■圏日王

第25巻第3号平成9年9月
内
原

容

著
ラオスにおけるメチシリン耐性黄色ブドウ球菌の出現（英文）一岩永 正明，Lay Sisavath，比嘉
恒花 シゲ

直美，本馬

恭子，

103−106

抗アレルギー薬，ケトティフェンはラットにおける八吻φOS加野1％S6瓶Sll歪6％傭感染によって誘導される肺好酸球浸潤を抑制する（英文）

一西田
稔，山田
稔，内川 隆一，松田．手越 達也，笹部 真人，有薗 直樹

信治，
10H12

北タイにおけるコガタハマダラカ固体群に対する20％フェニトロチオン
マイクロカプセル剤の残留噴霧野外試験（英文）一Wamapa Suwonkerd，Somsak
津田 良夫，高木 正洋

Prajakwong，

二酸化塩素溶液のC別ρ如砂on漉襯のオーシストに対する保存効果……………一…・一………松井 利博，藤野 隆志，小林富美江，辻会

報
1997年度日本熱帯医学会会員名簿113−115守康

117−120一121−147■■

103

Jpn. J. Trop. Med. Hyg., Vol. 25, No. 3, 1997, pp. 103 106

EMERGENCE OF METHICILLlN. RESISTANT
STAPHYLOCOCCUS A UREUS IN LAOSMASAAKI IWANAGAl'2, LAY SISAVATH', NAOMI HIGAl,

YASUKO HONMAI AND SHIGE KAKlNOHANA3
Received April 18, 1997/Accepted August 4, 1997

Abstract: Staphylococcus aureus isolated in Laos (People's Democratic Republic of Lao = Lao PDR) was
examined for the drug sensitivities and coagulase type. A total of 114 strains examined consisted of 32
isolated in 1995 and 47 isolated in 1996 from the infection focus, and 35 isolated from nasal mucosa of non‑

infected patients and nurses workingj in the wards. One strain with coagulase type IV was regarded asmethicillin‑resistant S. aureus (MRSA) depending on the minimum inhibitory concentration of the drugs
as > 100, 100, 100, > 100, and 25 /t g/ml of methicillin, ampicillin, tetracycline, erythromycin, and cefdinir,respectively. The mecA gene was detected in this strain but not in the others. This is the first report of

MRSA from Lao PDR.

INTRODUCTION
Although methicillin‑resistant S. aureus (MRSA)
was detected soon after the iritroduction of methicillin in

Hospital. Vientiane, Lao PDR was used. The organisrns
included 79 strains isolated from various clinical speci‑
mens in 1995 and 1996, and 32 strains isolated from thenasal mucosa of the staff nurse and in‑patients without

1960 (Barrett et al., 1968), a marked rise in the fre‑quency was seen in 1980s. In Japan, increasing of its

infectious disease in 1996.

isolation coincided with increasing use of third‑genera‑tion cephem antibiotics. Since the late 1980s, more than
50% of S. aureus isolates at the sophisticated hospitals
in Japan has been resistant to methicillin. Therefore,
the careful use of these antibiotics is now recommended.

produced by the isolates .was examined by a neutraliza‑

In contrast, MRSA is rarely isolated in communities
where antibiotic use is restricted. In People's Demo‑cratic Republic of Lao (Lao PDR) , all S. aureus isolated

at Mahosot Hospital (the most sophisticated hospital in
Lao PDR) in 1993 were sensitive to methicillin (Higa etal., 1994). However, urbanization of Lao PDR, espe‑
cially Vientiane the Capital of Lao PDR, has been rapid‑

ly advanced. And antibiotics are more available in themarket. In these situation, the ernergence of MRSA
should be carefully monitored. In this communication,

Coagulase typing: Antigenic type of coagulasetion test using the Coagulase Typing Immune Sera Kit(Denkaseiken Co. Tokyo) . Coagulation inhibition byadding antisera was examined using plastic microdilu‑
tion plates (Tajima et al., 1992).

Drug sensitivity test: The sensitivities of the iso‑lates against methicillin (DMPPC, Banyu) , ampicillin(ABPC, Meiji) , tetracycline (TC, Wako) , erythromycin(EM, Dainihon) , and cefdinir (CFDN, Fujisawa) were
exarnined by using the plate dilution method, and thedrug activities against the organisms were expressed byminimum inhibitory concentrations (MICs) of the drug.
Twofold dilution series of each antibiotics in heart
infusion agar (HIA) were prepared with the drug con‑centration ranging frorn 100 to 0.2 pg/ml. Cultures ofof S. aureus isolated at Mahosot Hospital in 1995 andthe isolates in heart infusion broth at 37'C for 6 hr werediluted I to 10 with normal saline solution (ca. 107/ml)

1996.

and were inoculated on the drug‑containing HIA plates

we described the drug sensitivities and coagulase typing

MATERIALS AND METHODS
Bacterial strains: S. aureus isolated at Mahosot

and drug‑free control plates by using Microplanter(Sakuma Co. model MITP # 00257) . MICs of each drug
were evaluated after 24 hr incubation at 37'C. For thesensitivities to methicillin, the concentration of NaCl in

1 Department of Bacteriology, 2 Research Center of Comprehensive Medicine, 3 Department of Fundarnental Nursing, Faculty of
Medicine, University of the Ryukyus, 207 Uehara, Nishihara, Okinawa 903‑01, Japan
4 National Institute of Hygiene and Epidemiology, Vientiane, Lao People's Democratic Republic

104

drug‑containing HIA was adjusted to 4%, and the cul‑
ture was carried dut at 30'C for 24 hr.

Detection of mecA gene: mecA was detected byusing PCR analysis. The 24‑hour cultured colonies onnutrient agar plates were suspended in distilled waterand then boiled for 20 min, which was regarded as the
samples for DNA of interest. The PCR primers used for

detecting mecA were 5'GAACCTCTGCTCAACAAGTT3'
and 5'‑GGATTTGCCAATTAAGTTTG‑3* as describedby Song et d. (1987) and re‑examined by Muraki et al.
(1993). These were designed to amplify a 630 bp frag‑ment derived from a portion of the mec'A gene encoding1 )BP2' which has been shown to be the entity of MRSA.

RES U l.TS

isolates, the growth of 3 isolates were inhibited at 12.5
/lg/ml or more, and I of them was resistant to methicil‑
lin at 100 ,tg/ml. MICs 'of the other drugs against the 2isalates were O.8 and 3.13 /lg/m[ of ABPC, ‑=0.2 and 0.8,eeg/ml of CFDN,

0.2 and 0.4 ;lg/ml of TC, and >lOO

and < +･=0.2 l'g/ml of EM, respectively. MICs of the one
isolate resistant to DMPPC at 100 ;tg/ml were 100 ,ugl

ml of ABPC, 25 /tg/mf of CFDN, 100 ;lg/ml of TC,
and > 100 ,zlg/ml of EM. Coagulase type of this strain
was type IV. These results are summarized in Tables l>
2, 3.

Detection of mecA gene: All isolates except one
were negative for mec'A. Only one strain, against which
the MIC of methicillin was > 100 ,ctg/ml, possessed thegene (Fig. l).

Coagulase type: The distribution of coagulase typevaried, but type V was predominant. In 1996, 63.8% of

MIC (; g/ml)

the isolates from infection sites revealed type V but only

TC

EM

<=0.2

o

6

9

27

ll

28.6% from the nasal mucosa of non‑infective individ‑o . 39

o

o

o

2

16

uals. Type 11 strains were faund in 4.3% of the isolateso . 78

o

4

o

o

4

from the infection, whereas 25.7% from the nasal

1 . 56

1

2

o

o

o

3.13

19

12

o

o

o

6 . 25

12

6

7

o

o

good susceptibilities agairist the drugs examined. A few

12 . 5

o

2

8

o

o

strains were highly resistant to erythromycin. The MIC

25

o

o

8

O

o

of methicillin vfas 6.25 ;xg/ml or less against almost all

50

O

o

o

o

1

l OO

o

o

o

O

o

> 100

O

o

a

3

o

mucosa of noninfected individuals.
Drug sensitivities: Generally, the isolates showed

12345678

Table lDrug sensitivities and coagulase type

DMPPC ABPC

CFDN

coagulase type I

II

III IVv

VI VII VIII UT

No, of strains

7

1

16

l

o

l

5

o

1

32 isolates from infection focus (1995)

Table 2

Figure l 630 bp fragment of mecA gene detected as the PCR

product. Lanes: I =molecular marker, 2=LC13 (>lOO feeg/inl, MRSA), 3=LC18 (12.5 pg/ml}, 4‑
95P1 (6.25 pg/mt), 5=96P5 (12.5 ,lg/ml), 6=Rl(> 100 ,lg/ml) . 7=R4 (100 / g/ml) , 8=R12 (>lOO'lg/ml). MICs of methicillin to each strain wete

Drug sensitivities and coagulase type

DMPPC ABPC

TC

EM

CFD N

̲ *‑..o.2

o

l

24

16

32

o . 39

o

a

l

l

14

o . 78

o

4

o

23

l

1 . 56

4

31

o

6

o

3 . 13

23

8

o

o

o

6 . 25

19

o

o

o

o

l

o

5

o

o

25

o

2

10

1

o

50

o

o

7

o

1

lOO

o

1

a

o

o

> 100

o

o

o

3

o

MIC (pg/m l)

12 .

