + cell ratio) induces the greatest facili‑

frorn two seronegative healthy donors were incubatedtation in cell proliferation in these conditions.

for 5 days with 8 pg/ml virions in the absence of IL‑2.

Proliferative response of CD8+ cells against irradiatedautologous CD4 + cells.

3H‑thymidine incorporation assay.

Cultures were maintained at 37 'C in a 5% C02
atmosphere. The cells were pulsed with 3H‑thymidine( [3H] TdR; 37 kBq/welD at 16 hr before terminating
the culture and then harvested on glass micro filter
papers. Incorporated radioactivity was measured in aliquid scintillation counter (TRI‑CARB‑4430; Packard
Instrument CO. Inc., IL) , and data are shown as themean SD. To evaluate the mitogenic activities of thepurified virions and irradiated CD4 + cells, stimulation
indices were calculated using the formula: mean cpm ofcultures with stimulation/mean cpm of cultures without
stimulation.

In contrast to the transformation of CD4 + cells, theproliferative response of CD8+ cells to autologous
HTLV‑ I ‑infected cells was not concomitant with theinduction of HTLV‑ I antigens in the proliferating cells(Ijichi et al., 1995). Preliminary studies have demon‑

strated that the in vitro proliferation of CD8 + cells
depends on the presence of viable CD4 + cells co‑cul‑tured, which include infected cells and may be supplying

growth factors, and the CD8 + cell response against
irradiated CD4 + cells requires preincubation of theCD4+ cells and supplementation of exogenous IL‑2 inthe medium (Ijichi et al., 1995; Nagai et al., 1995).
Because IL‑2‑induced proliferation of the CD8 + cells(alone) varies according to the in vivo expression of IL‑

RESULTS

2 receptors on the cells (Ijichi et al., 1995; Nagai et al.,Reconstituted late phase SP (Fig. 1) .
Cultured viable CD4+ cells (3.0 and 1.0 X 105 cells/

welD obtained from a representative patient with
HAM/TSP showed remarkable increase in [3H] TdR
incorporation without any mitogenic stimuli. In the1995) , the responsive proliferation was assessed as stim‑

ulation indices. To analyze the proliferative response of
uninfected CD8 + cells against autologous infected cells,

the proliferative potential of CD8 + cells cocultured
with precultured and irradiated CD4+ cells was inves‑[3H]TdR incorporation (cpm)ceo

3.0 x 105O

1.0 x 105

O

1 .O x 105

2.5 x 104

1.0 x 105

5.0 x 104

1.0 x 105

1.0 x 105

1.0 x 105

2.0 x 105O

3.0 x I 05

1 oooo 20000 30000

40000

Figure 1. Reconstituted cultures to show the each role of CD4 + cells andCD8 + cells for the HTLV‑ 1 Iate phase SP. Fractionated viable
cells were cultured in the absence of IL‑2 for 7 days. The results are

expressed as the means

SD in triplicate cultures.

146

lrradiated Culture [3H]TdR incorporation (cpm)
C cells duration da s
6000

1,0 x 105 O 3

1,0 x 10 O 5
1.0
O 73
O I x.O 10
x 105
5
5

O 1,0 x 105 5
O 1,0 x 105 7

1.0 x 105 1 .O x 105 3
1.0 x5 10 1 .O x 105 5

1,0 x5 10 1,0 x 105 7
Figure 2. CD8 + cell response against the autologous irradiated CD4 + cells(precultured) in the presence of xogenous IL‑2. The results are
expressed as the means i SD in triplicate cultures.

tigated (Fig. 2). The representative case in Fig. 2 is of exogenous IL‑2. Control cultures showed low[3H]

HAM3 in Table 1, whose CD8 + cells alone did not TdR incorporation into the viable CD8 + cells and theexhibit high incorporation of thymidine in the presence irradiated CD4+ cells cultured alone for 3 to 7 days in

Table I Cell responses against purified HTLV‑ I virions or irradiated autologous
CD4 + cells possessing HTLV‑ I antigens[3H] TdR incorporation (cpm) into cells stimulated by
Cells

Purified HTLV‑ I vrrrons

lrradiated CD4 + cells(Cultures in the absence of IL‑2)

NDI PBMCS

2539

556 (7.7)n.d.

5.0 x 10*ND2 PBMCS

8077 i 1221 (16.3)

n.d.

24207

13183 (69.8)

n.d.

16412

4529 (61.2)

n.d.

5.0 X 10'

NDI CD4+ cells
1.0 >< 10

ND2 CD4+ cells
1.0 X 10=

(Cultures in the presence of IL‑2)

NDI CD8+ cells

24311 i 1141 (0.8)

n.d.

52756

7780 (0.9)

n.d.

4553

439 (1.0)31232

5148 (7.2)

22516

3794 (0.8)

603945007 (2.0)

1.0 X 10ND2 CD8+ cells
1.0 X 10=

HAMI CD8+ cells
5.0 x 10"

HAM2 CD8+ cells
5.0 X 10'

HAM3 CD8+ cells

1956 i 510 (1.1)

17723 i 2539 (10.1)

5714 i 1884 (1.0)

11799 d : 470 (2.0)

22178 i 3177 (0.8)

45531

5.0 X 10+

HAM4 CD8+ cells
5.0 X 10'

HAM5 CD8+ cells

3227 (1.7)

5.0 X 10+

The results are expressed as the means SD (stimulation indices) in triplicate cultures.
Significant stimulation indices are underlined. Abbreviations: NDI and ND2, normal donors;

HAM1‑HAM5, patients with HAM/TSP; n. d., not done.

147

this patient. The CD8 + cells cocultured with theed cells requires the CD2/lyrnphocyte function‑associat‑

irradiated autologous CD4 + cells exhibited an increase

ed antigen 3 (LFA‑3) pathway for the activation ofin thymidine incorporation, and the proliferation of
CD8 + cells was found to be most remarkable in late

responder T cells (Dodon et al., 1989; Wucherpfennig etal., 1992; Kimata et al., 1993), and the subsequent auto‑phase of culture (day 7) .

crine or paracrine secretion of IL‑2 from responding

The late phase responsive proliferation of theCD8+ cells cocultured with irradiated autologous

cells is essential for cell proliferation (Dodon and Gaz‑

CD4 + cells was confirmed in a total of 5 patients with

HAM/TSP (Table I and Figure 2). Cell free purified
HTLV‑ I virions were found to have mitogenic activity

on normal donor PBMCS and CD4+ cells. However, thestimulation index data demonstrated that the virions
had essentially no stimulatory effect on the CD8 + cells

zolo, 1987; Wucherpfennig et al., 1992). In addition, it
has been suggested that other soluble factors may con‑tribute to the SP (Lal and Rudolph, 1991; Lal et al.,1991). Other studies have suggested that responsive T

cell proliferation in HTLV‑ I SP may be a human
lymphocyte antigen (HLA) haplotype‑1inked response
against HTLV‑ I virions (Usuku et al., 1988; Sonoda,

in these patients (Table 1). The quantity of purified1990) , and it had been suggested that an inhibitory effectvirus virions employed (8 pg/mD was found to be
optimal to activate normal donor PBMCS (data notof CD8+ cells on the in vitro HTLV‑ I expression in
infected autologous CD4+ cells is a HLA haplotype‑linked response (Sonoda, 1990). Moreover, cellshown) .

responses in HTLV‑ I SP have recently been shown toactivator, and antigens, the infection is characterized byinvolve a different signal transduction pathway from thegrowth of HTLV‑ I ‑infected cells (Mann et al., 1994) .
Therefore, the proliferation of CD8 + cells in the HTLV‑I SP has received much attention as an in vitro epit‑
ome of the cellular inflarnmation in the central nervous

the viral latency in circulating infected cells in the hostsystem (CNS) of HAM/TSP patients (Eiraku et al.,(SchUpbach, 1989). Peripheral blood lymphocytes

1992) , and the view supports the proposed implication ofobtained from HTLV‑ I ‑infected individuals exhibit
late phase SP accompanied by in vitro HTLV‑ I activa‑
tion, and patients with HAM/TSP are characterized byhigh and altered late phase SP as mentioned above.
Because purified T Iymphocytes can exhibit the latethe anti‑HTLV‑ I cytotoxic T Iymphocytes in the

DISCUSSION
Although HTLV‑ I antigens have a variety of viro‑

immunological properties as a mitogen, a trans‑

phase SP, this phenomenon is different from theautolegous mixed lyrnphocyte reaction (AMLR)(Minato et al., 1989; Lal et al., 1992), and recently, the

HTLV‑ I SP has been reported as a T cell colony‑forming cell abnormality (Lunardi‑Iskandar et al.,1993). In vitro spontaneous expression of HTLV‑ I
RNA and viral antigens in cultured PBMCS has beenpathogenesis f HAM/TSP (Moore et al., 1989; Jacob‑
son et al., 1990).

In the present study we could demonstrate the fol‑
10wing. (i) Not only the in vitro activation of infected
CD4 + cells but also the proliferative response of CD8 +cells against infected cells is involved and is one of the

major components of the HTLV‑ 1 Iate phase SP. (ii)
The presence of viable CD4 + cells is critical for the
HTLV‑ I virion‑related T cell activation. (iii) The
CD8 + cell response to infected cells requires either thepresence of viable CD4+ cells or exogenous IL‑2.

demonstrated in healthy HTLV‑ I carriers and patients

The HTLV‑ 1 Iate phase SP might bear an impor‑with HAM/TSP (Hinuma et al., 1982; Minato et al.,tant relationship to the observed cellular inflammation

1988; Mann et al., 1994), and HTLV‑ I viral particles
have been demonstrated in extracellular spaces of cul
tured PBMCS (Gessain and Gout, 1992) . As such, the IL‑
2‑independent growth of infected clones (H61lsberg etal., 1992), mitogenic activity of HTLV‑ I virions(Gazzolo and Dodon, 1987; Dodon and Gazzolo, 1987;
Dodon et al., 1989; Maguer et al., 1993; Cass et al.,1994), and T cell activation induced by cell‑to‑cellcontact with the infected cells (Wucherpfennig et al.,
1992; Kimata et al., 1993) may be involved in the HTLV‑ I SP. The mitogenic activity of HTLV‑ I virions andthe mitogenic surface structure (s) on HTLV‑ I ‑infect‑

in situ around HTLV‑ I ‑infected cells in HAM/TSP,
and may be involved in the critical pathological processof this disorder (Ijichi et al., 1993) . Although it has been

extremely difficult to detect HTLV‑ I antigens in theaffected tissues of patients (Gessain and Gout, 1992) , thepresence of integration and activation of HTLV‑ I in

the CNS of HAM/TSP patients has been demonstrated
by in situ hybridization techniques (Hara et al., 1994;
Kashio et al., 1994; Kuroda et al., 1994; Lehky et al.,1995). The cell responses involved in the HTLV‑ I Iate
phase SP presented here may be an in vitro equivalentof the cellular inflammation in the affected tissues in148patients with HTLV‑ I ‑associated diseases.

REFERENCES
1 ) Arneborn, P. and Biberfeld, G. (1983): T‑lymphocyte

subpopulation in relation to immunosuppression inmeasles and varicella. Infect. Irnmun., 39, 29‑37

2 ) Brinkman, C.J.J., Nillesen, W.M. and Hommes, O.R.
(1984): Lymphocyte subpopulations in multiple sclero‑sis: spontaneous and mitogen‑induced activity. Clin.
Immunol. Immunopathol. 31, 364‑370
3 ) Cass6, H., Girerd, Y., Gazzolo, L. and Dodon, M.D. (1994) :Critical involvement of human T cell leukaemia virus
type I virions in mediating the viral mitogenic effect. J.
Gen. Virol., 75, 1909‑1916

4 ) Crowther, D.. Fairley, G.H. and Sewell, R.L. (1969):
Lymphoid cellular responses in the blood after immuni‑
zation in man. J. Exp. Med., 129, 849‑869
5 ) deShazo, R.D., Morgan, J.E., Daul, C.B., Andes, A.,
Hyslop, N. and Palmer, C. (1989) : Studies of spontane‑ous proliferation of mononuclear cells in human im‑
munodeficiency virus (HIV‑ I ) ‑infected individuals. J.
Allergy Clin. Immunol., 84, 34‑39
6 ) Dodon, M.D. and Gazzolo, L. (1987): Loss of interleukin‑2 requirement for the generation of T colonies defines

an early event of human T‑1ymphotropic virus type I
infection. Blood 69, 12‑177 ) Dodon, M.D.. Bernard, A. and Gaz olo, L. (1989):
Peripheral T‑1ymphocyte activation by human T‑cellleukemia virus type I interferes with the CD2 but notwith the CD3/TCR pathway. J. Virol., 63, 5413‑5419
8 ) Eiraku, N., Ijichi, S., Yashiki, S.. Osame. M. and Sonoda,

S. (1992): Cell surface phenotype of in vitro proliferat‑

ing lymphocytes in HTLV‑ I ‑associated myelopathy. J.
Neuroimmunol., 37, 223‑2289 ) Epstein, L.B. and Brecher, G. (1965): DNA and RNA
synthesis of circulating atypical lymphocytes in infec‑
tious mononucleosis. Blood 25, 197‑203
10) Gavosto, F., Pileri, A. and Maraini, G. (1959): Incorpo‑
ration of thymidine labeled with tritium by circulating
cells of infectious mononucleosis. Nature 183, 1691‑1692.
11) Gazzolo, L. and Dodon, M.D. (1987) : Direct activation ofresting T Iymphocytes by human T‑1ymphotropic virus
type I. Nature 326, 714‑717

12) Gessain. A. and Gout, O. (1992): Chronic myelopathy
associated with human T‑lymphotropic virus type I(HTLV‑ I ) . Ann. Int. Med., 117, 933‑946
13) Griffin, D.E., Ward, B.J., Jauregui. E.. Johnson, R.T. andVaisberg, A. (1989) : Immune activation in measles. N.
Engl. J. Med., 320, 1667‑1672
14) Hall, W.W., Kubo. T., Ijichi, S., Takahashi, H. and Zhu,

S.W. (1994): Human T cell leukemia/lymphoma virus,
type 11 (HTLV‑II ) : emergence of an important newly
recognized pathogen. Semin. Virol., 5 165‑178
15) Hara. H., Morita, M., Iwaki, T.. Hatae, T., Itoyama, Y.,

Kitamoto, T., Akizuki. S., Goto, I. and Watanabe. T.

(1994) : Detection of human T Iymphotropic virus type I(HTLV‑ I ) proviral DNA and analysis of T cell rece‑
ptor Vp CDR3 sequences in spinal cord lesions of HTLV
‑ I ‑associated myelopathy/tropical spastic paraparesis.
J. Exp. Med., 180, 831‑839
16) Hinuma, Y.. Gotoh, Y., Sugarnura, K., Nagata, K.. Goto,

T., Nakai, M., Kamada, N., Matsumoto. T. and Kino‑
shita, K. (1982): A retrovirus associated with humanadult T‑cell leukemia: in vitro activation. Jpn. J. Cancer.

Res. (Gann) 73, 341‑344
17) H61lsberg, P., Wucherpfennig K. W., Ausubel, L.J.,Calvo,
V., Bierer B. E. and Hafler, D.A. (1992): Characteriza‑tion of HTLV‑ I in vivo infected T cell clones: IL‑2‑

independent growth of nontransformed T cells. J. Im‑
munol., 148, 3256‑3263
18) Ijichi, S., Ramundo, M.B., Takahashi, H. and Hall. W.W.
(1992) : In vivo cellular tropism of human T cell leuke‑
mia virus type 11 (HTLV‑ 11 ) . J. Exp. Med., 176, 293‑296

19) Ijichi, S., Izumo, S., Eiraku, N., Machigashira, K.,
Kubota, R., Nagai, M., Ikegami, N., Kashio, N., Umehar‑a, F., Maruyarna, I. and Osame, M. (1993): An autoag‑gressive process against bystander tissues in HTLV‑ I‑infected individuals: a possible pathomechanism of
HAM/TSP. Med. Hypoth., 41, 542‑547
20) Ijichi. S., Izurno, S., Nagai, M., Shinmyozu, K., Hall. W.

