An Outbreak of Acute Enteritis Due to Campylobacter fetus Subspecies jejuni at a Nursery. School in Tokyo

Transcription

1 Microbiol. Immunol. Vol. 24(5), , 1980 An Outbreak of Acute Enteritis Due to Campylobacter fetus Subspecies jejuni at a Nursery School in Tokyo Takeshi ITOH, Kahiko SAITO,* Tsutom MARUYAMA, Senzo SAKAI, Makoto OHASHI, and Aiko OKA * Department of Microbiology, Tokyo Metropolitan Research Laboratory of Public Health, Shinjuku-ku, Tokyo 160, and Higashikurume Health Center, Higashikurume, Tokyo (Accepted for publication, January 28, 1980) Abstract An outbreak of acute enteritis due to Campylobacter fetus subspecies jejuni involving a total of 35 out of 74 children occurred at a nursery school in Tokyo in January 1979 and lasted for 7 days. It was the first case of a community outbreak of the disease to be recognized in Japan. The major symptoms observed in the patients consisted of diarrhea (88%), fever (82%), abdominal pain (39%), and vomiting (6.1%). The rate of isolation of the organism from the patients was 39%. Sera of four patients showed elevated agglutinin titers against the organism ranging from 1: 80 to 1: 320. Although it is suggested that the outbreak was caused by a communal lunch or between-meal snacks prepared by and provided at the nursery school, the incriminated food, source and route of contamination could not be pinpointed. Vibrio fetus, known as an etiologic agent for infectious abortion or placentitis in livestock such as cattle and sheep has been transferred to the genus Campylobacter because it fails to ferment glucose and the guanine plus cytocine content of its DNA differs considerably from that of Vibrio cholerae, the type species of the genus (15). This organism has also been recognized as infecting man and as causing abortion (21), septicemia (7), meningitis (6), and arthritis (9). In addition, a group of organisms in this species, which have been classified as Campylobacter fetus subspecies jejuni by Smibert (17), has often been detected in diarrhea of human patients in European countries (4, 16), the United States (10) and Australia (18) recently, and has been reported to cause acute enteritis. During the course of a survey for enteropathogenic bacteria responsible for outbreaks of diarrhea in Tokyo, C. fetus subsp. jejuni was detected as the sole pathogen from fecal specimens of patients with diarrhea in an outbreak that developed at a nursery school in January of An elevation of agglutinin titers against one of the isolated strains was found in the convalescent serum of the patients. This fact may provide additional evidence to support the etiologic significance of the organism. 371

2 372 T. ITOH ET AL This was the first case of an outbreak of enteritis due to the organism to be recognized in Japan. This communication presents an outline of the outbreak, and the bacteriological and serological findings obtained. OUTLINE OF THE OUTBREAK During the period January 14 to 20, 1979, 35 of 74 children, 20 boys and 15 girls, ranging in age from one to five at a nursery school in Higashikurume City, a suburb of Tokyo, complained of diarrhea and/or fever. The first case occurred on the afternoon of the 14th and the oubreak peaked on the 16th and 17th (Table 1). Stool cultures from 13 of 33 patients among the nursery school children yielded C. fetus subsp. jejuni. No other enteropathogenic bacteria were found. All the patients from whom the organism was recovered had attended the school on January 13. No carrier of the organism was found among the children who were absent on that day. In addition to the children, one of the 19 staff members of the school had slight diarrhea on the 16th, and a member of an ill child's family developed diarrhea on the 17th. The former was a 45-year old woman working in the catering area at the Table 1. Campylobacter enteritis attack rates among children and staff members of a nursery school and distribution of dates of onset of the illness Table 2. Major symptoms of the 33 children with Campylobacter enteritis in nursery school

