FURTHER STUDIES ON ENTEROPATHOGENIC ESCHERICHIA COLI ASSOCIATED WITH DIARRHOEAL DISEASES IN CHILDREN AND ADULTS

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1 Japan. J. Med. Sci. Biol., 27, 7-18, 1974 FURTHER STUDIES ON ENTEROPATHOGENIC ESCHERICHIA COLI ASSOCIATED WITH DIARRHOEAL DISEASES IN CHILDREN AND ADULTS RIICHI SAKAZAKI, KAZUMICHI TAMURA AND AKIKO NAKAMURA The 1st Department of Bacteriology, National Institute of Health, Kamiosaki, Shinagawa-ku, Tokyo 141, Japan (Received : October 31, 1973) SUMMARY : Following the study of Sakazaki, Tamura and Saito (1967), search was made for previously unrecognized enteropathogenic OK groups of Escherichia coli among 756 strains isolated from children and adults with diarrhoea. Eleven OK groups of E, coli were recognized as possible enteropathogens associated with diarrhoea in children and adults, based on their frequency of isolation, ligated gut-loop reactions in rabbits and keratoconjunctival tests in guinea pigs, together with published reports by other workers. These were : 06:K.; 06:K62; 018 : K76 ; 018:K77; 020:K84; 025 : K 1; 078:K80; 0114:K90; 0142:K86; 0148:K.; and 0152 : K. Of the 11 OK groups, 025 : K1 and 0152:K., possessed Shigella-like enteropathogenicity, and the remaining 9, most of which had already been described as causative agents of infantile diarrhoea, were included in the hsalmonella-like group h of enteropathogenic E, coli by Sakazaki et al. (1967). The criteria for determination of the possible enteropathogenicity of a given E. coli OK group are discussed. INTRODUCTION Sakazaki, Tamura and Saito (1967) studied enteropathogenic Escherichia coli (EEC) associated with diarrhoea in children and adults. They listed 18 OK groups of E. coli and four Alkalescens-Dispar biotypes as possible enteropathogenic organisms for children and adults. Their findings were introduced into routine bacteriology laboratories in various parts Of Japan; since then many sporadic cases and community outbreaks of infection with these EEC OK groups have been reported. It is clear, however, that EEC strains are not limited to the 22 OK groups listed by Sakazaki et al. (1967). Although 789 of about 1,000 cultures in their collection bonged to these groups, most of the remainder were strongly suspected of being the cause of diarrhoea in the patients from whom they were isolated. This work has continued and several OK groups not included in the previous 22 OK groups have since been recognized as possible EEC strains based on their frequency of isolation from cases of diarrhoea and on the results of ligated rabbit gut-loop and guinea pig eye tests. The present paper 7

2 8 SAKAZAKI et al. Vol. 27 describes these additional E. coli OK groups thought to be enteropathogenic for children and adults. MATERIALS AND METHODS Strains studied : A total 756 strains, all isolated from children or adults with enteritis, enterocolitis or dysentery, were examined. In each infection, search for recognized enteropathogens such as Salmonella, Shigella, Vibrio parahaemolyticus, Staphylococcus, and Clostridium perfringens, was negative. Of the 756 cultures, 338 had been left over from the previous study of Sakazaki et al. (1967) and the remaining 418 collected since They were identified as E. coli by their biochemical reactions. Although they did not belong to any of the 22 OK groups previously reported, they were presented in large numbers in diarrhoeal stools. Physiological and biochemical tests: These included : motility and indole production in SIM medium (Eiken) ; H2S production in peptone iron agar (Difco) ; citrate and acetate utilization on Simmons' agar base; citrate utilization in the presence of organic nitrogen compounds and glucose in Christensen's citrate agar ; urease production on Christensen's urea agar ; phenylalanine deaminase on phenylalanine agar (BBL) ; lysine and ornithine decarboxylases and arginine dihydrolase in Miller's broth; fermentation of carbohydrates including arabinose, glucose, lactose, maltose, raffinose, rhamnose, sucrose, trehalose, xylose, adonitol, dulcitol, mannitol, sorbitol, salicin, and inositol; and Ĉ-galactosidase production. Serological tests: Strains biochemically identified as E. coli were tested for their O antigens by tube agglutination tests using antisera of E. coli of O groups 1 to 156. The test was carried out with cultures heated at 100 C for 2 hr. Determination of their H antigens was carried out by tube agglutination tests with overnight broth culture of actively motile organisms against antisera for E. coli H antigens Additional O or H antisera were prepared by immunizing rabbits with such strains that were not agglutinated with any of the antisera for O groups or H antigens The presence of K antigens was determined first by inagglutinability of the living culture with its corresponding O antiserum, and then by agglutination tests with a limited number of K antisera. Cultures with other unidentified K antigens were submitted to the WHO International Escherichia Centre for precise K antigen determination. The serological methods of identification employed were as described by Kauffmann (1954) and Edwards and Ewing (1955). Ligated gut-loop tests in rabbits: These were carried out by a modification of the method of De and Chatterjee (1953), and are described by Sakazaki et al. (1974). Each animal was killed and examined 18 to 24 hr after laparotomy and inoculation of test cultures for the presence or absence of dilatation and histopathological changes in the loop segments. Three or four animals were used to test each culture. Keratoconjunctival tests in guinea pigs: One drop of a saline suspension containing 108/ml viable cells of the test culture, grown on heart infusion agar (Difco), was dropped into the conjunctival sacs of guinea pigs. The animals were examined for a period of 5 days for evidence of keratoconjunctivitis (Seleny, 1954).

