Serotypes of verocytotoxigenic Escherichia coli isolated from cattle and buffalo calf diarrhoea

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1 FEMS Microbiology Letters 35 (1986) Published by Elsevier FEM Serotypes of verocytotoxigenic Escherichia coli isolated from cattle and buffalo calf diarrhoea (Verocytotoxin; Shiga-like toxin; Escherichia coli; serotypes; diarrhoea; calf) 1. SUMMARY A. Mohammad a, J.S.M. Peiris a,, and E.A. Wijewanta b a Department of Microbiology, Faculty of Medicine, and b Department of Veterinary Para-Clinical Studies, Faculty of Veterinary Medicine and Animal Science, University of Peradeniya, Sri Lanka A total of 45 verocytotoxin (VT)-producing Escherichia coli (VTEC) strains obtained from diarrhoeic cattle and buffalo calves were serotyped. These strains were classified into 27 'O'-groups, many of them being associated with VT for the first time. The frequently isolated VTEC serotypes were 086 : H26, O128 : H31, and OX6 : H2. However, the great diversity of VTEC O-groups is to be noted, those associated with VT production being 03, 05, 06, OX6, 07, 020, O21,049, 075, 076, 080, 084, 086, 088, O103, Ol16, Ol17, Ol18, O137, O153 and O159. It is interesting that 11 of the 41 O-groupable strains belong to well recognized human enteropathogenic E. coli (EPEC) O-groups. 2. INTRODUCTION Isolation of VTEC has been reported in humans [1-4], cattle [1,5,6] and pigs [1,3,6,8]. Most reports, however, have been based on selected Received 25 March 1986 Accepted 26 March 1986 isolates from diseased animals without data from corresponding controls, making valid association with disease difficult. VTEC have been isolated from outbreaks of human haemorrhagic colitis [9] and sporadic cases of haemolytic uraemic syndrome [10] with strong evidence for an aetiological relationship. A study of cattle and buffalo calves where E. coli isolates from diarrhoeic and healthy control animals were compared with respect to VT production was reported by us [7] and was the first field study to show significant association of VTEC with calf diarrhoea. We report here the serotyping data from 45 VTEC strains isolated in the course of the above study. Serotyping of VTEC strains from humans [1-3,9,10] and pigs [1,3] suggested that a limited number of serotypes were implicated. Data on cattle VTEC is limited; Sherwood et al. [5] reporting serotypes of 10 strains isolated from diarrhoeic calves in Scotland, and other workers [6,11] reporting serotypes of 2 more strains, making 12 strains in total. This communication adds to the known serotypes of VTEC and is the first such report from a tropical country. * To whom correspondence should be addressed /86/$ Federation of European Microbiological Societies