;

indicated in parenthesis. Lanes 2 to 5 are of Laostrains and lanes 6 to 8 are of Japanese strains.

coagulase type I

ll

III IVv

VI VII VIII UTOnly one Lao strain (LC13) of 114 examined was

No. af strains

2

4

30

o

positive for mecA .

o

2

3

1

5

47 isolates from inf, ection focus (1.996)

105

possessing mecA gene is the first reported MRSA in Lao

Table 3 Drug sensitivities and coagulase type

MIC (pg/ml) DMPPC ABPC TC

EM CFDN
30

14

o

1

19

o

o

o

6

o

o

1

24

13

1

o

o

6 . 25

4

3

7

o

o

12 . 5

1

2

5

o

o

25

o

o

3

o

1

50

o

1

o

o

o

100

o

1

1

1

o

> 100

1

o

o

3

o

0.2

1

o . 39o . 78

3

18

o

1

1

5

1 . 56

3

3 . 13

coagulase type I 11 111 IV V VI VII VIII UT

No. of strains O 9 4 2 10 O 5 2 3

35 isolates from nasal mucosa (1996)

DISCUSSIONMRSA is a major organism involved in npsocomial
infection. It is recognized that the increased isolation
frequency of MRSA is associated with the use of antibi‑

otics, especially the third generation cephems. We
examined S. aureus isolated in Lao PDR in 1993, and
MRSA was not found (Higa et al., 1994). According to
the hospital and drug stores in the city (we got the
information through the questionaire) , the restrictedkinds and amount of antirnicrobials such as penicillin (Gand V), ampicillin, tetracycline, gentamicin, eryth‑romycin, sulfamethoxazol‑trimethoprim cornpound andnalidixic acid, were used in the country before 1993.
However, since then, the types and amount of consumed
antimicrobials increased with amoxicillin, chloram‑
phenicol, cloxacillin, doxycycline, Iincomycin, and oflox‑

acin. The drug susceptibility of S. aureus looks getting
resistant little by little. A11 54 isolates from the infection

focus in 1993 were inhibited by methicillin at the concen‑

tration of 6.25 pg/ml including 7% of the isolates inhib‑

ited at 6.25 pg/ml, and most strains were inhibited at

3.13 pg/ml. However, 37% of the 32 isolates in 1995
were inhibited at 6.25 pg/ml, and there was no strain towhich the MIC of methicillin was 12.5 pg/ml or more.
In 1996, 42% of the 47 isolates were inhibited at 6.25 pgl

ml or more, including one strain inhibited at 12.5 pglml

of methicillin. However, the 35 isolates (in 1996) from

non‑infected patients and the nurses working at thewards included 4 strains (11%) inhibited at 6.25 pglml,
one strain at 12.5 pg/ml, and one highly resistant strain(resistant at 100 pg/ml). This highly resistant strain

PDR.
The coagulase types of MRSA recently isolated inJapan were mostly type II, but that of the first reported

MRSA from Lao PDR was type IV. It reminds us of thefact that, in the beginning of 1980s when the problem of

MRSA was recognized in Japan, the coagulase type of
MRSA was mainly type IV (Matsumoto et al., 1984).
The small percentage of coagulase type 11 in Lao PDR
may be due to the rare occurrence of MRSA at present
in this country.

This emergence of MRSA in Lao PDR is likely aresult of the increased use of a various antimicrobials.

In this study, only one isolate of MRSA was found in a
non‑infective patient, but not in the patient with Sta‑phylococcal infection. It may be due to small number ofparameter (Number of Patient examined) , because theisolation frequency of drug resistant organism supposed
to be higher in the patients with antibiotic treatment
than those without antibiotics. Therefore, it is now
important to start surveillance to control MRSA in thiscountry.

ACKNOWLEDGEMENTS
We thank the staff of Mahosot Hospital for collect‑
ing bacterial strains.

REFERENCES
1 ) Barrett, F.F., McGehee, R.F. and Finland, M. (1968):
Methicillin‑resistant Staphylococcus aureus at Boston
City Hospital. New Engl. J. Med., 279, 441‑448
2 ) Higa, N., Sithivong, N. and lwanaga, M., (1994): A
comparative study on Staphylococcus aureus isolated in
Lao PDR and in Japan. Jpn. J. Trop. Med. Hyg., 22,
129‑1313 ) Matsumoto, K., Kudo, K., Uzuka, Y., Watanabe, K.,
Nagatake, T., Rikitomi, N., Takahashi. A. and Suzuki,
H. (1984): The pathogenic strains of Staphylococcus
aureus lately isolated in Japan. Chemotherapy, 32, 527‑
5334 ) Muraki, C., Taishi, K., Yarnashita, K., Otsuka, N.,

Kagawa, S. and Matsuoka, A. (1993): Detection ofmethicillin‑resistant Staphylococcus aureus using PCR
and non‑radioactive DNA probes. Jpn. J. Clin. Pathol.,41, 1159‑1166
5 ) Song, M. D., Wachi, M., Doi. M., Ishino, F. and Matsuha‑shi, M. (1987) : Evolution of an inducible penicillin‑
target protein in methicillin‑resistant Staphylococcus
aureus by gene fusion. FEBS Iett., 167‑171

6 ) Tajima, Y., Nagasawa. Z., Tanabe, I., Yamada, H.,

Kusaba, K., and Tadano, J. (1992): An irnproved

106

method for the serotyping of free coagulase from Sta‑phyl ococcus aureus. Microbiol. Immunol., 36, 1233‑1237

J pn. J. Trop. Med

Hyg Vol 25 No 3 1997, pp. 107‑112

107

KETOTIFEN, AN ANTIALLERGIC AGENT,SUPPRESSES PULMONARY EOSINOPHILIAINDUCED BY THE NEMATODENIPPOSTRONG YL US BRASILIENSIS IN RATS
MlNORU NISHIDA, MlNORU YAMADA, RYUICHI UcHIKAWA, SHlNJI MATSUDA,TATSUYA TEGOSHI, MASATO SASABE AND NAOKI ARIZONO
Received May 14, 1997/Accepted July 17, 1997

Abstract: Certain parasitic infections such as ascariasis and hookworm diseases occasionally cause a typeof Loffler's syndrome. This syndrome, which consists of transitory and migratory pulmonary infiltrations
with eosinophilia, is presumed to be triggered by hypersensitivity reaction. Previously, we showed that
infection with the' nematode Nippostrongylus brasiliensis induces similar lung lesions, that are markedly
enhanced after challenge infection. To examine the role of mast cells in nematode‑induced pneumonitis, N.
brasiliensis‑primed Brown Norway rats received continuous intravenous administration of the mast cellstabilizer ketotifen (6 mg/day/kg body weight) from one day prior to 3 days after challenge infection.
Significantly larger nurnbers of rat mast cell protease (RMCP) II‑positive mast cells were identified in thelungs of ketotifen‑administered animals than in saline‑perfused animals, suggesting that ketotifen treatment
significantly suppressed mast cell activation. Morphological analysis of lung sections showed that ketotifen
adrninistration significantly suppressed eosinophil infiltration. Areas of lung granulomas, also triggered bynematode infection, were smaller in ketotifen‑treated than in non‑treated animals, although this effect was
not statistically significant. The worm recoveries from the srnall intestine did not differ between ketotifen‑
treated and non‑treated animals, indicating that the agent did not hinder lung migration of worms. These
results suggest that mast cells play an importaht role at least in the development of mematode‑induced lungeosino philia.

