Jpn. J. Infect. Dis., 58, 2005

Laboratory and Epidemiology Communications

Outbreak of Enterohemorrhagic Escherichia coli O121 among School Children
Exposed to Cattle in a Ranch for Public Education on Dairy Farming
Yoko Akiba*, Tomoko Kimura, Miyoshi Takagi, Toru Akimoto,
Yoshio Mitsui, Yoshihiro Ogasawara and Masayoshi Omichi
Chiba City Institute of Health and Environment, Chiba 261-0011, Japan
Communicated by Haruo Watanabe(Accepted May 24, 2005)
In 2004 June, there was an outbreak of enterohemorrhagic
Escherichia coli (EHEC) O121 infections among school
children exposed to infected cattle in a ranch for public education on dairy farming in Chiba Prefecture, Japan.
The first two patients had diarrhea and abdominal pain onJune 13 and 14. VT2 producing EHEC was isolated but
the O serogroup was untypable (OUT). A total of 63 school
children in the 6th grade of the U primary school developedsymptoms. They were among 110 persons (101 children and9 teachers) that attended a summer school in K City in Chiba
Prefecture from June 9 to 11. During the same period oftime, there was a report of EHEC infection (OUT) from akindergarten. The common exposure point for all of theaffected school and kindergarten children was the R ranch
for public education on dairy farming in Chiba Prefecture.
The investigation of the public authority of Chiba Prefecture
revealed that the stool samples of all six cattle kept at theranch and soil specimens from the cattle pen contained EHEC
OUT, and that some of both specimens also contained EHEC
O157. An investigation by the Akita Prefectural Institute*Corresponding author: Mailing address: Chiba City Iustitute ofHealth and Environment, 1-3-9 Saiwai-cho, Mihama-ku, Chiba261-0001, Japan. E-mail: yoko-akiba@city.chiba.jp
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Jpn. J. Infect. Dis., 58, 2005of Public Health identified that the EHEC OUT previously
isolated from the school children was EHEC O121.
We inoculated CHROMagar O157, CT-SMAC and DHL
plates with specimens directly and also after amplificationin TSB broth. The isolated EHEC O121 developed whitecolonies on DHL plates, and they appeared to be a mixtureof lactose-fermenting and non-fermenting bacteria. A VTproduction test using polymyxin B was performed as
described previously (1). EHEC O121:H19 (VT2) was isolated from 15 patients and 2 asymptomatic individuals, andEHEC O157:H7 (VT1 and 2) was isolated from 2 affectedchildren. Among them, one patient had both EHECs.
Pulsed-field gel electrophoresis (PFGE) (2) of XbaIdigested chromosomal DNA revealed that all the EHEC O121
isolates had the same PFGE pattern, except one patientderived isolate that differed from the others by three bands,
and two soil-derived isolates that differed from the others bytwo bands (Fig. 1). The EHEC O157 isolates had similar
PFGE patterns; one patient isolate and the bovine fecal
isolate (lanes 21 and 23, Fig. 1) had identical patterns. The
other patient-derived isolate and the soil-derived isolate (lanes22 and 24, Fig. 1) differed from the former two by one (notidentical) band. The above results are summarized in Table
1. Antimicrobial susceptibility testing done by the KB method(3) revealed that all the isolates were sensitive to 12 antibiotics, i.e., ampicillin, cefotaxime, kanamycin, gentamicin,
streptomycin, tetracycline, chloramphenicol, norfloxacin,
nalidixic acid, fosfomycin, trimethoprim, and trimethoprimsulfamethoxazole.
Investigation by the public authority of Chiba Prefecture
revealed the following. First, the school children made butter

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 M 21 22 23 24 M

Kb
452.
336.
244.
173.
104.
54.

O12

O15

Fig.1. PFGE patterns of XbaI restriction fragments of chromosomal
DNA of EHEC O121:H19 and O157:H7 isolates.
Lanes 1-17: specimens derived from children in U primary school(O121), Lane 18: cattle stool specimens (O121), Lane 19: soil specimen
from the cattle pen (O121), Lane 20: specimen derived from patientin K city (O121), Lanes 21-22: specimens derived from children inU primary school (O157), Lane 23: cattle stool specimens (O157),
Lane 24: soil specimen from the cattle pen (O157), M: Salmonella
Braenderup H9812.

and soybean powder candies after lunch at the ranch. Second,the children were permitted to touch the cows through a fence.
Third, the ranch had only a limited number of hand-washingfacilities, which were insufficient for all the children. Fourth,cattle dung was scattered in the surroundings of the cattle
pens; the sanitary measures were insufficient. From thesecircumstances and also from the isolation of EHECs fromthe environment, it was concluded that the outbreak was

Table1. EHEC strains isolated from patients and bovine sources
Strain No.