W. and Osame, M. (1995): Antiviral and irn‑
munomodulatory effects of interferon‑a on cultured
lymphocytes from patients with HTLV‑ I ‑associatedmyelopathy (HAM/TSP) . J. Neuroimmunol., 61, 213‑
22021) Itoyama, Y., Minato. S., Kira, J., Goto, I., Sato, H.,

Okochi, K. and Yamamoto, N. (1988): Spontaneous
proliferation of peripheral blood lymphocytes increasedin patients with HTLV‑ I ‑associated myelopathy. Neu‑rology 38, 1302‑1307
22) Jacobson, S., Zaninovic, V., Mora, C., Rodgers‑Johnson,

P., Sherernata, W.A., Gibbs, C.J., Gajdusek, C. and
MacFarlin, D.E. (1988) : Immunological findings in neur‑

ological diseases associated with antibodies to HTLV‑I : activated lymphocytes in tropical spastic parapar‑
esis. Ann. Neurol., 23 (Suppl.), S196‑S200
23) Jacobson, S., Shida, H., McFarlin, D.E., Fauci, A.S. andKoenig, S. (1990): Circulating CD8+ cytotoxic T
lymphocytes specific for HTLV‑ I pX in patients with
HTLV‑ I associated neurological disease. Nature 348,
245‑248
24) Kashio, N., Izumo, S., Ijichi, S., Hashimoto, K., Umehara,

F.. Higuchi, I. and Osarne, M. (1994): Detection of
HTLV‑ I provirus by in situ polymerase chain reac‑tion in mononuclear cells infiltrating spinal cord lesions

from HAM/TSP patients. Brain. Pathol., 4, 498(abs ract) .

25) Kimata, J.T., Palker, T.J. and Ratner, k. (1993): The
mitogenic activity of human T‑cell leukemia virus type

I is T‑cell associated and requires the CD2/LFA‑3
activation pathway. J. Virol., 67, 3134‑3141
26) Kuroda, Y.. Matsui, M.. Kikuchi, M., Kurohara. K.. Endo,149

C.; Yukitake, M., Matsuda. Y., Tokunaga, O.. Kornine‑
Sakaki, A. and Kawaguchi, R. (1994): In sutu demon‑
stration of the HTLV‑ I genome in the spinal cord of apatient with HTLV‑ I ‑associated myelopathy. Neurol‑ogy 44, 2295‑2299
27) Lal, R.B. and Rudolph, D.L. (1991): Constitutive produc‑tion of interleukin‑6 and tumor necrosis factor‑a fromspontaneously proliferating T cells in patients withhuman T‑cell lymhotrcipic virus type‑ I /II. Blood 78,
571‑574
28) Lal, R.B., Rudolph, D.L., Folks, T.M. and Hooper, C.
(1991) : Role of insulin‑Iike growth factor‑1 in spontane‑ous proliferation of HTLV‑ I ‑infected lymphocytes. J.
Acquir. Immune Defic. Syndr., 4, 1165‑1166
29) Lal, R.B., Hjelle, B. and Rudolph, D.L. (1992): Spontane‑ous proliferation of HTLV‑ 11 ‑infected peripheral bloodmodel of virus‑induced, cytotoxic T‑cell‑mediateddemyelination? Ann. Neurol., 26, 523‑530
38) Nagai, M., Ijichi, S., Hall, W.W. and Osame, M. (1995) :Differential effect of TGF‑pl on the in vitro activationof HTLV‑ I and the proliferative response of CD8 + T

lymphocytes in patients with HTLV‑ I ‑associatedmyelopathy (HAM/TSP). Clinical Immunol. Im‑
munopathol., 77, 324‑331

39) Prince, H., Kleinman, S., Doyle, M., Lee, H. andSwanson, P. (1990): Spontaneous lymphocyte prolifera‑tion in vitro characterized both HTLV‑ I and HTLV‑II infection. J. Acquir. Immune Defic. Syndr., 3, 1199‑
120040) Prince, H.E., Golding, J. and York, J. (1994):
Lymphocyte subset alterations associated with in‑creased spontaneous lymphocyte proliferation in human

lymphocytes: HLA ‑ DR ‑ driven, IL‑2‑dependent

T Iymphotropic virus (HTLV) infection: distinctive

response. Microbiol. Immunol., 36, 865‑872
30) Lane, H.C. and Fauci. S. (1985) : Immunologic abnormal‑
ities in the acquired immunodeficiency syndrome. Annu.

patterns for HTLV. ‑ I versus HTLV‑ I I infection. J.

Rev. Immunol., 3, 477‑500
31) Lehky, T.J., Fox, C.H., Koenig, S., Levin. M.C., Flerlage,

N., Izumo, S., Sato, E., Raine, C.S., Osame, M. andInfect. Dis., 169, 1409‑1410

41) Rinaldo. Jr., C.R., Black, P.H. and Hirsch, M.S. (1977):

Interaction of cytornegalovirus with leukocytes frompatients with mononucleosis due to cytornegalovirus. J.
Infect. Dis., 136, 667‑678

lymphotropic virus type I (HTLV‑ I ) tax RNA in the42) Schupbach, J. (1989): Introduction and history. In: R.W.
Compans et al. (Eds.) , Hurnan Retrovirology: Facts andcentral nervous system of HTLV ‑ I ‑ associatedConcepts (Current Topics in Microbiology and Im‑

myelopathy/tropical spastic paraparesis patients by insitu hybridization. Ann. Neurol., 37, 167‑175
32) Lunardi‑Iskandar, Y., Gessain, A., Lam, V.H. and Gallo,

munology 142), Springer‑Verlag, Berlin, 1‑543) Sonoda, S. (1990): Genetic and immunologic determi‑

R.C. (1993) : Abnormal in vitro proliferation and differ‑entiation of T cell colony‑forming cells in patients withner (Ed.), Human Retrovirology: HTLV. 315‑326,
Raven, New York, NY

tropical spastic paraparesis/human T Iymphocyte virus44) Usuku, K., Sonoda. S., Osame, M., Yashiki, S., Takaha‑shi, K., Matsumoto, M., Sawada, T.. Tsuji, K., Tara, M.

Jacobson, S. (1995): Detection of human T ‑type I (HTLV‑ I ) ‑associated myeloencephalopathy

nants of HTLV‑ I ‑associated diseases. In: W.A. Blatt‑and healthy HTLV‑ I carriers. J. Exp. Med., 177, 741‑
75033) Maguer, V., Cass6‑Ripoll, H., Gazzolo, L. and Dodon, M.
D. (1993): Hurnan T‑cell leukemia virus type I‑induced

and lgata, A. (1988) : HLA haplotype‑linked highimmune responsiveness against HTLV‑ I in HTLV‑ I

proliferation of human immature CD2+ CD3 ‑thymocytes. J. Virol., 69,5529‑5537
34) Mann, D.L., Martin, P., Hamlin‑Green, G., Nalewalk, R.
and Blattner, W. (1994) : Virti prbdtidtion and spontane‑45) Wiktor, S.Z., Jacobson, S., Weiss, S.H., Shaw, G.M.,
Reuben, J.S., Shorty, V.J., McFarlin, D.E. and Blatt‑ner, W.A. (1991): Spontaneous lymphocyte prolifera‑tion in HTLV‑II infection. Lancet 337, 327‑328

ous cell proliferation in HTLV ‑ I ‑ infected46) World Health Organization (1989): Virus diseases.

lymphocytes. Clin. Immunol. Immunopathol., 72, 312‑320

human T ‑ Iymphotropic virus type I, HTLV ‑ I35) Minato, S., Itoyama, Y.. Goto, I. and Yamamoto, N.
(1988): Expression of HTLV‑ I antigen in cultured47) Wucherpfennig, K.W., Hbllsberg, P., Richardson. J.

peripheral blood mononuclear cells from patients with
HTLV‑ I associated myelopathy. J. Neurol. Sci., 87, 233‑24436) Minato, S., Itoyama, Y., Fujii, N., Kira, J., Goto, I. andYamamoto, N. (1989): Activated T cells in HTLV‑ I ‑associated myelopathy: autologous mixed lymphocyte
reaction. Ann. Neurol., 26, 398‑40137) Moore, G.R.W.. Traugott, U., Scheinberg, L.C. andRaine. C.S. (1989): Tropical spastic paraparesis: a‑associated myelopathy: comparison with adult T‑cellleukernia/1ymphoma. Ann. Neurol., 23 (Suppl.) , 143‑150(abstract) . WHO Weekly Epidemiol. Rec., 49, 382‑383
H., Benjamin, D. and Hafler, D.A. (1992): T‑cell activa‑tion by autologous human T‑cell leukemia virus type I‑infected T‑cell clones. Proc. Natl. Acad. Sci., USA 89,

2110‑2114
48) Yasuda, K., Sei, Y.. Yokoyama. M.M., Tanaka, K. andHara, A. (1986): Healthy HTLV‑ I carriers in Japan:
the haematological and immunological characteristics.
Br. J. Haematol., 64, 195‑203

Jpn. J. Tro p. Med Hyg Vol 24 No 3 1996, pp. 151‑155

151

HEMOLYSIN PRODUCTION BY VIBRIOCHOLERAE AS EXAMINED BY HEMOLYTIC
AND ANTIGENIC ACTIVITIESISABEL CHlNENl, CLAUDIA TOMA2, YASUKO HONMA2
NAOMI HIGA2, AND MASAAKI IWANAGA2,3
Received June 17, 1996/Accepted July 30, 1996

Abstract: The hemolytic and antigenic activities of 36 strains of Vibrio cholerae O1 biotype E1 Tor,and 63 strains of Vibrio cholerae non‑OI isolated in Argentina were studied. Only 8% in stationary

culture and 11% in shaking culture were hemolytic when the heart infusion broth (HIB) culture
supernatants of V. cholerae O1 were examined. However, these percentages increased to 94% and
22% respectively when the strains were grown in HIB supplemented with 1% glycerol. On the other
hand, most strains of V. cholerae non‑OI (97%) were hemolytic in both stationary and shaking HIB
culture supernatants. The antigenic activity detected by reversed passive latex agglutination
method (RPLA) varied markedly from strains and culture conditions; ranging from below thedetection limit to 16,000 ng/ml. The optimal condition of hemolysin production varied with thestrain, but a stationary culture was preferable to obtain high hernolytic activity and shaking culture

to obtain a large amount of hemolysin antigen. The comparison of the hernolytic and antigenic
activities showed that the hemolysin is readily inactivated in the culture supernatant, especially inthe case of V. cholerae O1. These findings suggested the presence of some potent inactivation factor(s) in E1 Tor vibrios.

INTRODUCTION

rapidly spread through the continent and Central Amer‑

Since Gotschlich discovered the hemolytic prop‑

ica by Weekly Epidemiological Record(1992). Accord‑
ing to the weekly epidemiological record from WHO, inerties of Vibrio cholerae O1 in 1905 (Pollitzer, 1959), V.

Argentina there were 553 cases in 1992, 2080 in 1993, andcholerae O1 (cholera vibrio) was classified into two

889 in 1994. In the epidemiological studies for cholera inbiotypes, non‑hemolytic classical biotype and hemolytic
El Tor biotype. On the other hand, most V. cholerae non‑Argentina, a number of V. cholerae O1 El Tor wereO1 are hemolytic (Sakazaki, et al., 1967). The

with watery diarrhea. Among the V. cholerae non‑O1

hemolysin of V. cholerae non‑OI and O1 El Tor were
purified by Yarnamoto et al. (1986), and they proved

isolates, one strain of non‑toxigenic V. cholerae 0139
was found (Rivas et al., 1993). In this communication,
we describe the production of hemolysin by V. cholerae
strains isolated in Argentina as quantitatively examinedby hemolytic as well as antigenic activities.

that both hemolysins were identical. Although the roleof hemolysin in the illness is not elucidated, it is cytotox‑

ic and enterotoxic (Ichinose et al ., 1987; McCardell etal., 1985). Recently identified new cholera vibrios ( V.

cholerae 0139 synonym BengaD are also hemolytic andthe hemolytic activity is neutralized by anti‑EI Tor
hemolysin antisera (Higa et al., 1993). The hemolytic
activities vary with the strain and culture condition.
Hemolytic activities of V. cholerae non‑OI are morepotent than those of V. cholerae O1 E1 Tor. E1 Torcholera invaded South American Continent in 1991, andisolated as well as V. cholerae non‑OI from the cases

MATERIALS AND METHODS
1. Bacterial Strains: Vibrio cholerae non‑OI strain N037
isolated from a diarrheal patient in Thailand was used
for purification of hemolysin. Thirty‑six strains of V.
cholerae O1 El Tor and 63 strains of V. cholerae non‑O1

isolated in Atgentina were used to study hemolysin

1 Instituto Nacional de Microbiologia "Dr. Carlos G Malbran" Velez Sarsfield 563(1281), Buenos Aires, Argentina
2 Department of Bacteriology and 3 Research Center of Comprehensive Medicine, Faculty of Medicine, University of the Ryukyus 207
Uehara, Nishihara, Okinawa 903‑01, Japan

152

production.

precipitate was suspended in 10 mM phosphate buffered

2. Culture conditions: Heart infusion broth (HIB) wasused in stationary and shaking conditions to determine
hemolysin production. The organisms were cultured at
30'C for 18 hr stationary in a large test tube or withshaking in a 100 ml Erlenmyer's flask containing eachsaline pH 7.4 (PBS) and dialyzed against PBS. This
crude lgG fraction was adjusted to OD=0.7 at a wave‑
length of 280 nm, and I ml of the lgG solution wasapplied to the hemolysin conjugated Hi Trap Affinity
Colutnn equilibrated with PBS. The flow rate of lgGsolution was 2 drops/sec. The column was washed with
PBS until OD280 returned to the base line. Anti‑

10 ml of HIB. HIB supplemented with 1% glycerol(HIBG) was used for El Tor strains, producing a verysmall amount of hemolysin, to confirm the productivity.

hemolysin specific lgG was eluted with elution buffer[0.2M glycine, 0.5M NaCl, pH 2.7]. The eluted sample

was neutralized with 0.1M NaOH.
3. Purlfication of hemolysin: The strain N037 was cul‑
tured in a 3L Erlenrnyer's flask containing 300 ml ofheart infusion broth (HIB) at 37'C for 18 hr with shak‑ing. The culture supernatant was salted out with 60%
saturated ammonium sulfate. The precipitate was dis‑solved in TEA buffer [50mM Tris‑HCl, ImM EDTA,
3mM NaN3, pH 8.0] and dialyzed against the same
buffer. The sample was fractionated by Sephadex G‑
100 column chromatography using TEA buffer for elu‑tion. The fractions with hemolytic activity were pooled

and concentrated by ultrafiltration using an Amicon
PMIO filter membrane. The concentrated material was
fractionated by Sephacryl S‑200 column chromatogra‑
phy using TEA buffer for elution. When the purity of

7. Preparation of sensitized latex: SLD 59 Iatex (Takeda)

prepared in a 0.1% suspension was mixed with an equalvolume of 5% glutal aldehyde and incubated overnightat 4'C with shaking. The latex was washed and suspend‑
ed in PBS to a final concentration of 0.5%. The affinity

purified anti‑hemolysin lgG was dialyzed against 3%
sucrose in PBS. The latex and lgG were mixed at theconcentrations of 0.1% and 5 to 10pg/ml, respectively.
The mixture was incubated at 4 'C for I hr with shaking.
Then the sensitized latex was washed with PBS contain‑

hemolysin was not satisfactory, Sephacryl S‑200 column

ing 0.8% bovine serum albumin (BSA) and 0.1% sodium
azide. Finally, the latex was suspended in adjusting
buffer [PBS containing 0.8% BSA, 0.1% sodium azide,and 0.0005% polyvynylalcohol] at the concentration ofchromatography was repeated once again.

0.5%.

4. Preparation of anti‑serum: A rabbit was immunizedwith 100 pg of purified hemolysin every 2 weeks. One

blood agar (1.3% agar, 7% sheep blood, 0.03% sodium

8. Determination of hemolytic activities: Fifteen ml ofazide in 10mM PBS) was prepared in a plastic petri dishmilliliter of antigen emulsified with an equal volume ofFreund's complete adjuvant was injected multisite‑sub‑

9 cm in diameter, and about 20 wells each 3 mm incutaneously. For the boosting injection, 100 pg ofhemolysin and Freund's incomplete adjuvant was used.

diameter were made within one plate. The HIB culture
supernatants of V. cholerae were applied to the wells

5. Preparation of hemolysin coupled affinity gel column:

hr. The diameter of the hemolytic zone was measured.

