The Role of Goats and Pigs in the Maintenance and Transmission of VTEC Including EHEC O157:H7

Final report summary: Enterohaemorrhagic Escherichia coli O157:H7 infections of man have been associated with consumption of unpasteurised goat¡¯s milk and direct contact with kid goats on petting farms and yet little is known about colonisation of goats with this organism. To assess coloniosation and transmission in goats a series of deliberate oral inoculation studies were performed with goats of differing ages.
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Four conventionally reared goats aged 6 days were inoculated orally with approximately 1010 colony-forming units (cfu) of a non-verotoxigenic strain of Escherichia coli O157:H7. All remained clinically normal. Tissues were sampled under terminal anaesthesia at 24 (two animals), 48 and 72 h post-inoculation (hpi). E. coli O157:H7 was cultured from ileum, caecum, colon and rectum from all animals, but the number of bacteria recovered at these sites varied between animals. Attaching effacing (AE) lesions associated with O157 organisms, as confirmed by immunolabelling, were observed in the ileum of one of the two animals examined at 24 hpi, and in the ileum, caecum and proximal colon of an animal examined at 72 hpi. E. coli O157 organisms were detected at ¡Ý 105 cfu/g of tissue at these sites. In addition, AE lesions associated with unidentified bacteria were observed at various sites in the large bowel of the same animals. Lesions containing both E. coli O157 and unidentified bacteria (non-O157) were not observed. Non-O157 AE lesions were also observed in the large bowel of one of two uninoculated control animals. This indicated that three (one control and two inoculated) animals were colonized with an unidentified AE organism before the commencement of the experiment. The O157-associated AE lesions were observed only in animals colonized by non-O157 AE organisms and this raises questions about individual host susceptibility to AE lesions and whether non-O157 AE organisms influence colonization by E. coli O157.
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Eight-week-old conventionally-reared goats were inoculated orally in separate experiments with 1 x 1010 colony-forming units (cfu) of a non-verotoxigenic strain of E. coli O157:H7 (strain NCTC12900 Nalr), an aflagellate derivative (DMB1) and an intimin deficient derivative (DMB2). At 24h after inoculation the three E. coli O157:H7 strains were shed at approx. 5 x 104 cfu per gram of faeces from all animals. Significantly fewer initimin deficient bacteria were shed only on days 2 (p=0.003) and 4 (p=0.014) whereas from day 7 to 29 days there were no differences. Tissues from three animals inoculated with wild-type E. coli O157:H7 strain NCTC 12900 Nalr were sampled at 24, 48 and 96 hours after inoculation and the organism was cultured from the large intestine of all three animals and from the duodenum and the ileum of the one animal examined at 96 hours. Tissues were examined histologically but AE lesions were not observed at any intestinal site of the animals examined at 24 or 48 hours. However, the animal examined at 96 hours, which uniquely shed approximately, 1 x 107 E. coli O157:H7 per gram of faeces for the preceding three days, showed a heavy, diffuse infection with cryptosporidia and abundant, multifocal AE lesions in the distal colon, rectum and at the recto-anal junction. These AE lesions were confirmed by immunohistochemistry to be associated with E. coli O157:H7.
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Five 5-year-old Nanies with kids at foot were dosed orally with 1010 cfu O157:H7 at about six days after parturition. Four 4 of the 5 nannies were colonised and that faecal shedding was variable up to day 11 after dosing. Several samples containing greater than 1 x 104 E. coli O157:H7 cfu per gram. This short lived suggested antagonistic factors may have prevented colonisation. One possible route for kid goats to become infected with E. coli O157:H7 is by sucking at their nanny on teats contaminated with faeces. In this study the cleanliness of the teats and udders may be attributed to sucking and, if it is assumed that some faeces containing E. coli O157:H7 organisms had been on the teats, even transiently, it is probable that the kids were challenged. Faeces containing E. coli O157:H7 organisms shed into the environment were a source of infection of the kids also. No kids became positive for E. coli O157:H7 organisms. Thus, it may be argued that sucking kids are refractive to colonisation by E. coli O157:H7 possibly because the challenge dose was low and the kids had access to milk, that is known to be highly protective. However, these hypotheses need to be examined further.
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Isolation of Shiga-toxin (Stx) positive Escherichia coli O157:H7 from commercially-grown pigs has been reported and experimental infection studies have demonstrated that this pathogen can persist in 12-week-old experimentally orally inoculated conventional pigs for up to 2 months. Intimin is not required for persistence, but we have shown that the flagellum of Stx-negative E. coli O157:H7 does not
have a role to play in pathogenesis in ruminant models whereas, in poultry, the flagellum of E. coli O157:H7 was important for long-term persistent infection. The contribution of the flagellum of Stx-negative E. coli O157 in the colonisation of pigs was investigated by adherence assays on porcine (IPI-21) cell line and IVOC and experimental oral inoculation of conventional 14-week-old pigs. E. coli O157:H7 NCTC12900nalr and isogenic aflagellate and intimin deficient mutants adhered equally well to IPI-21 cell line cells. In porcine IVOC association assays E. coli O157:H7 NCTC12900nalr was associated in significantly higher numbers to tissues from the caecum and the terminal rectum than other sites. The aflagellate and intimin deficient mutants adhered to tissues from the duodenum, jejunum and ileum in significantly higher numbers than the parent. Groups of 14-week-old pigs were dosed orally with 1010 CFU/10ml of either E. coli O157:H7 NCTC12900nalr or isogenic aflagellate and intimin deficient mutants and recovery of each test strain was similar. Histological analysis of pig tissues at post mortem examination revealed that E. coli O157 specifically stained bacteria were associated with the mucosa of the ascending and spiral colon . These data suggest that for Stx-negative E. coli O157:H7 to colonise and persist in pigs, involves mechanisms that do not require the flagellum or intimin.

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