2 MATERIALS AND METHODS VT-producing E. coli isolates were those from diarrhoeic and healthy control cattle (Bos taurus) and buffalo (Bos bubalus bubalis) calves [7]. VT production was tested on Vero cells by a microplate modification of the method of Konowalchuk et al. [1]. The O, K and H-antigen serotyping of the strains was done at the Statens Serum Institute [12] for 35 strains, and the remaining 10 strains were serotyped at the Central Public Health Laboratory, Colindale, London, and the Rijksinstituut, Bilthoven, Netherlands. 4. RESULTS AND DISCUSSION Table 1 shows the serotypes of the VTEC strains, the farms, species of animal, and clinical status of the calves from which isolates were obtained, and whether independent confirmation of VT toxigenicity is available. The ages of calves ranged from 1 day to 6 months. 22 of the VTEC strains were independently confirmed to be VTproducing at the reference laboratories. 9 strains were not retested for VT production whereas 14 strains found to be VTEC by us on initial isolation were found to be negative when tested subsequently at the reference laboratories. This could be due to differences in the techniques used or may be the result of the loss of the VT-converting bacteriophage [13] on prolonged storage of the strains (the serotyping was done approximately 2 years after the first isolation and VT testing. The strains were maintained at 4 C on sugar-free agar [14] with periodic subculture). The majority (except for one strain each of OX6:H2 and O86:H26) of the strains whose VT status was subsequently unconfirmed, initially produced a CPE on Vero cells neutralizable by antiserum to purified E. coli Shiga-like toxin (kindly provided by Dr. A.D. O'Brien). This suggests that the initial designation of these strains as VT-positive was not based on non-specific toxicity. VTEC strains were obtained from 12 farms in different geoclimatic regions of the country, i.e., the wet zone (annual rainfall mm) and the dry zone (annual rainfall mm). The isolates were obtained all year round with no clear seasonality. The present study documents 27 O-groups to be VT-positive. Although the same serotype of VTEC was isolated from different animals from one farm on a given visit on some occasions, (e.g., OX6:H2 from farm I, O86:H26 from farm H and O128:H31 from farm B), there were many instances where different VTEC serotypes were isolated within the same farm on a given visit (e.g., 07 : H40, 08 : K44 : H from farm H; 086 : H26, O103 : H- and O153 : Hll from farm F; O139 : H51 and O128 : H31 from farm C). Further, the same serotype of VTEC was found in different farms (e.g., O3:K- in farms B and C; O128:H31 in farms A, B and D; O86:H26 in farms D and H) in widely separated parts of the country. The literature 3n serotypes of VTEC from calves is limited. Sherwood et al. [5] have reported the O-groups 04, 08, O111, O19, 026 and O149 to be associated with VT toxigenicity. The first 3 of these O-groups were found to be positive for VT production in this study as well. It is noteworthy that the group Olll VTEC has been implicated in outbreaks of haemolytic uraemic syndrome in human patients [10]. Group 026 was shown to be VT-positive by Wilson and Bettelheim [11] and Kashiwazaki et al. [6], but was not detected in the present study. This serogroup has been implicated in outbreaks of haemorrhagic colitis in man [9]. The present study documents 24 new O-groups of calf E. coli which are VT-positive. Of these, O128 VTEC has been isolated from man [1-3,15] and implicated in haemorrhagic colitis and the haemolytic uraemic syndrome [9] O141 and O139 VTEC have been isolated from pigs [3,6,8,23]. This communication is the first to our knowledge to document the VT toxigenicity of O-groups 03, 05, 06, OX6, 07, 020, O21, 049, 075, 076, 080, 084, 086, 088, O103, Ol16, Ol17, Ol18, O137, O153 and O159. It may be noted that of the O-groups found to be VT-positive in the present study, 07, O137 and Olll have been isolated from buffalo calf diarrhoea [16,17] and 086 isolated from calf septicaemia [18] in the South Asian subcontinent. The enterotoxigenic status of these isolates was not

3 263 Table 1 Serotypes of VTEC isolated from cattle and buffalo calves Serotype of Source of isolation Reconfirmed as VTEC Species a Clinical Farm VTEC at reference status b laboratory 03 : K- B d C not retested 03 : K- C d B not retested 04 : H11 B d F yes 04 : K3 : H5 B ic A no (05) : K? : H32 C ic H no 06 : H27? B d A no OX6 : H2 C d I yes OX6 : H2 B d K yes OX6 : H2 C d I no 07 : H40 C d H yes 08 : K44 : H- C d H yes 08, 075 : H49 B d A no 020 : K? C d B not retested O21 : K? B d H not retested 049 : K? : H12 C d L yes 075 : K+ : H19 B c J yes (076) : H48 C d B no 076 : K?H20 B d C no 080 : H31 B d C yes 084 : K? C d G not retested 086 : H26 C d H yes 086 : H26 B ic D yes 086 : H26 C c H yes 086 : H28 B d F yes 088 : H8 B d F yes O103 : H- B d F yes O103 : H21 C d B no Olll : H- C ic H yes O116 : H- C d K yes Ol17:K+ :H7 C d E yes Ol17 : H? B d A no Ol18 : K- B d A not retested O128 : H31 B d D yes O128 : H31 C d B yes O128 : K- B d B not retested O128 : H31 B d A no O137 : H41 C d H no O139 : H51 B d C yes O141 : K85AB B d A not retested O153 : Hll B d F yes O159 : H31 B ic F no O? : H34 B ic A yes O? : H16 B d A no Rough : H7 B d D no Untypeable C d B not retested Other references to VTEC of the same O-group Calf Pig Human [5] [51 [51 [1,1Ol [3,6,23] [3,8] a B, buffalo; C, cattle. b d, Diarrhoeic; ic, non-diarrhoeic calves housed in the same pen as diarrhoeic animals; c, non-diarrhoeic control. c A to G, farms situated in the 'dry' zone; H to L, farms situated in the 'wet' zone.

