第20巻第3号

平成4年9月15日
内

原

容

著

糞線虫症の治療効果に及ぼすATLウイルス混合感染の影響（英文）・・佐藤 良也，城間 祥行，喜友名 進，當真

小林

弘，

潤

183−192

航空輸送機関および外国各地公共施設における飲料水の水質（英文）・・奥村悦之，橋本 恵介，水田 成彦，秦
奥村

泰之，岡村

光孝，

宜典

193−201

実験的リーシュマニア症一エクアドルで分離された，L廊h㎜吻吻翻o朋α感染
ゴールデンハムスターの組織学的検討一（英文）一A．M．Bhutto，野中薫雄，E．A．Gomez

L．，

橋口 義久，古谷正人
短203−215報

無鉤条虫感染症における血清CRP値（英文）・・大西健児，村田三沙子

217−218

会報・記録

1992年度第1回幹事会記録 …一……・……・…一………………………・…………・………

219−220

1992年度会員名簿……一…………・……一一……・・……………・…・……一…………・……

221−246

第4回熱帯医学と医療を語る会のお知らせ……………・一・一・……………・………………

247

第17回日本熱帯医学会九州支部大会のお知らせ（第1報）

・…・……………・………………

248

投稿規定…………・…・・……・…・………・………………一・一………………・…………＿＿…

24g

日本熱帯医学会

Jpn J Trop Med Hyg., Vol. 20, No. 3, 1992, pp. 183‑192

183

REDUCED EFFECT OF CHEMOTHERAPY OF
STRONGYLOIDIASIS IN PATIENTS WITH
CONCURRENT HTLV‑1 INFECTION IN

OKlNAWA, JAPANYOSHIYA SATO*'2, *, YOSHIYUKI SHIROMA SUSUMU KIYUNAHIROMU ToMA AND JUN KOBAYASHll
Received March 9 1992/Accepted May 7 1992

Abstract: The effect of concurrent HTLV‑1 infection on the efficacy of anti‑Stron‑
gyloides chemotherapy has been tested in a prognostic study on 96 patients with uncom‑
plicated strongyloidiasis. The efficacy of treatment with pyrvinium pamoate and thiaben‑dazole was found to be significantly low in the HTLV‑1 seropositive patients, as comparedto that in the seronegative group. The cure rates in the HTLV‑1 seropositive patients,
when assessed by both faecal examination and serological testing, were only 8.6% forpyrvinium pamoate treatment and 35.0% for thiabendazole treatment. On the other hand,the cure rates were 31.3% for pyrvinium pamoate treatment and 57.1% for thiabendazole
treatment in the seronegative group.
Although antibody response against Strongyloides did not decrease among the HTLV‑I seropositive patients, the total serum lgE Ievels, as well as eosinophil counts in peripheral

blood, were relatively low in the seropositive group. Abnormal lymphocytes were demon‑
strated in 38.5% of the patients with concurrent HTLV‑1 infection. The presence ofabnormal lymphocytes, however, appeared not to correlate with the therapeutic efficacy.
A possible connection between the poor efiicacy and depres ed immune responses provided

by the concurrent HTLV‑1 infection is discussed. The low efficacy in the HTLV‑I
seropositive patients also provides a causal explanation for the significant accumulationof patients with concurrent HTLV‑1 infection in Okinawa.

INTRODUCTION
Strongyloidiasis caused by Strongyloides stercoralis infection still remains prevalent in

Okinawa Prefecture, Japan, where other parasitic diseases have been almost entirely
eradicated in recent years (Sato, 1986). Because of its opportunistic nature, the great
majority of patients who are immunocompetent are generally chronic, producing no symptom
clearly attributable to the presence of the parasite. On the other hand, under the conditionof depressed immune competence, the chronic infection progresses to a massive and often
fatal systemic infection as a consequence of increasing autoinfection with larval parasites.

1 Department of Parasitology, and 2 Research Center of Comprehensive Medicine, School of
Medicine, University of the Ryukyus, 207 Uehara, Nishihara, Okinawa 903‑01, Japan (*corre‑
spondent author)
3 Izumizaki Hospital, 1‑11‑2 Izumizaki, Naha, Okinawa 900, Japan

184

Okinawa Prefecture is also known to be an endemic area for human T‑cell leukemia
virus (HTLV‑D infection (Clark et al., 1985). The HTLV‑1 is aetiologically associated withadult T‑cell leukemia (ATL) which leads to severe deficiencies in immunological responses.
Recently, it has been demonstrated that the patients with an asymptomatic Strongyloides
infection in Okinawa are highly accompanied by the ATL viral infection (Nakada et al.,

1984; Fujita et al., 1985; Sato and Shiroma, 1989). Under the condition of concurrent
Strongyloides and ATL viral infection, the progression of asymptomatic Strongyloides infec‑
tion to a fatal disseminated state has been often observed among the patients who developed
ATL (Takara et al.. 1980; Matsui et al.. 1982; Oura et al.. 1986) . A possible explanation forthe high frequency of such a complication, however, has not yet been offered. In this paper
we present results showing the depressed efficacy of treatment of strongyloidiasis in patientswith concurrent HTLV‑1 infection and thus discuss the possibility that the low therapeutic
efiicacy may be attributed to the depressed immune responses provided by the viral infection
and also may be responsible for a significant accumulation of strongyloidiasis patients withconcurrent HTLV‑1 infection for a long period in Okinawa.

MATERIALS AND METHODSPatients
A total of 96 patients with strongyloidiasis received medical' treatment for stron‑
gyloidiasis in the lzumizaki Hospital, Okinawa, Japan. They were consisted of 47 males and

49 females. Their age ranged from 30 to 74 years (mean=53.3 years) . They werea ymptomatic or mildly symptomatic cases. Among them, positive rate of antibodies against

ATL‑associated antigen (ATLA) was as high as 67.7%.

Treatments
Fifty‑one patients were treated with pyrvinium pamoate suspension (PoquiD , which wasadministered in a dosage of 5 mg per kg body weight daily for 3 to 5 consecutive days. For
the other 27 patients, thiabendazole (25 mg/kg for 4 days) was administered. The above
treatment schedules have long been used in Okinawa for treatment of strongyloidiasis. The
remaining 18 patients were left without any treatment as a control group. The duration fromtreatment to follow‑up faecal examination was different for each cases, but it was more than
2 years for all cases.

Faecal examination
The follow‑up faecal examination after the treatment was performed repeatedly forconsecutive 3 days by three different method, e.g. direct faecal smear, formol‑ether concen‑

tration method and faecal culture (Harada‑Mori method) .
Antibodies to S, stercoralls

In order to assess serologically the efficacy of treatment, the serum antibodies to S.

stercoralis were measured by an enzyme‑linked immunosorbent assay (ELISA). The
antigens used were prepared from S. stercoralis filariform larvae collected from faeces ofstrongyloidiasis patients (Sato et al.. 1983). The conventional technique for the micro‑
ELISA using a microtiter plate was the same as that described in a previous paper (Sato et185al., 1985). The sera were tested at a single dilution of 1:50 and the intensity of antibody

response was expressed as the absorbancy (OD) at 500 nm. On the basis of the previous
criteria, the OD value of over 0.5 was regarded as being antibody positive (Sato et al.. 1990b) .

Detection of antibody to ATL virus
The individuals having anti‑ATLA antibodies have been known to be infected with
HTLV‑1 in their peripheral lymphocytes (Gotoh et al.. 1982).
A kit for the particle agglutination test to detect anti‑ATLA antibodies was kindly
supplied from Fujirebio Inc., Tokyo. A usual indirect agglutination test using gelatin
particles coated with antigens prepared from culture fluid of the virus‑producing cell line wasdone in U‑bottomed wells of a plastic microplate (Ikeda et al., 1984). The mixtures of theantigen‑coated particles and serially diluted sera in the wells were allowed to stand for 3 hr
at room temperature and the resulting patterns formed on the bottom of the wells were read.
The final serum dilution of 1:16 or higher showing agglutination was interpreted as positive.

Serum total lgE
Serum lgE was measured by a standard radioimmunosorbent test using Phadebas lgEtest kit (PRIST; Pharmacia Fine Chemicals, Uppsala, Sweden) . Serum lgE Ievels wereexpressed in ng/ml of serum, assuming that I unit/ml corresponds to approximately 2 ngl
ml.

Haematological examinations
Eosinophils from peripheral blood were counted in Wright's‑stained smear and werereported as percentage in 100 Ieukocytes. Morphologically abnormal lymphocytes having
lobular or indented nulei were also counted in the sarne srnears.
Statistics

The data were analyzed by X2 test and Student's t test, as appropriate. A P value ofmore than 0.05 was considered not to be siguificant.

RESULTSThe results of follow‑up examination after treatment are shown in Table 1. The total

cure rates, as estimated by both faecal and serological examinations, were 15.7% forpyrvinium pamoate treatment and 40.7% for thiabendazole treatment, respectively. Although
three patients in the untreated group were negative in the follow‑up faecal examination, they

were serologically equivocal for complete cure and the spontaneous cure determined by
exclusion of the equivocal cases was not detected in the untreated group. When the efficacy
were compared between the HTLV‑1 seropositive and seronegative patients, the cases with ̲complete cure were significantly fewer in the HTLV‑1 seropositive group than in the ser‑onegative group in both treatments.
Table 2 represents total lgE Ievels in sera of 39 patients who were still positive after thetreatment. The lgE Ievels were relatively lower in the HTLV‑1 seropositive group than in
the seronegative group. Although the difference was not significant statistically, the meanlgE Ievel in the seropositive group was as low as one‑third that of the seronegative group.