3 1974 ENTEROPATHOGENIC ESCHERICHIA COLI 9 RESULTS The 756 strains of E. coli studied were divided into a number of O groups. Of these O groups, the majority were represented by only one or two strains isolated from sporadic cases. None of them gave any reaction in either the ligated rabbit gut-loop or guinea pig eye test. They had not been reported previously in connection with diarrhoea in man and their enteropathogenicity was therefore unknown in this study. On the other hand, 13 OK groups including cultures of O groups 6, 18, 20, 25,. 27, 78, 114, 142, 151, and 152 were each represented by four or more strains, the majority of which yielded positive reactions in ligated rabbit gut loops; only two OK groups caused keratoconjunctivitis in the guinea pig eye test. The number and source of strains of these enteropathogenic OK groups in the collection are shown in Table I. The results of the ligated rabbit gut-loop and guinea pig eye tests with these OK strains are summarized in Table II. The biochemical characteristics of the 149 strains of these OK groups described further are summarized in Table III. E. col i 06 :K. group During the period of , eight strains of this OK group were received. Two of these were representative strains from two separate community outbreaks of gastroenteritis reported by Sakai et al. (1970 a) and by Sasagawa (1968, personal communication). The remaining six strains were isolated from sporadic cases of gastroenteritis. The two strains from the outbreaks gave positive reactions in ligated gutloop tests, but did not induce keratoconjunctivitis in guinea pigs. Two serotypes, 06 :K. : NM and 06 :K. : Hl, were found among the eight strains. Although the K antigen of this OK group was not designated, it was not agglutinated with any of the K antisera tested. E. coli 06:K62 group This group comprised of representative isolates from a family outbreak of gastroenteritis and from two sporadic cases of diarrhoea. The first strain was tested in animals and gave positive reactions in ligated rabbit gut-loop tests but did not produce keratoconjunctivitis in guinea pig eyes. Only one serotype, 06 :K62 :NM, was found among three strains in this OK group. E. col i 018:K76 group Five cultures, all isolated from sporadic cases of gastroenteritis, belonged to this group. They represented one serotype, 018 :K76 :H4. Two of the five strains were tested in animals and both produced positive rabbit gut-loop reactions but no kerato conjunctivitis in guinea pig eyes. E. coli 018:K77 group A total of 21 strains belonged to this group. Three of them were representative strains from outbreaks of food poisoning that occurred in 1958, 1963, and The. remaining 18 strains were isolated from sporadic cases of gastroenteritis. The former three strains were tested in animals, and two yielded positive results in rabbit gutloop tests but no reaction in guinea pig eyes. The 21 strains of this group included four different serotypes, 018 :K77 :NM, 018 :K77 :H7, 018 :K77 :H10, and 018 :K77 :H14