Key words: eosinophil, mast cell, Iung, rat, Nippostrongylus, nernatode, ketotifen, L ffler's syndrome

INTRODUCTION
Infection with certain intestinal nematodes, such as
Ascaris lumbricoides. Strongyloides stercoralis and hook‑worms occasionally causes pulmonary infiltration with
peripheral blood eosinophilia recognized as a type ofL6ffler's syndrome (Gelpi and Mustafa, 1968; Phills etal.. 1972; Spillmann, 1975). The lung lesions appear to
be associated with migrating nematode larvae, as thelarvae transiently pass through the lungs and trachea

parasites, infection with the rodent intestinal nematode
Nippostrongylus brasiliensis induces marked infiltrationof eosinophils, an increase in the numbe of alveolar

macrophages, development of granulomas, and anincrease in the number of mast cells in the lungs(Talliaferro and Sarles, 1939; Arizono et al., 1987;
Ramaswamy et al.. 1991). Therefore, N. brasiliensis
infection in rodents provides a suitable model in which

to examine the mechanisms of nematode‑induced
pneumonitis.

before infesting the final habitat, the intestine. These

In a previous study, we showed that geneticallypatients usually have elevated lgE antibody levels andsymptoms consisting of dyspnea, often of an asthmatic
type, suggesting that hypersensitivity mechanisms might
be involved in the development of nematode‑associatedlung lesions (Phills et al.. 1972). Like these humanmast cell‑deficient Ws/ Ws rats exhibited significantly

lower levels of eosinophil infiltration and granuloma
formation in the lungs than in normal + / + rats after N.

brasiliensis infection, although elevated levels of lgEantibody did not differ between Ws/ Ws and + / + rats,

Department of Medical Zoology, Kyoto Prefectural University of Medicine, Kawaramachi‑Hirokoji, Kyoto 602, Japan

108

suggesting that mast cells play an important role forcells and shows H1‑selective histamine antagonism

inserted. A sterilized silicone tube with a 0.5‑mm innerdiameter was inserted into the jugular vein between the
sutures and advanced into the superior vena cava. The
silicon tube was then secured with the silk sutures. The
free side of the silicone tube was passed through the
subcutaneous tissue into the occipital region where askin incision had been made, and secured to the exit siteusing a specifically constructed stainless steel appara‑(Martin and Roemer, 1977; Craps and Nay, 1984).

tus. The tube outside the animal was covered by atriggering the lung lesions (Arizono et al.. 1996) . In thepresent study, we further investigated whether an anti‑
allergic drug, ketotifen, would suppress the nematode‑
induced lung lesions in rats. Ketotifen { 4‑ (1‑methyl‑4‑
piperidylidene) ‑4H‑benzo[4,5]cyclohepta [1, 2‑b] thio‑phen‑10 (9H) ‑one hydrogen fumarate} stabilizes mastspring coil, and the free end of the tube was attached toSea) rats were purchased from Seiwa Experimental

a swivel from which a second tube was connected to an
infusion pump (STC‑501; Terumo. Tokyo. Japan) . Thismethod allowed the rat to move freely during infusion.
Animals were infused with ketotifen (fumarate salt,
Sigrna Chemical Co.. St. Louis. Mo., USA) dissolved in

Animals Co. (Fukuoka, Japan) . The animals, 8 weeks
old (body weight 180‑190 g) , received a subcutaneous

saline at a dose of 6 mg/daylkg, or saline as a control,
at a rate of 0.2 ml/hr., Twenty‑fours hour later, these

injection of 2,000 infective‑stage larvae of N. brasiliensis

animals were infected subcutaneously with 2.000 N.

as described previously (Arizono et al.. 1996). A groupof animals were sacrificed 28 days after infection. Other

continued for a further 72 hr and then animals wereanimals were divided in 2 groups, one subjected tosacrificed by overdose of ether.

ketotifen infusion and the other to saline infusion as acontrol (see below) , and the latter 2 groups received aHisto logy

MATERIALS AND METHODS
Animals and nematode infection
Specific pathogen‑free, male Brown Norway (BN/challenge infection with 2,000 infective‑stage larvae on
day 28 after the primary infection.

Passive cutaneous anaphylaxis (PCA) reaction
Naive rats were injected intradermally with 50 pl ofserially diluted test sera obtained 4 weeks after N.
brasiliensis infection or with uninfected rat sera. Forty‑

six hours later, these animals received intravenous injec‑

tions of various doses of ketotifen. Two hours after
ketotifen administration, each animal received intra‑
venous injection of I mg of N. brasiliensis adult worm
somatic antigen (Yamada et al.. 1991) and 100 mg ofEvans blue dye dissolved in I ml of saline. The blueingof sensitized skin was measured 30 min later and spots
more than 5 mm in diameter were regarded as positive.

brasiliensis L3 Iarvae. Ketotifen or saline infusion was

Lungs were removed and fixed for 3 hr in Carnoy's
fluid or for 48 hr in 4% formalin in 0.1 M phosphatebuffer, pH 7.3. Formalin‑fixed paraffin‑embedded lungsections were subjected to staining with hematoxylin

and eosin (H‑E) , or with carbol‑chromotrope for
eosinophil identification (Lendrum, 1944). Mast cells
were stained in Carnoy's fluid‑fixed paraffin‑embedded
sections for 30 min with 0.5% alcian blue dissolved in
0.5 N HCl.
Immunohistochemical staining of rat mast cell protease(RMCP) II
RMCP 11 immunostaining was carried out as de‑scribed previously (Arizono et al.. 1994). Briefly, thescribed by Steiger et al. (1972) with some modifications.

lung tissues were fixed in phosphate‑buffered 4% forma‑lin (pH 7.3) for 48 hr. Paraffin sections were cut at
4 pm and endogenous peroxidase activity was blockedwith 0.3% H202' Sections were incubated in 10% nor‑mal goat serurn and then successively incubated withrabbit anti‑RMCP 11 serum, and with horseradish perox‑

Animals which had been infected with N. brasiliensis 27

idase‑conjugated goat anti‑rabbit lgG serum (Zymeddays previously were anesthetized with Nembutal R(intraperitoneal injection, 25 mglkg) and small skinLab. Inc., San Francisco, CA, USA) . Finally, the DAB
reaction was performed. As a negative control, tissue
sections obtained from congenitally mast cell‑deficientWs/ Ws rats were used.

Continuous intravenous infusion of ketotzfen

Long‑term intravenous infusion of ketotifen inunrestrained rats was carried out by the method de‑incisions were made in the occipital as well as in theright cervical region. The right jugular vein was iso‑lated and ligated with a silk suture. A second suture
was placed around the vein 1.0‑1.5 cm proximal to theheart and was left untied until the catheter had beenCounts of al cian blue positive‑ or RMCP II positive‑
mast cells in lung sections

109

Fields of vision in the alcian blue‑stained or RMCPSALINE I KETOTIFEN

II immunostained lung sections were transcribed todigital images using video microscopy (HC‑2000D, FUJI
FILM, Tokyo, Japan) . Alcian blue positive‑ or RMCP
II‑positive mast cells were counted using Mac Scope

400

400

E
300
E

300

¥
J
J
UJ
u 200
<
=:

image processing software (MITANI Co., Fukui,
animal.

E:
ao
:
:D

UJ 100Counts of eosinophils in lung sections

phils were counted on the photographs and numbers of
eosinophils/mm2 were determined. A total lung area of
0.86 mm2 was analyzed per animal.

200

1 OO

z

Ten percent buffered formalin‑fixed, carbol‑
graphed and printed at a magnification of X 180. Eosino‑

rTl

'l)

Japan) . A total lung area of 5.99 mm2 was processed per

chromotrope‑stained lung sections (4 pm) were photo‑

B

o

o

Figure 1 Nurnbers of alcian blue‑positive (A) and RMCP II‑
positive (B) mast cells in the lungs of rats 3 days

after challenge infection with N. brasiliensis.
Ketotifen (6 mg/daylkg) or saline was infusedfrom one day before to 3 days after the challenge

infection. Columns and bars represent means

Measurement of lung granulomas
H‑E‑stained lung sections were photographed andprinted at a magnification of >< 13. The areas of
granulomas were measured with an electric digitizer(Two‑dimensional measuring software, Nikon cosm‑
ozone; Nikon, Tokyo. Japan) . Granuloma areas wereadded and total granuloma area/mm2 of lung section
was determined.
Counts of worms in the small intestine
The small intestine was removed from infected rats,
opened longitudinally and incubated at 37'C in normal

saline for 3 hr. The numbers of emerging worms werecounted under a dissecting microscope.

