Secretion of hydrogen peroxide by phagocytic cells from bovine non-lactating mammary glands.

Phagocytic cells from non-lactating bovine mammary glands have the capacity to secrete hydrogen peroxide when exposed to the soluble membrane stimulant phorbol myristate acetate (PMA). Unfractionated cell suspensions, containing mainly neutrophils and macrophages, and cell monolayers enriched for macrophages secreted hydrogen peroxide. A correlation was observed between the amount of hydrogen peroxide secreted, the antibacterial activity of the cells and the number of neutrophils present in the cell suspensions. Pre-exposure of cells to PMA significantly impaired their antibacterial activity against Staphylococcus aureus suggesting the importance of oxygen metabolism in the bactericidal capacity of these cells.

Phagocytic and bactericidal properties of bovine macrophages from non-lactating mammary glands.

Macrophages were isolated from the mammary glands of non-lactating (dry) cows and their ability to phagocytose and kill staphylococci in vitro assessed. Normal bovine serum enhanced the uptake of staphylococci and was required for optimal killing in the bactericidal test. Dry gland secretion interfered with uptake. Secretions taken progressively into the dry period became more inhibitory. The phagocytic ability of macrophages was significantly less than that of neutrophils present in the same gland preparation when tested in the presence of dry gland secretion. A marked variation in the antibacterial activity of macrophages from different cows was noted.

Heat-stable enterotoxins from Escherichia coli P16.

Escherichia coli P16 infant mouse active heat-stable enterotoxin may be fractionated into two distinct active moieties by ion-exchange chromatography, Sephadex G-25 chromatography, and isoelectric focusing.

The ability of cholestyramine resin and other adsorbents to bind Escherichia coli enterotoxins.

Several adsorbent materials were evaluated for their ability to bind Escherichia coli enterotoxins. Cholestyramine, a strong anion-exchange resin, bound the heat-labile and the heat-stable types of enterotoxin and reduced significantly their effects in some animal models. However, its efficacy in the treatment of diarrhoeic piglets appeared to be adversely affected by the presence of milk in the alimentary tract.

Assay of the heat-labile enterotoxin of Escherichia coli in infant rabbits.

Infant rabbits were shown to respond to Escherichia coli heat-labile enterotoxin by a consistent increase in intestinal fluid content, which was maximal 5 h after oral dosing. Infant rabbits could be used in a simple quantitative assay for heat-labile E. coli enterotoxin based on the ratios of gut weight to remaining body weight 5 h after oral dosing. Infant rabbits remained responsive to heat-labile enterotoxin up to 14 days of age, after which their gastric pH became low enough to destroy the enterotoxin. Rabbits that had been deprived of food before being dosed had a reduced gastric pH and a reduced response to the enterotoxin. Lincomycin andmitomycin C were found not to increase th e yield of heat-labile enterotoxin from E. coli strain P307.

Effect of Escherichia coli heat-stable enterotoxin on cyclic GMP levels in mouse intestine.

Partially purified heat-stable enterotoxin obtained from Escherichia coli strain F11/P155 caused an accumulation of cyclic GMP in the intestines of 8-day-old mice.

Biological evaluation of a methanol-soluble, heat-stable Escherichia coli enterotoxin in infant mice, pigs, rabbits, and calves.

Escherichia coli P16 was shown to produce two heat-stable toxins (ST) with differing biological activity. The toxins were separated by methanol extraction, and the first, STa, was methanol soluble, partially heat stable, active in neonatal piglets (1 to 3 days old) and infant mice, but inactive in weaned pigs (7 to 9 weeks old); the second, STb, was methanol insoluble, active in weaned pigs and rabbit ligated loops, but inactive in infant mice. It is therefore suggested that use of suckling mice as indicators of ST production will fail to identify certain ST-producing strains.

Characterization of a partially purified methanol-soluble heat-stable Escherichia coli enterotoxin in infant mice.

While studying the involvement of cyclic adenosine 3',5'-monophosphate (cAMP) in the fluid secretion caused by heat-stable enterotoxin (ST) from Escherichia coli P16 in infant mice, it was noted that the culture filtrate containing ST also contained large amounts of cAMP. The present paper details attempts to obtain a cAMP-free ST preparation. The organisms were grown in a defined medium, and the heated culture filtrate was concentrated by reverse osmosis. After methanol extraction of the filtrate, which removed 80% of the nonactive solids, the methanol-soluble ST was further purified by gel filtration through a Sephadex G-10 column. The first fraction recovered after gel chromatography contained ST with a negligible amount of cAMP. Treatment with methanol did not adversely affect the enterotoxic activity. Certain parameters of the infant mouse model have been investigated, and using our ST preparation it has been found that animals remain responsive up to 15 days of age with an optimum assay time of 2 h after toxin challenge.

Protection against gram-negative infections with antiserum to lipid A from Salmonella minnesota R595.

The ability of antisera to lipid A, induced in rabbits by immunization with lipid A complexed to various carriers, to protect mice against gram-negative infection and to inhibit the fluid loss caused by an enteropathogenic strain of Escherichia coli in the piglet ligated gut was investigated. No significant protection was obtained in either case, although passive hemolysis and quantitative precipitation tests showed the presence of antilipid A antibodies in the sera. Fluorescent antibody studies suggest that the lipid A is in a cryptic position on the surface of smooth strains of gram-negative bacteria.