186

Table I Effect of concurrent HTLV‑1 infection on treatment of strongyloidiasis with pyrvinium

pamoate and thiabendazole
Faecal examination

Anti‑ATLA
No. (not cured)
Treatment antibody
examined
Positive

Positive

Pyrvinium Negative

pamoate Total(equivocal)( cured )35 30 (85.7)
16 10 (62.5)
51 40 (78.4)

2 (5.7)1 (6.3)3 (8.6)3 (5.9)8 (15.7)

9 (45.0)
2 (28.6)

4 (20.0)
1 (14.3)

7 (35.0)
4 (57.1)

27 Il (40.7)

5 (18.5)ll (40.7)7 (70.0)8 (lOO)

3 (30,0)18 15 (83.3)

3 (16.7)

o (o)
o (o)
o (O)

Positive

20

Thiabenda‑ Negative

7

zole

Total
Positive

108Untreated Negative
Total

Negative Significance*
Sero‑positivet sero‑negativeto (o)5 (31.3)

P<0,05

N .S.

N.S.

* Significance between HTLV‑1 seropositive and seronegative groups; t anti‑Strongyloides
antibody
Although the difference of cure rate between HTLV‑1 seropositive and seronegative
groups was not significant (P<0.1) in thiabendazole treatment, the total cure rate of both
treatments was significantly lower (P<0.05) in the HTLV‑1 seropositive group.

Table 2 Comparison of serum total lgE Ievels between HTLV‑I
seropositive and seronegative patientslgE Ievel (ng/ml)

Anti‑ATLA antibody
Positive (n=38)

Negative (n=12)< I , OOO

34 (89.5)

8 (66.7)

1 , OOO ‑ 3, OOO

4 (10.5)

2 (16.7)

3, 100 ‑ 5 ,OOO

O (O)
O (O)

1 (8.3)l (8.3)415.8:!:523.2

l , 300 d: I , 795> 5 , OOO

Mean d: SD

N.S.**Significance between HTLV‑1 seropositive and seronegative groups.

Table 3 also shows eosinophil counts in peripheral blood of 25 patients with Strongyloides
infection. Eosinophilia of over 6% was detected in 57.8% of patients who were negative foranti‑ATLA aptibody, while the percentage of patients with eosinophilia was 35.0% in the
HTLV‑1 seropositive patients. On the other hand, when anti‑Strongyloides antibodies werecompared between the HTLV‑1 seropositive and seronegative groups, the antibody levels
were relatively higher in the seropositive group, showing no depressed antibody response toStrongyloides among the seropositive group (Fig. 1) .
The presence of abnormal lymphocytes in peripheral blood was determined in 52 patients

who were positive for anti‑ATLA antibody and atypical lymphocytes were detected in 20

187

Table 3

Eosinophil counts in peripheral blood of strongyloidiasis patientswith or without concurrent HTLV‑1 infection

Anti‑ATLA antibody

Bloodeosinophils (%)

Positive (n=40)‑6

26 (65.0)

Negative (n = 19)8(42 . 1)

7 ‑ 107 (17.5)

7 (36. 8)ll ‑ 15

6 (15.0)

2 (10. 5)

16 ‑

1 (2.5)

2 (10. 5)

Eosinophilia ( > 6% )

14 (35.0)ll (57.8)

N.S.**Significance between HTLV‑1 seropositive and seronegative groups.
P‑ms

.

e
e
O

(38.5%) patients. The relation between the pres‑

,D
c::]

ence of atypical cells and the therapeutic efflcacy

is shown in Table 4. The positive rate of atypical

e
1 .5

1

cells was relatively higher in the group of unsuc‑

dL

<
J
UJ
u)

cessful treatment than in the group of complete
cure in pyrvinium pamoate treatment, but it were
almost similar in thiabendazole treatment.

$

i

1 .O

‑

DISCUSsloN

i

A
t

a

‑.

Strongyloidiasis and ATL are presently high‑ly prevalent in Okinawa Prefecture, Japan; the

0.5

i.

prevalence levels appear to be 5 to lO% for.
8Strongyloides (Sato, 1986; Sato et al., 1990b) andabout 20% for HTLV‑1 infection (Clark et al..
1985). Recently, it has been pointed out by sev‑

eral researchers that Strongylo,ides carriers in

Positive Negative
Anti‑ATLA antibody

Okinawa are frequently accompanied by HTLV‑I

infection (Nakada et al., 1984; Fujita et al.. 1985; Figure I Comparison of anti‑Strongyloides

ELISA values between HTLV‑I

Sato and Shiroma, 1989). In these studies, thepositive rates of anti‑ATLA antibody as high asseropositive and seronegative
patients with strongyloidiasis.

Table 4 Correlation between efficacy of treatment and presence of abnormal
lymphocytes in peripheral blood of patients with concurrent HTLV‑I
inf ection

Efficacy of treatment

Treatment
Not curedEquivocal

Cured

Total

Pyrvinium pamoate 13/28 (46.4) 0/2

(o)

1/3

(33 . 3)

14/33 (42.4)

Thiabendazole

3/9 (33.3) l/3(33 . 3)

2/7

(28 . 6)

6/19

(31 . 6)

Total

16/37 (43.2)

3110(33 . 3)

20/52

(38 . 5)

No. positive/No. examined (%)

1/5 (20.0)

188

57.8 to 73.6% were detected among the strongyloidiasis patients.
Several explanations have been proposed for the severe complication of these infections.

The participation of antigenic components common to Strongyloides and ATLA which may
produce high positive rate of anti‑ATLA antibody among the Strongyloides carriers has been

excluded by the authors (Sato and Shiroma, 1989). Possible participation of any
epidemiological factors to produce the severe overlap of the two infections has also been
excluded in a previous paper (Sato et al.. 1990a).
Alternatively, it can be supposed that the concurrent HTLV‑1 infection may affect on theseverity of Strongyloides infection through the depressed immune competence of host during
the viral infection. The data presented in this paper demonstrate that the anti‑parasitic effectof the anthelmintics is greatly reduced in strongyloidiasis patients with concurrent HTLV‑I
infection. In Okinawa, intractable cases of strongyloidiasis, in which relapse occurs repeated‑
ly after various treatment over a period of many years, have been often observed (Shiroma
et al., 1990) . Although the factor responsible for such a resistance to anthelmintic treatment
is unclear, it can be postulated that the poor efiicacy of treatment may be attributed to thedepressed immune response which provided by the viral infection. As well documented, ATL
is characterized by an unique T‑lymphocyte malignancy which leads to severe deficiencies in
immune responses, and it is also known that the disorder to a T‑cell mediated immune system
has already begun in the stage of virus carriers (Imai and Hinuma, 1983; Yasuda et al.. 1986;
Tanaka et al., 1989; Prince et al.. 1990). Because of the opportunistic nature of the parasitic

pathogen, it is reasonable to suppose the immune dependence of anti‑parasitic chemotherapy
in strongyloidiasis.

In the past decade, substantial evidence for the immune dependence of chemotherapy has
accumulated. It has been known that immunosuppression reduces the efiicacy of chemother‑apy in several parasitic diseases, such as malaria (Lwin et al.. 1978), trypanosomiasis(Frommel, 1988), onchocerciasis (Bianco et al., 1986) and schistosomiasis (Doenhoff andBain, 1978). In the case of strongyloidiasis, it is also well documented that the patients withsevere infection under the immunocompromised condition often fail to respond to anthel‑mintic treatment, and that repeated courses of treatment are necessary to obtain a complete
cure for such a severe case (Scowden et al.. 1978; Weller et al., 1981; Shelhamer et al.. 1982;

Morgan et al.. 1986). Although the immune factor involved in influencing the drug efficacy
is not yet determined, it has been reported that antibodies specific for parasite, when it wereadministered simultaneously with drug, enhanced the efflcacy of drug against schistosomes
and malaria parasite (Doenhoff and Bain, 1978; Targett, 1985; Brindly and Sher, 1987).

When the antibody titers to Strongyloides were compared between HTLV‑1 seropositive andseronegative patients in the present study, however, we could not find any evidence to suppose

a depressed antibody response against Strongyloides in the seropositive group. While the drug
efiicacy in onchocerciasis reduced markedly in T‑cell deprived mice but not in congenitally

B‑cell deficient mice, suggesting that cellular immune effector mechanism other than theserum antibody may contribute to the efficacy of chemotherapy in this parasitic disease(Bianco et al., 1986). With respect to strongyloidiasis, significance of cell‑mediated immu‑
nity in controlling and preventing Strongyloides infection has also been suggested in several
clinicopathological studies (Purtilo et al.. 1974; Scowden et al.. 1978; Cohen and Spry, 1979) .

As already mentioned above, HTLV‑1 carriers are known to have immunological abnormal‑
ities, such as increased spontaneous lymphoproliferation, decreased mitogenic responses and189

increased expression of IL‑2 receptor on the surface of T Iyinphocytes. In the present study,morphologically atypical lymphocytes were detected in about 40% of HTLV‑1 seropositive
patients examined. In their study on 36 patients, Nakada et al. (1987) has reported that
abnormal lymphocytes, as well as monoclonal integration of HTLV‑1 proviral DNA, weredetected in many patients and indicated that the presence of abnormal cells and monoclonal
integration of proviral DNA correlated with a trend for greater severity of the parasitic
infection. However, we could find no correlation between the presence of abnormal cells andthe e cacy of treatment in the present study. On the other hand, it was noted that total serumlgE Ievels and eosinophil counts in peripheral blood were relatively low in the HTLV‑I
seropositive patients. The lgE and eosinophilic responses are well known to play an impor‑tant role in protective immunity to parasitic helminths (Capron et al.. 1981; Dessein et al..