(about 15 pl to each welD and incubated at 37 'C for 18

The hemolysin solution was dialyzed against coupling

buffer [0.2M NaHC03, 0.5M NaCl, pH 8.3] and I ml
containing 9 mg of hemolysin was applied to I ml
volume of Hi Trap Affinity Column NHS‑activated(Pharmacia Biotech.). Then, the column was washed

9. Titration of hemolysin antigen: The amount ofhemolysin antigen was quantitatively determined byreversed passive latex agglutination (RPLA) . The

with buffer A [0.5M ethanolamine, 0.5M NaCl, pH 8.3]
and successively with buffer B [0.1M sodium acetate,
0.5M NaC1, pH 4.0]. The column was equilibrated withstorage buffer [0.05M Na.HP0+, 0.1% NaN3, pH 7.0],

the diluent [lOmM PBS containing 0.8% BSA, 0.01%

culture supernatants were serially diluted 2‑fold withanti‑serum was heated at 56'C for 30 min and was salted

polyvinylpyrrolidone, and 0.1% sodium azide] in the U‑
bottom microdilution plates. An equal volume (25pD of
0.025% sensitized latex was added to each well, mixed
on the plate vibrator and incubated overnight at room
temperature. The titers were defined as the reciprocalof the highest dilution in which agglutination was obser‑
ved or the number of wells with agglutination. Purified

out with 20% saturated ammonium sulfate. The super‑
natant was salted out again with 33% saturation. The

hemolysin protein (1 or 0.1pg/mD was used as thestandard in each plate to determine the amount ofand kept in a cold room until use.

6. Preparation of anti‑hemolysin lgG: Anti‑hemolysin

153

hemolysin.

detected from the non‑hemolytic culture supernatants.

10. Protein assay: The protein concentration was deter‑than the non‑OI strains (Table 2, Figure 1) . Generally,

The E1 Tor strains produced more hemolysin antigen
mined by Lowry's method (Lowry et al., 1951).

the shaking culture produced more hemolysin antigen
than the stationary culture, except for a few strains.

RESULTS

Table 2 Hemolysin production in HIB

1. Sensitization of latex: Various concentrations of anti

Organisms‑hemolysin lgG were examined at a constant concentra‑(No. examined)

tion of latex to find the optimal concentration to sensi‑
tize the latex. At an lgG concentration lower than I pgl

O1 E1 Tor
(n=36)

ml, the detection limit of hemolysin was higher than 10
ng/ml. However, IgG at a concentration higher than 10
pglml tended to cause autoagglutination. Therefore, tosensitize latex we used lgG at a concentration of 5pgl
ml. The detection limit of hernolysin was I to 2 ng/ml.

2. Hemolytic activities of V. cholerae: Among 36 V.
cholerae O1 (EI Tor strains) , hemolytic activity was

Cultureconditions

Hemolysin antigen

ng/ml (RPLA)

ST

min.

6.25

max.

6400

mean

764

SH

min.

40

max.

6400

mean

1744

ST

non‑O1(n=63)

min.

o

max.

6000

mean

224

SH

min.

6.25

in stationary culture and 4 strains (11%) in shaking

max.

6400

culture. However, 61 out of 63 V. cholerae non‑O1

mean

903

found in the culture supernatants of only 3 strains (8%)

strains (97%) in both culture conditions were hemolytic.

Hemolytic activities of non‑OI V. cholerae in station‑
ary culture were generally greater, than those in shak‑ing culture (Table 1).
Comparing each strain, stronger activity was see‑nin stationary culture for 43 strains, but in shaking cul‑abbreviations: same as in Table 1

20
"
oo 15

Z

ture for 10 strains. The other 10 strains showed equal
10

activity in both culture conditions.

3. Antigenic activities of hemolysin: Although most El
Tor strains were not hemolytic in the culture conditions

5

used (HIB), Iarge amounts of hemolysin antigen wereO

Table I Hemolytic activities of the culture supernatantp̲iame}̲ rs of̲̲h̲ mol̲Y ic z9̲̲̲(̲ }Il).

8non ‑4 6‑ ‑10
10‑O1, E1 Tor ST 33 1 2 O O O(n = 36) SH 32 O 2 2 O O‑ I OO,oJnon‑Ol
Lli
LL
L

o

40

30

O

6

O

23

O

Numerals indicate number of strains (36 O1 and 63 non‑O1
strains in totaD, ST: stationary test tube culture, SH: shaking
flask culture. HIB: heart infusion broth, HIBG: HIB supplement‑

ed with 1% glycerol, (EI Tor strains were also cultured in
HIBG) . Diameter of hemolytic zone includes the size of well

(3mm)

ng/ml

50

O1, E1 Tor ST 2 2 30 O O

HIBO

1 OOO ‑

60

20

(n=63)‑ I OOO' ,,,ST 2 12 28 15
SH 2 14 14 10

non‑O1

‑ 500

lO

‑100

‑ 500

‑ I ooorooo‑ ng/ml

Figure 1 The amount of hemolysin produced in heart infu‑
sion broth as determined by reversed passive latex

agglutination (RPLA) method. Solid column indi‑cates stationary test tube culture, shaded column
indicates shaking flask culture. Non‑OI strains do
not include 0139.

154

Table 3 Hernolysin production(36 E1 Tor strains in HIBG)

Hemolysin antigen

Cultureconditions

ng/ml (RPLA)

ST

SH

min.

31.3

max.

16000

mean

1282

min.

7.8

max.

16000

mean

1849

abbreviations: same as in Table 1

4. Hemolysin production in HIBG: Since most El Tor
strains were not hemolytic in HIB culture, HIB was
supplemented with 1% glycerol (HIBG) . In HIBG, 34
strains of 36 (94%) were hemolytic by stationary test
tube culture, but 8 strains (22%) were hemolytic byshaking flask culture. A Iarger amount of hemolysin
antigen was produced than in HIB. The average amountof hemolysin produced in the stationary test tube cul‑tures was 1282 ng/ml in HIBG and 764 ng/ml in HIB,
and that in the shaking flask cultures was 1849 ng/ml in

HIBG and 1744 ng/ml in HIB (Table 2, Table 3, Figure2). However, the amount of production varied marked‑
ly from strain; ranging from 7.8 ng/ml to 16,000 ng/ml.

25

5. Inactivation test of hemolytic activity: Hemolytic activ‑"; 20

ity of non‑OI culture supernatant was markedly inhib‑
ited by mixing El Tor culture supernatant (Figure 3) .

d
15

DISCUSSIONlO

The present study revealed that the hemolysin
produced by V. cholerae is readily inactivated in the5o rooo‑ ng/ml‑ 500‑ I oo‑ roooFigure 2 The amount of hemolysin produced by E1 Tor
vibrios in heart infusion broth supplemented with

1% glycerol as determined by reversed passive
latex agglutination (RPLA) method. Solid col‑umn indicates stationary test tube culture, shadedcolumn indicates shaking flask culture.

14

S

hO

O1 El Tor. Therefore, the hemolytic activity of theculture supernatant is not correlated with the amount ofhemolysin as examined by antigenic determination.
There was no hemolytic activity in the 18 hr culture
supernatant of most El Tor vibrios, but, a large amountof hemolysin protein was antigenically detected in thenon‑hemolytic culture supernatant. Besides, when E1
Tor vibrios were cultured for 4‑6 hr, most culture
supernatants were hemolytic (Data not shown) . Thismeans that some inactivation factors appear in the
The factor(s) that
culture fluid within 18 hr.
inactivates the hemolysin of El Tor vibrios has not yet

been identified, and it is unknown why the hemolysin
produced by V. cholerae non‑OI (identical to El Tor

12'lculture supernatant, especially in the case of V. cholerae,1 O

hemolysin) maintained its hemolytic activity for 18 hr.
The inactivation is not clearly understood but there may

8

be a certain protease specific for El Tor vibrios,

64

jeo ¥('
oeF ci'vC;

Figure 3 Inactivation test of hemolytic activity. The culture
fluid of non‑OI vibrio strain (Arg 45) was incubat‑
ed with fresh HIB at 37'C for I hr (volume ratio of
2 : 1) . The diameter of hemolytic halo was reduced
by adding the culture fluid of E1 Tor vibrio strain,
Arg 57 (solid line) , or Arg 62 (broken line) .

although non‑OI produces a protease similar to E1 Tor
vibrios (Honda et al., 1987). The non‑OI V. cholerae
has stronger hemolytic activity than El Tor vibrios up
to 18 hr of culture. Nevertheless, more hemolysin was
produced by E1 Tor vibrios than by non‑OI V. cholerae.
These findings also suggest the presence of some potent
inactivation factor(s) in El Tor vibrios. The inactiva‑tion test of hemolytic activity supported this theory(Figure 3). Most El Tor vibrios cultured in heart infu‑

sion broth supplemented with 1% glycerol, became
hemolytic even in an 18 hr culture but less hemolytic

155

than non‑OI V. cholerae strains in HIB culture, and in(1993): Characterization of Vibrio cholerae 0139 syno‑

addition, El Tor vibrios cultured in HIBG produced

nym Bengal isolated from patients with cholera‑1ike

more hemolysin than those cultured in HIB. This
inactivation mechanism is characteristic of E1 Tor vi‑

brios. V. cholerae non‑OI in shaking culture gave alarger amount of hemolysin and weaker hemolytic activ‑
ity than in stationary culture, which indicated the pres‑ence of a certain factor which inactivates hemolysin tosome extent but not completely.
The antigenically determined, biologically inactive
hernolysin of E1 Tor vibrios consisted of a protein withthe molecular weight of 60kDa and some other proteins
with srnaller molecules in SDS‑PAGE (data not shown) .
The molecular weight of 60kDa is coincident with thatof mature El Tor hemolysin. E1 Tor vibrios have an
epidemic potential but non‑OI V. cholerae (excluding0139) including cholera toxin producing strains do not.
Although the factors involved in the epidemic potential
are unknown, it is attractive to speculate the relation‑

ship between the hemolysin inactivation mechanism andepidemic potential.

The optimal condition of hemolysin production variedwith the strain, but a stationary culture was preferable

to obtain high hemolytic activity and shaking culture
was preferable to obtain a large amount of hemolysin
antigen. If the inactivation mechanism could be blocked
by some techniques in̲ shaking culture, a large amountof biologically active hernolysin should be obtained.

REFERENCES
1 . Higa, N., Honma, Y., Albert, M.J., and lwanaga. M.

disease in Bangladesh. Microbiol. Immunol., 37, 971‑974
2 . Honda, T., Booth, B.A., Finkelstein, M.B., and Finkel‑
stein R.A. (1987) : Comparative study of Vibrio choler‑ae non‑OI protease and soluble hemagglutinin withthose of Vibrio cholerae O1. Infect. Immun., 55, 451‑454
3 . Ichinose, Y., Yamamoto, K., Nakasone, N., Tanabe, M.,

Takeda, T., Miwatani, T., and lwanaga, M. (1987):
Enterotoxicity of E1 Tor‑like hemolysin of non‑O1
Vibrio cholerae. Infect. Immun., 55, 1090‑1093
4 . Lowry, O.H., Roserlbrough, N.J., Farr. A.L., and Randall,

R.J. (1951): Protein measurement with the folin phenol
reagent. Biol. Chem., 193, 265‑275
5 . McCardell. B. A., Madden, J.M. , and Shah D.B. (1985):
Isolation and characterization of cytolysin produced byVibrio cholerae serogroup non‑O1. Can. J. Microbiol.,31, 711‑720
6 . Pollitzer, R. (1959): Cholera. Monograph series, N0.43,
147, Wld. Hlth. Org., Geneva.
7 . Rivas, M., Toma, C., Miliwebsky, E., Caffer, M., Galas,
M.. Varela. P., Tous, M., Bru. A.M., Binsztein, Norma.
(1993): Cholera isolates in relation to "eighth pan‑demic". Lancet, 342, 926‑927

8 . Sakazaki, R., Tamura, K., and Sebald, M. (1967):
Taxonomical studies of the so‑called NAG vibrios. Jpn.
J. Med. Sci. Biol. 20, 265‑280

9 . Weekly Epidemiological Record (1992): N0.6. Wld. Hlth.

Org. Geneva.
10. Yamamoto, K., Ichinose, Y., Nakasone, N., Tanabe, M.,

Nagahama, M., Sakurai, J., and lwanaga, M. (1986).
ldentity of hemolysin produced by Vibrio cholerae non‑O1 and V. cholerae O1, biotype El Tor. Infect. Immun.,
51, 927‑931

157

Jpn. J. Tro p. Med. Hyg., Vol. 24, No. 3, 1996, pp. 157 161

DESCRIPTION OF A NEW SPECIES OFSIMULIUM (SIMULIUM) FROM SABAH,
MALAYSIA (DIPTERA= SIMULIIDAE)
HIROYUKI TAKAOKA
Received May 15, 1996/Accepted June 14, 1996

Abstract: The fernale and male of Simulium (Simulium) beludense sp. nov. are described based on reared
adult specimens collected from Sabah, Malaysia. This new species was previously misidentified as S. (S.)
nigripilosum Edwards of the melanopus‑group because of a great similarity of their external morphology.
The female genitalia examined in this study easily separate this new species from S. (S.) nigripilosum. It
is also indicated that S. (S.) beludense sp. nov. is not assignable to the melanopus‑group, but is rather closely

related to S. (S.) argentipes Edwards from Peninsular Malaysia, S. (S.) bidentatum (Shiraki) frorn Japanand China, and S. (S.) canlaonense Delfinado from the Philippines, all of which belong to the different groupso far undetermined.
Key words: black fly, Simuliidae, Simulium, Sabah, Malaysia

Smart and Clifford (1969) gave the descriptions of
the male, pupa and mature larva, as well as the rede‑scription of the female of Simulium (Simulium) ni‑
gripilosum originally described by Edwards (1933) frorn

female and male

speclmens collected from Sabah,

Malaysia. Takaoka (1983) illustrated for the first time

the genitalia of the holotype female of S. (S.) ni‑
gripilosum loaned from British Museum (Natural His‑
tory) , London (abbreviated hereafter BMNH) and as‑
signed it to the melanopus‑group on the basis of thecharacteristic genitalia, particularly paraproct. The
male genitalia of this species were also illustrated at thesame time by Takaoka (1983) based on the reared adultspecimen (also loaned from BMNH). This was one of
the male adult specimens reared from the pupae col‑lected by Smart in 1964 and determined by Smart andthought as "S. (S.) nzgripllosum" by Smart and Clifford(1969) are regarded as a new species, and the female
and male are described below. It should be also notedthat the illustration of the male genitalia given by
Takaoka (1983) was not of S. (S.) nigripilosum but of
the new species.

Simulium (Simulium) beludense sp. nov.
Figs. 1‑13

DESCRIPTION. Female. Body length ca. 2.5 mm.
Head. Narrower than width of thorax. Frons brownish
black, shiny, with a few dark stout hairs along eachlateral margin and above antennal base; frontal ratio
1.15:1.00:1.10; frons‑head ratio 1.0:4.2. Fronto‑oculararea (Fig. 1) narrowly developed, with round lateral tip.

Clifford (1969) . While revising the melanopus‑group of
the subgenus Simulium s.str. in Southeast Asia, I foundthe pupa of S. (S. ) nigripilosum described by Smart andClypeus brownish black, shiny, thickly white pruinose,

Clifford (1969) was atypical among the melanopus

Antenna (lost in holotype) composed of 2 + 9 segments,
brown except scape and pedicel orange in one paratype‑group by having the eight‑filamented gill and cor‑largely bare medially, covered moderately with darkstout hairs only near and along lateral margins.

quent examination of the reared female specimensfrom Pegalan River, but yellow on basal 1/2 andbrown on apical 1/2 in the other paratype
from
Tuaran River. Maxillary palp brownish black, com‑bicular cocoon in place of the six‑filamented gill andsimple shoe‑shaped cocoon, respectively. The subse‑
10aned again from BMNH revealed that their genitalia

posed of 5 segments with proportional length of 3rd, 4thwere completely different from those of the holotype S.
(S.) nigripilosum illustrated by Takaoka (1983).

moderate size; sensory vesicle medium in size, elliptical,

In this paper, all the pupae, Iarvae and reared adults

with rugged surface, 0.24 x length of 3rd segment, withand 5th segments 1.0:1.0:2.5; 3rd segment (Fig. 2) ofDrvrslon of Medical Zoology, Oita Medical University, Hazama, Oita, Japan 879‑55

158

medium round opening medially. Maxillary lacinia with
11 or 12 inner and 12 or 13 outer teeth. Mandible withca. 22 inner and 10 outer teeth. Cibarium (Fig. 4) withca. 20 minute tubercles. Thorax. Scutum dark brown,
shiny, thinly white pruinose, covered moderately withfine copper‑colored pubescence, interspersed with long,
upstanding dark hairs on prescutellar area. Scutellum
dark brown, with long dark hairs and copper‑coloredpubescence. Postscutellum dark brown, shiny, whitepruinose, without hairs. Pleural membrane bare. Kate‑
pisternum longer than deep, bare. Legs. Foreleg: coxa
pale yellow, trochanter and femur brown; tibia whitish
yellow with base somewhat darkened and distal 1/5 or
1/4 brown, and with median outer portion largely white
sheeny in lights; basitarsus entirely brownish black,dilated (W:L = 1.0:4.1); rest tarsal segments brownish
black. Midleg: coxa, trochanter and femur brown; tibia

produced ventrally and ventroanteriorly, covered withca. 12 short stout hairs in lateral view; surface of antero‑ventral corner densely covered with pale setae; insidesurface largely concave anteriorly and with several
minute setae. Cercus semilunar in lateral view, covered

with numerous short hairs. Spermatheca nearly globu‑
lar, well sclerotized with no definite reticulate pattern,with minute internal setae; tube and small area of tubalbase unsclerotized.

