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Surmmary Some plate-grown strains of Helicobacter (Campylobacter) pylori that were harvested into phosphate-buffered saline and left for 1 h released soluble haemagglutinins. These caused high-titre agglutination of human and guinea-pig erythrocytes, whereas chicken, sheep and bovine erythrocytes were agglutinated at various titres. Six of 10 strains which had been subcultured repeatedly did not possess soluble haemagglutinins. Slide agglutination of bacterial suspensions demarcated the strains into two groups; Group 1 gave strong agglutination with most types of erythrocyte, Group 2 did not. By microtitration assay, all Group-1 strains but only two Group-2 strains produced a soluble haemagglutinin. Cell-associated haemagglutinins were found by microtitration assay in all strains of H. pylori, but higher titres were found within Group-1 strains. The supernates of broth-grown, shaken cultures also showed the presence of soluble haemagglutinins, with higher titres for recently isolated strains. Pre-treatment of human erythrocytes with neuraminidase from Arthrobacter ureafaciens and Clostridium perfringens abolished haemagglutination by the soluble, but not by the cell-associated haemagglutinin. The soluble haemagglutinin was inhibited by sialoproteins containing predominantly the N-acetylneuraminyl (2–3) galactopyranosyl [NeuAc(2–3)Gal] structure, fetuin, glycophorin and bovine N-acetylneuraminyl-lactose (NeuAc-Lac). Transferrin and human NeuAc-Lac, which contain predominantly the N-acetylneuraminyl (2–6) galactopyranosyl [NeuAc(2–6)Gal] structure were not inhibitory. However, bovine submaxillary mucin (BSM) was strongly inhibitory; it contains several structures with sialic acid linked 2–6 to oligosaccharides. These results suggest that the soluble haemagglutinin recognises a NeuAc(2–3)Gal structure, but has high affinity for another, as yet undetermined, sialic acid-containing structure.

SUMMARY Culture filtrates of micro-organisms isolated from the upper intestinal secretions of malnourished children and grown in pure culture were shown to impair the intestinal absorption of water and electrolytes in live rats. Decreased net movement out of the intestinal lumen, or actual secretion of water, sodium or potassium into the intestinal lumen, was found with culture filtrates of single isolates of Staphylococcus epidermidis, Escherichia coli 055, Escherichia coli B7A, Shigella sonnei, Klebsiella pneumoniae, Candida albicans and Candida tropicalis. These organisms have been found to contaminate upper intestinal secretions in malnourished children and it is suggested that the effects observed in these experiments might be relevant to the production of the diarrhoea that is a dominant clinical feature of childhood malnutrition.

A soluble haemagglutinin has been identified in cell-free culture supernates of human diarrhoeal isolates of Aeromonas sobria, A. hydrophila and A. caviae. It was oligomeric; a major peak of haemagglutinating activity had an apparent mol. wt of 780,000 but there was haemagglutinating activity throughout the mol. wt range less than 40,000- greater than 10(6). Human group O, A and B, horse, rabbit, chicken and rat erythrocytes, but not those of sheep and cow, were agglutinated by the soluble haemagglutinin, in contrast to the cell-bound agglutinin. Agglutination was inhibited by fetuin, a complex glycoprotein, but not by simple sugars. The haemagglutinating activity was not affected by 0.5 M NaCl, dithiothreitol or the presence or absence of Ca++. It was unrelated to the haemolytic, enterotoxigenic and proteolytic activities present in cell-free extracts of A. sobria. All A. sobria, 73% of A. hydrophila and 68% of A. caviae strains tested produced this soluble haemagglutinin. A. caviae does not appear to be an enteric pathogen, therefore this soluble haemagglutinin alone is unlikely to be a virulence factor in Aeromonas spp.