1981; Kojima et al.. 1985). Recently, it was also demonstrated in a study with murine
infection model that IL‑5‑dependent eosinophilic response was important for protective
immunity to Strongyloides infection (Korenaga et al.. 1991). The relative decrease of lgElevel and peripheral eosinophils in the HTLV‑1 seropositive patients may participate in thepoor efficacy of chemotherapy. Further investigations on a possible connection between the
immune responses and drug e :cacy in strongyloidiasis should be intended.

Finally, the reduced efiicacy of drug treatments in HTLV‑1 seropositive patients may
provide another explanation for the high concurrency of HTLV‑1. Due to resistance toanthelmintic treatments, which has long been used for strongyloidiasis in Okinawa, thepatients with concurrent . HTLV‑1 infection might harbour the infection for many years,
resulting a significant accumulation of such patients.

ACKNOWLEDGMENTS
This study was supported in part by grants from the Ohyama Health Foundation and the
Chiyoda Mutual Life Foundation. The authors are also grateful to Fujirebio Inc., Tokyo,
Japan (Mr. S. Hanzawa) for supplying kits for detection of anti‑ATLA antibody (Serodia‑

ATLA) .

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Okinawa, Japan, Jpn. J. Parasitol., 39, 277‑283
32) Takara, M., Hirata, R., Maeshiro, H., Nakamura, M., Shiroma, Y., Akaboshi, N., Nakaji, S.
and Murata, S. (1980) : Five cases of malignant lymphoma be6ame overt during the course ofstrongyloidiasis, Okinawa Med. J., 18, 129‑131 (in Japanese)33) Tanaka, Y., Oda, S., Nagata, K., Mori, N., Sakamoto, H., Eto, S. and Yamashita, U. (1989):
Immunological functions and phenotypes of peripheral blood lymphocytes from human T‑cellleukemia virus‑1 carriers, J. Clin. Immunol., 9, 477‑484

34) Targett, G.A.T. (1985): Chemotherapy and the immune response in parasitic infections,
Parasitology, 90, 661‑673
35) Weller, I.V., Copland, P. and Gabriel, R. (1981): Strongyloides stercoralis in renal transplant
recipients, Brit. Med. J., 1, 52436) Yasuda, K., Sei, Y., Yokoyama, M.M., Tanaka, K. and Hara, A. (1986): Healthy HTLV‑I
carriers in Japan: the haematological and immunological characteristics, Brit. J. Haematol.,
64, 195‑203

192

糞線虫症の治療効果に及ぼすATLウイルス混合感染の影響
佐藤良也 ・2・＊・城間 祥行3・喜友名 進3・當真

弘1・小林

潤1

沖縄では糞線虫保有者の多くに，ATL病原ウイルスの混合感染が見られることが知られている。ATLウイルスの混合感染が，糞線虫に対する駆虫効果にどのような影響を与えているかについて，96名の糞線虫保有者を対象に検討した。治療はピルビニーム・パモエート（PP）とサイア

ベンダゾール（TB）を用いて行い，いずれの場合でもATLウイルスの感染が陽性である糞線虫保有者において，治療後の治癒率が著明に低いことを確認した。すなわち，ATLウイルス陽性者
での治癒率は，PP治療でわずか8．6％，TB治療の場合で35．0％であったのに対し，ATLウイル
ス陰性の糞線虫保有者では，おのおの3L3％，57．1％であった。
これらの糞線虫保有者について，抗一糞線虫抗体値を比較したところ，ATLウイルス陽性群において抗体値が低いことを確認することはできなかったが，末梢血好酸球と血清総IgEレベル

は，ATLウイルス陽性群において低い傾向が認められた。また，ATLウイルス陽性の対象者において，その約40％に異型リンパ球の出現を認めたが，異型リンパ球の存在と駆虫効果の間には特に関連を認め得なかった。かかる治癒率の低下は，ATLウイルスの感染による何らかの免疫低下状態によってもたらされた可能性と，これが長年にわたりATLウイルス陽性の糞線虫保有者を選択的に蓄積させ，今日の高い混合感染状態をもたらした，主要な原因である可能性が考えられた。

琉球大学医学部寄生虫学教室（＊別冊請求先：〒903−01沖縄県中頭郡西原町上原207）

琉球大学医学部付属地域医療研究センター
泉崎病院

Jpn. J. Trop. Med. Hyg., Vol. 20, No. 3, 1992, pp. 193‑201

193

ANALYSIS OF DRlNKING WATER IN AIRPORTS,
AIRPLANES AND PUBLIC WATER FACILITIESETSUSHI OKUMURA , *, KEISUKE HASHIMOT02, NARUHIKQ MizUTA'

MITSUTAKA HATA', YASUSHI OKUMURA' AND YOSHISUKE OKAMURA
Received April 8 1992/Accepted June 4 1992

Abstract: The results indicated that drinking water samples from 4 (44.4%) of 9
airports and from 2 (22.2%) of 9 airplanes gave a positive qualitative test for at least any

one of ammoniacal nitrogen, nitrite nitrogen and nitrate nitrogen. Samples from 8(57.1%) of 14 airports, from 8 (66.7%) of 12 airplanes and from 10 (62.5%) of 16 public

facilities showed a value for the consumption of potassium permanganate of 20 mg/1 orabove. Those from 5 (31.3%) of 16 airports, from 3 (18.8%) of 16 airplanes and from 3(15.0% ) of 20 public facilities had a hardness of 200 mg/1 or above. A total bacterial countof 1.0 x 102/ml or above was obtained for samples from 6 (66.7%) of 9 airports, from 7(63.6%) of 11 airplanes and from ll (61.1 ) of 18 public facilities; a positive coliform
group culture was noted for samples from 5 (62.5%) of 8 airports, from 6 (60.0%) of 10
airplanes and from 4 (36.4%) of ll public facilities. Residual chloride was not detectedin any measurable amount in samples from 9 (56.3% ) of 16 airports, from 2 (40.0% ) of 5
airplanes and from 4 (57.1%) of 7 public facilities.

Bacteriological examination of 29 samples positive for total bacteria and coliform

bacilli revealed that Escherichia coli was detected most frequently, followed by
A cinetobacter calcoaceticus. Aeromonas hydrophilia. Pseudomonas spp., Klebsiella pneumoniae.

Proteus mirabilis. Enterobacter cloacae and Citrobacter freundii in that descending order.

Striking correlation were noted to exist between negativity for residual chloride andpositivities for total bacteria or coliform group; and then positivities for ammoniacal
nitrogen, nitrite nitrogen, nitrate nitrogen, a heightend value of the consumption ofpotassium permanganate and positivities for total bacteria or coliform group.

INTRODUCTION
The number of Japanese traveling abroad has reportedly been increasing steadily to
exceed 10 million per year in these latest years. As a natural consequence, persons who are
affected with a diarrheal or feverish disease while staying abroad or after returning home arealso on the increase and this growing population with so‑called imported infections poses a3

Department of Health Science, Kochi Gakuen Junior College, 292‑26 Jingamori, Asahi‑Tenjin‑machi, Kochi 780, Japan (*correspondent author)
Association of Tropical Medicine, Osaka Medical College
Department of Clinical Technology, Minami Osaka General Hospital

5

Okumura Medical Clinic
Department of Animal Health Service Center, Kochi

1

2

194

serious socio‑medical ploblem (Okumura and Nunode, 1980; Kimura et al., 1987; Weinke etal., 1988). Of these infectious diseases, the main source of those involving the digestiveorgans, needless to say, is insanitary foods and drinks, plays a major etiologic role (Sunaga

and Sawada, 1986; Okumura et al.. 1990).
From this viewpoint, we have carried out analysis of drinking water offered to Japanese
residents as well as natives in under‑developed countries and reported the results thus
obtained during the past decade (Okumura and Watanabe, 1985; Okumura, 1986) . In thepresent study, we undertook analyses, with bacteriological examination, of those types ofdrinking water to which travelers in general can easily gain access, i.e. offered in airplanes,in airports and via public water facilities in noted places for sight‑seeing in an effort tocontribute to the prevention of water‑borne infections occurring during overseas traveling.
The purpose of this communication is to present the finding thus yield.

MATERIALS AND METHODS
Chosen by us for examination were samples of drinking water serviced in airplanes, fromwater coolers at the airports and from water faucets at railway stations, parks, noted places

and historic sites. These samples were analyzed or determined for ammoniacal nitrogen,nitrite nitrogen, nitrate nitrogen, chloric ion, residual chloride, consumption of potassium

permanganate, fluorine and total hardness in accordance with "Method of Water Analysis" ‑Explanation of Hygienic Test Methods ed. the Pharmaceutical Society of Japan.
For quantification of total bacteria and coliform group, water specimens were filteredthrough membrane filters (Millipore, Water‑tester MT 25 & MC 25, Millipore Corp.),washing of the filters were incubated in media for 24‑48 hr at 37'C and colonies formed werecounted. Specimens were also incubated in blood agar, BTB and Butzler's medium for theformation of colonies, which submitted themselves t() identification procedures utilizing
media for identification (TSI‑agar, etc.) and simple identification kits (Thermo Co., Ltd.,BL‑E21 Enterogram, BL‑Nl8 Nonfagram, etc.) (Pharmaceutical Society of Japan, 1983) .