SE of 4 rats. *Significantly different (P <0.01).
Statistical analysis

Student's t‑test was used to evaluate the signifi
cance of differences.

RESULTSThe effective suppressive dose of ketotifen on
immediate hypersensitivity was examined using PCA
reaction. Two hours after ketotifen injection, PCA
reaction was suppressed most significantly with 0.2 or O.

5 mg/kg ketotifen (Table 1). The suppressive effect of
ketotifen became less distinct when ketotifen was ad‑
ministered 3 or 7 hr before the antigen challenge, and it

Table 1

Suppression of PCA reaction by ketotifen.
which had received intradermal injection of seri‑Thus, a continuous intravenous perfusion method was
employed with ketotifen at 6 mg/daylkg, a dose corre‑

ally diluted N. brasiliensis‑infected rat sera 46 hr

sponding to administration of 0.5 mg/kg ketotifen every

previously. These animals were challenged with

2 hr.

Ketotifen was intravenously injected into rats

intravenous injection of the worm somatic

N. brasiliensis‑primed animals were perfused con‑antigen 2 hr later, and PCA titers were deter‑
mined. Injection of uninfected‑rat sera showedtinuously with 6 mg/day/kg of ketotifen from 24 hr
before challenge infection to 3 days after challenge

PCA titers less than 24

AnimalNo.
1

was totally abolished when ketotifen was injected 24 hr
before challenge, indicating that the suppressive effectof ketotifen was rather short‑1ived (data not shown) .

Ketotifen Dilution of sera

(mglkg) 5 26 2' 8 29
O2

'O . 05

3

0.2

4

0.5

5

1.0

6

2.5

24

2

+
+
+
+
+
+

+
+
+
+
+
+

2

+
+

+

+

infection. The nurnbers of alcian blue‑stained lung mast
cells in ketotifen‑treated animals were not significantly

different from those in saline‑perfused animals (Fig.
IA) . In contrast, numbers of RMCP II‑positive mast
cells in ketotifen‑treated animals were significantly
larger than in control animals (Fig. IB) , suggesting that++ketotifen treatment suppressed mast cell activation thatallowed more mast cells to retain RMCP 11 in thecytoplasmic granules. In fact, high power microscopic

l 10

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* '=

'

' ";{ "I '̲:̲ ̲̲ ̲̲''; / ' ; '*"*'*""*'**' " '/'i"'i'/=' *i:* '* *" ** **"*"" '= "'""*"/i;=..

* '=***"'･" ･･:･ . i:: ･.. *,･S;."* *+Figure 2 Immunostaining of RMCP IIpositive mast cells in the lungs after challenge
infection with N. brasiliensis. Animals received saline (A or ketotifen (B)from one day before to 3 days after the challenge. infection. In saline‑

perfused animals, some of the mast cells showed degranulation (arrowheads) , while mast cells in ketatifen‑treated animals did not. Magnifica‑tion, x600.

observations of RMCP 11‑immunostained lung sections Viability of L3 Iarvae was also not affected in thefrequently showed mast cell degranulation in saline‑ presence of ketotifen.

perfused animals, while not in ketotifentreated anhTlals

j SALiNE

i KETOTIF N(Fig. 2) .

Eosinophil infiltration in the lungs was examined onreoocarbol‑chromotrope‑stained lung sections. After chal‑

**

*

rl
,:<{i;=' i'*';:; ( * :;;*lenge infection, numbers of eosinophils in the lungs wereEsignificantly increased in salineperfused animals, whileeosinophil numbers did not increase in ketotifen‑treatedanimals (Fig. 3).

V¥
SJ 400l:O: '+"**="++*+'*

Ci ; :"""*. ' ;' ‑Q*

Lung granulomas developed after N. br( ;siliensis
challenge infection (Fig. 4) . The lesions consisted ofhistiocytic cells, fibroblasts, and lymphoid cells withoccasional appearance of multinucleate giant cells.

a:itions (Fig. 5).

*!* i** * ,, ',i='*:'+;'; ' *200+**=* <==Z:)*/;;=:#!:,:= ;{: =*;:;i; 'i'

+= '

o

Some of the eosinophils also appeared in thesignificant because of the large variations in the reac‑

:

(:)Lu
+

granulomatous lesions. A'reas of lung granulomas in
ketotifentreated animals were sualler than in saline‑
perfused animals, although this was not statistically+*;i>i==*= *'**=o

3

DAYS AFTER INFECTION

Figure 3

Numbers of eosinophils in the lungs befcre and 3days after challenge infection with N. brasil iel esis.
Ketotif en(6 mg/daylkg) or saline was infused

f rom one day before to 3 days after the challenge

directly affected N brasiliel sis and suppressed themigration of worms into the lungs, numbers of worms in

The data before challenge infection
obtainedfrom rats at 28 days after primarywere
inf ection. Coiumns and bars represent means i SE
of 4 rats. Significance of differences (*p < O.05,

the small intestine 3 days after challenge infection were** P < O.Ol) .

To exclude the possibility that ketotifen might haveinf ection.

counted. L4 Iarvae were recovered from the small
intestine without siguificant differences in the numbers

between ketotifen‑treated and non‑treated animals(Fig. 6). Further. N. brasilie zsis L3 Iarvae and adultworms were incubated at 37'C with various doses of
ketotifen dissolved in saline. Approximately 90% of
adult worms showed active mobility 8 hr after incuba‑tion and 80% after 24hr incubatl'on regardless of thepresence or adsence of ketotifen from O to 2 mg/ml.

DISCUSSION
The principal pharmacological eftect of ketotifen
has been reported to be a stabilization of mast cells,
prohibiting release of chemical mediators such as his‑
tamine and leukotrienes from the cells. In addition,ketotifen is an I･･11‑selective histamine antagonist(Martin and Roemer, 1977; Craps and Nay, 1984) . In the

111

Figure 4 Granulomatous response in the lungs after challenge infection with N.
brasiliensis. Animals received saline (A) or ketotifen (B) from ane day
before to 3 days after the challenge infection. Multinucleate giant cells(arrows) were observed in the granuloma. In ketatifen‑treated animals,
small granuloma.s were more frequently found than in saline‑perfused ani‑mals. H‑E, x60.
Soo

Soo
SALIN

‑ 400

400

KETOTIFEN
Q

: :

o :

O

300

<

:

O
J:
:)

vv

L

o
o
Lo ZOOLuo::

2 r fl

:
:

Zoo

z

o I oo1 OO

o+KETOTiFENoDAYS AFTER fNFECTION

Figure 5 Granuloma areas in the lungs before and 3 days
after challenge infection with N. brasillensis.

Ketotifen (6 ng/daylkg) or saline was infusedfrom one day before to 3 day8 after the challenge
infection. Data before challenge infection, wereobtained from rats at 28 days after primary infec‑

tion. Data shown are means of total granuloma
areas

SE of 4 rats.

Figure 6 Worm recovery fram the small intestine of rats 3days after challenge infection with N. brasille csis.

Ketatifen (6 mg/daylkg) ar sali,ne vas infusedfrom one day before to 3 days after the challenge
infection. The majority of recovered worms were
L4 Iarvae. Data shown are means SE of 4 rats.

of mast cell activation by this agent. In iact, it has beenreported that anaphylactic secretian of RMCP 11 fcllow‑

ing intravenous challenge of primed rats with worm
present study, the numbers of RMCP II‑positive mast

antigen was accompanied by significant depletion of thiscells were significantly larger in ketotifen‑treated than

enzyme and decreased numbers of RMCP II‑positivein non‑treated animals despite the similar levels ofalcian blue‑positive mast cell numbers between the twogroups of rats. Since Rlvl:CP II, a preformed intra‑
granular mediator, is readily released together withmast cells in the tissues (King et al., 1986) . The present

histami,ne and other chemical mediators after mast cell
activation (Woodbury et al.. 1984), the appearance ofresuits showed that ketotifen treatment induced signifi‑
cant suppress,ion of eosinophil iufiltration into the lungs,

Granulomatous reaction also seems to have been sup‑pressed, although not to a statistically significant level

larger numbers of RlviCP IIpositive mast cells in

because of the large variation in the reaction. On theother hand, the worm recoveries from the small intes‑

ketotifen‑treated animals appears to reflect suppression

tine did not differ between ketotifen‑treated and112

nontreated animals, indicating that the agent did nothinder lung migration of worms. These results suggestthat mast cells have an important role at least in thedevelopment of nematode‑induced lung eosinophilia.
Mast cells are the major effector cell for type I
hypersensitivity, in which lgE antibody‑mediated mastcell activation induces prompt mediator release. In fact,