4 264 determined. The O137:H41 serotype has been recognized as a strain causing calf colisepticaemia in both field and experimental studies [19,20]. Of the 26 O-groups of E. coli (from 103 diarrhoeal calves) isolated in Iraq [21], O-groups 020, O21, 086, Ol17, 03, 08, O103 and O141 were found in our study to be VT-positive. Although the first 4 groups were found in Iraq to be heat-stable toxigenic strains, the latter 5 O-groups had no identifiable virulence factor. No data on the VT toxigenicity of the Iraqi isolates was available, and the possibility that VT was the virulence factor in these latter strains remains worthy of consideration. In reviewing E. coli O-groups isolated from cattle worldwide, 08, 020, Ol17 and O137 (found to be VTEC in this study) are among those commonly isolated from diarrhoeic and septicaemic calves [22], although the authors point out that these same O-groups have also been isolated from healthy animals. It is to be noted that 5 VTEC strains in our collection (belonging to the serotypes OX6: H2, O88:H8, Ol16:H-; Ol17:K + :H7 and O141:K85AB) produced VT-like CPE on Vero cells that was not neutralizable by antibody to purified E. coli Shiga-like toxin. This indicates that VT as detected on Vero cells is antigenically heterogeneous [24,25]. Human EPEC are a cause of infantile enteritis and are recognized by their O-groups. The virulence factors and mechanism by which these strains cause diarrhoea are at present unclear, although there is some evidence that VT may be one such factor. It is interesting to note therefore, that in this study, of the 41 O-groupable strains, 11 belong to well-recognized human EPEC O-groups (i.e., 020, 086, Ol11, O128 and O159) [22]. However, when the H antigen is also considered, the O:H serotypes of these isolates are not identical to those of well-known EPEC strains. ACKNOWLEDGEMENT We wish to thank Drs. I. and F. iorskov, Statens Serum Institute, Copenhagen, Denmark, for their critical review of the manuscript and for serotyping the E. coli strains; Dr. B. Rowe, Central Public Health Laboratory Colindale, London, and Dr. P.A.M. Guinee, Rijksinstituut, Bilthoven, Netherlands, for serotyping some strains of E. coli; and Dr. A.D. O'Brien, Uniformed Services University of the Health Sciences, Maryland, U.S.A. for providing us with antiserum to purified E. coli Shiga-like toxin. This work was supported by a grant from the Wellcome Trust, U.K., given to Dr. J.S.M. Peiris. REFERENCES [1] Konowalchuk, J., Speirs, J.J. and Stavric, S. (1977) Infect. Immun. 18, [2] Scotland, S.M., Day, N.P. and Rowe, B. (1980) FEMS Microbiol. Lett. 7, [3] Smith, H.W., Green, P. and Parsell, Z. (1983) J. Gen. Microbiol. 129, [4] Wade, W.G., Thom, B.T. and Evans, N. (1979) Lancet ii, [5] Sherwood, D., Snodgrass, D.R. and O'Brien, A.D. (1985) Vet. Rec., 116, [6] Kashiwazald, M., Ogawa, T., Nakamura, K., Isayama, Y., Tamura, K. and Sakasaki, R. (1981) Natl. Inst. Anim. Health Q. (Jpn.) 21, [7] Mohammad, A., Peiris, J.S.M., Wijewanta, E.A., Mahalingam, S. and Gunasekara, G. (1985) FEMS Microbiol. Lett. 26, [8] Gonzalez, E.A. and Blanco, J. (1985) FEMS Microbiol. Lett. 26, [9] O'Brien, A.D., Lively, T.A., Chen, M.E., Rothman, S.W. and Formal, S.B. (1983) Lancet i, 701. [10] Karmali, M.A., Petric, M., Steele, B.T. and Lim, C. (1983) Lancet ii, [11] Wilson, W.M. and Bettelheim, A.K. (1980) Lancet i, 201. [12] Orskov, F. and Orskov, I. (1984) in Methods in Microbiology, Vol. 14, (Bergan, T., Ed.) pp Academic Press, London. [13] O'Brien, A.D., Newland, J.W., Milem, S.F. Holme, R.K., Smith, H.W. and Formal, S.B. (1984) Science 226, [14] WHO/UNEP Report (1977) Guideline for health related monitoring of coastal water quality. Rovinj, Yugoslavia, Feb [15] O'Brien, D., LaVeck, G.D., Thompson, M.R. and Formal, S.B. (1980) J. Infect, Dis. 146, [16] Kistwaria, R., Misra, S.K. and Chaudhuri, P.C. (1982) Sri Lanka Vet. J. 30, [17] Verma, P.C. and Kalra, D.S. (1975) Ind. Vet. J. 62, [18] Triphathi, R.D. and Soni, J.L. (1984) Ind. Vet. J. 61, 4-8. [19] Rees, I.A. (1958) J. Comp. Pathol. Ther., 68, 388. [20] Gay, C.C., McKay, K.A. and Barnum, D.A. (1964) Can. Vet. J. 5,

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