Anti-K 1 phages were more active in vitro and in vivo against an 018:K1:H7 ColV+ Escherichia coli strain, designated MW, than were other phages. A single intramuscular dose of one anti-K1 phage was more effective than multiple intramuscular does of tetracycline, ampicillin, chloramphenicol, or trimethoprim plus sulphafurazole in curing mice of a potentially lethal intramuscularly or intracerebrally induced infection of MW; it was at least as effective as multiple intramuscular doses of streptomycin. When MW and the phage were inoculated into different gastrocnemius muscles of the same mice, a rapid reduction in numbers of MW organisms occurred in the MW-inoculated muscle and in other tissues; the numbers of phage particles in the MW-inoculated muscle increased rapidly and greatly. MW failed to proliferate in the brains of intracerebrally infected mice that had been inoculated intramuscularly with the phage at the same time; many more phage particles were found in the brains of these mice than in other sites. The few phage-resistant mutants of MW found in the phage-treated mich were K1-; previous studies had shown such mutants to be of greatly reduced virulence. The phage administered intramuscularly 3-5 d before challenge with a potentially lethal intramuscularly induced infection of MW was protective, the protective effect varying between phage propagated on different bacterial strains.

A mixture of two phages, B44/1 and B44/2, protected calves against a potentially lethal oral infection with an O9:K30,99 enteropathogenic strain of Escherichia coli, called B44, when given before, but not after, the onset of diarrhoea; a mixture in which phage B44/3 was replaced by phage B44/3 was effective after the onset of diarrhoea. Calves that responded to phage treatment had much lower numbers of E. coli B44 in their alimentary tract than untreated calves. Usually, high numbers of phage B44/1 and rather lower numbers of phage B44/2 or B44/3 were present in the alimentary tract of these animals. At death, most calves that had not responded to treatment with phages B44/1 and B44/2 had high numbers of mutants of E. coli B44 resistant to phage B44/1 in their small intestine. Phage-treated calves that survived E. coli infection continued to excrete phage in their faeces, at least until the numbers of E. coli B44 also excreted were low. The phages survived longer than E. coli B44 in faecal samples taken from phage-treated calves and exposed to the atmosphere in an unheated animal house. Calves inoculated orally with faecal samples from phage-treated calves that contained sufficient E. coli B44 to cause a lethal infection remained healthy. A mixture of two phages, P433/1 and P433/2, and phage P433/1 alone cured diarrhoea in piglets caused by an O20:K101,987P strain of E. coli called P433. The numbers of the infecting bacteria and phages in the alimentary tract of the piglets resembled those in the calves. Another phage given to lambs 8 h after they were infected with an O8:K85,99 enteropathogenic strain of E. coli, called S13, reduced the numbers of these organisms in the alimentary tract and had an ameliorating effect on the course of the disease. No phage-resistant mutants of E. coli S13 were isolated from the lambs. The only mutants of E. coli B44 and P433 that emerged in the calves and piglets were K30- or K101- and resistant to phage B44/1 or P433/1 respectively; those tested were much less virulent than their parent strains.

Animal Health Trust, Stock, Essex0 NE characteristic common to enteropathogenic strains of Escherichia coli of porcine origin is the ability to produce enterotoxin. A large proportion of these strains also produce a-haemolysin. Many possess a common K antigen, K88ab or ac, in addition to the one by which E. coli are usually classified. In some enteropathogenic strains, enterotoxin, haemolysin and K88 production are known to be governed by transmissible plasmids, designated Ent (Smith and Halls, 1968a), Hly (Smith and Halls, 1967) and K88 (0rskov and 0rskov, 1966) respectively, in the same manner as are antibiotic resistance (R factors) and colicine production (Col).By plasmid transmission and by " curing " with cytoplasmic poisons (Watanabe, 1963), we created strains of E. coli that differed only in regard to the particular combination of the Ent, Hly and K88 plasmids they possessed. We then used them in infection experiments with the object of determining the part played by enterotoxin, haemolysin and K88 antigen in the pathogenesis of E. coli diarrhoea in the pig. The results of these experiments are reported in this paper. So are some of the more general pathological effects of haemolysin and the K88 antigen in mice and pigs; those of enterotoxin have been reported previously (Smith and Halls, 1968a). MATERIALS AND METHODSTransfer of Ent, Hly and K88 plasmids. Essentially the same technique was employed for transferring all three plasmids, the basic principle being to grow donor and recipient organisms together in nutrient broth for conjugation and plasmid transfer to occur and then to culture on media that suppressed the donor organisms and, if possible, permitted the identification of those recipient organisms that had acquired the character under study.Ent transfer. This was performed by the method of Smith and Halls (1968a), the recipient strains being mutants resistant to streptomycin or sodium nalidixate. The mixed culture of donor and recipient organisms, after incubation at 37°C for 24 hr, was subcultured twice in broth containing streptomycin or sodium nalidixate, to eliminate donor organisms, and then once in plain broth. This final culture was submitted to the ligated-intestine test in a pig. If it produced dilatation, cultures of colonies obtained from the final culture were also tested. Those that produced dilatation were designated Ent + and those that did not, Ent -.The Ent status of wild strains used in this work was also determined by the ligated-intestine Hly and K88 transfer. The method of Smith and Halls (1967) for demonstrating Hly transfer was employed. Wherever possible, mutants resistant to sodium nalidixate or streptomycin were used as recipients; the mixed culture of donor and recipient, after incubation at 37°C for 24 hr, was inoculated so as to yield as many as possible well-isolated colonies on " washed blood " agar containing sodium nalidixate or streptomycin. The plates were incubated at 37°C for 24 hr and any colonies that were haemolytic or agglutinated in slide tests with se...