4 10 SAKAZAKI et tl. Vol. 27 E. col i 020 :K84 group Twenty-nine strains belonging to this group were recognized : four were representative isolates from small community outbreaks of food poisoning during the past 10 years, and the remaining 25 were isolated from sporadic cases of gastroenteritis Three strains, one from an outbreak and the other two from sporadic cases, were examined by the ligated gut-loop and guinea pig eye tests; two of the three strains gave positive reactions in the former test. The 29 strains comprised three serotypes, 020 :K84 :NM, 020 :K84 :H7, and 020 :K84 :H26. E. col i 025 :K1 group A total of 21 strains of this OK group were identified : 16 were isolated patients clinically diagnosed as dysentery, other three were from cases of enterocolitis, and the other two from gastroenteritis. All the incidents were sporadic. Three cultures were tested and each produced dilatation of the rabbit gut-loop segments and keratoconjunctivitis in guinea pigs. All of 21 strains comprised a single serotype,. from TABLE I Additional serotypes of Escherichia coli isolated from children and adults with diarrhoea during the period *

5 1974 ENTEROPATHOGENI C ESCHERI CHIA COLI 11 TABLE I (Continued) * Twenty-two other E. coli OK groups associated with diarrhoea in children and adults were reported by Sakazaki, Tamura and Saito (1967) during the period TABLE II Activity of Escherichia coli cultures studied on ligated pig eyes rabbit gut loops and guinea * The dominator indicates the number of strains tested; the numerator indicates the number of positive strains.

6 12 SAKAZAKI et al. Vol. 27 TABLE III Biochemical properties of Escherichia coli strains studied Note: Ind=indole ; Ly=lysine; Ar=arginine ; Or=ornithine ; Muc=mucate ; Lac=lactose; Figure indicates the number of positive strains in the tests. 025:K1:NM. When reciprocal agglutination and absorption tests were performed, it was found that the O and K antigens of this serotype were apparently identical with those of Alkalescens-Dispar serotype 01:K1. However, they differed from the latter in producing gas from glucose. All 21 strains gave negative results in lysine and ornithine decarboxylase, arginine dihydrolase, and mucic acid utilization tests. E. coli 027:K. group Eighteen of these strains were identified. Although most strains were isolated from sporadic cases of gastroenteritis, two came from separate community outbreaks of food poisoning reported by Sakai et al. (1970 b) and Sasagawa et al. (1968, personal communication). A strain from one outbreak and two of three strains from sporadic cases produced positive rabbit gut-loop reactions but not keratoconjunctivitis in guinea pigs. K antigens of the B type were recognized in all strains but they have been given no antigenic symbol. Four serotypes, 027 :K. : NM, 027 :K. : H6, 027 :K. : H18, and 027 :K. : H2O were recognized in this group. E. coli 078 :K80 group There were six cultures isolated from sporadic cases of gastroenteritis in this group. Two strains tested gave positive ligated gut-loop reactions but keratoconjunctivitis in guinea pigs was not observed. Four serotypes, 078 :K80 :NM, 078 :K80 :H6, 078:1(80 :H12, and 078 :K80 :H35, were found among these six strains. E. coli 0114:K90 group Twenty-one strains were included in this group : one represented a community

7 1974 ENTEROPATHOGENI C ESCHERI CHIA COLI 13 Suc = sucrose; Rham = rhamnose ; Xyl = xylose ; Dul= dulcitol ; Sorb = sorbitol; outbreak of food poisoning and the remaining 20 were from sporadic cases of diarrhoea. Only the former single strain was tested with animals, and it produced positive rabbit gut-loop reaction, but not keratoconjunctivitis in guinea pigs. The majority strains were serotype 0114 :K90 :H2, but seven belonged to serotype 0114 :K90 :H26. EK coli 0142:K86 group Only two strains, isolated from sporadic cases of gastroenteritis, belonged to this group. One of them was tested and gave positive reaction in the ligated gut-loop test but did not cause keratoconjunctivitis in the guinea pig. Both strains were serotype 0142 :K86 :H18. K coli 0148:K. group Nine strains were found in this group; one represented the community outbreak of food poisoning reported by Ikemura et al. (1971, personal communication) ; the remaining eight strains were from sporadic cases of gastroenteritis. When tested in animals, three of the nine strains gave positive rabbit gut-loop reactions, but none of them induced keratoconjunctivitis in guinea pigs. All the nine strains belonged to a single serotype, 0148 :K. : H28. Their K antigens were of the B type, but no symbol has been assigned to them. E. coil 0151 :K. group This group consisted of a representative from the community outbreak of gastroenteritis reported by Asakawa et al. (1968, personal communication) and two from sporadic cases. Each strain was tested in the rabbit gut loop and guinea pig eye