N. brasiliensis infection induces marked elevation of
specific lgE antibody (Yamada et al.. 1991) . Further, N.

brasiliensis‑sensitized rat lungs showed immediate
release of histamine and leukotriene C4 upon worm‑
antigen stimulation in vitro (Nishida et al., manuscript

in preparation) , suggesting that at least in part
nematode‑induced pneumonitis might be an expressionof type I hypersensitivity. On the other hand, it has beenreported recently that Arthus reaction, the type 111
hypersensitivity mediated by lgG immune complexes, isalso dependent on mast cells (Ramos et al.. 1994; Sylves‑tre and Ravetch, 1996). Further, mast cells also aug‑

ment various non‑allergic inflammatory responses(Galli, 1993) . Thus, the precise pathogenetic mecha‑cell proliferation following normal and heterotopic
infections of the nematode Nippostrongylus brasiliensis inrats. APMIS, 102, 589‑596
4 ) Craps, L.P. and Ney, U.M. (1984) : Ketotifen: current
views on its mechanism of action and their therapeutic

implications. Respiration, 45, 411‑421 .
5 ) Galli, S.J. (1993) : New concepts about the mast cell. N.

Engl. J. Med., 328, 257‑265
6 ) Gelpi. A.P. and Mustafa, A. (1968) : Ascaris pneumonia.

Am. J. Med., 44, 377‑389

7 ) King, S.J., Miller, H.R.P., Woodbury, R.G. and New‑lands, G.F.J. (1986) : Gut mucosal mast cells in Nippo‑
strongylus‑primed rats are the major source of secreted
rat mast cell protease 11 following systemic anaphylaxis.
Eur. J. Immunol., 16, 151‑155
8 ) Lendrum, A.C. (1944): A stain for eosinophil cells. J.
Pathol. Bacteriol., 56, 441

9 ) Martin, U. and Roemer, D. (1977): Ketotifen: a his‑
tamine release inhibitor. Monograph in A1lergy, 12,
145‑149
10) Phills, J.A., Harrold, A.J., Whiteman. G.V. and Perel‑mutter, L. (1972): Pulmonary infiltrates, asthma andeosinophilia due to Ascaris suum infestation in man. N.
Engl. J. Med., 286, 965‑970

nisms of the development of nematode‑induced11) Ramaswamy, K., De, S.G., Green, F. and Befus, D.

penumonitis and the role of mast cells remain to be(1991): Pathology of pulmonary parasitic migration:
morphological and bronchoalveolar cellular responses

elucidated.

Taken together, the present findings suggest thatdevelopment of nematode‑induced lung eosinophilia isdependent on mast cell activation, and mast cell stabi‑
lizers appear to be partially, if not completely, effective

for suppression of the lung lesions.

ACKNOWLEDGMENTS
This study was supported in part by research grants
from the Ministry of Education, Science and Culture ofJapan, the Toray Research Institute, and the Kyoto
Medical Science Institute.

REFERENCESl ) Arizono, N.. Koreto, O., Nakao, S., Iwai, Y., Kushima, R.

and Takeoka, O. (1987) : Phenotypic changes in mast
cells proliferating in the rat lung following infection
with Nippostrongylus brasiliensis. Virchows Arch. B, 54,
1‑72 ) Arizono, N., Nishida. M., Uchikawa, R., Yamada, M.,
Matsuda, S., Tegoshi, T., Kitamura, Y. and Sasabe, M.
(1996): Lung granulomatous response induced by infec‑

following Nippostrongylus brasiliensis infection in rats.
J. Parasitol., 77, 302‑312

12) Ramos, B.F., Zhang, Y. and Jakschik, B.A. (1994):
Neutrophil elicitation in the reverse passive Arthus

reaction. Complement‑dependent and ‑independent
mast cell involvement. J. Immunol., 152, 1380‑1384
13) Spillmann. R.K. (1975): Pulmonary ascariasis in tropi‑
cal communities. Am. J. Trop. Med. Hyg., 24, 791‑800

14) Steiger, E.. Vars, H.M. and Dudrick, S.J. (1972): A
technique for long‑term intravenous feeding in unre‑strained rats. Arch. Surg., 104, 330‑332

15) Sylvestre, D.L. and Ravetch, J.V. (1996): A dominantrole for mast cell Fc receptors in the Arthus reaction.

Immunity, 5, 387‑390
16) Talliaferro, W. and Sarles, M. (1939): The cellular
reactions in the skin, Iungs and intestine of normal andimmune rats after infection with Nippostrongylus muris.
J. Infet. Dis., 64, 157‑192

17) Woodbury, R.G., Miller, H.R.P., Huntley, J.F., New‑lands, G.F.J., Palliser, A.C. and Wakelin, D. (1984):
Mucosal mast cells are functionally active during spon‑taneous expulsion of intestinal nernatode infection in
rat. Nature, 312, 450‑452

18) Yamada. M., Nakazawa, M., Matsumoto, Y. and Ar‑
izono, N. (1991): IgE antibody production in rats

tion with the intestinal nematode Nippostrongylus

against multiple cornponents of excretory‑secretory

brasiliensis is suppressed in mast cell‑deficient Wsl Ws

products of the nematode Nippostrongylus brasiliensis.

rats. Clin. Exp. Immunol., 106, 55‑61

Immunology, 72, 104‑1083 ) Arizono, N.. Yamada, M., Tegoshi, T., Okada, M.,
Uchikawa, R. and Matsuda, S. (1994): Mucosal mastJ pn. J. Trop. Med

Hyg Vol 25 No 3 1997, pp. 113‑115

113

A FIELD STUDY ON THE EFFECTS OF RESIDUALSPRAY OF ENCAPSULATED FENITROTHION
ON ANOPHELES MINIMUS POPULATION IN
PHARE PROVINCE, NORTHERN THAILAND
WANNAPA SUWONKERD , SOMSAK PRAJAKWONG1, YOSHIO TSUDA2
AND MASAHIRO TAKAG12
Received June 9, 1997/Accepted July 17, 1997

Abstract: A field study was carried out in 2 villages of Phare Province, Thailand to evaluate effect of theresidual spraying of a microcapsulated formulation of 20% fenitrothion (Sumithion 20 MC R) on Anopheles
minimus populations. In the treatment village, houses were sprayed with I g/m2 of fenitrothion, except for
2 houses which were selected to spray with 0.5 g/m' of fenitrothion for comparative bio‑assay test. The

results of bio‑assay test showed that mortality of An. minimus was 100% in I g/m2‑30 miniutes until 4months after the spray. The growth rate of An. minimus population during the first 4 months of the study
period in the treatment village was lower than that in the control area. These results suggested that theresidual spray of fenitrothion microcapsules at the beginning of the dry season was effective at least for 4

months after the spray and could suppress the density of An. minimus.
Key words: Residual spray, Fenitrothion, Microcapsule, Anopheles minimus. Thailand

INTRODUCTION
DDT has played an important role in the vector
control until recent years in developing countries.
However, the behavioral and/or physiological resis‑

results suggested the presence of behavioral resistance(Suwonkerd et al., 1990). In this study effects of theresidual spray of a microcapsulated formulation of 20%

fenitrothion (Sumithion 20 MC R ) on An. minimus
were examined in northern Thailand.

tance of mosquitoes have been developed by the continu‑

ous application of DDT (WHO, 1992). It has become a

MATERIALS AND METHODS

serious problem in vector control in many countries, inaddition to the side effects of DDT on the surrounding
environment through the biological concentration (Cur‑The study was carried out from October 1995 toSeptember 1996 in 2 villages in Saeipt Canton, Songtis, 1994).