RESULTS
Drinking water samples from water coolers installed in 4 (44.4% ) of 9 airports (including

their boarding lounges) , from 2 (22.2%) of 9 airplanes and from 10 (56.2%) of 16 public water

facilities (railway station and noted place) studied were found positive, both qualitatively

and quantitatively, for all or any one or two of ammoniacal nitrogen, nitrite nitrogen andnitrate nitrogen.

High values for the consumption of potassium permanganate in excess of 20 mgll wereobtained for drinking water samples from 8 (57.1%) of 14 airports, from 8 (66.7%) of 12
airplanes and from 10 (62.5%) of 16 public water facilities studied.

A total hardness of 200 mgll or greater was noted for drinking water samples from 5(31.3%) of 16 airports, from 3 (18.8%) of 16 airplanes and from 3 (15.0%) of 20 public water
facilities.

Drinking water samples from 6 (66.7%) of 9 airports, from 7 (63.6%) of ll airplanes and11 (61.1%) of 18 public water facilities were shown to contain 1.0 X 10'/ml of total bacteria.

Drinking water samples from 5 (62.5% ) of 8 airports, from 6 (60.0% ) of 10 airplanes and from

195

Table lRatio of abnormality on chemical and biological test in drinking water
Airport

Airplane(%)(%)

Water facilrtles Total(%) (%)

NH3‑N, N02‑N, N03: positive

4/9

2/9

(44 . 4)(22 . 2)

KMnO*: over 20 mg/l

8114

8/12

10/16

26/42

(57 . l)(66 . 7)(62 . 5)(61 . 9)Hardness: over 200 mg/l
Total bacteria: positive

Coliform group: positive
Residual Cl: negative

9/16

15/34

(56 . 2)

(44 , l)

5/16

3/16

3/20

ll/52

(31 . 3)(18.8)(15.0)(21.2)

6/9

7/11

11/18

24/38

(66 . 7)(63 . 6)

(61 . 1)(63 . 2)

5/8

6/10

4111

15/29

(62 . 5)(60 . O)

(36.4)(51 . 7)

9/16

2/5

417

15/28

(56 . 3)(40 . O)

(57 . 1)(53 . 6)4 (36.4%) of ll public water facilities proved positive for coliform group.
Also samples from 9 (56.3%) of 16 airports, from 2 (40.0%) of 5 airplanes and 4 (57.1%)of 7 public water facilities were noted to be negative for residual chloride on qualitative
testing (Table l).
Bacteriological examination of colonies of total bacteria and coliform group with culture
media for their identification and simple identification kits revealed that E. coli was detected

in drinking water samples from 11 sources (5 airports, 4 airplanes and 2 public water
facilities) , A. calcoaceticus from 6 sources, A. hydrophila from 4 sources, Pseudomonas spp.

from 4 sources, K. pneumoniae from 3 sources, P. mirabilis from 2 sources, E. cloacae from
2 sources, C. freundii from 2 sources, and non‑haemolytic streptococci, Bacillus spp., Sta‑phylococcus epidermidis, NF‑GNR (dextrose non fermentant Gram‑negative rods) ,Campylobacter jejurd. Chromobacterium spp., Klebsiella oxytoca. Hafnia alvei andAcinetobacter lwoffii from I source each (Tables 2, 3, 4 and 5).
Table 2 Bacterial dose and isolated stains from drinking water in airports

AirportDose (CFU/ml)

A

3 . O X 106

Acinetobacter calcoaceticus, Escherichia coliB

1 . 6 x 106

A, calcoaceticus, non‑haemolytic streptococcii

C

2 . 7 x 106

Aeromonas hydrophila, E. co li, Bacillus sp.

D

2 . O X 105

E, coliE

8 . 2 x 105

Pseudomonas sp., Proteus mirabil isF

7 . 4 x 103

Pseud omonas ce paciaG

1 . O x 104

E. coli, Pseudomonas sp.

H

2 . 8 x 104

E. coliI

3 . O x 103

Enterobacter cloacae(mediaIsolated strains

MacConkey‑agar, YCA, DHL, BTB)

196

Table 3Bacterial dose and isolated strains from drinking water in airplanes

Airpl ane

Dose (CFU/ml)

A

7 . O X 102

E. coli. Staphylococcus e pid ermid is. Citrobacter freundii

B

1 . O X 102

E. coliC

5 . O X 106

A. calcoaceticus. A. h yd ro philaDE

5 . O X 107

Pseudomonas sp., Klebsiella pneumoniae

3 , O X 102.E. coli, E. cloacae

F

7 . O X 102

E, coliGH

4 . 3 x 105

Campylobacter jejuni, Chromobacterium sp.

7 . 8 x 105

P, mirabllrs

Isolated strains

NF‑GNR*(media : MacConkey‑agar, YCA, DHL, BTB)': dextrose non fermentant Gram‑negative rods

Table 4Bacterial dose and isolated strains from drinking water in public water facilities

Place

Dose (CFU/ml)

Isolated strains

Bus stop in Thailand

3 . 2 x 103

K. pneumoniae

National park in Moritania

2 . O X 103

A cinetobactor lwoffii, Hafnia alvei

Oasis in Argeria

2 . O X 104

E. coliNational park in Ethiopia

l . 5 x 103

A. hydrophila

Water job in Nigeria

2 . O X 106

A. calcoaceticus. C, freundii

Central park in Tanzania

2 . O x 103

Klebsiella oxytoca

Central park in lraq

6 . 4 x 104

A calcoaceticus, E. coli

Market in Saudi Arabia

2 . O X 103

A. calcoaceticus

Central park in Israel

5 . O X 106

A. hydrophila

Park in Pakistan

1 . 4 x 103

K. pneumoniae(mediaMacConkey‑agar, YCA, DHL, BTB)

The relationships between positivities for total bacteria and coliform group vs.
nitrogenous compounds, consumption of potassium permanganate and residual chloride areshown in Table 6, indicating that the ratio of drinking water positive to negative forammoniacal nitrogen, nitrite nitrogen and nitrate nitrogen was 9:5 for samples positive fortotal bacteria and 6:2 for those positive for coliform group. Similarly, the ratio of drinking

water samples showing a consumption of potassium permanganate of
20 mg/1 to those witha corresponding value of > 20 mgll was 10:6 for samples positive for total bacteria and 7:3
for those positive for coliform group. The positive‑to‑negative ratio of samples for residual
chloride was 9:1 and 8:O, respectively, for those positive for total bacteria and coliform group

197

Table 5

Total of isolated strains in each place
AirportAir plane

Public water facilities

Total

E. coli

5

4

2

ll

A . calcoaceticus

2

1

3

6

1

A. hydrophila

24Pseudomonas sp.

3

1

o

4

K. pneumaniae

o

1

2

3

P. mirabilis

1

o

2

E. cloacae

l

o

2

C, freundii

o

2

l

non‑haemolytic streptococcii
Bacillus sp.

O

o

1
1

O

o

S. epidermidls

o

1

o

NF‑GNRo

1O

C. jejuni

o

1

o
o

1

Chromobacterium sp.

o1

K. oxytoca

o

O

1

H, alveioO

1

A. Iwoffii

o

O

1

Table 6

Correlation between chemical test and isolated bacteria
Airport

NH3‑N, N02‑N,l

PublicAirplaneTotalwater facilities

Total

Colif orm

Total

Colif orm

Total

Colif orm

Total

Colif orm

Bacteria

group

Bacteria

group

Bacteria

grQu pBacteria

grou ppositive

3

2

l

2

5

2

9

6

N03‑N '

negative

1

l

3

l

1

o

5

2

KMn0+ 20 mg/1:

over
under

3

3

2

3

5

1

lO

7

3

2

1

l

2

O

6

3

negative

6

5

1

2

2

l

9

8

positive

o

o

O

o

l

O

1

o

Residual Cl:

DlscussroN
While the main source of intestinal infection in Japanese travelers and residents inforeign countries is contaminated foods and drinks in many instance, it is an undeniable factthat contaminated drinking water also plays a causative role not infrequently. From thisviewpoint we have carried out analyses of drinking water mainly in underdeveloped countries
during the past 10 years and, based on the results thus obtained, have referred to possible
relations between regional peculiarities and the quality of drinking water in climatic zones
of vegetation (Okumura, 1986) .

In the present study which was conducted over a 10 year period from 1981 to 1991,

198

samples of drinking water readily accessible to Japanese tourists while traveling abroad, e.g.,offered in airplanes, airports and sight‑seeing place of various countries, were collected,
analyzed and examined bacteriologically in an attempt to contribute to the prevention ofdrinking water‑bone bacterial infections occurring in Japanese travelers in foreign countries.

Testing for the quality of drinking water is done in two ways, i.e. by an environmental
research to investigate water fiow, geographic and geologic features and the way of using andtreating water and by physicochemical tests of water, and on the basis of the results of thesestudies combined the potability of water and the most appropriate methods of treating andusing it are determined. Therefore, with the intention of supplying water of good quality,

Article 4 of Japanese Water Works Law (Health & Welfare Ministry's Ordinance No. 56,
August 1978) stipulates quality standards of drinking water. While offlcial quality standardsof drinking water very somewhat from one country to another, the Japan's law requires thatthe content of nitrite nitrogen and of nitrate nitrogen be below 10 mg/1, although there is no

regulation content of ammoniacal nitrogen. The consumption of potassium permanganate
must be below 10 mg/1, free residual chloride must be 0.1 mg/1 or above and the hardness beless than 300 mg/1. The absence of coliform bacteria and less than 100 of total bacteria per
mililiter are mandatory.