8 14 SAKAZAKI et al. Vol. 27 with negative results. All the strains belonged to the same serotype, 0151: H52. When examined in the authors' laboratory, they were not agglutinated by any recognized O or H E. coli antiserum. Later, one of the strains was sent to the WHO International Escherichia Centre where new antigens, 0151 and H52, respectively, were designated by orskov, orskov, and Furowicz (1972). The presence of K antigen was not established in these strains. E. coli 0152:K. group Three cultures of this group, all isolated from patients with dysentery, were received during All strains gave positive reactions in ligated rabbit gutloop and guinea pig eye tests. All were non-motile, and accordingly included in a single serotype, 0152 :K. : NM. The presence of K antigens in these strains was uncertain. These strains were not agglutinated with any of recognized E. coli O antisera; a new antigen 0152 was assigned by orskov, orskov, and Furowicz (1972). They gave negative reactions in lysine and ornithine decarboxylase, arginine dihydrolase, and mucic acid fermentation tests. DISCUSSION The majority of E. coli OK groups described in this paper have already been reported by others as causative agents of infantile diarrhoea. Strains of E. coli. O group 6 were reported from cases of diarrhoea by Nestorescu (1954), Ewing, Davis, and Montague (1963) and Polotsky (1972, personal communication). Dulaney and Mickelson (1935) and Mickelson and Dulaney (1936) described E. coli 018 strains in outbreaks of infantile diarrhoea under the name Bacterium coli-mutabile. Later, their E. coli cultures were characterized and oriented in the Escherichia antigenic schema by Ewing, Tanner, and Tatum (1956). Mushin and Ashbourner (1964) also reported outbreaks of infantile diarrhoea associated with E, coli 018. Strains of E. coli 020 isolated from infantile diarrhoea were described by Tatum, Ewing, and Davis (1958), Lachowicz and Torum (1959), Tawil and El-Kholy (1959), and Ewing, Davis, and Montague (1963). Andreoni and Grosso (1957) reported the isolation of E. coli strains of 027 from infantile diarrhoea. As mentioned, two outbreaks of food poisoning associated with this O group were described independently by Sakai et al. (1970 b) and Sasagawa et al. (1968, presonal communication). Wramby (1948) reported members of E. coli 078 as a causative agent of calf scours. He designated this O group as 41 w, and later orskov (1952) confirmed that this was identical with 078 in Escherichia antigenic schema. Linzenmeier and Metz (1960) and Linzenmeier et al. (1962) described the isolation of E. coli 078 strains from human infantile diarrhoea. Members of E. coli 0114 were also isolated from calf scours by Wramby (1948), and designated as 26 w; this was later redesignated 0114 by Bokhari and orskov (1952). Charter (1956), Rogers and Cracknell (1956), and Linzenmeier et al. (1962) also isolated strains of this O group from infantile diarrhoea. E. coli 0142 culture was first reported by orskov et al. (1960) from diarrhoea in children. E. coli 0148 was described by Rowe, Taylor, and Bethelhaim (1970) as a causative agent associated with traveller's diarrhoea. Strains of this O group were also isolated from American soldiers in Vietnam (DuPont et at., 1971). Ikemura et al. (1971, personal communication) encountered a large community outbreak of food poisoning associated with this serotype. The isolation of 025 strains