Male. Body length 2.6 mm. Head. Slightly wider thanthorax. Upper eye consisting of large facets in 15
horizontal rows and 15 vertical columns. Clypeus black,silvery pruinose, strongly iridescent when illuminated,sparsely covered with dark hairs. Antenna composed of
2 + 9 segments, dark brown except scape, pedicel and a
few basal flagellar segments yellowish; Ist flagellomere

brown with posterior surface along basal 4/5 or a little

1.6 x as long as 2nd one. Maxillary palp composed of
5 segrnents, proportional lengths of 3rd, 4th and 5th

more white and largely white sheeny when illuminated;segments 1.0:1.2:2.1; 3rd segment (Fig. 3) of normal size,

tarsi brownish with basal 2/3 of basitarsus yellow. Hindwith small sensory vesicle. Thorax. Scutum brownish

leg: coxa, trochanter and femur brown; tibia (Fig. 5)
brown with posterior surface along basal 2/3 white, andlargely white sheeny when illuminated; tarsi (Fig. 5)

black, with silvery and bluish iridescent pattern com‑posed of an anterior pair of large spots on shoulders, alarge transverse spot on prescutellar area contiguous tobrown with basal 2/3 of basitarsus whitish yellow;

anterior spots by broad band along lateral margins;basitarsus parallel‑sided. W:L = 1.0:5.3, calcipala short,anterior pair of spots widely separated from each other

L:W ratio 1.0:1.4; pedisulcus distinct. A11 tarsal claws

in middle, curved posteromedially with posteromedial
end somewhat pointed; scutum uniformly covered withbrown pubescence, interspersed with long upright darkhairs on prescutellar area. Scutellum dark brown with
10ng upright dark hairs. Postscutellum dark brown,(Fig. 7) with small subbasal tooth. A11 femora andtibiae covered with scale‑1ike setae on outer surface.
Wing. Length 2.0 mm; costa with spinules and hairs;
subcosta haired except distal 1/3 or 1/4 bare; basal

section of vein R bare; hair tuft of stem vein darkbrown; basal cell absent. Abdomen. Basal scale brown‑

white pruinose, shiny and bare. Katepisternum andsilvery iridescent spots broadly connected to each otherpleural membrane as in. Legs. Foreleg: coxa yellow;
trochanter pale brown; femur brown to brownish black;tibia brownish black with outer surface mostly white,iridescent when illuminated; tarsi blackish; basitarsus

in middle; tergites 3, 4 and 5 small and dull, tergites 6‑8

somewhat dilated, W:L = 1.0:6.1. Midleg: coxa, tro‑

large, shiny, with dark hairs. Genitalia (Figs. 8 & 9).
Ventral surface of abdominal segment 7 without devel‑
oped sternite. Sternite 8 well sclerotized, bare medially
but with 10‑13 dark long stout hairs laterally on eachside; anterior gonapophysis tongue‑1ike in shape, mem‑chanter and femur brownish black; tibia brownish blackwith posterior surface along basal 1/3 white, iridescenton basal 1/2 when illuminated; tarsi brownish black withbasal 1/2 or a little more of basitarsus and basal 1/2 ofbraneous, bent ventrally, covered with 9‑11 somewhat
long hairs as well as numerous microsetae except nar‑row portion along inner margin bare; inner borders
concave, widely separated in middle from each other.
Genital fork of inverted‑Y form, with well sclerotized

except basal 1/2 of basitarsus yellow; tibia white irides‑

ish black with fringe of pale hairs; all segments brown‑
ish black; tergite 2 with a pair of large, dorsolateral

2nd tarsal segment yellow. Hind leg: brownish black
cent at base when illuminated; basitarsus enlarged, W:
L = 1.0:4.7, and ca. 0.7 x as wide as tibia and femur(1atter two segrnents subequal in width) ; calcipala
small, pedisulcus distinct. Wing. Length 1.9 mm; otherfeatures as in except subcosta bare. Abdomen. Basal

stem; arms narrow, each with strongly sclerotized distalbulge and with or without very short projection directed
anterodorsally (if present, discernible when viewed fromscale brownish black with long dark hairs. Tergites
brownish black with dark hairs; segments 2, 4‑7 eachside) . Paraproct enlarged, somewhat longer than widewith a pair of silvery and bluish iridescent spots dor‑in ventral view; nearly as long as wide, somewhat

solaterally; those on segment 2 broadly connected in

159?tls "Llti"i '

;

C ‑ i
.1r ' :･ Ptr44':Lf':;;1 l 'a';:} ."', ; i‑{ :i iiJ"'

: ' *

.::r4
t'

: ""

*t=" .. ri:i:?
s j ':Ii :: ;; "" et:‑:* :s! *: +::i"Y:s::'1

4

2

;

1
'l

f

i}

; ; ,t: aC)
Q oOcoOoo'OOoOQ (2

/

/

/

o
) aOooOoo
'

3

e

8

/

lI ･t
't b

a

/

el ,
11

Ll IL
L
t

C.¥b'l "

11'c

ll

l･:

///[

/ l//l‑/;‑*j, : i::Ei?::

11

/

:

b

f'

a '

Ib

¥.. .. ' "': *:.,!'I J/Jl
" t'}'
l]

12

10

I

t':: f': I t i(':' : :'6 : J l: a' i' /7/" i:'c. :4='; : /: 1::.;'

: 'i:

L

'

i'

' "

d

(‑'

:;{,:'::*::;'r"'{::}' .:=̲:;i ;{ ;̲:‑ i
E S*:':' I:'‑:i' ::4x" s '‑d" 't ! i : !111
a

l

J

7‑f'

b

¥::^

. {

ll II

I l [

e/ ==' ";' =, ,',.

･･*'*1:1.f;'

5

9

Figs. 1‑13. Female and male of Simulium (Simulium) beludense sp. nov. 1, fronto‑ocular area of; 2 & 3,3rd segment of maxillary palp (a, sensory vesicle) (2,; 3, c l ) ; 4,cibarium; 5,hind tibia andtarsi; 6, c l hind basitarsus and 2nd tarsal segment; 7, claw; 8 & 9, genitalia in situ (a, 8th sternite;b, anterior gonapophyses; c, genital fork; d, paraproct; e, cercus; f, spermatheca) (8, ventral view; 9,lateral view) ; 10, c l genitalia in situ (a, coxite; b, style; c, ventral plate) (ventral view) ; 11 & 12,
ventral plate (11, Iateral view; 12, end view) ; 13, paramere (a) and median sclerite (b) (end view) .

160

middle. Genitalia (Figs. 10‑13). Coxite nearly qua‑

and with apical spine; style flattened dorsoveritrally andFig. 77 in Takaoka, 1983).
S. (S.) beludense appears to be very close in theadult to S. (S.) bidentatum (Shiraki) from Japan and
China (Takaoka, 1976) by sharing many characters, eg.,having no basal protuberance. Ventral plate Y‑shaped

characteristic genitalia of both sexes, the female

in ventral view, having median process directed ventral‑ly with ca. 10 fine setae on each side; posteroventral

unpatterned shiny scutum and tarsal claws with a small
basal tooth. The pupa and larva of this species, thoughsurface of median process with 2 marginal rows ofseveral small spines converging apically; basal arms

not available in this study, also appear to resemble thoseof S. (S.) bidentatum; according to the illustrations of S.

diverged from each other, with apical tip curved inward.

(S.) beludense (="S. (S.) nigripilosum nec Edwards")

drate, somewhat longer than wide. Style elongate, 1.6 x
as long as coxite, narrowed from basal 1/3 to apical tip,Parameres wide, each with several hooks. Median scle‑
rite wide, thin, almost transparent except basal portion
brownish.

TYPE SPECIMENS. Holotype(BMNH 1969‑492),

depinned, dissected, cleared in KOH and slide‑mounted(see Figs. 11A, 10A and 4C of Smart and Clifford, 1969) ,the pupal gill, cocoon, and larval ostgenal cleft arealmost the same as those of S. (S.) bidentatum (Taka‑oka, 1976). However S. (S.) bidentatum differs from S.
(S. ) beludense in the female by the predominant yellow‑

in Berlese; reared from pupa, collected from Riverish leg coloration and in the male by the shape of theventral plate and the abdominal iridescent spots present

Wariu, ca. 15 m in altitude, Kota Belud, Sabah, 10.III.
1964, by J. Smart. Paratypes: I c l , slide‑mounted, same

similarities of the female genitalia, tarsal claws anddata as holotype; I c?1 , pinned, other data same as

scuturn, this new species also seems to be closely related

holotype; I, pinned, reared from pupa, collected fromto S. (S.) argentipes Edwards from Peninsular Malaysia

a stream on Tenom‑Keningau Road, Pegalan Valley, ca.
180 m in alt., Sabah, 5‑7.III.1964, by J. Smart; I ,pinned, reared from pupa collected from a small tribu‑(Takaoka and Davies, 1995) and S. (S.) canlaonense
Delfinado from Negros Island of the Philippines (Ta‑kaoka, 1983) , both of which have been known only fromfemale adults. However S. (S.) beludense differs fromtary of Tuaran River, ca. 60 m in alt., Tuaran, Sabah, 2.

III.1964, by J. Smart; I c71, Pinned, reared from pupa
collected from Tuaran River, above Tamparuli, ca. 14 m

only on the segments 2, 5‑7. From the morphologicalboth species by the much darker leg coloration, and also

in alt., Tuaran. Sabah, 28.II.1964, by J. Smart. A11 typefrom the former species by the copper‑colored pubescen‑ce on the scutum and from the latter species by the muchspecimens examined in this study had been previously

produced paraproct.

labelled as "S. (S.) nigripilosum" and "Brit. Mus. 1969‑492" by Smart and Clifford (1969) .

ACKNOWLEDGEMENTS

ECOLOGICAL NOTE. According to Smart and Clifford

I am grateful to Dr. A. Shelley and Ms. C. Lowry,(1969), the pupae and larvae of this species were col‑Department of Entornology, British Museum (Naturallected on dead leaves caught among the stones in theriver which was, at place of collection, unshaded andrelatively wide and shallow.

History) , London, U.K. for their kind arrangement of
the loan of the reared adult specimens collected by Dr.
J. Srnart. This study was in part supported by the Grant‑

in‑Aid from the Japan Society for the Promotion of

DISTRIBUTION. Sabah.

Science.

ETYMOLOGY. This new species beludense was named

REFERENCES

after the locality from where the holotype specimen wascollected.

1 ) Edwards, F. W. (1933): Diptera Nematocera from

REMARKS. This new species has a great resemblance

Mount Kinabalu. J. Fed. Malay States Mus., 17, 223‑296
2 ) Smart, J. and Clifford, E. A. 1969. Simuliidae (Diptera)in external characters to S. (S.) nigripilosum, but dis‑tinctly differs from the latter species by the genitalia of

the female. The paraproct of this new species is ofnormal form and lacks a ventrointernal plate (Figs. 8d& 9d) while that of S. (S.) nigripilosum is remarkably
produced ventrally and bears a ventrointernal plate (seeof Sabah (British North Borneo). Zool. J. Linn., Soc.,
48, 9‑47

3 ) Takaoka, H. (1976) : Studies on black flies of the
Nansei Islands, Japan (Simuliidae; Diptera) II. On six

species of the ‑ subgenera Gomphostilbia Enderlein,
Morops Enderlein, Odagmia Enderlein and Gnus Rubzov,
with the description of Simulium ( Gomphostilbia) okina‑

161
wense sp. nov. Jpn. J. Sanit. Zool., 27, 385‑398

4 ) Takaoka, H. (1983): The blackflies (Diptera:
Simuliidae) of the Philippines. Japan Society for the
Promotion of Science, Tokyo, xi + 199p.

5) Takaoka, H. and Davies, D. M. (1995): The black flies(Diptera: Sirnuliidae) of West Malaysia. Kyushu Univer‑sity Press, Fukuoka, viii + 175p.

163

Jpn. J. Trop. Med. Hyg., Vol. 24, No. 3 1996 pp 163 169

A NEW SPECIES AND NEW RECORDS OF
BLACK FLIES (DIPTERA=SIMULIIDAE) FROM THAILAND
HIROYUKI TAKAOKAI and KATSUMI SAIT02
Received May 24, 1996/Accepted June 21, 1996

Abstract: Simulium (Byssodon) siripoomense sp. nov. is described based on two mature larval specimens
collected from Thailand. This species is distinguished from the other known species of this subgenus by the
pupal gill histoblast with six slender filaments. Four other simuliid species are newly recorded from thiscountry: i.e., S. (Gomphostilbia) decuplum Takaoka and Davies, S. (G. ) parahiyangum Takaoka and Sigit,

S. (Simulium) brevipar Takaoka and Davies, and S. (S.) quinquestriatum (Shiraki) . Two known species,
which had been tentatively identified as S. (S.) sakishimaense Takaoka and S. (S.) nitidithorax Puri, aretreated to be conspecific to S. (S.) fenestratum Edwards and S. (S.) tani Takaoka and Davies, respectively.
Simulium (G.) sp. C, one of the five unnamed species previously reported, is found to conform to S. (G.)

asakoae Takaoka and Davies.
Key word : black fly, Simuliidae, Simulium, Diptera, Thailand

Takaoka and Suzuki (1984) reported 19 species of
black flies (Diptera: Simuliidae) from Thailand, includ‑ing seven new species. No other simuliid species havesince been recorded from this country.
The present paper reports the result of black flycollections made in 1989 by the junior author. The

scribed as a new species on the basis of two maturelarvae, which show remarkable characters as noted
below.

DESCRIPTION. Female, male and pupa. Unknown.
Mature larva. Body length 3.3‑3.8 mm. Body pale

identified are a total of 16 simuliid species, including one

yellowish, somewhat brownish dorsally on posterior
abdominal segments. Head capsule pale yellowish withweak positive head pattern on cephalic apotome; cer‑

new and four newly recorded species. Three of the otherknown species, previously reported as Simulium ( Gom ‑vical sclerites separated and faint. Antenna longer thanstem of labral fan, with 3 segments and apical sensillum;

phostilbia) sp. C. S. (Simulium) nitidithorax Puri, and S.

(S. ) sakishimaense Takaoka, are newly identified as S.

proportional lengths of 3 segments frorn base to tip 1.0
1.2 : 1.0. Labral fan with 35 main rays. Mandible (Fig.

(G.) asakoae, S. (S.) tani and S. (S.) fenestratum,
respectively, the former two described from Peninsular
Malaysia (Takaoka and Davies, 1995) and the last from1) with comb‑teeth decreasing in size from Ist to 3rd;
mandibular serrations composed of I Iarge and I small
teeth, without supernumerary serrations. Hypostomium

Sumatra (Edwards, 1934).

(Fig. 2) with a row of 9 apical teeth; each corner tooth
prorninent; Iateral margin serrate; hypostomal bristles 4material examined consists of larvae, pupae and reared

adult specimens preserved in 70 % ethanol. HereGENUS SIMULIUM LATREILLE
SUBGENUS YSSODON ENDERLEIN

in number lying subparallel to lateral margin on eachside. Postgenal cleft (Fig. 3) deep, reaching posteriorborder of hypostomium. Thoracic cuticle bare. Histo‑
blast of pupal gill with 6 slender filaments in pairs; all

1. Simulium (Byssodon) siripoomense sp. nov.

filaments tapering toward apex and with minute tuber‑cles on surface. Abdominal cuticle almost bare except

The adult female, male and pupal specimens of thisspecies were not available. However this is here de‑sides of anal sclerite of last segment moderately covered

with colorless setae; abdominal segments 1‑4 each with

1 Division of Medical Zoology, Oita Medical University, Hazama, Oita 879‑55, Japan
2 Department of Parasitology, School of Medicine, Yokohama City University, Kanazawa‑ku, Yokohama 236, Japan

164

l

o

1

o
!