However, ammoniacal nitrogen which results from contamination with excreta, factorydrainage, etc. is a valuable index for estimation of water contamination. In addition, nitrite

nitrogen which is generated mainly by oxidation of ammoniacal nitrogen produced bycontamination with excreta and sewage and nitrate nitrogen which is an end‑product ofoxydation of various nitrogenous compounds are also hallmarks of hygienic importance. The
fact that drinking water samples from 15 (44.1% ) of 34 facilities studied were positive for all

three or any one or two of these forms of nitrogen (even though a substantial number ofsamples gave less than the regulation value) may be considered as suggesting the possibility

that samples were contaminated to some extent.
The Germany's standards require that the consumption of potassium permanganate be
below 20 mg/1. Drinking water samples from 26 (61.9%) of 42 facilities showed higher values
even compared to this rather conservative standard level. The consumption of potassium
permanganate usually is increased by contamination with sewage, factory drainage or urine
and feces, though occasionally by reasons of geological factors.

The presence of residual chloride in demonstrable quantities is an indication of theefnciency with which drinking water has been sterilized. The fact that samples from 15(53.6% ) of 28 facilities were negative for residual chloride can be constructed as evidencingthat chlorination of drinking water as an administrative procedure was not practiced at allor only incompletely.
Moreover, a more than 1.0 X 102 cell count was noted of total bacteria for drinking water
samples from 24 (63.2%) of 38 facilities and coliform group for those from 15 (51.7%) of 29

facilities. This fact implies that tap water offered at these facilities was inadequately
sterilized or was contaminated with excreta of man and/or animal or some other agents ororganisms in the water distribution system. A compar,ative study of positivities for total
bacteria and coliform group vs. nitrogenous compounds, consumption of potassium perman‑
ganate and residual chloride showed that drinking water samples that were found positive fornitrogenous compounds as well as those showing higher values for the consumption ofpotassium permanganate gave higher positive rates for total bacteria and coliform group199

bacilli. As a matter of course, drinking water samples without detectable free residual
chloride were found to contain a larger number of total bacteria and coliform bacteria. A
total bacteria count of 2.0 X 103/ml was obtained for a drinking water sample from a public
water facility that was positive for residual chloride and this was considered as due presum‑

bly to contamination occurring in the water distribution or delivery system.
Bacterial species detected in the present study were as listed in Table 5. As can be seen,
E. coli was most frequently, probably as a result of contamination with urine and feces fromunknown causes. In addition, A. calcoaceticus. A. hydrophila. Pseudomonas spp., K.
pneumoniae. P. mirabilis. E. cloacae and C. freundii were also detected. All these bacterial

species were found to bear a close resemblance to strains detected from drinking water inIndonesia by Okuwaki et al. (Okuwaki et al.. 1982, 1985).
Bacterial infection depends for its establishment upon the virulence of the infecting
strain, the pathogenicity of the infecting bacterial species and quantity of the infecting
organisms. In other words, its occurrence is determined by the onset dose‑to‑host response

relationship, hence in accordance with dose‑response curve. Therefore, when infecting
organisms are weak in virulence and minimal in amount and when there are enough hostresistance and acquired immunity, a clinically manifest infection, e.g., of intestinal tract, cannever be caused. However, it would seem important to recognize drinking water from which
infecting agents have been detected to serve at least as a source or route of contaminationeven if it does not constiture a source or route of infection. In fact, according to Fujita et al..

there was a atriking correlation between long‑term intact of coliform bacteria‑positive
drinking water and the positive rate of intestinal parasitosis (Fujita et al.. 1982) . Traveler's

affection with diarrheal disease spoken of so often can be constructed as due to biologicalfactors (i.e. the amount and virulence of infecting agents introduced and decreased immune
response of host mentioned above) , biochemical changes in vivo caused by intake of drinking

water of high chlorine concentration and/or of high Mg++ concentration (high hardness) andpsycho‑psychological factors (inadaptability to environmental changes and alien culture) .
Thus, in order to get safe and good drinking water, it is most recommendable to boil
water, which is the most reliable and easy‑to‑use method of obtaining potable water. Boilingtime is 5 min at level land, increasing by I min for each 1,000 m increment of altitude. Boilingof water for 30 min at 60'C will usually be destructive to ordinary pathogenic organisms. If
no fuel is available for boiling water, use of water filter (activated charcoal covered withsilver carbon) or sodium hypochlorite (the simplest method of purifying drinking water) is

highly advisable as a convenient alternative. To add one drop of commercialized sodium
hypochlorite solution to approximately 180 ml of drinking water and just wait for 5 min will
suffice for making it potable. Instead, addition of 4‑5 drops of 2% iodine tincture to one literof drinking water and subsequent allowing it to stand for about 15 min will also serve thesame purpose. The resulting aqueous solution will surely be destructive to parasitic ova andcysts, if present at all, without doing any harm to the body (Okumura, 1989) .

In summary, our present study revealed that drinking water offered in airplanes and via

public water facilities surprisingly was not sanitary enough but contaminated to somevarying degrees. It is earnestly hoped that health authorities concerned will make all‑out
efforts to supply the public with safe drinkable water. Man craves for water by instinct whenthirsty. Even though healthy persons are confident of their resistance to casual infection ofintestinal tract, it is of utmost importance from the standpoints of preventive medicine and200

personal hygienics to know that drinking water offered by readily accessible public facilities

is likely to be contaminated with pathogenic organisms. Such awareness will certainly proveof great aid in making a safe and pleasant overseas tour as well as in living a sound andmeaningful life in foreign countries.

REFERENCESl ) Fujita, K., Sugiyama, M., Tsukidate, S., Okuwaki, Y., Ikeda, T., Oda, T. and Asakura, T.
(1982) : Correlation of infection rate of intestinal helminths in Japanese inhabiting tropicalcountries with degree of pollution of drinking water, Jpn. J. Trop. Med. Hyg., 10, 41‑48
2 ) Kimura, A., Minekawa, Y., Kitaura, T., Nakano, H.. Goto, I., Ikeda, N., Abe, H., Ono, T. andNakabayashi, T. (1987): Incidence of intestinal infection in traveller's diarrhea, Jap. Assoc.
Infec. Dis., 61, 789‑795

3 ) Okumura, E. and Nunode, Y. (1980): The problem and situation of anti‑malarial drug inJapan; On the difficulties in obtaining anti‑malarial agents in our three cases, Jpn. J. Trop.

Med. Hyg., 8, 187‑196
4 ) Okumura. E. and Watanabe, M. (1985): Analysis of drinking water in Southeast Asia; Reseachon diarrhea seemingly caused by drinking water, Minami Osaka Med. J., 33, 32‑39
5 ) Okumura, E. (1986): Characteristics of drinking water in tropical areas; Quality analysis ofwater used by local inhabitants, Asian Med. J., 29, 582‑589
6 ) Okumura, E. (1989): How to cope with epidemics and secure household water in emergencies,

Minami Osaka Med. J., 37, 155‑162
7 ) Okumura, E., Nakajima, T., Hata, M. and Mypa, L.E. (1990) : Community outbreaks of food‑borne infection with so‑called Vibrio furnissii in Japan and Philippines, Jpn. J. Trop. Med.
Hyg., 18, 23‑30
8 ) Okuwaki. Y., Fujita, K., Sugiyama, M., Yanai, H., Yutaka, K., Tsukidate, S. and Asakura, T.
(1982): Bacteriological and chemical study of the drinking water in Indonesia, Jpn. J. Trop.

Med. Hyg., 10, 33‑39
9 ) Okuwaki, Y., Yanai, H., Yutaka, K., Fujita, K., Tsukidate, S., Kurokawa. K.. Sugiyama, M.
and Asakura, T. (1985) : Calculation of total colonies and coliform group counts, and detectionof enteric and related bacteria from drinking water in Indonesia. Trop. Med., 27, 67‑74
10) Pharmaceutical Society of Japan (1983) : Standard Method of Analysis for Hygienic Chemists,Vol. 5, 921‑1019, Kanehara Co., Tokyoll) Sunaga, H. and Sawada, K. (1986): The water in tropical environment, Vol. 1, 87‑98,
Association of Tropical Medicine. Tokyo
12) Weinke, Th., Friedlich‑J nicke. B. und Janitschke, K. (1988): Bedeutung von Entam6ba
histolytica bei Tropenruckkehren. Dtsch. Med. Wschr., 113, 678‑682

201

航空輸送機関および外国各地公共施設における飲料水の水質
奥村 悦之1・橋本 恵介2・水田 成彦2・秦奥村 泰之4・岡村 宜典5

光孝3・

1981年より1991年の10年間，一般旅行者がごく手軽に飲料水の提供をうけ易い航空機内や空港
内，また観光地など公共水飲み施設の飲料水について水質検査を実施し，特に細菌学的検査成績
を通して海外旅行中における飲料水による感染病防止について考察した。

アンモニア性窒素，亜硝酸性および硝酸性窒素のいずれかが定性試験で陽性を示したものは，空港では9空港中4空港で44．4％，航空機は9機中2機で22．2％，公共施設は16施設中9施設で56．2％であった。過マンガン酸カリ消費量20mg／」以上を示したものは，空港では14空港中8空港
57．1％，航空機は12機中8機66．7％，公共施設は16施設中10施設62．5％であった。