9 1974 ENTEROPATHOGENIC ESCHERICHIA COLI 15 has been reported by many investigators (Ocklitz and Schmidt, 1952; Cefalu and Brancato, 1953; Braun, 1954; Christov and Ne j tcheff, 1957; Lackowicz and Torum, 1959; Linzenmeier et al., 1961). A number of E. col i cultures of O groups 25 isolated from various sources were studied by orskov (1954). He concluded, however, that there was not sufficient information to decide whether serotypes of this O group played a significant role in infantile diarrhoea. In fact, cultures of E. coli 025 are frequently isolated not only from patients with diarrhoea but also from other sources including normal stools. The serotype 025 :K1 : NM reported in this study was not included in the collection of orskov. Although the strains of this serotype studied were not members of the Alkalescens-Dispar biotype, it was demonstrated that their antigens were identical with those of Alkalescens-Dispar serotype 01:K1. The latter has been reported as a Shigella-like EEC (Mikata, 1933; De Assis, 1947; Sakazaki et al., 1967). This is the first report of the isolation of new E. coli serotypes, 0151:H52 and 0152 :NM. The latter possessed enteropathogenicity similar to that of Shigella. From experiences with other groups of Shigella-like EEC such as 0136 :K78, 0143 :K., and 0144:K., the serotype 0152 :NM may well be found in other countries if the diagnostic antiserum would be used in clinical diagnostic laboratories. There are many problems in determining whether E. coli serotypes, other than those already recognized, are enteropathogenic. It seems that Shigella-like EEC serotypes are relatively easy to identify, because the ligated rabbit gut-loop and guinea pig eye tests yield reliable reactions for these types. On the other hand, with Salmonella-like EEC, the lack of a reliable test is the main difficulty in ascertaining potential enteropathogenicity. In the ligated gut-loop test in rabbits, many factors, including host condition and resident flora in the intestine are known to influence the response of segment inoculated with Salmonella-like EEC cultures (Taylor, Maltby, and Payne, 1958; Ogawa, Nakamura, and Sakazaki, 1968). In the present study, however, the ligated gut-loop tests, in which three or four animals were tested for the response with each culture by a modification of the original method of De and Chatterjee (1953), gave useful results concerning enteropathogenicity in this group of EEC. In addition to ligated rabbit gut-loop tests, the frequency with which a given E. coli serotype is found to be associated with diarrhoea is important in assessing possible enteropathogenicity. Complete serological identification of strains is also necessary, as discussed by Ewing et al. (1963). The presence of a dominant strain in large numbers in stools in patients with diarrhoea is also important in assessing potential enteropathogenicity. These criteria were applied by Sakazaki et al. (1971) in their study in Calcutta. Indeed the enteropathogenicity of a serotype may only be established in this way. Hence, decision on the enteropathogenicity of 0151:H52 has been deferred. The epidemiological data of Asakawa et al. (1968, personal communication) may be significant, but cultures of this serotype gave negative reaction in ligated gut-loop tests, and further work is needed. From the present and the previous study (Sakazaki et al., 1967), the following 34 groups of E. coli associated with diarrhoea in children and adults are considered enteropathogenic.

10 16 SAKAZAKI et al. Vol. 27 O6:K. O44:K74 O124:K72 O144:K. O6:K62 O55:K59 O125:1(70 O146:K. O18:K76 O78:K80 O126:1(71 O148:K. O18:K77 O86:K61 O127:K63 O152:K. O20:K84 O86:K62 O128:K67 AD 01:K1 O25:K1 O111:K58 O136:K78 AD 02:K1 O26:K60 O112a,112c:K66 O142:K86 AD 03:1(2 O27:K. O114:K90 O143:K. AD 04:K3 O28a,28c:K73 O119:K69 Of these 34 serotypes, it was clear that 025:K1, 028a,28c:K73, 0112a,112c: K66, 0124:1(72, 0136:K78, 0143:K., 0144:K., and 0152:K., and the four serotypes of the Alkalescens-Dispar biotype were included in the Shigella-like (invasive) EEC (Sakazaki et al., 1967), because of the clinical picture they produced and the results of ligated rabbit gut-loop and guinea pig eye tests with these organisms. Antigenic difference from non-invasive E. coli cultures were also demonstrated in the K antigens of invasive serotypes by immunoelectrophoresis of K antigen (orskov et al., 1971). The remaining OK groups may belong to the Salmonella-like (non-invasive) group of EEC, because the clinical picture of infection was mainly of gastroenteritis, and they failed to induce keratoconjunctivitis in guinea pigs. The relationship among enterotoxin production, enteropathogenicity, and serotype in E. coli strains is discussed separately (Sakazaki et al., 1974). REFERENCES ANDREONI, O. AND GROSSO, F. (1957): L'Escherichia coli O27 in un episodio di gastroenteriti infantile. Boll. Ist. Sieroter. Milano., 36, BOKHARI, S. M. H. AND ƒórskov, F. (1952): O grouping of E. coli strains isolated from cases of white scours. Acta Pathol. Microbiol. Scand., 30, BRAUN, O. H. (1954): Uber die Bedeutung der E. coli-stð mme aus der O-Gruppe 25 fð¹r die Sð uglingsenteritis. Zschr. Hyg. Infekt.-Kr., 139, CEFALð, M. AND BRANCATO, P. (1953): Ann. Saint. Pub., 14, 671 (Quoted by Kauffmann, F. 1954: Enterobacteriaceae. 2nd ed. Munksgaard, Copenhagen). CHARTER, R. E. (1956): Escherichia coli type O114 isolated from infantile diarrhea and calf scours. J. Pathol. Bacteriol., 72, CHRISTOV, G. AND MEJTCHEFF, S. (1957): Dyspepsie-Coli-Bakterien in Bulgarien. Zbl. Bakt., I. Abt. Orig., 168, COSTIN, I. D. (1966): Diarrheal disease in children and adults associated with Escherichia coli strains not belonging to enteropathogenic serotypes. Pathol. Microbiol., 29, DE, S. N. AND CHATTERJEE, D. N. (1953): An experimental study on the mechanism of action of Vibrio cholerae on the intestinal mucous membranes. J. Pathol. Bacteriol., 66, DE ASSIS, A. (1947): Shigella tið te, novo tipo serologio de bacilo dysenterico. O'Hospital, 32, DULANEY, A. D. AND MICKELSON, I. D. (1935): A study of Bacterium coli mutabile from an outbreak of diarrhea in the newborn. Am. J. Public Health, 25, DUPONT, H. L., FORMAL, S. B., HORNICK, R. B., SNYDER, M. J., LIBONATI, J. P., SHEA-