Escherichia coli 0157:H7 strain 933 contains two distinct toxin-converting phages (933J and 933W). The biologic activities and antigenic relationship between the toxins produced by 933J and 933W lysogens of E. coli K-12, as well as the homology of the genes that encode the two toxins, were examined in this study. The 933J and 933W toxins, like Shiga toxin produced by Shigella dysenteriae type 1, were cytotoxic for the same cell lines, caused paralysis and death in mice, and caused fluid accumulation in rabbit ileal segments. The cytotoxic activity of 933J toxin for HeLa cells was neutralized by anti-Shiga toxin, whereas the activity of 933W toxin was not neutralized by this antiserum. In contrast, an antiserum prepared against E. coli K-12(933W) neutralized 933W toxin but not 933J toxin or Shiga toxin. For E. coli 933, most of the cell-associated cytotoxin was neutralized by anti-Shiga toxin, whereas most of the extracellular cytotoxin was neutralized by anti-933W toxin. However, a mixture of these antisera indicated the presence of both toxins in cell lysates and culture supernatants. Among 50 elevated cytotoxin-producing strains of E. coli, we identified 11 strains isolated from cases of diarrhea, hemorrhagic colitis, or hemolytic uremic syndrome that produced cell-associated cytotoxins which were neutralized by the 933W antitoxin. Southern hybridization studies showed that the cloned toxin structural genes from phage 933J hybridized with DNA from phage 933W under conditions estimated to allow no more than 26% base-pair mismatch. These findings indicate that E. coli produces two genetically related but antigenically distinct cytotoxins with similar biologic activities which we propose to name Shiga-like toxins I and II. Strains of E. coli that produce elevated levels of Shiga-like toxin I or Shiga-like toxin II, or both, have been associated with the clinical syndromes of diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome.

Seven phages highly active in vitro and in vivo against one or other of seven bovine enteropathogenic strains of Escherichia coli belonging to six different serotypes were isolated from sewage. Severe experimentally induced E. coli diarrhoea in calves could be cured by a single dose of 10(5) phage organisms. It could be prevented by doses as low as 10(2), by spraying the litter in the calf rooms with aqueous phage suspensions or simply by keeping the calves in uncleaned rooms previously occupied by calves whose E. coli infections had been treated with phage. Microbiological examinations of calves used in these experiments revealed that the phage organisms multiplied rapidly and profusely after gaining entry to the E. coli-infected small intestine, quickly reducing the E. coli to numbers that were virtually harmless. The only phage-resistant E. coli that emerged in the studies on calves infected with one or other of the seven E. coli strains were K-. These organisms were much less virulent than the K+ organisms from which they were derived and did not present a serious problem in calves given adequate amounts of colostrum. Infections produced by oral inoculation of a mixture of six strains of the E. coli could be controlled by administration of a pool of the six phages that were active against them but, in general, the control was less complete than that observed in the single-strain infections. K+ phage-resistant bacteria emerged in some of the calves used in these mixed infections and they were as virulent as their parent organisms; evidence from in vitro studies suggested that they might have arisen by genetic transfer between organisms of the different infecting strains. Infections produced by these K+ mutants and their parents could be controlled by the use of mutant phages derived from phages that were active on their parents. During the experiments with mixed E. coli infection, an extraneous phage active against one of the six E. coli strains suddenly appeared in calves kept in the same rooms. Microbiological examinations revealed that this phage was effectively controlling the multiplication of organisms of that particular strain of E. coli in the small intestines of the calves.