一般細菌がLO×102／mJ以上の集落を認めたものは，空港では9空港中6空港66．7％，航空機は11機中7機63・6％，公共施設は18施設中11施設61．1％であった。大腸菌群が陽性を示したものは，空港では8空港中5空港62、5％，航空機は10機中6機60．0％，公共施設は11施設中4施設36．4％であった。残留塩素の存在が証明し得なかったものは，空港では16空港中9空港56．3％，航空機

は5機中2機40。0％，公共施設は7施設中4施設57．1％であった。
一般細菌および大腸菌群陽性の29検体について細菌学的検査を行ったところ，Esoh碗oh勿60」づが最も多く検出され，次いでAoづn6 oδ40孟砂6αJooαo召漉％s，、4召m吻o微s勿伽oρh吻，R躍40吻oπ硲
SPP・，κZ6加勿1勉1）％％吻o％勿θ，P 陀郷吻1勉わ〃廊，E磁卯oδαoセ70Joαoπθ，C髭m加o陀7ル2観4JJの順であった。

一般細菌や大腸菌群が陽性を示した飲料水と，アンモニア性窒素，亜硝酸性および硝酸性窒素が陽性を示した飲料水，過マンガン酸カリ消費量が高値の飲料水，そして残留塩素が陰性を示し
た飲料水との相関は高かった。

高知学園短期大学保健科（別冊請求先：〒780高知市天神町字陣ヶ森292−26）
大阪医科大学熱帯医学研究会
南大阪病院臨床検査科
奥村胃腸科外科
高知県東部家畜保健衛生所

Jpn. J. Trop. Med. Hyg., Vol. 20, No. 3, 1992, pp. 203‑215

203

HISTOPATHOLOGICAL OBSERVATIONS OF
GOLDEN HAMSTERS INFECTED WITH
AN ECUADORIAN ISOLATE OF
LEISHMANIA MEXICANAABDUL MANAN BHUTTO1' *, SHIGEO NONAKA2, EDUARno A. GOMEZ L.3
YOSHIHISA HASHIGUCHI' AND MASATO FURUYA5
Received May 18 1992/Accepted June 30 1992

Abstract: An experimental study was performed to investigate the Leishmania
mexicana infection in golden hamsters. The animals were infected with L. mexicana from
Ecuador. At the autopsy 6 months after inoculation, the inoculated sites were shallow,
ulcerative and covered with thick crusts. No cutaneous metastasis was observed on other¥exposed parts of the body. Histologically, specimens of both the nose and footpads
showed large numbers of amastigoteS with extensive infiltration of histiocytes andlymphocytes and, to some extent, of neutrophils, eosinophils and plasma cells. Large
numbers of mast cells were evident in the upper and lower dermis of granulomatous
lesions. Amastigotes were found in the macrophages inside the large parasitophorous
vacuoles, mostly at the central part of the lesion. Amastigotes were also observed in theliver and spleen by electron microscope but the number was fewer in visceral than in
cutaneous sections. Regular destruction of parasites was observed within macrophages inall the cutaneous and visceral sections indicating the phagocytizing role of these cells
against parasites.

INTRODUCTION
American cutaneous leishmaniases, caused by Leishmania mexicana and L. (Leishmania)
braziliensis complex, are widely endemic in Central and South America. Among the many
species of the New World Leishmania. L. mexicana ( = L. mexicana mexicana) is well known
to be responsible for a variety of disease forms that show everything from localized simple
mild lesions to generalized diffuse lesions and that are distributed widely from lowlands toAndean highlands. In Ecuador, the species L. mexicana was isolated from both Andean
highland and Pacific lowland patients (Armijos et al.. 1990; Hashiguchi et al.. 1991).
How ver, the clinical manifestations caused by L. mexicana had a tendency to vary depend‑ing on several unknown factor (s) , such as the geographical distribution and immunological

1 Department of Dermatology, Nagasaki University School of Medicine, 1‑7‑1 Sakamoto‑machi, Nagasaki 852, Japan (*correspondent author)
2 Department of Dermatology, School of Medicine, University of the Ryukyus, 207 Uehara,
Nishihara, Okinawa 903‑12, Japan
3 Departamento de Medicina Tropical, Facultad de Medicina, Universidad Catolica Santiago de
Guayaquil, Guayaquil, Ecuador
4
Department of Parasitology, Kochi Medical School, Nankoku, Kochi 783, Japan
5 Institute for Laboratory Animals, Kochi Medical School, Nankoku, Kochi 783, Japan204or physiological conditions of human hosts (Hashiguchi et al., 1991 our unpublished data) . In

order to investigate the factor (s) involved, we launched a comparative study of Ecuadorianleishmaniasis between the lowlands and highlands, carrying out histopathological andelectromicroscopical observations on biopsy materials from humans and experimental ani‑mals. As a first step, an attempt was made in the current study to obtain baseline data

(information) on the pathology of experimental (animaD Ieishmaniasis caused by L.
mexicana isolated from the Pacific lowlands of Ecuador. Together with the ultrastructural
study of the Ecuadorian forms of leishmaniasis reported previously (Bhutto et al., 1992) , theinformation reported here will provide a clearer understanding of this disease in Ecuador.
The present report focuses mainly on the results of histopathological findings of the dermal
lesions (ulcers) of hamsters infected experimentally and the visceral organs invaded by theparasite L. mexicana.

MATERIALS AND METHODS
Animals:
Male golden hamsters, 100 to 150 g and 7 weeks of age, were used. The animals were
fade a standard pellet diet and given water ad libitum.

Parasites and mode of infection:
Parasites were isolated from an Ecuadorian leishmaniasis patient and maintained invitro for several months before animal inoculation. They were identified as L. mexicana(MHOM/EC/INH690) using isoenzyme electrophoresis by Dr. T. Agatsuma, Department of
Parasitology, Kochi Medical School, Japan. The promastigotes of log phase were inoculated
to the nose and hind footpads of 10 hamsters. Each hamster received I x 107 promastigotes.
The development of lesions was observed and recorded periodically during infection.

Histopathology:
At about 6 months after post‑infection the animals were sacrificed and specimens weretaken from central and peripheral parts of the lesions. Specimens were also taken from theliver and spleen and divided into two parts. One part was fixed in 10% formalin, from whichparafiin sections were made and 5 pm sections were stained with hematoxylin‑eosin. For thedifferentiation of mast cells, sections were also stained with toluidine blue (pH 5.0).

Electron Microscopy:
The other part of autopsy materials was cut into 2‑3 mm pieces and fixed with 2%paraformaldehyde and 2% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4). The tissues
were then washed with 0.1 M cacodylate buffer and post7fixed in 2% osmium oxide for 2 hr.
After dehydration in different concentrations of alcohol, specimens were embedded in epon
812. One micron semi‑thin sections were cut with a glass knife on an LKB ultrotome andstained with toluidine blue. Ultra‑thin sections were cut with a diamond knife, stained withlead citrate and uranyl acetate, and examined under a JEM 1200 EX electron microscope(JEOL, Japan) .

RESULTS
Almost all the hamsters inoculated with L. mexicana promastigotes developed eryth‑ematous lesions at the site of inoculation. The lesions gradually changed into nodules,

205** , 'a';*':. *Figure 1+ *"='*:(*"'+:=b

The inoculated sites of the golden hamsters infected wit.h L. mexicana isolated from

Ecuadorian patient. Shallow ulcers are visible on the (a) 1 rose and (b) both hindfoot pads,

ultirnately resulting in ulcers. At the time of autopsy, the inaculated sites of the nose and bothhind footpads were abserved as swallen and large ulcerations (Figs. Ia, b) . The ulcers werecovered with thick crusts. There was nO metastasis or nodule formation on other areas of
the body surface.
Light microscopic findings:
No significant epidermal changes were seen in either the nasal or the footpad sections.
The epidermis was intact and thin at the center of the lesion, and crusts and parakeratasis
were present in some parts of the specimens. In the dermis, histiocytes were predominant in.he sections, and most of these contained numerous amastigotes in the large vacuole8,
particularly at the central part of the lesions (Figs. 2a, b). Huge numbers of lymphocyte8
accumulated around the parasitiz d macrophages in the peripheral part of the lesions (Figs.
3a, b) . Neutrophils, eosinophils and plasma cells were al o seen throughaut the specimens.
Many mast cells were observed in the upper dermis in both the nasal and fcotpad sections(Figs. 4a, b) . Mast cells were located in the areas of the dermis free from parasites but ahsentbaFigure 2a, Light photomicrograph of fonualin embedded section from the nose of a golden
hamster infected with L. mexicana, showing the exten<sive infiltration Of histiocytes

and the fonnation of parasitophorous vacuoles. b, Many parasites invaded bymacrophage8 are visible inside the parasitophorous vacuoles. (H & E stain x 40,
x 160)

206

ab

Figure 3a, Footpad section, shawhng the pre '. ence of large number of lymphocytes

and the parasitized macrophages. b, high magnification of 3a, showing theperivascular infiltration of lymphocytes. H & E stain x 16, x40)ba#Figure 4a, Huge number of mast calls in the upper and lewer dermis. b, High magnificationof 4 . (Taluidine blue stain x 16, x 160)

in t.he areas where macrophages were occupied with parasites. Focal necrosis was also
recognized in the deep dermis.
In the liver and spleen sections, it was difficult to find the parasite8 because of the verysmall number.
Electron microscopic findings:
The epidermis of inoculated sites was free from the invasion of parasites. In the dermis,

parasites were found both intracellularly and extracellularly. Amastigotes in the cytoplasmof macrophages were located either inside or outside vacuoles (Figs. 5 and 6) . More than one

vacuole was seen in most of the macrophages and large numbers of amastigotes were present
in most of the vacuoles. Some parasite‑free vacuoles were also seen, and these connected207;* *;* * "' = ; +"'* ' ;^* :**;

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Electron micrograph. A group of L. mexican:a parasites are evident inside

the parasitophorous vacuole of macrophage. bar‑2 ftm

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Figure showing the parasites inside the cytoplasm of macrophage without
farmation of vacuole. Mitochondria of the macrophage are aiso visible.
bar=2 ,;im

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Electron micrograph of the section frcu n the nose. Parasites are in various

stages o･f degeneration. bar=5 ;Im** ･ {=>=' /** {: : "'. .. < " i=

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210

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Figure 11 Section from left footpad. A11 the parasites are in various phases ofdegeneration inside the vacuole of macrophage. bar=2 ,Imwith parasitophorous vacuoles (Fig. 7). Parasites were neither inside the mast cells nor in
the eosinophils or neutraphils.