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12 18 SAKAZAKI et al. Vol. 27 Scand., 48, ROGERS, K. B. AND CRACKNELL, V. M. (1956): Epidemic infantile gastroenteritis due to Escherichia coli type O114. J. Pathol. Bacteriol., 72, ROWE, B., TAYLOR, J. AND BETHELHEIM, K. A. (1970): An investigation of traveller's diarrhoea. Lancet, 1, 1-5. SACK, R. B., GORBACH, S. L., BANWELL, J. G., JACOBS, B., CHATTERJEE, B. D. AND MITRA, R. C. (1971): Enteropathogenic Escherichia coli isolated from patients with severe choleralike disease. J. Infect. Dis., 23, SAKAI, S., ITO, T., MARUYAMA. T., SAITO, K. AND ZEN-YOJI, H. (1970a): Outbreak of acute enterocolitis ascribed to the infection with Escherichio coli O6:K.:H-. Annual Rept. Tokyo Metropol. Res. Lab. Public Health, 22, 1-6. (text in Japanese). SAKAI, S., ITO, T., MARUYAMA, T., SAITO, K. AND ZEN-YOJI, H. (1970b): Outbreak of acute enteritis ascribed to the infection with Escherichia coli O27:K.:H7. Annual Rept. Tokyo Metropol. Res. Lab. Public Health, 22, 7-14 (text in Japanese). SAKAZAKI, R., TAMURA, K. AND SAITO, M. (1967): Enteropathogenic Escherichia coli associated with diarrhoea in children and adults. Japan. J. Med. Sci. Biol., 20, SAKAZAKI, R., TAMURA, K., PRESCOTT, L. M., BENCIC, Z., SANYAL, S. C. AND SINHA, R. (1971): Bacteriological examination of darrheal stools in Calcutta. Indian J. Med. Res., 59, SAKAZAKI, R., TAMURA, K., NAKAMURA, A., KURATA, T., GOHDA, A. AND TAKEUCHI, S. (1974): Enteropathogenicity and enterotoxigenicity of human enteropathogenic Escherichia coli. Japan. J. Med. Sci. Biol., 27, SELð NY, B. (1955): Experimental Shigella keratoconjunctivitis (A preliminary report), Acta Microbiol. Acad. Sci. Hung., 2, TATUM, H. W., SWING, W. H. AND DAVIS, B. R. (1958): Escherichia coli O group 20 cultures possessing K antigen 61 (B7) in common with O86:K61 (B7) strains. Public Health Lab., 16, TAWILL, G. S. AND EL-KHOLY, S. (1959): Serological types of Escherichia coli in association with infantile gastro-enteritis. J. Bacteriol., 78, TAYLOR, J., MALTBY, M. P. AND PAYNE, J. M. (1958): Factors influencing the response of ligated rabbit gut segments to injected Escherichia coli. J. Pathol. Bacteriol., 76, WRAMBY, G. (1948): Investigations into the antigenic structure of Bact. coli isolated from calves. Appelbergs, Uppsala. 196p.