I

2

i
I
,
l
l

l

,

*'‑.*..**...i

,

t
,

,

4 4

I
l
I
I

,
,

Figs. 1‑4. Larva of Simulium (Byssodon) siripoomense sp. nov. 1, apex of mandible; 2,hypostomium; 3, head capsule (ventral view) , showing deep postgenal cleft; 4, wholebody (1ateral view) , showing protuberances on the abdomen.

6 pairs of subconical protuberances (1 dorsally, I dor‑ and S. (S.) chamlongi Takaoka and Suzuki. The water
solaterally, 2 Iaterally, I ventrolaterally and I ventral‑temperature of this stream was 18'C. The paratypely) , although those on segment I are much less distinct;
larva was collected from a slow‑flowing, unshaded

segments 5 and 6 each with 3 pairs of similar protuber‑
ances (1 dorsally, I dorsolaterally and 1 Iaterally) ;stream, 3‑4 m wide and 0.1‑0.2 m deep. The water

segments 7 and , 8 each with I or 2 pairs of similar

with S. (G.) asakoae Takaoka and Davies, S. (G.)
decuplum Takaoka and Davies, S. (G.) parahiyangum

protuberances (1 dorsally and I dorsolaterally) (Fig. 4) .

Rectal papilla compound, each of 3 Iobes with 7 or 8
finger‑like secondary lobules. Anal sclerite of usual X‑
shape, with anterior arms ca. 1/2 x as long as posteriorarms, broadly sclerotized between arms at base. Last
abdominal segment somewhat bulged laterally but with‑temperature was 24'C. This species was caught together

Takaoka and Sigit, S. (S.) nodosum Puri, S. (S.) quin‑
questriatum (Shiraki), and S. (S.) tani.

DISTRIBUTION. Thailand.

out ventral papillae. Posterior circlet with 66 rows of up

ETYMOLOGY. The species name siripoomense refers

to 14 hooklets per row.

to the place name Siripoom from where this species wascollected.

TYPE SPECIMENS. Holotype mature larva (BMNH) ,slide‑mounted, Siripoom Water Fall, near Chiang Mai,
14.VIII.1989. Paratype I mature larva, in alcohol, Ban
Tapa, near Chiang Mai, 15.VIII.1989.

REMARKS. This new species is tentatively assigned tothe subgenus Byssodon, defined by Crosskey (1969),
because of its very large postgenal cleft reaching theposterior border of the hypostornium, and subconical

ECOLOGICAL NOTES. The holotype larva of thisprominent protuberances at least ventrally on thespecies was collected from a rapidly‑flowing mountain‑

abdominal segments I to 4, as shown in Figs. 3 and 4.

ous stream which was 3‑6 m wide and 0.3‑0.5 m deep,
together with S. (Himalayum ) nl rogilvum Summers, S.

The larva of this species appears to be indistinguishable(S.) rufibasis Brunetti, S. (S.) tani Takaoka and Davies

from that of S. (B. ) maculatum (Meigen) and its vari‑eties of the meridionale‑group (Rubtsov, 1959‑1964),

165

from which it differs by the pupal gill histoblast with six

SPECIMENS EXAMlNED. I

filaments, instead of 22‑26.

Ban Tapa, 15.VIII.1989; I c l, Huai Kaeo, 15.VIII.1989

The subgenus Byssodon is widely distributed in the

Palaearctic, Nearctic and Ethiopian Regions. In the
Oriental Region only one Byssodon species, S. (B.)
languidum , was described from Sri Lanka (Davies andand 4 mature larvae,(both localities near Chinag Mai) .

DISTRIBUTION. Penmsular Malaysra and Thailand(new record) .

Gy6rk6s, 1988) , which showed several characters resem‑
bling this new species, including the deep postgenal cleft.

REMARKS. The female, pupae and mature larvae

However, S. ( . ) Ianguidum lacks protuberances on thecollected from Thailand are in accordance with theoriginal description given by Takaoka and Davies

larval abdomen, and bears three somewhat inflated
pupal gill filaments on each side.
The larva of this species resembles that of S. (S.)(1995). This species is readily separated from the othernobile de Meijere by having the deep postgenal cleft,

Gomphostilbia species from Thailand by the 10 short
pupal gill filaments per side. The description of thedorsal protuberances on the abdomen, and the pupal

male of this species will be given elsewhere on the basis

histoblast with six slender filaments, but the latter
species lacks ventral and lateral protuberances on theabdomen and has prominent spinous scales on the dorsal
surface of the abdomen. By sharing the deep postgenal
cleft and dorsal protuberances on the abdomen in thelarval stage, this species is also similar to S. (G.)
parahiyangum originally described from Java (Takaoka
and Sigit, 1992). However, S. (B. ) siripoomense differsfrom the latter by the shape and angle of the main toothof mandibular serrations, the serrate lateral margins of

the hypostomium, the pupal gill histoblast with six
filaments and the absence of the ventral papillae.

of specimens recently collected from Peninsular
Malaysia (unpublished data) .

4. Simulium ( Gomphostilbia) parahiyangum Ta‑
kaoka and Sigit, 1992

Simulium ( Gomphostilbia) parahiyangum Takaoka andSigit, 1992 : 135‑142 (female, male, pupa and maturelarva); Takaoka and Davies, 1995: 50‑51.

SPECIMENS EXAMlNED. I mature larva, Ban Tapa,
15.VIII.1989; I mature larva, Hoy Nam Kam, 15.VIII.
1989 (both localities near Chiang Mai).

SUBGENUS GOMPHOSTILBIA ENDERLElN

DISTRIBUTION. Java, Peninsular Malaysia andThailand (new record) .

(A) batoense‑group

REMARKS. The morphological characters of the
2 . Simulium ( Gomphostilbia) siamense Takaoka andSuzuki, 1984

Simulium ( Gomphostilbia) siamense Takaoka and Suzu‑ki, 1984: 14‑18 (female, male, pupa and mature larva) .

SPECIMENS EXAMlNED. 2, I pupa and 2 pupal

mature larvae collected from Thailand agree well withthe original description given by Takaoka and Sigit(1992). This species is readily distinguished from mostof other Gomphostilbia species in the pupa by the eightshort, slender filaments per side, and antennal sheathwith nine well marked ridges on the outer margin, eachcovered with several cone‑shaped tubercles, and in the(both localities near Chiang Mai) .

larva by the prominent dorsal protuberances on theabdominal segments 1‑5, several dark stout spines on
the abdominal segrnents 6‑8, and the deep postgenal

DISTRIBUTION. Thailand

cleft reaching the posterior margin of the hypostomium(Takaoka and Sigit, 1992).

exuviae, Ban Thung Pong, 16.VIII.1989; I pupa, I pupal

exuvia and I mature larva, Loaw River, 17.VIII.1989(B) ceylonicum‑group

3. Simulium ( Gomphostilbia) decuplum Takaoka andDavies. 1995

5 . Simulium(Gomphostilbia) asakoae Takaoka andSimulium ( Gomphostilbia) decuplum Takaoka and Davies,
1995: 46‑50 (female, pupa and mature larva) .

Davies, 1995

Simulium ( Gomphostilbia) asakoae Takaoka and Davies,

166

1995: 55‑60 (female, male, pupa and mature larva) ;Takaoka, Davies and Dudgeon, 1995: 192‑193.

Simulium (Gomphostilbia) sp. C Takaoka and Suzuki,
1984: 21‑22 (male).

larva) .

Simulium (Eusimulium) aureohirtum: Puri, 1933: 1‑7(female, male, pupa and mature larva); Takaoka,
1976: 170‑171; Takaoka, 1979: 382‑384 (female, male,pupa and mature larva) ; Takaoka, 1983: 15‑16.

SPECIMENS EXAMlNED. I c l, Doi Inthanon, 14.VIII.

Simulium (Nevermannia) aureohirtum: Ogata, 1956: 61

1989; I c l, Ban Tapa, 15.VIII.1989; I mature larva, Hoy‑62; Ogata, 1966: 129; Takaoka and Roberts, 1988: 194

Nam Kam, 15.VIII.1989; 6‑195; Takaoka and Davies, 1995: 87‑88.

, 6 c l, I pupa, 6 pupal

exuviae and 10 mature larvae, Ban Thung Pong, 16.VIII.
1989 (all localities near Chiang Mai).

SPECIMENS EXAMlNED. 11, 13 c, I Pupal exuvia

and 10 mature larvae, Ban Mae Kanin, near Chiang Mai,

DISTRIBUTION. Peninsular Malaysia, Hong Kong

16.VIII.1989.

and Thailand.

DISTRIBUTION. China, Hong Kong, India, Japan,
REMARKS. The male of S. (G.) sp. C reported fromThailand (Takaoka and Suzuki, 1984) has proved to be
the same as that of S. ( G. ) asakoae described from
Peninsular Malaysia (Takaoka and Davies, 1995). It
should be however noted that the present reared malespecimens slightly differ from those of the MalaysianJava, Peninsular Malaysia, Philippines. Sabah, Sri
Lanka, Sulawesi, Sumatra, Thailand and Taiwan.

SUBGENUS SIMULIUM LATREILLE

specimens by having the large eye facets in 15 horizontal(A) multistriatum‑grouprows and in 12 vertical columns (not in 13 horizontal
rows and in 10 or 11 vertical columns as in Malaysianspecimens and S. (G.) sp. O .

8. Simulium (Simulium) fenestratum Edwards, 1934
Simulium (Simulium) fenestratum Edwards, 1934: 110‑111 (female, male, pupa and mature larva)

SUBGENUS HIMALA YUM LEWIS

Simulium (Simulium) sakishimaense (nec Takaoka,
1977): Takaoka and Suzuki, 1984: 39‑40.

6. Simulium (Himalayum) nigrogilvum Summers,
SPECIMENS EXAMlNED. I

1911

1989; I

Simulium (Himalayum) nigro ilvum Summers, 1911:
586‑588 (female) ; Takaoka and Suzuki, 1984: 22‑27(female, male, pupa and mature larva) .

SPECIMENS EXAMlNED. I, Chiang Dao, 13.VIII.

and I c71. Hoy Nam Kam, 15.VIII.1989; I cfl and2 mature larvae, Huai Kaeo, 15.VIII.1989; I, Loaw

River, 17.VIII.1989; I c !, Doi Steps, 17.VIII.1989; 2 pupal

exuviae, Ban Pongyame, 16.VIII.1989 (A11 Iocalities near
Chiang Mai) .

, I c71 and 6 maturelarvae, Siripoom Water Fall, near Chiang Mai, 14.VIII.

DISTRIBUTION. Sumatra and Thailand.

1989.

REMARKS. Takaoka and Suzuki (1984), while
DISTRIBUTION. Thailand.

identifying this species as S. (S. ) sakishimaense Takao‑

ka originally described from the Ryukyu Islands,

SUBGENUS NEVERMANNIA ENDERLElN

pointed out several characters of the Thailand speci‑
mens differing from those of the specimens frorn the(A) ruficorne‑grouptype locality. The most remarkable difference wasfound in the male hind basitarsus which was mostly

Simulium aureohirtum Brunetti, 1911: 283‑288 (male) ;darkened except for the basal 1/3 sornewhat paler oryellowish in the Thailand specimens but darkened only
on the distal 1/2 in the Ryukyu specimens. The differ‑ence was also found in the larval stage: the Thailand
larvae had more secondary lobules of the rectal papilla

Edwards, 1934: 134‑137 (female, pupa and mature(10‑15 vs. 5‑8 per each lobe) . In the original description

7. Simulium (Nevermannia) aureohirtum Brunetti,
1911

167

of S. (S.) fenestratum (Edwards, 1934) the male hindbasitarsus was not observed for its coloration, although

the nurnber of secondary lobules of the larval rectal
papilla was given as 15‑20. Recent collections made in
Sumatra by the senior author have revealed that theThailand specimens are almost the same as those of S.

(S.) fenestratum from Sumatra in many characters,
including the male and larval characters mentioned
above (unpublished data) . The male of S. (S.) hirtiner ‑middle from each other (their apices not so converged
and approximating as originally illustrated) . The male
genitalia of the Thailand specimens are also the same as
illustrated by Datta (1992). The larval characters, suchas the dorsal protuberances and chisel‑shaped spines on
the abdomen present in the Thailand specimens (Takao‑
ka and Suzuki, 1984) but not mentioned in the originaldescription, have been suggested to be present also in
the Indian specimens according to Datta (1992).

vis Edwards described from Peninsular Malaysia (Ta‑(O striatum‑groupkaoka and Davies, 1995) has a hind basitarsus similar toS. (S.) sakishimaense. Simulium (S.) malayense, also

described from Peninsular Malaysia (Takaoka and10. Simulium (Simulium) chiangmaiense Takaoka

Davies, 1995), shows a darker coloration on the male

alld Suzuki. 1984hind basitarsus as in S. (S.) fenestratum according to thereared male specimens recently collected (unpublished

Simulium (Simulium) chiangmaiense Takaoka and Suzu‑

data) , but differs from the latter by having its cocoon

ki, 1984: 38‑39 (female, male and pupa) ; An, 1989: 186.

without windows anteriorly.

SPECIMENS EXAMINED. 2(B) nobile‑groupand 5 mature larvae,

Chiang Dao, 13.VIII.1989; I , Loaw River, 17.VIII.1989(both localities near Chiang Mai) .

9. Simulium (Simulium) nodosum Puri, 1933

DISTRIBUTION. China and Thailand.
Simulium (Simulium) nodosum Puri, 1933: 813‑817(female, pupa and mature larva) ; Takaoka and Suzu‑ki, 1984 : 38 (pupa and mature larva); Datta, 1988: 6(male) ; An, 1989: 187; Takaoka, Davies and Dudgeon,

11. Simulium (Simulium) nakhonense Takaoka and1995: 195.

Simulium (Simulium) nakhonense Takaoka and Suzu‑

SPECIMENS EXAMINED. 2

Suzuki, 1984

ki, 1984: 33‑37 (female, male, pupa and mature lar‑vae); Chen, 1985: 308.

, 2 c; , 4 Pupae and 3

mature larvae, Chiang Dao, 13.VIII.1989; I pupa and 3

mature larvae, Hoy Mae Aem, 14.VIII.1989; 3 mature

SPECIMENS EXAMINED. I pupal exuvia, Hoy Maelarvae, Ban Tapa, 15.VIII.1989; I , 3 c , 2 pupae, 1

Aem, 14.VIII.1989; I pupal exuvia, Water Fall, 14.VIII.
1989; I pupal exuvia, Maeklang Fall, 14.VIII.1989; I ,pupal exuvia and 2 mature larvae, Hoy Nam Kam, 15.
VIII.1989; I, I cfl and 5 mature larvae, Ban Mae
Kanin, 16.VIII.1989; 5, 9 c l and 6 mature larvae, Ban

Mae Ha, 16.VIII.1989; 2 pupae, 3 pupal exuviae and 2

Hoy Nam Kam, 15.VIII.1989; I

and 3 c l, Ban Pon‑gyame, 16.VIII.1989; 4
and 7 c7! , Mae Sa Water Fall,
Ban Nie, 16.VIII.1989 (all localities near Chiang Mai).

mature larvae, Bang Thung Pong, 16.VIII.1989; I cf! , 3

12

pupae, 2 pupal exuviae and 3 mature larvae, Ban Pon‑

VIII.1989 (both localities in Nakhon Nayok).

gyame, 16.VIII.1989; 3 and 2 c71 , Mae Sa Water Fall,
Ban Nie, 16.VIII.1989; 2
and 9 c71, Ban Sobpong, 17.
VIII.1989; I
and I mature larva, Loaw River, 17.VIII.
1989 (all localities near Chiang Mai).

and 8 (71 , Saeng, 20.VIII.1989; I c?1 , Nan Long, 20.

DISTRIBUTION. China and Thailand.
12. Simulium (Simulium) quinquestriatum (Shira‑ki, 1935)

DISTRIBUTION. China, Hong Kong, India and Thailand.
Stilboplax 5‑striatum Shiraki, 1935: 27‑33 (fernale) .

REMARKS. The reared female and male specimens of
this species were obtained for the first time fromThailand. The female characters are almost the sarne

Simulium (Simulium) quinquestriatum: Anonym, 1974:

as originally described (Puri, 1933) except the posterior‑pupa and mature larva); An, 1989: 187; Takaoka,

ly produced round robes which are well separated in theDavies and Dudgeon, 1995: 195‑196.