Parasites were also faund in the liver and spleen of hamsters infected experimentallywith L, mexicana from Ecuador. They were lOcated within the macrophages but very few innumber. Regular degeneration of parasites was nated within, macrophages inside or outside
the vacuoles in all the cutaneous and visceral sections (Figs. 8, 9, 10 and ll) . M:orphological‑

ly, the normal amastigotes were rounded or elongated, surraunded by two layers of mem‑ranes and containing a rounded nucleus with a small nucleolus. The flagellum, fiagellar
pocket, kinetoplast, mitochondria, golgi apparatus, Iysosomes, vacuoles and electron‑dense
granules in vacuoles were distinguishable (Figs, 12a, b) .

21 1

Figure 12a, b Ultrastructure of the I.. mexicana parasite. F=flagellum, FP=ffagel‑lar pocket, K=kinetoplast, M=mitochondria, N = rtucleus with small

nucleolus, G*golgi apparatus, L*lysosomes, V=vacuoles and F̲g =electron‑dense granule inside the vacuole. bar=500 nm

212

DISCUSSION
After the autopsy of hamsters 6 months after inoculation with L. mexicana, we observed
shallow ulcers covered by thick crusts over the nose and both hind footpads in all the animals,confirming the finding that the hamster is a good animal model for L. mexicana infection(Wilson et al.. 1979; Bretana et al., 1983).

An understanding of the relationship between L. mexicana and the cells of the hostEcuadorian patient is essential to the elucidation of the pathogenesis of the disease in thatcountry. Observations of our specimens revealed that once the parasite is phagocytized bymacrophage it is enclosed by phagolysosome. Then, the phagolysosome starts to distend andinside it multiplication of parasites takes place (Berman et al.. 1979).

With regard to the development of experimental cutaneous lesions, Bretana et al. (1983)
proposed l) that toxic factors from the amastigote inhibit the action of lysosomal enzymes;2) that this causes the phagolysosome to become enlarged and new vacuoles to be invadedwith parasites; 3) that extensive enlargement of the vacuoles and certain other factors cause
the rupture of cell membranes; and 4) that repetition of this process results in ulceration at

the sites. From the observations of many stained specimens in this study, we concluded thata similar process of ulceration takes place in hamsters infected with L. mexicana from
Ecuador.
In the present light microscopic sections, Iarge numbers of L. mexicana amastigotes
inside the parasitophorous vacuoles were observed in the nose and footpad sections but it was
difiicult to find the amastigotes in the liver and spleen sections. The amastigotes in the liverand spleen were confirmed in ultra‑thin sections but the number was limited, indicating theactive multiplication of L. mexicana (dermatophilic) parasites in the inoculated sites (noseand footpads) but not in the liver and spleen. These findings suggest that although L.
mexicana inoculated to the nose and footpads of hamsters can disseminate from the cutaneoussites to the liver and spleen, they do not replicate in these organs.

Similar results have also been presented by Hill (1988) , who showed the possibility ofdissemination of L. amazonensis to the liver and spleen in two different strains of mice.
However, the metastasis (visceral dissemination) was not observed in C3H mice infectedwith L. mexicana (Grimaldi et al., 1980) . When the five different strains of mice were infectedwith L. mexicana, different responses were observed including the appearance of metastatic
lesions in the tail of BALB/C mice (Perez et al.. 1979) . Recently, L. amazonensis, well known

as a causative agent of human cutaneous leishmaniasis, was isolated from the bone marrowof a Brazilian patient with visceral leishmaniasis (Barral et al.. 1986) . With regard to L.
mexicana infection, therefore, visceral dissemination may be observed in humans in future
examinations as it was in the present animal (hamster) model.
There is controversy about the role of the macrophage in killing parasites intracellularly.

Previously, we found the degeneration of parasites inside the macrophages in human speci‑
mens (Bhutto et al.. 1992). In the present study, Iarge numbers of parasites also showeddegeneration within macrophages inside and outside the vacuoles in all of the cutaneous andvisceral specimens. This indicates the active role of macrophages in the destruction ofparasites intracellularly, although the destruction mechanism is unknown. Intracellulardegeneration of parasites inside macrophages has been reported in other human cases(Sandbank, 1976) and experimental works (Mauel et al.. 1978) . On the other hand, however,213

Bretana et al. (1983) showed that macrophages are incapable of destroying the parasites
intracellularly.

Immunocytochemical and electron microscopic studies in animals have suggested that T‑
cells provide lymphokines (cytokines) that can activate the host macrophage to destroy theparasites intracellularly, or that T‑cells play a cytotoxic role, killing the infected macro‑phages and helping to destroy the liberated extracellular parasites (McElrath et al., 1987) .
Sypek et al. (1984) believed that intracellular destruction of L. tropica can take place onlyby the direct cell contact mechanism between lymphocytes and macrophages. Moreover,
Murray et al. (1982) demonstrated the killing of L. donovani amastigotes inside macrophagesby the oxygen dependent mechanism and concluded that non‑activated phagocytes may
display effective microbicidal activity against intracellular parasites utilizing the oxygen

dependent mechanism.
From the present hamster and L. mexicana model, we propose that some unknown
factor/factors (as discussed above) under host immune mechanism (s) play an important roleand promote the destruction of parasites inside the macrophages, and therefore that effective
killing of the parasites finally might result in healing of the lesions. However, the precise
conditions and mechanisms of the destruction of parasites were not elucidated in the present

study. In our specimens, severe degeneration of amastigotes in large numbers of macro‑phages may represent a forward step in the healing process and may allow us to suggest apossible mechanism in the self‑healing of the disease. We did not, however, observe thecomplete healing of the lesions. Our experimental study using hamsters infected with L.
mexicana isolated from an Ecuadorian patient is a model of classic self‑healing of the
cutaneous form that adds to our understanding of the mechanism of the pathogenesis of
Ecuadorian leishmaniasis. On the basis of the results obtained, we conclude that macrophagecan destroy the parasites intracellularly under certain mechanism (s) and play an effective
and major role in the spontaneous healing of the disease.

ACKNOWLEDGEMENTS
We thank Professor Hikotaro Yoshida, Director of the Department of Dermatology,
Nagasaki University, for helpful discussions.

REFERENCES
1 ) Armijos, R.X., Chico, M.E., Cruz, M.E., Guderian. R.H., Kreutzer, R.D.. Berman, J.D., Rogers,

M.D. and Grogl, M. (1990): Human cutaneous leishmaniasis in Ecuador: Identification ofparasites by enzyme electrophoresis, Am. J. Trop. Med. Hyg., 42, 424‑428
2 ) Barral, A., Petersen, E.A., Sacks. D.L. and Neva. F.A. (1983): Late metastatic leishmaniasis

in the mouse. A model for mucocutaneous disease, Am. J. Trop. Med. Hyg., 32, 277‑285
3 ) Berman, J.D., Dwyer, D.M. and Wyler, D.J. (1979) : Multiplication of Leishmania in humanmacrophages in vitro. Infect. Immun., 26, 375‑379
4 ) Bhutto, A.M., Okada.S., Nonaka, S., Gomez L, E.A. and Hashiguchi, Y. (1992) : Ultrastructural

studies on cutaneous leishmaniasis in Ecuador, Jpn. J. Trop. Med. Hyg., 20, Il‑215 ) Bretana, A., Avila J.L., Lizardo, G., Convit, J. and Rondon, A.J. (1983): Leishmania species:

Comparative ultrastructure of experimental nodules and diffuse human cutaneous lesions in
American leishmaniasis, Exp. Parasitol., 55, 377‑385

2146 ) Grimaldi, G. Jr., Moriearty, P.L. and Hoff, R. (1980): Leishmania mexicana: Immunology andhistopathology in C3H mice, Exp. Parasitol., 50, 45‑56
7 ) Hashiguchi, Y., Gomez L, E.A., Coronel, V.V., Mimori, T., Quizhpe, A.M., Grimaldi, G. Jr.,