3 CAMPYLOBACTER ENTERITIS OUTBREAK 373 school and the latter was the 43-year old father of a child whose illness began on the 16th. He had not eaten any food at the nursery school. C. fetus subsp. jejuni was also detected in his feces. As shown in Table 2, the children's major symptoms were diarrhea (88%), fever (82%), abdominal pain (39%), and vomiting (6.1%). The stools from most of the patients with diarrhea were watery and those from several contained mucus and blood. Twenty-one among 29 patients had five or fewer diarrheal bowel movements a day (72%) ; four patients had six and two patients had eight. The highest temperatures of 11 children (41%) were below 38.0 C, those of nine (33%) were C, and those of seven others (26%) were 39.0 C or higher. Of the total of 35 ill children, 24 were treated by physicians and cured by three or four days of medication. There were no fatalities. This nursery school is in a residential quarter of Higashikurume City and looks after children 5 years old or younger who live in the area within a radius of about 1 km. All the water in the area including the school is furnished by the city water supply. Although a few rabbits are kept in cages at the school, the children had no direct contact with them and they had no contact with other animals such as cattle and sheep. All of the school children had been provided a common lunch and between-meal snacks prepared at the school with pasteurized market milk. No outbreak of acute enteritis was observed among neighboring residents or at other nursery schools, kindergartens or primary schools in the adjacent area. BACTERIOLOGICAL AND SEROLOGICAL EXAMINATIONS Materials and Methods 1. Materials examined. On January 19 and 20, the first bacteriological examinations were made on the stools of 33 of the 35 ill children, and the staff member and the family member, who were in the acute phase of the illness. The stools of the six healthy staff members of the nursery school were also examined. The second stool examination was carried out on February 16, about a month after the occurrence of the disease, in order to study the carrier state of the children and the two adults. Parallel to the stool examinations, blood was drawn from four patients, at the acute and convalescent stages, in order to study rise in levels of serum agglutinins against the isolated organism. Additionally, blood from 71 and 37 persons, both patients and nonpatients, which was taken about one and two months, respectively, after the peak of the outbreak, were tested for agglutinin titers. 2. Method for isolation of pathogenic bacteria. A search for Shigella, Salmonella, Yersinia, enteropathogenic Escherichia coli, Vibrio parahaemolyticus, Vibrio cholerae including the so-called NAG vibrio, Clostridium perfringens and Bacillus cereus, in the stools was carried out according to the methods described previously (12). For culturing Campylobacter, the selective technique described by Skirrow (16) was followed. Fecal specimens were inoculated directly onto Skirrow's selective agar plates. Blood Agar Base No. 2 (Oxoid) was supplemented with 7% horse

4 374 T. ITOH ET AL blood, vancomycin (10 Đg/ml), polymyxin B (2.5 IU/ml), and trimethoprim (5 Đg/ ml) (16). Inoculated plates were incubated at 37 C in an atmosphere of 5% oxygen, 10% carbon dioxide, and 85% nitrogen in an anaerobic jar. The plates were examined daily for three days. Identification of Campylobacter. Biochemical and biological characteristics of suspicious nonhemolytic colonies appearing on the selective agar plates were determined by the method described by King (8) and Dekeyser (5). For the biochemical characterization test, Albimi Brucella broth (Pfizer Diagnostics, New York) supplemented with 0.15% agar was used as the basal medium unless otherwise stated. For identification and subspeciation of isolates as C. fetus subsp. jejuni, the definition described by Smibert (17) was adopted. 4. Serological methods. Paired sera from patients, collected at the acute and convalescent stages, 26 and 18 patients' sera collected one and two months, respectively, after the outbreak, and nonpatients' sera collected one month after the outbreak were tested for their agglutinin titers. Sera from 10 healthy school children and 12 healthy adults were also tested as controls. Agglutinin titers against C. fetus subsp. jejuni were determined by the tube method using heat-killed cells of strain CF-1, an isolate from one of the patients, as the antigen. The antigen was prepared by heating for 2 hr a saline suspension of the organisms harvested from a 3-day culture of the strain grown on a blood agar plate at 121 C. The heated cells were washed three times by centrifugation and resuspended in saline. The results were read after incubation for 18 hr at 50 C. RESULTS I. Detection of Campylobacter in Stools The results of the bacteriological examination of the fecal specimens are summarized in Table 3. C. fetus subsp. jejuni was detected in the fecal specimens taken at the acute stage of the illness of 13 (39%) of the 33 sick children. The same organism was recovered from the family contact who had diarrhea. Although it was not recovered from the ill staff member, the organism was detected in two of six Table 3. Results of bacteriological examination of the fecal specimens