District, Phare Province, 245 km east of Chiangmai City,

In Thailand DDT has been used on a large scale forthe malaria vector control since 1952. The physiologicalThailand. One village (No. 8 Ban Tawa) where 67
houses were situated and the population = 272 wasand behavioral resistance to DDT have been observed insome anopheline species in Thailand (WHO, 1970, 1992) .

selected as the treatment area. The other village (No.
5 Ban Mae Ten) was selected as the control area where
89 houses were situated and the population was 340.
Among the houses in the treatment area, 2 houses
were selected to spray with 0.5 g/m2 of encapsulated
fenitrothion for the cornparative bio‑assay test and theother houses were sprayed with I g/m2 in October, 1995.
Before the insecticide spray, mosquito collections

The screening of alternative insecticide to DDT has
become an important subject in the vector control,
though it has been believed that the major malaria
vectors, Anopheles minimus and An. dirus, have been
still susceptible to DDT (Ismail et al., 1974, 1975;
Nutsathapana et al., 1986). Since 1982 fenitrothion has
been introduced to some areas as another candidate ofinsecticide. In 1988 a field study on the response of An.

dirus to DDT and fenitrothion was conducted and thewere performed in 4 successive nights. Monthly mos‑quito collection (4 nights) was conducted by 3 different
methods for I year after the spray. Using human baits,

1) Office of Vector Borne Disease Control, Region 2, Ministry of Public Health, Thai Government, Amphur Muang, Chiangmai 50200, Thailand
2) Department of Medical Entomology, Institute of Tropical Medicine, Nagasaki University, 1‑12‑4 Sakamoto, Nagasaki 852, Japan114

2 different collections, indoor and outdoor human baitcollections were made from 18:OO to 24:OO at 2 fixedhouses and landing mosquitoes were collected by an

Table 1 The result of bio‑assay test of An. minimus on
the residual deposits of 20% microcapsulated
fenitrothion sprayed from October 1995 to Sep‑
tember 1996 in Phare, Thailand

aspirator hourly for 50 min. In the 3rd collection method(the animal bait collection) , a cow was tethered inside aTime after 0.5 g/m2*

1 g/m2*spray (month) 3 min** 30 mm**

3 min ** 30 min**houses. A pair of collectors sat inside and outside of thegauze net (4x4x2 m) , which was similar to the one

100
100
100
100

O

described by Service (1993), and one collector caught

12

mosquitoes landing in and out of the net using an aspir‑

3
4
5
7
9

ator at every 15 min. During the collection period
relative humidity and temperature were recorded.

Following WHO (1970) the bio‑assay test was

9
7

9
10
11
14

10
11

made every month for 2 different dosage (0.5 and I glm2of fenitrothion) and 2 different periods of exposure (3

100
100
100
100
52

100
100
100
100
49
44
40
25
35
34

12
14
17
15
18

100
100
100
100
100
95
78
47
48
40

The total number of mosquitoes used in each test was 100‑200

and 30 min) using 100‑200 An. minimus adults reared in
the laboratory. The test was duplicated in each combi‑

and mortality rate of the control mosquitoes was O in all the bio‑

nation.

** Exposure time

assay test.

* Dosage of fenitrothion

For the bio‑assay test the mortality was calculated
for each replication and the average of the 2 replications

was shown in the table.' The average number of An.
minimus per half night was calculated for each collec‑
tion method and the growth rate of An. minimus popu‑
lation during the first 4 months of the study period wasestimated by applying log regression analysis to the

1 g/m2, was about I month, and the higher dosage

average number of An. minimus + 1.

in Fig. 1. The density of An. minimus declined after thealways showed the higher mortality.

The temporal changes in the number of An.
minimus collected by 3 different methods are depicted
insecticide spray, except for the result of animal baitcollection in the control area, and started to increase in

RESULTS AND DISCUSSION

May 1996. Although the observed temporal changes inthe density may be partly ascribed to the seasonal

The results of bio‑assay test showed that mortalityof An. minimus was 100% until 3 months after the sprayin all combinations (Table 1). Only the combination ofprevalence of this species in northern Thailand (Takagi

et al.. 1995; Suwonkerd et al.. 1995), the density ofindoor and outdoor collection in the treatment area

the higher dosage with 30 min exposure showed 100% of

decreased more rapidly from November to December

mortality until 4 months after the spray. The differencein the effective period between 2 dosage, 0.5 g/m' and

(,D
Q)o

1 OO

1995 than the control area.

OutdoorIndoor1 OO

Animal Bait

1 OOO

h
*:l

cc')

o
H

o

10

'

+
F,

;

It , ,'l10

1

O OND J F MA M J J A S

1 995 1 996

1 oO
lf

¥I

l

II

¥

ll

I .l

I

･i
ll'l
1

ll. .

il

10

!
t ¥

t ¥

1

O ONDJ FMAMJ JA S O OND J F M A M J J A S

1 995 1 996 1 995

1 996

Figu*e 1 Temporal changes in the number of An. minimus/half night collected by 3
different methods from October 1995 to September 1996 in 2 villages in Phare,
Thailand. The solid and dashed line shows the result in the treatment andcontrol village, respectively. The arrow shows the day of residual sprayingof I g/m2 of microcapsulated fenitrothion in the treatment village.

115

Table 2 Results of log regression analysis of the temporal changes in the number of An. minimus
collected by 3 different methods in the control and treatment area* from October 1995 to

February 1996, Phare, Thailand
IndoorTreatment Control
Slope (b)OutdoorAnimal Bait
Treatment
Control Treatment Control‑ O . 35

‑ O . 123‑o. 468‑0.111

O . 059

S.E.

O . 126

O . 017o. 132

O . 023

O . 153

O . 09Growth rate/month (10b)O . 447

O . 753o. 340O . 661

O . 775

1 . 146

Correlation

O . 721o . 944o . 808o . 953

O . 149

O . 125‑0.18

coefficient (r)*Houses were sprayed with I g/m2 of microcapsulated fenitrothion in October 1995.

Because the bio‑assay test showed that the sprayed
insecticide was effective at least for the first 4 months

REFERENCES

of the study period, the growth rate of the average
number of An. minimus during the first 4 months (from1 ) Curtis C.F. (1994) : Should DDT continue to be recom‑

October 1995 to February 1996) was estimated andEntomol., 8, 107‑1122 ) Ismail, I.A.H., Notananda, V. and Schepens, J. (1974):
Studies on malaria and response of Anopheles balabacen‑
sis and Anopheles minimus to DDT residual spraying incompared in Table 2. Both indoor and outdoor collec‑
tions, the slope of log regression line (b) in the treat‑ment area was siguificantly smaller than that in thecontrol area. The growth rate in the control area wasestimated as 0.753 and 0.661/month for indoor andoutdoor collections, respectively, and that in the treat‑ment area was 0.447 and 0.340/month for indoor andoutdoor collections, respectively. Though the log regres‑sion was not significant in the animal bait collection, thedifference between the treatment and control area wasclear; the density of An. minimus in the control area
showed gradual increase whereas that in the treatmentarea showed decreasing tendency during the first 4
months of the study period (Fig. 1).
A11 the results suggested that the residual spray ofcapsulated fenitrothion at the beginning of the dry sea‑
son was effective at least for 4 months after the sprayand could suppress the density of An. minimus. How‑ever the present study started from the declining periodof An. minimus population, thus, the effect of the insec‑

ticide sprayed might be overestimated in this study.
Additional field studies in the increasing period of An.

minimus population will be needed to evaluate the effectof the residual spray of microcapsulated fenitrothion in

malaria vector control throughout the year.

ACKNOWLEDGEMENT
We thank Sumitomo Co. Ltd for financial support
and all field staffs of Office of Vector Borne Disease
Control, Region 2, Chiangmai, Thailand for their contin‑uous help for the field study.

mended for malaria vector control? Med. Vet.

Thailand. Part 1; Pre‑spraying observations. Acta.
Trop., 31, 129‑1643 ) Ismail, I.A.H., Notananda. V. and Schepens, J. (1975) :Studies on malaria and response of Anopheles balabacen‑

sis and Anopheles minimus to DDT residual spraying in
Thailand. Part 2; Post‑spraying observations. Acta.
Trop., 32, 206‑231

4 ) Nutsathapana, S., Sawasdiwongphorn, P., Chitprarop,
U. and Cullen. J.R. (1986): The behavior of Anopheles
minimus Theobald (Diptera: Culicidae) subjected todiffering levels of DDT selection pressure in northern
Thailand. Bull. Entomol. Res., 76, 303‑312

5 ) Suwonkerd, W., Amg‑Ung, B., Rimwangtrakul, K.,
Wongkattiyakul, S., Kattiyamongkool, B., Chitprarop,
U. and Takagi, M. (1990) : A field study on the responseof Anopheles dirus to DDT and Fenitrothion sprayed tohuts in Phetchabun Province, Thailand. Trop. Med., 32,
1‑56 ) Suwonkerd, W., Takagi, M., Arng‑Umg, B. and Praja‑
kwong, S. (1995) : Seasonal and spatial distribution of 3

malaria vectors at three mountainous villages in northThailand. Jpn. J. Trop. Med. Hyg., 23, 183‑187