Kreutzer, R.D. and Tesh, R.B. (1991): Andean leishmaniasis in Ecuador caused by infection
with Leishmania mexicana and L. major‑like parasites, Am. J. Trop. Med. Hyg., 44, 205‑217
8 ) Hill, J.O. (1988) : Pathophysiology of experimental leishmaniasis: The role of parasite
physiology in the development of metastatic disease, Am. J. Trop. Med. Hyg., 39, 256‑260
9 ) Mauel, J., Buchmuller, Y. and Behin, R. (1978) : Studies on the mechanisms of macrophageactivation. I. Destruction of intracellular Leishmania enriettii in macrophages activated bycocultivation with stimulated lymphocytes, J. Exp. Med., 148, 393‑407
10) McElrath, M.J., Kaplan, G., Nusrat, A. and Cohn, Z.A. (1987): Cutaneous leishmaniasis. The
defect in T cell influx in BALB/C mice, J. Exp. Med., 165, 546‑559
11) Murray, H.W. (1982) : Cell‑mediated immune response in experimental visceral leishmaniasis.
II. Oxygen‑dependent killing of intracellular Leishmania donovani amastigotes, J. Immunol.,129, 351‑357
12) Perez, H., Labrador, F. and Torrealba, J.W. (1979): Variations in the response of five strainsof mice to Leishmania mexicana. Int. J. Parasitol., 9, 27‑32
13) Sandbank, M. (1976): Cutaneous leishmaniasis. Ultrastructural study of 3 cases, Arch. Derm.
Res., 257, 195‑201

14) Sypek, J.P., Panosian, C.B. and Wyler, D.J. (1984): Cell contact‑mediated macrophageactivation for antileishmanial defence. II. Identification of effector cell phenotype and genetic

restriction, J. Immunol., 133, 3351‑3357
15) Wilson, H.R., Dieckmann, B.S. and Childs, G.E. (1979): Leishmania braziliensis and Leishmania

mexicana: Experimental cutaneous infections in golden hamsters, Exp. Parasitol., 47, 270‑283

¥

215

実験的リーシュマニア症
一エクアドルで分離されたL廊h㎜η勿御観昭no感染ゴールデンハムスターの組織学的検討一

A．M．BHuTTo1・野中薫雄2・E．A．Gomez，L．3・橋口

義久4・古谷正人5

実験はゴールデンハムスターに，L廊h㎜n勿卿ε短の磁を感染させて行った。接種後6カ月目に屠殺した時，接種部位は厚い痂皮を付す浅い潰瘍となっていた。躯幹の他の部位には，病変を認めなかった。接種部位（鼻，足踪）標本の病理組織学的所見は，多数のamastigotes（AG），組織球，リンパ球の広範な浸潤が見られ，好中球，好酸球，プラズマ細胞を軽度混在していた。真

皮の肉芽腫性病変の上下では，マスト細胞の出現が著明であった。AGは病巣中央部のマクロファージにある，大きな空胞内に存在していた。また電顕的には，肝臓，脾臓にもAGが認められたが，皮膚に比べると数は少なかった。皮膚，および内臓のマクロファージ内で観察された寄生虫の破壊は，この細胞の貧食作用によるものと思われた。

長崎大学医学部皮膚科学教室（別冊請求先：〒852長崎市坂本1−7−1）
琉球大学医学部皮膚科学教室
Depaftamento

deMedicina

Guayaquil，Ecuador
高知医科大学寄生虫学教室
高知医科大学動物実験施設

Tropica1，Facultad

de Medicina，Universidad Catolica，SantiagodeJpn. J. Trop. Med. Hyg., Vol. 20, No. 3, 1992, pp. 217‑218

217

Short communication

SERUM C‑REACTIVE PROTEIN (CRP) VALUES OF
PATIENTS INFECTED WITH TAENIA SAGINATA
KENJI OHNISHI* AND MISAKO MURATA
Received April 3 1992/Accepted May 26 1992

Elevated serum C‑reactive protein (CRP) values are identified in patients with infec‑
tions, some tumors or various types of tissue destruction, and clinical measurement of serum
CRP is valuable as a screening test for organic diseases and as a sensitive index of diseaseactivity and response to therapy in the disease mentioned above (Pepys, 1981) . But, to ourknowledge. CRP has been given but scant attention in cases of tapeworm infection. Human
infection with Taenia saginata is uncommon in Japan and clinical manifestations of thistapeworm infection are mild or symptomless, but infection with T. saginaia is an importantparasitic diseases because of its world wide distribution.

From April 1987 to March 1991, we treated 5 patients with T. saginaia infection. All of

the patients were Japanese males aged 23 to 66 years old. None of them had a previous
history of the tapeworm infection. All of the infections were asymptomatic, except for the
discomfort and embarrassment occasioned by the crawling of proglottids from the anus. The
place of contraction of 3 patients was Ethiopia, I patient Japan and I patient unknown. A11of the patients expelled a single T. saginata with intraduodenal gastrographin injection ororal administration of paromomycin sulphate. Before the therapy, serum CRP Ievels were
measured with a Behring Nephelometer Analyzer with CRP‑Latex reagent kit.
The serum CRP value of all patients was under 0.3 mg/dl and this value was in normal
range. The results are shown in Table 1.
The results reported here show that CRP values of patients infected with a single T.
saginata are in the normal range. It has been reported that CRP was found in a human liver

Table I Pre treatment serum CRP value in patients
infected with a single intestinal T. saginata

Case Age (years) Sex Place of CRP (mg/dl)
contraction
34
2

32

3

23

4

26

5

66

M
M
M
M
M

Ethio pia

Japan
Ethio pia

Ethiopia

unknown

<0.3
<0.3
<0.3
<0.3
<0.3

Departtnent of Infectious Diseases, Tokyo Metropolitan Bokutoh General Hospital, 4‑23‑15
Kohtohbashi, Sumida‑Ku, Tokyo 130, Japan (*correspondent author)

218

culture but not in bone marrow，spleen，thyroid，mammary gland or thoracic duct
lymphocytes in culture（Hurlimann6」召乙，1966）and that interleukin−1（IL−1）andinterleukin−6（IL−6）both

stimulated

liver

synthesis

of

CRP

and

that

probably

Iレ6playedakeyroleinstimulationofCRP（Hazenbergα認，1988）．Theresultsofthesereportsindicate
that

by

the

liver

is

interleukins

animportantstimulated

by

organ

for

tissuedamage。T

intestinal

producing

CRP

and

that

the production

s4g初α如，whose headby attaching

to the small

sαg初α如maynotinjury totheintestinal

singleTsαgゴnα如issotapeworm

does not stimulate production

of

CRP

liver

has

is stimulated

four suckers

lives

wall．Our results suggest that single infections withT

induce interleukins sufncient to increase the CRP value．It is thought that
wall

by

a

in themild

thatintestinal

infectionbytheto clinically detectable levels．

Wethink

that ifanelevatedserumCRPvalueisobservedinanasymptomaticpersoninfectedwith a T

sα8 伽α如，other abnormal

disease shouldbeaccompanying

conditions

such

as

neoplasmaorcollagenandYap，SH。（1988）：The

considered．

REFERENCES
1）Hazenberg，B．P．C．，vaneffectandLeewen，M．A．，Limburg，P．C．，Aarden，LAof interleukin−1，interleukin−6andC−reactive protein inits

interrelation

primary cultures

of

on

adult

the

human

synthesis

of

serum

amyloidA

hepatocytes，Biochem．Biophys．

Res．Commun．，30，112−117
2）Hurlimann，J．，Thorbecke，G．J．andproteinMochwald，G．M．（1966）：The

liver

aもthe

site

ofC−reactive

formation，J．Exp．Med．，123，365−378

3）Pepys，M．B．（1981）：C−reactive

protein nfty years

on，Lancet，i，653−657

無鉤条虫感染症における血清CRP値大西

健児・村田三紗子

単数寄生の無鉤条虫感染症患者5人で，治療前血清CRP値を測定した。症例は全員日本人男性
で，片節の排出時の不快感以外に症状はなかった。結果は全例で＜0．3mg／dJであり，正常範囲にあった。このことから，単数の無鉤条虫の腸管寄生は，血清CRP値を上昇させる原因とはならな
いと考えられた。腸管の単数の無鉤条虫感染症で血清CRP値の上昇を認めた場合，合併症の検索が必要と考えられる。

東京都立墨東病院感染症科（〒130東京都墨田区江東橋4−23−15）

ISSN 0304‑2146

JAPANESE JOURNAL OF
TROPICAL MEDIClNE AND HYGIENE
September 1992

Vol. 20 No. 3

CONTENTS
Original article

Sato, Y., Shiroma, Y., Kiyuna, S., Toma, H. and Kobayashi, J.

Reduced Effect of Chemotherapy of Strongyloidiasis in Patientswith Concurrent HTLV‑ I Infection in Okinawa, Japan """""""""""

183‑192

Okumura, E., Hashimoto, K., Mizuta, N., Hata, M., Okumura, Y.

and Okamura, Y.
Analysis of Drinking Water in Airports, Airplanes andPublic Water Facilities """"""""" """"""""" """ """ " "" "193‑201

Bhutto, A. M., Nonaka, S., Gomez L., E. A., Hashiguchi, Y. and Furuya, M.

Histopathological Observations of Golden Hamsters Infected with an

Ecuadorian Isolate of Leishmania mexicana """"""""""""""""""""

203‑215

Short communication
Ohnishi, K. and Murata, M.
Serum C‑reactive Protein (CRP) Values of Patients Infected with

Taenia saginata """""""""""""""""""""""""""""""""""""""

Jpn. J. Trop.

Med. Hyg.

JAPANESE SOCIETY OF TROPICAL MEDIClNE

217‑218