5 C AMPYLOBACTER ENTERITIS OUTBREAK 375 healthy staff members. No other enteropathogenic bacteria were detected in these specimens. One month after the outbreak, stools taken from 72 nursery school children, 33 patients, and 39 nonpatients, were also cultured for Campylobacter. All of the 39 specimens from the uninvolved children were negative for C. fetus. However, it was reisolated from three of the 13 children who had been positive in the first test. Stool cultures of these three were negative 10 days later. Campylobacter was not found in any of the specimens taken from 66 persons (19 staff members, 18 of their family members and 29 children's family members) one month after the outbreak. Feces from the rabbits kept at the nursery school were investigated in an effort to trace the source infection, but were negative for this organism. 2. Characteristics of Isolated Strains The 19 strains isolated, 16 at the first and 3 at the second examination, were all slender curved gram-negative rods with a single polar flagellum at one or both ends of the cell and motile with a corkscrew-like motion. They grew under the microaerophilic conditions described above, although they failed to grow under aerobic conditions. They were catalase- and oxidase-positive, reduced nitrate to nitrite, were negative for hydrogen sulfide on triple sugar iron agar, and failed to decompose glucose, lactose and sucrose. They grew in the presence of 1 % bile but not of 3.5% NaCl. Thus they were identified as members of the genus Campylobacter. Hydrogen sulfide produced in a medium containing cysteine was detected with lead acetate-impregnated paper strips. They grew in the presence of 1% glycine, and at 42 C but not at 25 C on 7% horse blood agar plates. findings they were identified as strains of C. fetus subsp. jejuni. From these 3. Serum Agglutinin Titers Agglutinin titers in sera from four patients, collected at the acute (1st or 2nd day of the illness) and convalescent (21st or 22nd day of the illness) stages were determined, with the results shown in Table 4. The titers at the acute stage were 1: 40 or less, but increased, ranging from 1: 80 to 1: 320, about 2 weeks after onset of the illness. The results of the investigation of changes in the agglutinin levels of patient and nonpatient children as well as staff members of the nursery school are summarized in Table 5. Sera of 17 of the 26 patients (65%) had agglutinin titers of 1: 80 or more Table 4. Serum agglutinin titers of patients against Campylobacter fetus subsp. jejuni, strain CF-1

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7 CAMPY LOBACTER ENTERITIS OUTBREAK 377 one month after the outbreak. Moreover, in the nonpatient group, fifteen of the 45 individuals (33%) also showed agglutinin titers of 1: 80 or more. In the patients' sera collected two months after the outbreak, seven of the 18 cases still maintained agglutinin titers of 1: 80 or more. None of the children who were absent from the nursery school on January 13 had agglutinin titers as high as 1: 80. The antibody titers of 10 healthy school children and 12 healthy adults unrelated to the outbreak were 1: 20 or less except for two which were only 1: 40. DISCUSSION King (7) was the first to investigate human strains of Vibrio fetus in detail. She recognized that these strains failed to grow at 25 C and grew more luxuriantly at 42 C than at 37 C, and were serologically different from other strains of animal origin. She provisionally termed the organism a "related vibrio," but it is now called C. fetus subsp. jejuni by Smibert (17) or C. jejuni by Veron (20). It has also been pointed out that the most marked symptoms of patients infected with this organism are diarrhea, abdominal pain and fever (5, 10, 11, 18). Recent investigations in the United Kingdom suggest that the organism is responsible for from 4% to 14% of cases of diarrhea (2, 3, 13, 16). Steele and Mc- Dermott (18) reported its isolation from 13 (5.8%) of 224 patients with suspected infectious diarrhea in Australia. Recent surveys in the United States (1) and South Africa (14) revealed that C. fetus subsp. jejuni was recovered as the sole bacterial pathogen from 31 % of young children and 34% of babies with acute gastroenteritis. A large-scale waterborne outbreak of Campylobacter enteritis occurred in Vermont (10), resulting in about 2,000 cases, and a small milkborne outbreak of the disease in Colorado, U.S.A. (11, 19), in 1978 and 1979 has been reported. Very recently, a large outbreak of enteritis associated with Campylobacter sp. was reported in Scotland (22). Although subspeciation of the isolates has not been made in some of these studies, C. fetus subsp. jejuni now appears to play an important role in the etiology of acute enteric infection in man. In Japan, Yoshizaki et al ( J. J.A.Inf.D : Meeting abstract, text in Japanese) reported that it was found in sporadic diarrheal cases. However, no previous outbreak of enteritis due to this organism had been recognized in this country. In the outbreak described in the present paper, the symptoms of the patients closely resembled those previously reported, and consisted mainly of diarrhea, abdominal pain and fever. The clinical course of the infection was benign and there was no positive evidence to indicate continuous spread in the nursery school. C. fetus subsp. jejuni was recovered at a rate as high as 39.4% in cultures of the patients' feces, revealing that the disease was caused by the organism. It was additionally notable that the four patients from whom paired sera were obtained showed a marked rise in specific antibody titer against the isolated strain during the course of the illness. In other words, an immunological response to the organism was