7 ) Takagi, M.. Suwonkerd, W., Tsuda, Y. Kamboonruang,
C.. Chitpralop, U., Nakazawa, S., Kanbara, H. and
Wada, Y. (1995): Seasonal density and malaria vector

competence of Anopheles minimus and other ano‑phelines at a shallow valley in north Thailand. Jpn. J.
Trop. Med. Hyg., 23, 177‑1828 ) WHO (1970): Resistance of vectors and reservoirs of
disease to pesticides. WHO technical report series No.
737, Geneva

9 ) WHO (1992): Vector resistance to pe ticides. WHO
technical report series No. 818, Geneva

日熱医会誌，第25巻

117第3号，1997年，117−120頁

二酸化塩素溶液のC昭餌oερorε痂腐肌のオーシストに対する保存効果
松井

利博，藤野

隆志，小林富美恵，辻守康

平成9年6月30日受付／平成9年8月12日受理

緒論

硫吻o⑫oガ4伽解はコクシジウム類の中で最も小さく，
一般にオーシストの検出には薦糖液浮遊法が用いられている（Current，1990；Fujinoαα1．，1996）。しかし，オーシ

ストは乾燥や熱処理を受けると薦糖液でも浮遊せず，検出困難となる。さらにオーシストの生存には酸素が要求される（Davis，1973）ことから，その保存には酸素を消費する
糞便内の細菌を殺滅することが必要である。このために通常実験室内では2％から2．5％の重クロム酸カリ溶液中で保存されているが，クロムは産業廃棄物であることから環境汚染を防ぐために特別な廃棄処理が要求され（Current，
1990），安全で簡単に廃棄できる保存液の開発が望まれる。
そこで，某薬品会社が畜舎用消臭剤として，住宅や畜舎の消毒・消臭用に販売している水成二酸化塩素溶液（商品名「FC1736」），および重クロム酸カリ溶液の代用品として使用されている希硫酸（Frenkel and Dubey，1975；Ma・2％の重クロム酸カリ溶液を対照として用いた。
検査方法：

C耀溶またはニワトリ由来のオーシストを経口投与
後，新たなオーシストを排泄中のマウスまたはニワトリの新鮮な糞便を採集し，水を加えてよく撹搾した。これを50と100の二重メッシュで濾過し，濾液を2，000rpm7分遠沈した後上清を捨て，沈渣にほぼ同量の水を加えて撹拝した。この材料に7倍量の供試保存液を加えて混和した後，冷蔵庫内で保存した。

C吻％漉は1，2，3週間および1，2カ月間，ニワト
リ由来のオーシストは1および3週間放置後，水で6回遠心洗浄し，（二窺痂sは1群5頭のマウスに1頭当たり
2．0×106個のオーシストを，またニワトリ由来のものは1群3羽のニワトリに1羽当たり3．0×106個のオーシストをそれぞれ経口投与した。保存効果は，実験動物の糞便1g

中のオーシスト数（OPG値）を毎日計測し（松井ら，1992，1994），その推移により判定した。

tsui，1991）とにっいて，C勿ゆ如砂oπ4伽窺窺％万sとニワト

リ由来Cηψ嬬ρoガ4勿郷sp．とのオーシストの保存効果を

結

調べた。

果

C．muris＝

材料および方法
供試原虫：

C窺％漉のオーシストは自然感染ドブネズミの糞便から分離し（Iseki，1986），マウスで継代・増殖したものを，

またニワトリ由来C吻孟o幼oガ4伽窺sp．のオーシストは自然感染鶏の糞便から採集し（ltakura8孟磁，1984），その実
験感染鶏のファブリキウス嚢より分離した（松井ら，1992）
後，ニワトリで継代・増殖したものをそれぞれ供試した。
供試動物：

4週齢のICR雄のマウスと1週齢の白色レグホンの雄を供試した。いずれも実験前に数回薦糖液浮遊法による糞便検査を行い，オーシスト陰性であることを確認した後
coccidia−freeの管理下で飼育した。

供試保存液：

オーシストの保存効果を調べたFC1736は，水成二酸化塩素溶液より製剤化された商品（ClO2．Na2CO3）で，その1％と2％溶液を供試した。希硫酸は2％溶液を供試し，
杏林大学医学部・熱帯病・寄生虫学教室

保存オーシストを投与したマウスにおけるオーシストの排泄状況を比較するために，2％重クロム酸カリ溶液で3週間保存したオーシストを各102個から106個投与したマウスにおけるoPG値の推移（松井ら，1994）をFig．1のAに示した。1週問保存のオーシストを投与したマウスでは，いずれの群も106個台投与における典型的なOPG値の推移，すなわち，10日目に105個台を，その後下降した後14日目には再び上昇して105−106個台を示した（Fig．1，B）。2

週間保存では二酸化塩素溶液の2％群のみが若干低下して，105個台投与時と106個台投与時との中間の推移を，3週間
保存ではさらに希硫酸群が低下して，105個台投与時におけるOPG値の推移を示した（Fig．2）。

1カ月間保存では二酸化塩素溶液の1％が105個台投与時と類似した推移を示したが，重クロム酸カリ溶液群は106
個台投与時の推移を維持した（Fig．3，A）。2カ月間保存では，1％二酸化塩素溶液と重クロム酸カリ溶液群とが105
個台投与時の推移を示したが，2％二酸化塩素溶液群は104
個台投与時の推移を，希硫酸群は103個台投与時の推移をそ

118
O

P

O

G

P

G

107

107

樹
く1

｝

㍗

ノ

八ン

105

、戸
も

103
F

ロご

＼ll

＿＿2x1。5＝二：1委1望

1％［。s

104

／

一｝2弘t邸

103

−

10

15

20

l

Comparison

of

OPG

valuesinedwith106−1030．，nπ短s

oocysts

keep

and

in

mice

mean

after

sas

尋

15

10

Days
Figure

一一一2％

ρ」

v、●

2％POS

20

inoculat−in

PDS（A）

storedin4storage media for l week（B）．
F●：Oocyst positive by sugar flotation method．
CDS：Chlorine dioxide solution．SAS：Sulfuric acidO

P

oocysts

dichromate

solution．

F
10

15

Days

！r

ノ／

rノ＾写

／

孜／

刃

positive

P

5

10

15

ρつ、

戸

…

5

10

15

20

Day8a伽r

Figure3Mean

OPG

oocystsvaluesstoredinmice

in4storage

positivebyflotation

method．

1lmonth（A）

sugar

flotation

method．

CDS：Chlorine dioxide solution．SAS：Sulfuric
acid solution．PDS：Potassium dichromate solu−
tion．
れぞれ示した（Fig．3，B）。
ニワトリ由来Cryρtosρorldium sp．：

1週間保存ではいずれも106個台投与時の推移を示した。

3週間保存では，希硫酸群のみが若干低いOPG値の推移を示したが，二酸化塩素溶液群は双方とも重クロム酸カリ
溶液群と同様の推移を示した（Fig．4）。

『一一2％

s

s

I
103
5

10

D8y8a危er

inoculation

given2．0×106C灘漉

1％c鵬

一一2％［OS『I一 一一 ，腎z％ pos

20

for

、、

誇

104

昌Z％POS

media

スモ

《1「＼瓢
、！〆

and2months（B）．
F●：Oocyst

sugar

G

106

＼11＿＿1菱器：
v1＿＿2鴇SRS
F

by

tion．

105

103

afte駐inoculatiOB

CDS：Chlorine dioxide solution．SAS：Sulfuric
acid solution．PDS：Potassium dichromate solu−

1へ！
冷￥11．v

104

20

and3weeks（B）．
F●：Oocyst

105

猷

15

107

106
￥

10

POS

Figure2Mean OPG values in mice given2．0×106C吻漉soocysts stored in4storage media for2weeks（A）

B、

・・．・一・一一一2％

5

20

1％cns

一一2％εBS
一一一2％舘S

．●、

O

A

ヤ

1

G

107

1＼

in㏄ulation
mice

given2．0×106（二吻％万ssolution．PDS：Potassium

1

1

5

■

一
＝／1
八v1

》

1

ア

噛．】〉 ノ

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1

104

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1

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トz

106

vψ

鹸、

のr
ぽ

B

ノ

〈

ノ

106

105

A

B

A

Figure4MeanchickenOPG

valuesin

15

Inoo闘lation

chickensC哩）云o釦りoガ♂づ％〃zgiven3．0×106

sp．oocysts

storedin

4storage media for3weeks．
CDS：Chlorine dioxide solution．SAS： Sulfuric
acid solution．PDS：Potassium dichromate solu−
tion．