192; Takaoka, 1977: 205‑209 (female, male, pupa andmature larva) ; Takaoka, 1979: 396‑399 (female, male,

168

SPECIMENS EXAMlNED. I, 3 c71, I Pupa, I pupal
exuvia and 6 mature larvae, Hoy Mae Aem, 14.VIII.
1989; 2 and 3 mature larvae, Ban Tapa, 15.VIII.1989;4
and 2 c71, Mae Sa Water Fall, Ban Nie, 16.VIII.1989(all localities near Chiang Mai). I pupa and I pupal
exuvia, Nan Long, in Nakhon Nayok, 20.VIII.1989.

DISTRIBUTION. China, Hong Kong, Japan, Korea,
Thailand (new record) and Taiwan.

SPECIMENS EXAMlNED. 2 c ! , 2 pupae, 2 pupal
exuviae and I mature larva, Siripoom Water Fall, near
Chiang Mai, 14.VIII.1989.

DISTRIBUTION. China, India, Japan, Korea, Myan‑mar, Pakistan, Thailand and Taiwan.

15. Simulium (Simulium) tani Takaoka and Davies,
1995

REMARKS. The female, male, pupal and mature larval
specimens collected from Thailand morphologically

Simulium (Simulium) tani Takaoka and Davies, 1995:

agree well with the redescription

Simulium (Simulium) nitidithorax (nec Puri, 1932):
Takaoka and Suzuki, 1984: 40‑41 (mature larva) .

of this species (Takao‑ka, 1979). This species is distinguished from the otherthree Thailand species of the striatum‑group (i.e., S.

137‑141 (female, male, pupa and mature larva).

(S.) chiangmaiense. S. (S.) nakhonense and S. (S.)

SPECIMENS EXAMlNED. I pupa and 5 mature lar‑thailandicum) by the pupal gill with 10 slender filaments

vae, Siripoom Water Fall, 14.VIII.1989; 2

per side, which are all subequal in thickness, and are

exuviae and 2 mature larvae. Water Fall, 14.VIII.1989; 1, 2 c?1 , I pupa, I pupal exuvia, 10 mature larvae, Banarranged in 2 + 3 + 3 + 2 filaments.

(D) tuberosum‑group, I crl , 2 pupal

Tapa, 15.VIII.1989; 3 pupal exuviae, Huai Kaeo, 15.VIII.
1989; I (71 and 5 mature larvae, Doi Stops‑Tai, 17.VIII.
1989 (all localities near Chiang Mai).

13. Simulium (Simulium) brevipar Takaoka andDavies, 1995

DISTRIBUTION. Peninsular Malaysia and Thailand.

Simulium (Simulium) brevipar Takaoka and Davies,

REMARKS. This species was previously reported from1995: 132‑137 (female, male, pupa and mature larva) .

Thailand as S. (S.) nitidithorax Puri originally describedfrom India (Takaoka and Suzuki, 1984). This species

SPECIMENS EXAMlNED. I c71 and I mature larva

closely resembles the latter species, but differs from it

Doi Steps, near Chiang Mai, 17.VIII.1989.

by several characters, including the short‑stalked pupal
gill filaments (not sessile as in S. (S.) nitidithorax), as

DISTRIBUTION. Penmsular Malaysra and Thailand

already discussed by Takaoka and Davies (1995) .

(new record) .

(E) variegatum‑group

REMARKS. The morphological characters of the male,pupa and mature larvae collected from Thailand arealmost the same as those originally described (Takaoka
and Davies, 1995). The presence of a pit‑1ike cuticularorgan at the base of pupal gill filaments separates thisspecies from all the other simuliid species fromThailand.

14. Simulium (Simulium) rufibasis Brunetti, 1991
Simulium rufibasis Brunetti, 1911: 282‑288 (female) ;Rubtsov, 1959‑1964: 554.
Simulium (Simuliun ) rufi'basis: Puri, 1932: 899‑903

16. Simulium ( Simulium) chamlongi Takaoka and Suzu‑ki, 1984

Simulium (Simulium) chamlongi Takaoka and Suzuki,
1984: 27‑30 (female, male, pupa and mature larva) ;An, Zhang and Deng, 1990: 106.

SPECIMENS EXAMINED. 5, 7 cfl, 5 pupae, 7 pupal

exuviae and 3 mature larvae, Siripoom Water Fall, 14.
VIII.1989; 10 , 9 c71, 6 Pupae and 4 mature larvae,
Water Fall, 14.VIII.1989 (both localities near ChiangMai) .

(female, male and pupa); Takaoka, 1977: 213‑216(female, male, pupa and mature larva) ; Takaoka andSuzuki, 1984: 41‑42; An, 1989: 187‑188.

DISTRIBUTION. China and Thailand.

169

Appendix. The list of Sirnuliidae from Thailand

Genus Simulium Latreille
Subgenus Byssodon Enderlein
meridionale‑group
S. (B.) siripoomense Takaoka and Saito, sp. nov.
Subgenus Gomphostilbia Enderleinbatoense‑group
S. (G.) decuplum Takaoka and Davies, 1995
S. (G.) parahiyanagum Takaoka and Sigit, 1992
S. (G.) siamense Takaoka and Suzuki, 1984

ACKNOWLEDGEMENTS
This study was supported by the Grant‑in‑Aid from
the Ministry of Education, Science and Culture, Japanfor International Scientific Research Program titled"Studies on the medically important Diptera in South‑
east Asra" (no 63041058 representatrve Dr. K. Kami‑mura). We thank Ms C. Aoki, Division of Medical
Zoology, Oita Medical University, Oita for her help in
preparing the manuscript.

ceylonicum ‑grou p

S. (G.) asakoae Takaoka and Davies, 1995
C= S. (G.) sp. C Takaoka and Suzuki, 1984
S. (G.) inthanonense Takaoka and Suzuki, 1984

Subgenus Himalayum Lewis
S. (H.) nigrogilvum Summers, 1911
Subgenus Nevermannia Enderlein
feuerborni‑group
S. (N.) sp. B Takaoka and Suzuki, 1984
ruficorne‑group
S. (N.) aureohirtum Brunetti, 1911

vernum ‑group
S. (N.) sp. A Takaoka and Suzuki, 1984
Subgenus Simulium Latreillegriselfrons ‑group

S. (S.) digrammicum Edwards, 1928

REFERENCES
1) Crosskey, R. W. (1969): A re‑classification of the
Simuliidae (Diptera) of Africa and its islands. Bull. Br.
Mus. (Nat. Hist.) Entomol. Suppl., 14, 195pp.
2) Datta, M. (1988) : Simulium (S.) asishi spec. nov., a new
blackfly from the Terai of West Bengal, India (Diptera:
Sirnuliidae) . Opusc. Zool. Flumin., 31, 1‑83) Davies, D. M. and Gydrkos, H. (1988): The Simuliidae(Diptera) of Sri Lanka. Description of a new species ofSimulium (Byssodon) . Can. J. Zool., 66, 2150‑2154

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) Puri. I. M. (1933) : Studies on Indian Simuliidae. Part
VII. Descriptions of larva, pupa and female of Simulium

multistriatum ‑group
S. (S.) fenestratum Edwards, 1934

C = S. (S.) sakishimaense: Takaoka and Suzuki,
1984 (nec Takaoka, 1977))
nobil e‑grou p

S. (S.) nodosum Puri, 1933
striatum ‑grou p

S. (S.) chiangmaiense Takaoka and Suzuki, 1984
S. (S.) nakhonense Takaoka and Suzuki, 1984

nodosum sp. nov., with an appendix dealing with S.
novolineatum nov. nom. ( = S. Iineatum Puri). Indian J.

Med. Res., 20, 813‑817
6) Rubtsov, I. A. (1959‑1964) : Simuliidae (Melusinidae) .
In: Lindner, E., ed., Die Fliegen Palaearkt. Reg., 14, 1‑6897) Takaoka, H. (1979) : The black flies of Taiwan (Diptera:
Simuliidae) . Pac. Insects, 20, 365‑403
8) Takaoka, H. and Davies, D. M. (1995): The black flies(Diptera: Simuliidae) of West Malaysia. Kyushu Uni‑

S. (S.) quinquestriatum (Shiraki, 1935)

S. (S.) thailandicum Takaoka and Suzuki, 1984
tuberosum ‑grou p

versity Press, Fukuoka, viii + 175pp.

9) Takaoka. H. and Sigit, S. H. (1992): A new blackfly
species of Simulium ( Gomphostilbia) from Java, In‑
donesia (Diptera: Simuliidae) . Jpn.
Hyg., 20, 135‑142

S. (S.') brevipar Takaoka and Davies, 1995
S. (S.) rufibasis Brunetti, 1911

S. (S.) iani Takaoka and Davies, 1995
C = S. (S.) nitidithorax: Takaoka and Suzuki,
1984 (nec Puri, 1932)]

S. (S.) sp. D Takaoka and Suzuki, 1984
S. (S.) sp. E Takaoka and Suzuki, 1984
variegatum ‑group
S. (S.) barnesi Takaoka and Suzuki, 1984
S. (S.) chamlongi Takaoka and Suzuki, 1984

10)J

Trop. Med.

Takaoka, H. and Suzuki, H. (1984):

The blackf lies

(Diptera: Simuliidae) from Thailand.

J pn.

Zool., 35, 7‑45

J. Sanit.

171

Jpn. J. Trop. Med. Hyg., Vol. 24, No. 3, 1996, pp. 171 175

CASES OF OVALE MALARIA AND MALARIAE
MALARIA SUCCESSFULLY TREATED
WITH MEFLOQUlNE.
MIKIO KIMURAl, MANABU TANABEl, YOSHIHISA OGATA1,

AND YUSUKE WATAYA2
Received March 4, 1996/Accepted June 3, 1996

Abstract: Two Japanese with ovale malaria and a Cameroonian with malariae malaria were put on
mefloquine and were successfully treated as long as they were observed. Two of the 3 cases entered into
this study because the possibility of falciparum malaria was not excluded by the physicians who firstexamined their blood specimens. These case reports offer a promising alternative to chloroquine in thetreatment of ovale and malariae malaria.

CASE DESCRIPTIONINTRODUCTION
The antimalarial drug mefloquine, which was first

approved and sold in Switzerland in 1985, has been

Patient I was a 30‑years‑old Japanese male whostayed in Equatorial Guinea from June 25 to July 24,

widely distributed and has become a first‑line drug for1991. His body weight was 88kg. During and for 4

the treatment of uncomplicated falciparum malaria(Palmer et al.. 1993). Although the resistance ofPlasmodium falciparum to mefloquine is emerging and isposing therapeutic difficulty to a greater extent in the

weeks after the stay, he was on a weakly chloroquine asprophylaxis. On April 19, 1992, he began to have feverreaching up to 40'C and lasting for a few days. On April
22, he visited our hospital and the blood revealed toThai‑Cambodian and Thai‑Burmese borders, mostcontain a small number of malaria parasites. Because

cases acquired in other areas of the world can be cured
with this drug (Kimura, 1996). In addition to its excel‑

the species of malaria parasites could not be identified

by the first physician, 750 mg of mefloquine base
(8.5mglkg) was administered on April 22, considering

lent clinical efficacy in falciparum malaria, it wasproved to be also effective in vivax malaria (Dixon et

the possibility of falciparum malaria. On April 24, hisal.. 1985) , sometimes even with a smaller dosage

blood had turned negative for malaria parasites and(Harinasuta et al., 1985). Cases of chloroquine‑resis‑

since April 25, his body temperature has been under 37'C

tant vivax malaria have been reported in some areas(Rieckmann et al., 1989; Schwartz et al.. 1991), and(Figure D .

mefloquine could be a drug of choice in such settings.
To date, the antimalarial efficacy of mefloquine on
ovale or malariae malaria has never been studied exten‑
sively (Palmer et al.. 1993), because so far both of thetwo types of malaria can be successfully cured by chlor‑oquine. Recently, we experienced 2 cases of ovale
malaria and one case of malariae malaria who weretreated successfully with mefloquine. Herein we
describe the 3 cases and will discuss about the possible

use of mefloquine in the two kinds of malaria.
1

2

After the start of therapy, the blood smear was
examined by a more skilled microscopist. Some of theinfected eryihrocytes were oval‑shaped with fimbria‑tion, contained coarse Schtiffner'dots and the parasites

themselves were rather small and less amoeboid
compared to those seen in vivax malaria. These fea‑tures are typical of P. ovale. The diagnosis of ovale
malaria was further substantiated by a species‑specific

PCR assay which had been developed and extensively
used by our group (data not shown, Kimura et al.. 1995;
Arai et al.. 1996) . Unfortunately, the blood smears were

Department of Infectious Diseases & Applied Immunology, Institute of Medical Science, University of Tokyo, 4‑6‑1, Shiro‑
kanedai, Minato‑ku, Tokyo 108, Japan
Department of Medicinal Information, Faculty of Pharmaceutical Sciences, Okayama University, 1‑1‑1, Tsushima‑naka,

Okayama 700, Japan

172

Hb

1 6.7

1 6.9

1 6.7

1 6.0

.1 6,4

1 5.8

WBC

2,700

3,700

4,300

5,700

6;200

6,200

4.6

5. 1

4.0
71

1 5.4

1 9,1

24.4

82

75

82

85

2,9

s.3

Plt(XI 04)

PT(V.)FDP> I ,ooo

1 0.2

1 1 .7

LDH

622

527

<2.5

49 1

422

Parasites

+

+

40

u 39

e
,e

o

336

o

o

o o

LQ,

cc 38
La'

CL

E' 37
o'
'

*>

100 36co35

Apr ZZ 23 24 25 Da26y 27 28 29 30 May 1
Figure 1 Clinical course of Patient I with ovale malaria. A total of 750rng mefloquinebase ( o = 250mg of mefloquine base) was administered followed by a daily
dose of 15mg primaquine base ( o ) for 14 days. Values of Hb are g/dl, thoseof WBC, Plt ( x 10') and malaria parasites are per pl blood, and those of FDP

and LDH are pg/ml and IU/ml, respectively.

10st and the numbers of parasites were not counted.
From April 27, a daily dose of 15mg primaquine base
was administered for the following 14 days as a curative
therapy. He experienced no recurrence, and no signifi‑

cant subjective or objective adverse reactions werenoted during the entire observation period.

Patient 2 was a 46‑year‑old Japanese male whostayed in Mali from October 20, 1994, to January 17,

1995. His body weight was 65 kg. On Novernber 17,
1994, malaria was diagnosed although he was not
informed about the causative species, and was given
intravenous quinine for 2 days. For nearly one year

78kg. On April 14, he started to feel feverish although he

did not measure the body temperature. On April 20, his
fever was up to 38.5'C and he sought medical attention
at our hospital. Malaria parasites were detected micro‑
scopically by a physician who, however, could not iden‑
tify their species. On that and the following day, hereceived a total dose of 1,500mg mefloquine base(19.2mglkg) divided into two doses 8 hours apart(Figure 3). On April 22 he was no more febrile and on
April 26 he had no detectable parasitemia. During thewhole observation period until September 1993 when heleft for home, he experienced no recurrence, and noadverse effect to mefloquine was observed.

after returning to Japan, he was free of any symptoms.
On December 30, 1995, January I and 3, 1996, he devel‑
oped high fever with shaking chills. On January 5, hevisited our hospital and was diagnosed as ovale malaria

The first blood smear was submitted to a moreskilled microscopist who identified it as malariae
malaria since band‑form trophozoites were evident(Photo la, 124 parasites/pD. This was later sub‑inside the smaller than normal erythrocytes (Photo lb) .

stantiated by the results of the PCR assay (data not

This was also confirmed by the species‑specific PCR

shown) .

assay (data not shown) .

On the same and the following day, a total ofl,OOOmg of mefloquine base (15.4mglkg) was adminis‑
tered (Figure 2). Fever and the parasites were cleared
6 days and 5 days, respectively, after the treatment.
After this successful mefloquine therapy, 15mg daily

DISCUSSION
Most of malaria caused by species other than P.
falciparum had been successfully treated by chloroquine.

primaquine base was given for 14 days. No adverse

Recently, however, there appeared reports on vivax

reactions were noted.
Patient 3 was a 29‑year‑old Cameroonian male who
left his country on March 21, 1993. His body weight wasmalaria that were not cured by the standard course ofchloroquine, first from Papua New Guinea (Rieckmann
et al., 1989) and then from Indonesia (Schwartz et al.,

173

Hb

1 5.6

WBC

1 3.8

1 4.2

1 4.0

1 5.1 1 5.4

1 4.2

1 3.3

4,540
21 .9

6,220

4,600

4,400

4,650

4,980 5,s50

6,620

Plt(Xt 04)

7.7

4.7

9.2

1 1 .o 1 1 .8

1 7.3

PT(%)

89

9.6
91

70

1 oo

LDH

590

Parasites

1 24

41

89
662

506
851

1 76

ee
ee

U 40

' '

2
::

39

'as

La,

38

o.