8 378 T. ITOH ET AL observed. The antibody titers in the sera obtained one and two months after the outbreak were higher in the patients than in the nonpatients. The existence of individuals in the nonpatient group showing relatively high agglutinin titers suggests that they had suffered from subclinical infection by this organism. From these findings, it can be said that the serological method described here should provide a useful aid for laboratory diagnosis of the disease caused by this organism. As the Campylobacter-positive family member who was taken ill one day later than his child had not eaten any food provided at the nursery school, it is suspected that his was a case of secondary infection from his child. However, sufficient evidence to support this assumption could not be obtained. It has been strongly suggested from previous investigations in European countries and the United States that poultry and livestock are reservoirs of the organism which could eventually enter the human food chain through contamination of meat, milk or water, which would thus be a source of human infection, just as is the case with salmonellae. The possibility of direct transmission of the organism from livestock and pet animals to humans should also be considered. In this outbreak, however, the source of infection unfortunately could not be determined. Further studies into the ecology of Campylobacter will facilitate the establishment of countermeasures against enteritis due to the organism. REFERENCES 1) Bokkenheuser, V.D., Richardson, N.J., Bryner, J.H., Roux, J., Schutte, A.B., Koornhof, H. J., Freiman, I., and Hartman, E Detection of enteric campylobacteriosis in children. J. Clin. Microbiol. 9: ) Bruce, D., Zochowski, W., and Ferguson, I.R Campylobacter enteritis. Br. Med. J. 5: ) Brunton, W.AT., and Heggie, D (ampylobacter-associated diarrhoea in Edinburgh. Br. Med. J. 3: ) Butzler, J.P., Dekeyser, P., Detrain, M., and Dehaen, F Related vibrio in stools. J. Pediat. 82: ) Dekeyser, P., Gossuin-Detrain, M., Butzler, J.P., and Sternon, J Acute enteritis due to related vibrio: First positive stool cultures. J. Infect. Dis. 125: ) Eden, A.N Vibrio.fetus meningitis in newborn infant. J. Pediat. 61: ) King, E.O Human infection with Vibrio,fetus and a closely related vibrio. J. Infect. Dis. 101: ) King, E.O The laboratory recognition of Vibrio fetus isolated from cases of human vibriosis. Ann. N.Y. Acad. Sci. 98: ) King, S., and Bronsky, D Vibrio,fetus isolated from a patient with localized septic arthritis. J.A.M.A. 175: ) Morbidity and Mortality Weekly Report Waterborne ('ampylobacter gastroenteritis Vermont. 27: ) Morbidity and Mortality Weekly Report ('ampylobacter enteritis- -Colorado. 27: 226, ) Ohashi, M., Murakami, H., Kudoh, Y., and Sakai, S Manual for the laboratory diagnosis of bacterial food poisoning and the assessment of the sanitary quality of food. SEAMIC publication No. 12. SEAMIC/IMFJ, Tokyo. 13) Pearson, A.D., Suckling, W.G., Ricciardi, I.D., Knill, M., and Ware, E Campylobacterassociated diarrhoea in Southampton. Br. Med. J. 3: ) Richardson, N.J., and Koornhof, H. J Campylobacter infection in Soweto. S. Air. Med. J. 55:

9 CAMPYLOBACTER ENTERITIS OUTBREAK ) Sebald, M., and Veron, M Teneur en bases de l'adn et classification des vibrions. Ann. Inst. Pasteur Paris. 105: ) Skirrow, M.B Campylobacter enterutis: a "new" disease. Br. Med. J. 2: ) Smibert, R.M In Buchanan, R.E., and Gibbons, N.E.(eds), Bergey's manual of determinative bacteriology, 8th ed. The Williams & Wilkins Company, Baltimore. 18) Steele, T.W., and McDermott, S Campylobacter enteritis in South Australia. Med. J. Aust. 2: ) Taylor, P.R., Werstein, W.M., and Bryner, J.H Campylobacter fetus infection in humans: Association with raw milk. Am. J. Med. 66: ) Veron, M., and Chatelain, R Taxonomic study of the genus Campylobacter Sebald and Veron and designation of the neotype strain for the type species, Campylobacter fetus (Smith and Taylor) Sebald and Veron. Int. J. Syst. Bacteriol. 23: ) Vinzent, R., Dumas, J., and Picard, N Septicemie grave an cours de la grossesse, due a un vibrion. Avortement consecutif. Bull. Acad. Nat. Med. 131: ) Weekly Epidemiological Record, W.H.O Surveillance of foodborne outbreaks. 54: 151. (Received for publication, August 3, 1979)