考

察

一般にコクシジウム類では宿主におけるオーシストの排泄数は接種オーシスト数に依存しており（Long，1973），従来からその推移は宿主における感染状況の把握や治療薬の効果判定などに応用されている（DubeシandFrenkel，1974；
飯島ら，1989；Tsunoda6勧1．，1968）。C鯉ρ渉oψ07翅襯についてもC、郷％腐は感染14日目まで，ニワトリ由来の原119

虫は10日までに，接種オーシスト数と排泄オーシスト数との間に相関関係がみられている（松井ら，1992，1994）ことから，他のコクシジウム類と同様にオーシストの保存液の効果判定にも応用できる。

C窺癬sに対する保存液の効果を調べた結果，2％重ク
ロム酸カリ溶液では1カ月間保存まで，1％水成二酸化塩
素溶液では3週問保存まで，2％希硫酸では2週間まで，2％水成二酸化塩素溶液では1週間まで，その投与動物におけるOPG値の推移に変化が認められなかった。2カ月
保存では1％水成二酸化塩素溶液と重クロム酸カリ溶液群とが105個台投与時の推移を示し，2％水成二酸化塩素溶液
群は104個台投与時の推移を，希硫酸群は103個台投与時の推移を示したことから，それぞれ約90％，99％，99．9％のオーシストが死滅していたことが示唆された。ニワトリ由来のC乃ψ≠oψo万4伽窺sp．においては1週問および3週間
保存について調べたが，希硫酸で3週間保存した群を除き，他はいずれも106個台投与時におけるOPG値の推移を示し，C．吻癬sにおける成績と類似していた。一方，全実験群でオーシスト保存前と後に薦糖液添加法（松井ら，1992，
1994）を用いてその数を測定したが，いずれにも大きな差が認められず，オーシストの生死については判定できない
ものの，藤糖液浮遊法によるその検出は可能であった。・これらのことから，水成二酸化塩素（FC1736）の1％溶液における保存はC脚ホo砂o万4伽吻のオーシストの検出に少な

くとも2カ月間有効で，実験での使用には3週間まで可能であることが明らかになった。本溶剤は入手が簡単で，安全なために容易に廃棄できることを考慮すると，患者の検査用糞便の輸送などに際しての使用も好適であると考えた。

文

献1）Current，W．L．（1990）：

Techniquesandlaboratory

maintenance

of

man

animals（Eds，J．P．Dubey，C．A．Speer，RandCゆ∫o砂oγ躍づ％窺．Cryptospori（1iosis

of

Fayer），31・49，CRC Press，Flori（ia2）Davis，L．R．（1973）：Techniques．The coccidia（Eds，D．
M．Hammond and P．L．Long），411−458，University Park

Press，Baltimore
3）Dubey，J．P．and

Frenke1，」．K．（1974）：Immunity

toxoplasmosis：Modification

byadministration

tofelineofcor−ticosteroids．Vet．Path．，11，350−3794）Frenkel，」．K．and Dubey，」．P．（1975）： 施別郷onぬα
h召窺郷o裾歪gen．nov．，sp．nov．，fromdomestic

coccidianandrelatedtoTo％ρρZzsηzzcats，anew

Sσκoのオsあs．

Z．

Parasitenk．，46，3−125）Fujino，T．，Matsui，T．，Tsutsumi，Y．，Saito，Y．，Kobaya−

shi，F．andmunogenicity

Tsuji，M．一（1996）：Infectivity

toJapanease

and

im−

quails（Co伽魏㍍oo伽7η㍍

ノ勿on蜘）of C脚孟o砂oπ4伽卿sp．isolated
Japan．JPn．」．ParasitoL，45，139−143

fromchickensin6）飯島利彦，松井利博，森井勤，小林富美恵，藤野隆志（1989）：E吻61吻oα∂蜘感染モルモットにおけるSu1−

fadimethoxineとSulfamonomethoxineの効果．寄生虫誌，38，129−134
7）Iseki，M．（1986）：Two
rallyinfectinghouse

speciesofrats，ノ〜z〆∫z据C吻渉o砂o吻伽吻natu・
π07zノ昭づo％s・

JPn・J・

Parasitol．，35，521・526

8）Itakura，C．，Goryo，M．and
Cryptosporidial

infectioninUmemura，T．（1984）：
chickens．Avian

PathoL，13，

487−4999）Long，P．L．（1973）： Pathology and pathogenicity ofcoccidial infections．The coccidia（Eds，D．M．Hammondand P．L．Long），253・294，University Park Press，

Baltimore
10）Matsui，T．（1991）：Thetissuestagesof途o砂o名召hの40吻
in the guinea pig as an intermediate host． JpnJ．
ParasitoL，40，581−586

11）松井利博，森井勤，藤野隆志，Tadeja，S．L．，板倉智敏（1992）：℃吻∫o砂oガ協％窺sp．の土ウトリにおけるオーシス

トの排泄状況と免疫原性．寄生虫誌，41，24−29

12）松井利博，藤野隆志，小林富美恵，森井

勤，辻

守康

（1994）：C吻孟o砂07耀％郷〃z醜sの実験感染マウスにおけるオーシストの排泄状況と免疫原性．寄生虫誌，43，199−204
13）Tsunoda，K．，Imai，S．，Tsutsumi，Y．and

Clinicaleffectivenessfadimethoxine

inofInoue，S（1968）：

sulfamonomethoxine

spontaneous

coccidial

rabbits．Jpn．J．Vet．Sci．，30，109−117

and

sul−

infections

in

120

EFFECT OF STORAGE WITH CHLORINE DIOXIDE SOLUTION
ON CRYPTOSPORIDIAN OOCYSTTOSHIHIRO MATSUl, TAKASHI FUJlNO,
FUMIE KOBAYASHI AND MORIYASU TSUJI
Received June 30, 1997/Accepted August 12, 1997

Effects of storage in chlorine dioxide solution (CI02. Na2CO,, 1% and 2% w/v in water) and 2% sulfuric acidsolution on oocysts of Cryptosporidium muris and chicken Cmptosporidium sp. were examined. Fresh oocysts
were collected from the feces of infebted mice or chickens within I hr of oocyst shedding. They were poured
into 7‑fold volume of chlorine dioxide solution, sulfuric acid solution or 2% potassium dichromate solution
and stored at 4'C. After 1, 2, 3 weeks and I and 2 months, the stored oocysts were washed 6 times with waterby centrifugation and 106 oocysts from each storage were inoculated to a mouse or a chicken. Inoculated
animals were examined daily for oocyst discharge. When stored in 1% chlorine dioxide solution, C. murisand chicken Cmptosporidium oocysts remained viable and retained infectivity for at least 3 weeks. Viable
oocysts began to decrease after I month in 1% chlorine dioxide solution storage, though similar number ofoocysts were detected by the sugar flotation method. These results clarified that 1% chlorine dioxide
solution was usefull as a storage medium to keep oocysts viable for at least 3 weeks and detectable for 2

months.
Key words: cryptosporidian oocyst, chlorine dioxide, storage medium

Department of Tropical Diseases and Parasitology, Kyorin University School of Medicine

SEPTEMBER1997

VOL．．25NO．3

CONTENTS
Origiml

ArticleIwanaga，M．，Sisavath，L．，Higa，N．，Honma，Y．and
Emergenceof

Methicillin

Resistant

Kakinohana，S．

S勿勿loooo6％s側名郷in

Laos・・10HO6

Nishida，M．，Yamada，M．，Uchikawa，R．，Matsuda，S．，Tegoshi，T。，Sasabe，M．and
Ketotifen，antheAntiallergic

Agent，Suppresses

Nematode八厚●鋤osケon8夕1％s6π〜sllづ6郷乞sSuwonkerd

Pulmonary
inW．，Prajakwong，S．，Tsuda，Y．and

Eosinophilia

InducedArizono，N．

by

Rats一………・…・・…………・……・………………107−112

Takagi，M．

AFieldStudyontheEffectsofResidual，SprayofEncapsulatedFenitrothionon／4πρρh召Z8s郷初珈％sPopulation

in

Phare

Matsui，T．，Fujino，T．，Kobayashi，F．andEffectofStoragewith

Chlorine

DioxideProvince，Northem

Tailand．

・・113−115

Tsuji，M
Solutionon

Cryptosporidian

Oocyst（inJapanese）

一117−120