E
(!)

'

>1
10

o

Go

37
36
35

Jan. 5 6 7 1 O 1 1 1 Z
8

9

Da y

Figure 2 Clinical course of Patient 2 with ovale malaria. A total of 1,000mg meflo‑
quine base ( e = 250mg of mefloquine base) was adrninistered. Values of Hb
are g/dl, those of WBC, Plt ( x 10') and malaria parasites are per pl bloodand those of LDH are IU/ml.

Hb

1 1 .9 1 2.7

1 1 .91 1 .8WBC4,600 4,320

3,600

2,860

6.0 7.0482 5 1 2
3,358 1,469

7.9
44 8

1 1 .2LD H

Plt(XI 04)

Parasites

446
86

252

39
o*
c I

38

*Q,

CLE 37

o 36
*:h
1:'

o 35
co

Apr 20 21 22 23 z4 25 26
Da yFigure 3 Clinical course of Patient 3 with malariae malaria. A total of 1,500rng
mefloquine base ( e = 250mg of mefloquine base) was administered. Valuesof Hb are g/dl, those of WBC, Plt ( x 10') and malaria parasites are per plblood and those of LDH are IU/ml.

199D. Because the blood concentrations of the drug

as mefloquine against ovale and malariae malaria are

were high enough to eradicate the usual P. vivax, theselimited (Danis et al., 1982; Dixon et al., 1983; Coulaud etparasites are considered to be resistant. Thus, theal.. 1983). Thus, 3 (Coulaud et al., 1983) and 4 (Danissecond‑1ine antimalarials for the chloroquine‑resistantvivax malaria are of particular importance. In a pre‑marketing study performed in Thailand, mefloquine wasshown to be effective against vivax malaria (Dixon etal., 1985) even sornetimes with a smaller dosage (Har‑et al.. 1982) cases of ovale malaria were reportedly
treated with 25mg/kg and 1,000mg, respectively, of

inasuta et al.. 1985).

malariae malaria was treated with 1,500mg of meflo‑
quine base with the fever clearance of 4 days and theStudies on the efficacy of other antimalarials suchmefloquine base. A11 those reported cases were cured interms of the acute febrile episodes caused by intraeryth‑rocytic asexual parasites. In one report, one case of

174

: '>'

*""'*;{{i i{*<*i lil: *+* *+;;

S"' "' :'"'

f, :; =;:j:: '==: **=Photo la Plasmodium ovale seen in the blood of Patient

Photo lb

2. The infected erythrocyte is ovalshaped';'" = " :'/ //'(/';;=/;t'/'; ' SS i'

plasmodium malar:iae seen in the blood ofpatient 3' The infected erythracyte is snall‑
Sized without stiPPlings and contains a band
form trophozoite'

with fimbriati.on and coarse Schiiffner's stip‑

plings, and the parasite itself is smaller andless amoeboid than Plasmodium vivax.

parasite clearance of 7 days (Dixon et al.. 1983).

10ng‑term observation of the treated patients.

Here we added 3 cases to the literature, 2 with ovale

and one with malariae malaria. The former two wereACKNOWLEDGMENT

treated with low doses, 750mg and 1,000ng, respectively,of mefloquine base, followed by the standard course of
primaquine, and the latter with 1,500mg, a high dose. In
all cases, fever dissolved in several days. In one of thecases of ovale malaria (Patient 1) , the parasites were no
more detectable 2 days after the start of therapy. Thisis consistent with a low number of malaria parasites
before the start of therapy aithough not counted accu‑
rately. In the other case of ovale malaria (Patient 2),

fever and parasites were cleared 6 days and 5 days,

We are 1'ndebted to Ms. C. Oribe f or reviewing themanuscri pt.

REFERENCESl ) Arai. M.. Kunisada, K., Kim, H.‑S., Miyake, H., MizukoLshi, C., Kakutani, T., Yamane, A., Nakagami, S., Kawai,S., Nakano, H., Kawamoto, F. and Wataya, Y. (1996) : A

colorimetric DNA diagnostic method for falciparum

after the start of therapy. Patient I was followed up for

malaria and vivax malaria: a field trial in the Solomon
lslands, Nucleosides Nucleotides, 15, 338‑351
2 ) Coulaud, J.P., Felix, F., Penalba, C., Thimossat, P., Le
Bras, J. and Saimot. A.C.. (1983) : Traitement du paludis‑

3 years, and Patient 3 for 5 months until he left forme d'importation par la mefloquine, Ann. Soc. Belge

respectiveiy, after the start of therapy. In the malariae

malaria (Patient 3) microscopy turned negative 6 days

Cameroon. No recurrence and no adverse reactions

M6d. Trop., 63, 247‑257

were observed in all 3 cases during the entire observa‑

3 ) Danis, M., Felix, H.. Brucker, G., Druilhe, P., Datry, A.,tion periods. The clinical courses of the patients did not

Richard‑Lenoble, D. and Gentili･ni, M. (1982): Traite‑
ment curatif du paludisme a Plr ;smodium f'alciparum. P.
vivax et P, o.vale par la mefloquine, M6d. Trop., 42, 427=4324 ) Dixon, K.E., Pitaktong, U., Bamnetpandh, S., Teopipith‑seem to be different from those who were treated withchloroquine. Although more cases should be studied,mefloquine could be a promising substitute for chloro‑
quine in the treatment of ovale and maiariae malaria
especially when the latter drug is no longer effective dueto th, e emergence of drug resistance in future. Recrudes‑

cence of malariae malaria more than several years after

chloroquine treatment sometimes poses a diagnostic
problem, and is not attributed to the true resistance ofthe strain. Thus, it is worthwhile to determine whether

the recrudescence could be prevented by mefloquine
which has a long halflife, although the study requires

aporn, S, and Na‑Nakorn, A. (1983); Treatment of anacute case of Plasmod･ium ma:1･ariae malaria with meflo‑
quine, Am. J. Trop. Med. Hyg., 32, 631‑632
5 ) Dixon, K.E., Pitaktong, U. and Phintuyothin, P. (1985)i
A clinical trial of mefloquine in the treatment of Plas ‑modium vivax malaria, Am. J. Trop. Med. Hy*"., 34, 435
4376 ) Harinasuta, T.. Bunnag, D., Lasserre, R., Leimer, R, andVinijanont, S. (1985) : Trials of mefloquine in vivax ahd

175

Species‑specific PCR detection of malaria parasites by9 ) Palmer, K.J., Holliday, S.M. and Brogden, R.N. (1993):
Mefloquine. A review of its antimalarial activity, phar‑macokinetic properties and therapeutic efficacy, Drugs,
45, 430‑475
10) Rieckmann, K.H., Davis, D.R. and Hutton, D.C. (1989):
Plasmodium vivax resistance to chloroquine?, Lancet, ii,
1183‑1184
11) Schwartz, I.K., Lackritz, E.M. and Patchen, L.C. (1991):

microtiter plate hybridization: clinical study with

Chloroquine‑resistant Plasmodium vivax from In‑

malaria patients, J. Clin. Microbiol., 33, 2342‑2346

donesia, N. Engl. J. Med., 324, 927

of mefloquine plus <Fansidar' in falciparum malaria,
Lancet, i, 885‑888
7 ) Kimura, M. (1996): Current status of antimalarial ther‑

apy with mefloquine (in Japanese with an English
abstract) , Jpn. J. Trop. Med. Hyg., 24, 27‑33

8 ) Kimura, M., Miyake, H., Kim, H.‑S., Tanabe, M., Arai,

M.. Kawai. S., Yamane, A. and Wataya. Y. (1995):

Jpn. J. Tro p. Med. H yg

177

Vol. 24, No. 3, 1996, pp. 177 180

Clinical "*,"''A CASE REPORT OF AN OVERSEAS‑TRAVELER'SDIARRHEA PROBABLY CAUSED BY

CHILOMASTIX MESNILI INFECTION

NoRIHITO MORIMOTol, MASATAKA KORENAGA2*, CHIZU KOMATSUl, SIGEYOSHI SUGIHARA ,MASAAKI NISHIDA*, MARI YASUOKA3, HIDEO KUMAZAWA2MASAHIDE SASAKll'4 AND YOSHIHISA HASHIGUCH12
Received May 17, 1996/Accepted August 1, 1996

Abstract: Many active flagellates were detected in stool samples of a 22‑year‑old Japanese male who
traveled China and India. Microscopic observation showed that the organisms stained with Wright's solution

were pyriform or rounded in shape, measuring 11.7‑17.5 pm in length and 7.9‑f2.2 pm in width. The
posterior end was pointed. Three free anterior flagella were located in the front end. A spherical nucleus
was situated near the anterior pole. Based on these morphological features we identified the organisms as
trophozoites of Chilomastix mesnili, a common intestinal protozoon. Although we examined viral, bacterial
and parasitic infections other than C.mesnili, no pathogen was found. From these results obtained, it wasconsidered to be highly probable that C. mesnili infection was the cause of the diarrhea.

INTRODUCTION
Reflecting recent advancement in international
interchange and traveling abroad, imported cases with

infectious diseases have been increasing in Japan.
Annually most of these cases are bacterial diarrhea(Yoh and Honda, 1995). It has been pointed out thatJapanese inhabitants in tropical countries have a highrisk of infection with intestinal parasites, because ofcontamination of drinking water or food (Tsukidate etstay in India, he had fever on August 28th, followed bydiarrhea and nausea. On September Ist, he returned tohis home town, Kochi, and visited a physician because ofcontinuing diarrhea. Immediately he was transferred to
the Kochi General Hospital of Agricultural Cooperation.

The symptom of the patient was diarrhea and nausea.
The body temperature was 37.2'C at the time of admis‑
sion.

Laboratory data are shown in Table 1. C‑reactive

al., 1985). Chilomastix mesnili is a species of flagellate

protein (CRP) was 10.5 mg/dl. Other data of blood
chemistry were not remarkable except for a little

protozoa of the digestive tracts and is basically thought

increase of LDH and uric acid. The urine examination

to be a harmless commensal. However there is a reportsuggesting pathogenicity of C.mesnili (Mueller, 1959).
The prevalence of C. mesnili infection ranges from lessthan 1% to 10% or more in the area of warm climates

showed a little increment in acetone bodies. The periph‑(Beaver et al., 1984). In this paper, we report a case ofa patient with diarrhea possibly caused by C.mesnili.

Tokyo, Japan) . Results of fecal examination for bacte‑
ria and fungi are shown in Table 2. Feces appeared like

CASE REPORTThe patient was a 22 year‑old, male student living
in Kochi Prefecture, Japan. He traveled China and India
from the end of July to September Ist, 1995. During theeral blood picture was normal. We could not detect
antibody against Entamoeba histolytica using a hemagg‑

lutination test (Japan Lyophilization Laboratory,

mud with mucus. Escherichia coli and Citrobacter
amalonaticus, indigenous intestinal bacteria, weredetected by fecal culture. Diarrheagenic E.coli was notdetected. Serum antibodies against various viruses were

examined by neutralization tests (NT) or complement
fixation tests (CF) (Table 3). Antibodies against influ‑

1 Department of Clinical Laboratory, Kochi Medical School, Nankoku, Kochi 783, Japan
2 Department of Parasitology, Kochi Medical School (*corresponding author)
3 Department of Medicine, Kochi General Hospital of Agricultural Cooperation, Nankoku, Kochi 783, Japan
4 Department of Clinical Laboratory Medicine, Kochi Medical School

178

Table 1

Laboratory data on September Ist, 1995

1 ) Blood chemistryTP3 ) Peripheral blood cell counts
7.8g/dl

507 x 10'/uunf

1.69

Ht

46.1 %

Glu

95mg/dl

ALP

Hb

15.5g/dl

1581U/1

Plt

18.6 x 10'/num'

T‑Cho116mg/dly‑GTP

16lU/1WBC

7200/mm*

2.0%
62.0%
0.0%
0.0%
22.0%
12.0%
2.0%

4 ) Peripheral blood picture

T‑Bil

0.5mg/dl

Band

ChE

248mg/dl

AlbGlb

4.9g/dl

Seg
Eos
Baso

GPT
GOT

24lU/126lU/l431lU/1
90lU/l

2.9g/dl

LD H

CPK

BUN

Crn

UA

Amy

lgM

Aty‑Lymp
5 ) Serological test

20mg/dl

CRP6 ) Urine analysis

1260mg/dl
310mg/dl
13lmg/dl

lgG
lgA

Lymph
Mono

0.8mg/dl
8.6mg/dl
89lU/l2 ) Immunoglobulins

Table 2 Examination of bacteria and fungi of the fecal

10.5mg/dl

20mg/dl

Pro
Glu
Ket

Orng/dl

10mg/dl( )Occult7 ) HA test

E. histolytica antibodies ( ‑ )Table 3 Serological examinations of antibodies
against viruses

sample on September Ist, 1995
Bacteria and fungi

RBC

A/G

Antibody titers t

Culture media* Results
Viruses

Culture of feces
E. coli

C. amalonaticus
P. shigelloides

A
AA

Salmonella spp.

B, C

Shl ella spp.

B, C

Viblio cholerae

D, E

Viblio parahaemolyticus

D, ECampylobacter

F

Fungi

G

(+)
(+)
(‑)
(‑)
(‑)
(‑)
(‑)
(‑)
(‑)

Influenza B virus

Rotavirus
Enterovirus 72

Adenovirus l
Adenovirus 2

Adenovirus 3
Adenovirus 4
Adenovirus 7

Sept. 7 $

CF
CF
CF

8

8

<4
<4

<4
<4

NT
NT
NT
NT
NT
NT

32

32

64

64

64

64

4

4

<4
<4

4

<4

* CF: complement fixation tests; NT: neutralization test

Diarrheagenic E. coli tEnteropathogenic E. coli (EPEO
Enteroinvasive E. coli (EIEO
Enterotoxigenic E. coli (ETEOEnterohemorrhagic E. coli (EHEO

Influenza A virus

Methods *Sept. I t

(‑)
(‑)
(‑)
(‑)

* A: BTB agar; B: SSB agar; C: SS agar; D: TCBS agar; E:
Viblio agar; F: Skirrow zigar; G: Sabouraud agar.
t Agglutination test by serum groups.

enza A virus, enterovirus 72, adenovirus 1, adenovirus 2,

adenovirus 3, and adenovirus 4 were detected.
Parasitological examination was made by using fecal

4 or more is positivet Admission (Sept. 1), Discharge (Sept. 7)

samples. In a direct smear specimen of feces many
active and motile flagellates were observed under amicroscope (Fig. 1). When a fecal sample was diluted
tenfold with saline, the number of the organisms wasapproximately 60 per high power field (400 X ) . The
spiral form was recognized in organisms (Fig. 2). The
morphology of the flagellates stained with Wright's
solution was pyriform and rounded in shape, measuring
11.7 to 17.5 pm long and 7.9 to 12.2 pm wide. Three free179

anterior flagella were equal in length and located at thefront end. The posterior end was pointed. A spherical
nucleus, measuring 2.7 to 4.2 ;/m, was present near theanterior pole. A well‑difined cytostome was seen on one
side of the nucleus. The cytoplasm contained numerous
food vacuoles. But this flagellates had no undulating
membrane and axostyle. Based on these morphologicalfeatures, we identified the organ, isms (Fig. 3) as troph‑ozoites of Chilomastix mesnili (Wenyon, 1910).

Fecal samples were also examined by formalin‑ether
sedimentati,on (MGL method) , or were cultured in
Tanabe=Chiba medium. Neither protozoa nor helminths
Figure 1 Motile trophozoites with three flagella of
Chilomastix mespeili (arrows) were observed on a
direct smear of fecal specimen from the patient(400 X ) .

except C. mesnili were detected by these trials.

The patient was treated with 750 mg/day of
metronidazole and 2 g/day of cefmetazole sodium, and 4
g/day of anti‑diarrhoics from September Ist (Fig. 4) .
The parasites were not detected in fecal samples after
4th day of the treatment. No cysts of the parasite wereiound throughout the clinical course.

DISCUSSION
Chilomastix mespzili has a cosmopolitan distribution.