Cytotoxic enterotoxin of Aeromonas sobria was purified by affinity chromatography with monoclonal antibodies. The purified enterotoxin gave a single protein band in polyacrylamide gradient gel electrophoresis and its mol. wt estimated by this technique was 63,000; it had a pI of 6.2. The purified enterotoxin caused fluid accumulation in rat ileal loops and in infant mice, was cytotoxic to cultured cells, was haemolytic to human erythrocytes, and was lethal to mice after intravenous injection. The relative concentrations of enterotoxic, cytotoxic and haemolytic activities were approximately the same in a culture filtrate and in purified, electrophoretically homogeneous enterotoxin. The three activities were also inactivated to the same extent after incubation for 10 min at 56 degrees C. There was no immunological cross-reactivity with cholera toxin (CT) nor did antiserum to CT neutralise the biological effects of the toxin.

Isolation by affinity chromatography from crude culture filtrate of Aeromonas sobria of protein that cross reacted with cholera toxin (CT) revealed a toxin that produced fluid accumulation in rat ileal loops and in infant mice and caused rounding of Y1 adrenal cells. All these activities were neutralised by antiserum to CT. There was no haemolytic or cytotoxic activity associated with this CT-cross reactive cytotonic enterotoxin. CT-cross reactive material detected in enzyme linked immunosorbent assay (ELISA) was produced by 25% of Aeromonas isolates from faeces of children with or without diarrhoea-26% of A. sobria, 20.0% of A. hydrophila and 24% of A. caviae tested gave positive ELISA results. Most strains that produced this cytotonic enterotoxin but no cytotoxic enterotoxin were isolated from children without diarrhoea. Toxin preparations from Aeromonas spp. that completely inhibited adenosine-5'-diphosphate-induced platelet aggregation, an effect related to elevation of intracellular cAMP, were, with one exception, cross reactive with CT in ELISA.

BACTERIAL overgrowth occurs in the upper small intestine of malnourished children (Mata et al., 1972; Gracey et al., 1973; Heyworth and Brown, 1975) and species of Candida are found more frequently in the gastric and small-intestinal contents of such children (Gracey et al., 1974). The possible importance of Candida species as a cause of diarrhoea in malnourished children has been suggested by El-Gholmi, Aboul-Dahab and El-Essawi (1961); this is supported by our findings that there is decreased absorption of sugar (Burke, Houghton and Gracey, 1977) and water (Thelen, Burke and Gracey, 1978) in rat intestine perfused in vivo with cell-free broth cultures of Candida species isolated from the upper intestinal contents of malnourished children. We have now studied the effects on intestinal transport and activity of brush-border enzymes in rats experimentally infected with C. albicans to clarify the relationship between diarrhoea and intestinal candidiasis.

SUMMARY. The suckling-mouse assay was reliable for detecting enterotoxigenic strains of Aeromonas hydrophila when standard conditions for growth and toxin testing were used. Enterotoxins were produced by bacteria grown in tryptone soya broth supplemented with yeast extract and aerated by shaking in an environmental incubator or water bath. When culture supernates together with dye were administered intragastrically to mice less than 6 days old, the presence of enterotoxin was assessed on the basis of a scoring system that incorporated the ratio intestinal weight: remaining body weight, and production of diarrhoea. This method should facilitate the detection of enterotoxigenic strains of Aeromonas in epidemiological studies.

Summary Direct plating of faecal specimens on blood agar was compared with the use of enrichment culture for isolation of Aeromonas spp. from faeces during a large epidemiological study. Of enterotoxigenic strains isolated by direct plating, 89% were associated with acute diarrhoea and 7% with an episode of diarrhoea during the month before collection, but 79% of enterotoxigenic strains isolated only after enrichment were not associated with acute diarrhoea. With Aeromonas spp., as with intestinal pathogens, it appears that enrichment allows isolation of the bacteria when in low faecal concentrations likely to be found in convalescent patients, carriers and those with subclinical infection. The routine use of enrichment for isolation of faecal aeromonads, by detecting Aeromonas spp. in low numbers in patients without diarrhoea, is likely to confuse interpretation of epidemiological studies seeking to clarify the relationship between Aeromonas spp. and acute diarrhoea.