Isolation date

Age/Sex

12341)
5
6
7
8
9
10
11
12
13
14
15
16
17

2004/6/19
2004/6/19
2004/6/19
2004/6/21
2004/6/22
2004/6/22
2004/6/22
2004/6/22
2004/6/22
2004/6/22
2004/6/22
2004/6/22
2004/6/23
2004/6/24
2004/6/25
2004/6/25
2004/6/25

11/F
10/M
11/F
11/F
11/F
11/F
11/M
11/M
11/F
11/F
11/F
11/M
11/F
11/M
11/F
11/F
11/M

182)
193)
204)

2004/7/7
2004/7/7
2004/6/17

3/F

1)

2122

2004/6/21
2004/6/22

11/F
11/F

232)
243)

2004/7/7
2004/7/7

Gastrointestinal symptoms
Diarrhea
BloodyAbdominal
diarrhea
pain〇〇〇〇〇〇〇〇〇〇〇〇〇〇〇〇〇〇〇〇〇〇〇〇〇〇〇

None
None

〇〇〇

Characteristics of isolates
Sero type

toxin

PFGE typeO121:H19
O121:H19
O121:H19
O121:H19
O121:H19
O121:H19
O121:H19
O121:H19O121:H19O121:H19
O121:H19O121:H19O121:H19O121:H19O121:H19O121:H19O121:H19

VT2VT2VT2VT2VT2VT2VT2VT2VT2VT2VT2VT2VT2VT2VT2VT2VT2A
A
A
A
A
A
A
A
A
A
A
A´
A
A
A
A
A

O121:H19O121:H19O121:H19

VT2VT2VT2A
A´´A

O157:H7O157:H7VT & 2VT & 2B
B´

O157:H7O157:H7VT & 2VT & 2B
B´´Strain Nos. 1-17, 21-22: children in U primary school.
1): Same patient. 2): Bovine feces. 3): Soils around cowsheds. 4): Resident within K city.

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Jpn. J. Infect. Dis., 58, 2005
caused through contamination of the school children’s handswith EHEC O121 derived from the infected cattle.
Outbreaks caused by EHEC O121, though rare, have beenreported in Akita and Saga Prefectures (4-5). In situations inwhich humans and cows come into close contact, EHEC O121
infection can occur, and special care, such as environmental
sanitation and hand washing, should be taken. The present
outbreak was caused by EHEC of a rare serotype. The prompt
response of the medical facilities and quick response of theresponsible authorities in Chiba City and Chiba Prefecture
facilitated identification of the source of the outbreak.

specimens. Modern Med. Lab., 28, 231-236 (in Japanese).
2. Izumiya, H., Terajima, J., Wada, A., Inagaki, Y.,
Itoh, K., Tamura, K., et al. (1997): Molecular typing ofenterohemorrhagic Escherichia coli O157:H7 isolates inJapan by using pulsed-field gel electrophoresis. J. Clin.
Microbiol., 35, 1675-1680.
3. National Committee for Clinical Laboratory Standards(1993): Performance standard for antimicrobial disksusceptibility tests. 5th ed. Approved Standard. M2-A5.
Villanova, Pa.
4. Yatsuyanagi, J., Saito, S. and Ito, I. (2001): Case reportof HUS caused by STEC O121 whose infection source
was considered cattle. Infect. Agents Surveillance Rep.,
22, 141-142 (in Japanese).
5. Masumoto, K., Moriya, K., Kumamoto, H., Uziwara, Y.
and Yamaguchi, H. (2002): Outbreak of EHECO121: H19
in a kindergarten. Infect. Agents Surveillance Rep., 23,
143-144 (in Japanese).

We thank Dr. Hiroshi Yoshikura, Emeritus Researcher of
the National Institute of Infectious Diseases, for advice on
preparing the manuscript.
The present case communicated in Japanese appeared in
Infectious Agents Surveillance Report (IASR), vol. 25, no.
11, p. 302-303, 2004.
REFERENCES
1. Uchimura, M. (2000): Detection of verotoxin in stool

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