The infection occurs by swallowing the cysts of theparasite (Kreier,1978). C.mesleili infection is known to
be endemic in many tropical areas where sanitation andpersonal hygiene are bad (Clarke et al., 1974; Chacin‑
Bonilla et al.,1993) . It has been thought generally that C.

mesrdli is a normal inhabitant of the cecal region of thelarge intestine, where the trophozoites live on entericFigure 2 Spiral groove of C. mespeili was observed on a
direct smear specimen (lOOO X ) .

bacteria in the lumen of the glands, and that C.mesn'ili isa harmless commensal and is not responsible for symp‑
toms (Beaver et aJ., 1984) . However, infections with C.

mesp2ili have been observed in diarrhea of children

D ate 1 2

67(+) (‑) (‑) (‑)

(*)

3

4

5

Sept 1995

N'

Metronidazole 750 mg/day
Cefinetazole Sodium 2.0 g/day

Treatment

Antidiarrhoics 4.0 g/day

C.mesnili (+)
trophozoitc
3g

S
Figure 3 A specimen of C.mes;eili trophozoite stainedwith Wright's solution. Various organelles suchas flagella (AF1) , cytostome (CT), nucleus (N)and food vacuoles (FV) are observed (lOO0><).

37

BT('C)
36

nodefecation

Diarrhea

Property of feee

mud with mucus soft

Figute 4 Clinical courSe of the patient.

180(Cerva and Vetrovsk , 1958), homosexual patients(Peters et al., 1986), HIV (human immunodeficiency

Chapter 9: Flagellate protozoa of the digestive andurogenital tracts. In: Clinical Parasitology (9th edition) .

virus) positive populations (Mendez et al., 1994) and avisitor from the United States who stayed in a develop‑

ing country (Mueller, 1959). This suggests that C.
mesnili might be pathogenic to immunocompromised, orless resistant hosts.

In the present case, pathogenic intestinal bacteria,
helminths and protozoa were not detected except for C.
mesnili. Serum antibodies against viruses were detectedby complement fixation tests and neutralization tests.
A slight increase of titers was seen against adenovirus 4during hospitalization. We can not exclude the possibil‑

2 )pp.42‑43. Lea & Febiger, Philadelphia
erva, L. and Vetrovsk , G. (1958): Towards the ques‑tion of pathogenic character of the flagellate Chilomas‑
tix mesnili. eskoslov. Epidemiol. Mikrobiol. Immunol.,7, 126‑1353 ) Chacin‑Bonilla. L., Mejia. M. Y., Cano, G., Guanipa, N.,
Estevez J. and Bonilla, E. (1993) : CIOiptosporidium infec‑

tions in a suburban community in Maracaibo, Ven‑ezuela. Arn. J. Trop. Med. Hyg., 49, 63‑67
4 ) Clarke, M. D., Carney, W. P.. Cross, J.H., Hadidjaja, P.,
Oemijati, S. and Joesoef, A. (1974): Schistosomiasis andother hurnan parasitoses of Lake Lindu in Central Sul‑awesi (Celebes) , Indonesia. Am. J. Trop. Med. Hyg., 23,

ity that diarrhea in this case was caused by the viral
infection. However, it should be noticed that the scoreof titration was the lowest level at the recovery stage

385‑3925 ) Kreier, J. P. and Nohynkov , E. (1978): Flagellates of(Table 3). No cysts were found in fecal samples

Parasitic Protozoa, vol. 2, pp. 1‑138. Academic Press,

throughout the present observation. It is possible thatthe human intestine and of intestines of other species. In:

New York.

the metronidazole acted effective by on the troph‑6 ) Mendez O. C., Szmulewicz. G., Menghi. C., Torres. S.,

ozoites. In the literature, a similar case report was mede

Gonzalez, G. and Gatta, C. (1994): Comparacion deby Mueller (1959).

indices de infestaciQnes por enteroparasitos entre pob‑

In conclusion, we reported that the C. mesnili infec‑

tion accompanied the severe diarrhea in the Japanesepatient who possibly did not have immunity to the intes‑

tinal protozoa. When the international interchange of
personnel and the imported cases of diarrhea have been
increasing, C. mesnili should be considered as one of thepossible pathogens for so‑called traveler's diarrhea.

ACKNOWLEDGMENTS
We thank Dr. Takuro Endo, National Institute of
Health, Japan for his valuable comments. Thanks arealso due to Mrs. Imamura, Department of Parasitology

laciones HIV positivas y negativas. Medicina (Buenos
Aires) 54, 307‑310
7 ) Mueller, J. F. (1959) : Is Chilomastix a pathogen? J.
Parasitol., 45, 170

8 ) Peters, C. S., Sable, R. Janda, W. M., Chittom, A. L. andKocka, F. E. (1986): Prevalence of enteric parasites inhomosexual patients attending an outpatient clinic. J.
Clin. Microbiol., 24, 684‑685

9 ) Tsukidate, S., Fujita, K., Kurokawa, K., Nakanishi,H.,
Horii, Y., Ogasawara, Y. and Asakura. T. (1985): Intes‑
tinal protozoan and parasitic infections of Japanese
inhabitants in developing countries. Nettai, 18, 159‑167(in Japanese) .

Kochi Medical School, Kochi, Japan, for her supports.

10) Wenyon, C, M. (1910) : A new flagellate (Macrostoma
mesnili n. sp.) from the human intestine with some
remarks on the supposed cysts of Trichomonas. Par‑REFERENCES

11) Yoh, M. and Honda. T. (1995): Present status of bacte‑rial diarrhea diseases. Igakuno ayumi, 175, 839‑843 (inasitology, 3, 210‑216

1 ) Beaver, P. C.. Jung, R. C. and Cupp, E. W. (eds.) (1984):

Japanese) .

181

Jpn. J. Trop. Med. Hyg ., Vol. 24, No. 3, 1996, pp. 181 183

es**"*""

INFLUENCES OF THE PREVENTIVE USE
OF ANTIFILARIAL DRUGS ON THE

TRANSMISSION OF DIROFILARIA IMMITIS IN
HOUSE‑DOGS IN NAGASAKI CITY, JAPANTSUTOMU ODAl, MARIKO MlNE2, OSAMU SUENAGA3, KENJI KUROKAWA4,
KOICHIRO FUJITA5, KATSUTOMO KAT06 AND HIROYUKI TAHARA6
Received May 22, 1996/Accepted July 15, 1996

Keywords:

Dirofilria immitis, antifilarial drugs, preventive use, house‑dogs.

The microfilarial prevalence of Dirofilaria immitis

antifilarial drug against microfilaria (Mf) of D. immitis

in house‑dogs in the southern and northern parts ofwas estimated not to be important to the reduction,

Nagasaki City decreased for 27 years from 1968 to 1994

because the rate was found not high and about similar in(Oda et al., 1993, 1995). Oda et al. (1994a, 1996) report‑

questionnaire surveys in 1989 and 1993 (Oda et al.,1994b) . However, Oka et al. (1988) reported that the
Mf prevalence of D. immitis in house‑dogs examined ina domestic animal hospital has been decreasing gradu‑

ed that the decrease in the number of the main vector
mosquito, Culex pipiens pallens, in parallel with the
expansion of public sewage system and the increase ofindoor‑kept dogs were related to the reduction of theprevalence. The rate of dogs that was given theally for 30 years since 1956 in Tama district in Tokyo,
and the increase of ‑dog‑owners using the antifilarial

Rate of dog owne r s using drugs
D i ethy I ca rbamaz i ne* *50

40

l ve rmect i n(DEO Levamiso I e I emycln

and
D* *

30
20

̲¥‑ Io

l o
a)
(

a(

40

Mf positive rate of al I exam ined dogs***

30
20
10
o

94
93 94
66 67 68 6g 70 71 T2 73 74 T5 7G 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 922 93

Figure 1 Annual changes in rate of dog owners using antifilarial drugs (upper) and
Mf positive rate of Dirofilaria immitis in house‑dogs (10wer) in Nagasaki City.
*Data not available.
* *Data on new drugs are cited from Ohishi (1990) .
***Cited･ from Oda et al., 1995.
Department of General Education, the School of Allied Medical Sciences, Nagasaki University, 1‑7‑1 Sakamoto, Nagasaki 852, Japan
2

3
4
5
6

Scientific Data Center of Atomic Bomb Disaster, Nagasaki University School of Medicine, 1‑12‑4 Sakamoto, Nagasaki 852, Japan
Reference Center, Institute of Tropical Medicine, Nagasaki University, 1‑12‑4 Sakamoto, Nagasaki 852, Japan
Department of Bacteriology, Nagasaki University School of Medicine, 1‑12‑4 Sakamoto, Nagasaki 852, Japan
Department of Medical Zoology, Faculty of Medicine, Tokyo Medical and Dental University, 1‑5‑45 Yushima, Bunkyo‑ku, Tokyo
113, Japan
Department of Physical Therapy, the School of Allied Medical Sciences, Nagasaki University, 1‑7‑1 Sakamoto, Nagasaki 852, Japan182

Table I Cornparison of positive rates between dogs
with and without use of antifilarial drug*

Year

Dogs with drug‑use

Dogs without drug‑use

No. examinedNo, positive (%)

No. examined

No. positive (%)

1989

72

15 (20 . 8)

134

25 (18 . 7)

1993

90

5( 5.6)111

12 (10 . 8)

drug‑using owners as well as the increase of the small‑

sized dogs kept indoors played important roles in the
reduction of the Mf infection rate. The microfilarial
prevalence was reduced both in Tokyo and in Nagasaki,
but the relative importance of the factors reducing thetransmission of D. immitis seemed to be different in thetwo cities.

*Dogs with or without drug‑use were determinedaccording to questionnaire survey. (Oda et al., 1994b).

ACKNOWLEDGMENTS
drugs and of small‑sized dogs kept indoors became

Professor Y. Wada, Department of Medical Ento‑

important to the decrease of Mf prevalence. We reex‑

mology, Institute of Tropical Medicine, Nagasaki Uni‑
versity kindly read the manuscript and provided valu‑
able suggestions. Dr. T. Fukuda, President of the Soci‑amined whether or not the use of the drug by dog‑owners contributed to the reduction of the Mf positive
rate during recent 27 years in Nagasaki City.
Fig. I shows the year‑to year change in the rate of
dog‑owners using antifilarial drugs in relation to theappearance of the drugs and the Mf positive rate of dogs
in Nagasaki City. The rate of dog‑owners was utilized
from a record of a joint meeting of veterinarians in 1983,

ety of Veterinarians in Nagasaki City, cooperated in our

research. Dr. I. Ohishi (Professor Emeritus of Tokyo
University of Agriculture and Technology) kindly sup‑plied us with references related to antifilarial drugs
against D. immitis and gave valuable advice. We wish to
express our sincere gratitude to these persons.

question to 43 dog‑owners at blood examination for
Dirofilaria Mf in 1984 and questionnaire surveys in 1989

and 1993. Diethylcarbamazine became commercially

REFERENCES

available in 1966. During the decade starting from 1966,

1 ) Oda, T., Suenaga, O., Mori, A., Fujita, K., Zaitsu, M.,

the rate of dog‑owners who used the drugs in Nagasaki
City was very low (it wa assumed to be about 10% orless) , and the Mf positive rate of dogs did not change

Kurokawa, K., Nishioka, T., Itoh, T. and Mine, M.

markedly during the same period. Levamisole becamecommercially available in 1982, and ivermectin and mi‑lbemycin D in 1986. The rate of dog‑owners using the
drugs increased from 1984, and Mf positive rate of dogs

began to decrease from around 1983. To make clear arelationship between these rates, we compared the Mf
positive rates between dogs with drug‑use and those
without drug‑use according to questionnaire surveys in1989 and 1993 (Table 1). Positive rates were not signifi‑

cantly different between dogs with drug‑use and those
without drug‑use. Therefore, decrease in Mf positive
rate does not seem to be related to the increase in therate of dog‑owners who used drug. This may be due tothe low rate of dog‑owners using drugs for a sufficientperiod to allow complete elimination of D. immitis
infection in house‑dogs, because only about 20 % of thedrug‑using owners used these antifilarial drugs regular‑ly, according to questionnaire surveys (Oda et al.,1994b) .

On the other hand, Oka et al. (1988) showed thatthe infection raie of D. immitis in house‑dogs examinedin Tokyo was 33. 3 % in 1956 to 1957, and this rate
decreased gradually to 13. 6 % in 1984 to 1985. Oka etal. (1988) speculated that the increase in the rate of(1993) : Studies on epidemiology of Dirofilaria immitis
in house dogs in Nagasaki City, Japan, with considera‑
tions on yearly changes in microfilarial prevalence. Jpn.
J. Trop. Med. Hyg., 21, 231‑237
2 ) Oda, T., Suenaga, O., Fujita, K., Zaitsu, M., Kurokawa,

K., Ogawa, Y., Yamazaki, I., Iida, K. and Mine, M.
(1994a) : Comparison of population of vector mos‑quitoes of Dirofilaria immitis and their natural infection

rates in southern and northern parts of Nagasaki City,
Japan. Jpn. J. Trop. Med. Hyg., 22, 196‑202
3 ) Oda. T., Suenaga, O. and Mine, M. (1994b) : A question‑
naire survey of living environments of house dogs andmeasures taken by dog owners to prevent zoonotic
parasite, Dirofilaria immitis infection in Nagasaki City,
Japan. Bull. Sch. Allied. Med. Sci., Nagasaki Univ., 8, 9
‑164 ) Oda, T.. Suenaga, O., Zaitsu, M., Mori, A., Kurokawa,
K., Fujita K., Ogawa. Y., Yamazaki, I., Iida, K., Doi, K.,

Mine, M. and Kato, K. (1995) : Studies on annual changes
in microfilarial prevalence of Dirofilaria immitis amonghouse dogs for 27 years in Nagasaki City, Japan. Jpn. J.

Trop. Med. Hyg., 23, 133‑137
5 ) Oda, T., Mine, M.. Suenaga. O., Zaitsu, M., Kurokawa,
K., Fujita, K., Kato, K.. Ogawa, Y. and Yamazaki, I.
(1996) : Effect of indoor‑keeping of house‑dogs on thetransmission of Dirofilaria immitis in Nagasaki City,
Japan. Jpn. J. Trop. Med. Hyg., 24, 21‑26
6 ) Ohishi, I. (1986) : Inushijochu, ‑kiseichugaku no tatiba‑kara‑, 136, Bun‑eido, Tokyo (in Japanese) .

183

7 ) Ohishi, I. (1990) : Inushijochusho, 185, Bun‑eido, Tokyo(in Japanese) .

8 ) Oka, H., Kobayashi, S., Nakagaki, K., Hayasaki, M., Oh‑
ishi, I. and Fukutomi, K. (1988) : Tokyoto Tamachiku‑
niokeru inushijochu no ekigakuteki kenkyu ( I ) . The
proceeding of the 106th Meeting of Jap. Soc., Vet. Sci.,
255

Morimoto，N．，Korenaga，M．，Komatsu，C．，Sugihara，S．，Nishida，M．，

Yasuoka，M．，Kumazawa，H．，Sasaki，M．andACaseReportofanOverseas−Traveler
by

Chπo

Researchz硲云批

z8s％〃づInfection

Hashiguchi，Y．

sDiarrheaProbablyCaused

・…一……・…一・・…一……一・一…・……・一…

…177−180

Note

Oda，T．，Mine，M．，Suenaga，0．，Kurokawa，K．，Fujita，K．，

Kato，K．，and
InfluencesofTahara，H．
thePreventive

Z万名φ●伽吻」勉卿げ廊in

Use

of

House−Dogs

Antifilarial

inNagasaki

Dmgs

onCity，JapantheTransmissionof

一……………・……一…

181−1認September1996

VOL．24NO．3

CONTENTS
Original

ArticleIjichi，S．，Machigashira，K．，Nagai，M，Osame，M
Late

and

Phase

Spontaneous

ProliferationofLymphocytes

Associatedwith

Infection：

Involvementof

Cellular

HTLV−I

Responses

againstCulturedtheInfected

Chinen，1．，Toma，C．，Honma，Y．，Higa，N。and

Hemolysin
andProduction

Antigenic

Ha11，W．W，
Peripheral

Blood
一143−149

Cells…一Iwanaga，M．

by四乃吻ohoJ召勉6as

ExaminedbyHemolytic

Activities……………・……

・・151−155…一

Takaoka，H．
Description

of

a

New

Speciesof

S伽％伽窺（S7吻％1勉卿）from

Sabah，
・・157−161

Malaysia（Diptera：Simuliidae）・…………一……・・………・……
Takaoka，H．and Saito，K．
A New Species and New

fromClinicalRecordsofFlies（Diptera：Simuliidae）

Thailand…一……・………………一・・一……………・一………………・……一…

一163−169

ReportKimura，M．，Tanabe，M，Ogata，Y．and
Cases

Black

of

Treated

Ovale

with

Malaria

andMalariae

Wataya，Y．
Malaria

Successfully……・…一一171−175

Mefloquine．一………・……・……………

（Contents

continuedonreversedof

thiscover）