PLATES XXVIII AND XXIX NEONATAL diarrhoea in piglets follows the proliferation of certain strains of Escherichia coli in the small intestine (Smith and Jones, 1963). These enteropathogenic strains synthesise enterotoxins which produce diarrhoea and dehydration that frequently result in the death of the piglets (Kohler, 1968;Smith and Gyles, 1970;Smith and Linggood, 1971). The rapid proliferation of E. coli in the small intestine appears to be attributable to the ability of the bacteria to attach themselves to the intestinal epithelium (Arbuckle, 1970; Drees and Wader, 1970a and b; Bertschinger, Moon and Whipp, 1972) and thereby avoid removal from the small intestine by peristalsis (Dixon, 1960). Sojka (1965) noted that the majority of E. coli strains isolated from cases of neonatal piglet diarrhoea possess a surface antigen designated K88, and it has been shown that this antigen is an essential virulence determinant (Smith and Linggood, 1971 ;Jones and Rutter, 1972) apparently because it enabled K88-positive E. coli to attach to the intestinal lining (Jones and Rutter, 1972).To investigate the interaction between the host mucosal surface and K88-positive E. coli a convenient test system is essential. The K88 antigen agglutinates guinea-pig red cells (Stirm et al., 1967;Jones and Rutter, 1974), and attempts to study the chemical aspects of adhesion by means of this reaction have been reported (Gibbons, Jones and Sellwood, 1975). It would have been preferable to use a natural intestinal receptor, but the technique involving adhesion of E. coli to disks of intestinal tissue (Jones and Rutter, 1972) is laborious. This paper describes a simple in-vitro technique that demonstrates adhesion of K88-positive E. coli to brush borders from pig intestinal cells; a survey of pigs in respect of the ability of their brush borders to adhere to E. coli is also described. MATERIALS AND METHODS

The role of the K88 antigen of Escherichia coli in neonatal diarrhea of piglets was studied by comparing a K88-positive strain with three K88-negative strains derived from the K88-positive strain. K88 antigen was produced by the K88-positive strain in the intestinal tract of gnotobiotic piglets, whereas K88-negative strains did not regain the ability to synthesize K88 antigen. Synthesis of the antigen conferred different colonization characteristics on the four strains; K88-positive bacteria adhered to the mucosa of the small intestine, whereas K88-negative bacteria did not attach and were distributed throughout the lumen. Adhesion of K88-positive bacteria to tissue from the small intestine of gnotobiotic piglets was demonstrated in vitro and was inhibited by antisera that contained K88 antibodies. Attachment did not occur with bacteria grown at 18 C. Adhesion of cell-free K88 antigen was also demonstrated. The K88-positive strain and one of the K88-negative strains were equally virulent in gnotobiotic piglets. In contrast, the K88-positive strain killed 50% of conventionally reared piglets, whereas the K88-negative strain killed only 3%. Adhesion of the K88-positive strain, but not of the K88-negative strain, to the mucosa of the small intestine was demonstrated. Our results show that K88 antigen is responsible for attachment of K88-positive bacteria to the wall of the small intestine, and that adhesion is essential for the virulence of K88-positive bacteria in conventionally reared piglets. Neonatal diarrhea in piglets is characterized by the proliferation of certain strains of Escherichia coli in the small intestine (41). Colonization of the anterior small intestine by these strains followed by their multiplication to reach large numbers (41) and production of enterotoxins (21, 28, 39) results in clinical disease. The comparatively small numbers of non-enteropathogenic strains of E. coli in the small intestine of apparently healthy piglets (24, 41) may be due, in part, to the removal of bacteria by gut motility (1, 11, 18). Adhesion of E. coli to the intestinal epithelium has been demonstrated (2, 12, 13, 14, 40, 44, 46, 50), and it is possible that attachment allows enteropathogenic strains to overcome gut motility and to reach large numbers in the small intestine. Strains of enteropathogenic E. coli isolated from diseased piglets frequently possess K88 antigen (20, 45, 52). In contrast to the other K antigens, K88 is a protein component (48) that forms a substantial layer of fine filaments on the surface of the cell (47). Its synthesis is controlled by a plasmid that can be spontaneously lost (30). Ad

All of 108 strains of Escherichia coli that synthesized K88 antigen caused mannose-resistant and eluting (m.r.e.) haemagglutination of guinea-pig erythrocytes in a microhaemagglutination test; none of 23 representative strains did so in a tile haemagglutination test which requires firmer binding. It was concluded that the K88 antigen was the m.r.e. haemagglutinin since (i) only K88-positive strains caused m.r.e. haemagglutination (ii) K88-positive strains grown at 18 "C failed to produce both haemagglutinin and K88 antigen (iii) haemagglutinating activity was not detected in K88-negative mutants of a K88-positive enteropathogenic strain, and (iv) extracts of K88 antigen possessed haemagglutinating activity which could not be separated from the K88 antigen by the fractionation and serological procedures examined. Haemagglutination appears to resemble the attachment of K88-positive bacteria to the gut wall in enteric disease and the haemagglutination test may assist in characterizing this mechanism.