Compounds that prevent chloride transport in membrane vesicles have been tested for in vivo activity against the effects of intestinal secretory agents. Chloride channel blockers including diphenylamine-2-carboxylate, 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonate, 5-nitro-2-(2-phenylethylamino)benzoic acid, and alpha-phenylcinnamic acid were tested for effects on jejunal or ileal secretion in weanling pigs. Secretion was studied in ligated intestinal loops in a control state, during exposure to secretory concentrations of theophylline, and after prior treatment with cholera toxin. Increases in net fluid flux induced by either theophylline or cholera toxin were not prevented by adding chloride channel blockers into the intestinal lumen. Channel blocker concentrations that reduced chloride transport by > 50% in pig jejunal brush border vesicles did not cause significant changes in unidirectional blood to lumen chloride flux measured in situ. Several routes of administration of the specific chloride channel blocker alpha-phenylcinnamate failed to reduce fluid secretion induced by theophylline. Chloride channel blocker effectiveness appears to be significantly different between in vitro and in vivo experimental models. In contrast to the chloride channel blockers, loperamide significantly reduced net fluid and chloride flux in ileal loops secreting fluid in response to theophylline. Antagonism of the production or actions of second messenger by loperamide was more effective than the chloride channel blockers in reducing conductive chloride transport associated with intestinal secretion.

Pleuropneumonia is an important disease of swine caused by Actinobacillus pleuropneumoniae. Putative virulence determinants include capsule, lipopolysaccharide, and cytotoxin. We studied the virulence and virulence determinants of 2 strains: CM5 and CM5A of serotype 1. Strain CM5 was isolated from a pig with pleuropneumonia and passaged once in vitro; strain CM5A was a substrain of CM5 passaged 70 times in vitro. Pigs challenge exposed to an aerosol of 1.3 x 10(7) colony-forming units of CM5/ml died within 30 hours; pigs challenge exposed to an aerosol of 1.6 X 10(8) colony-forming units of CM5A/ml survived. The average thickness of the capsular layer was 137 nm in strain CM5 and 53 nm in strain CM5A in bacteria treated with homologous antibody and examined by transmission electron microscopy. Similarly, capsular material binding polycationic ferritin was found in colonies of strain CM5, but not in strain CM5A. The ratio of hexosamine to protein in extracted capsule of CM5 was more than twice that of CM5A. The polyacrylamide gel electrophoretic profile of the lipopolysaccharide, outer membrane proteins, and whole cell proteins did not differ between the 2 strains. Also, the amount of cytotoxin or endotoxin produced by the 2 strains during the logarithmic growth phase was not different. The electrophoretic profile of restriction endonuclease digested DNA was similar, with the exception of bands in the 750- and 620-basepair regions. It was concluded that attenuation of strain CM5A during in vitro passage was a result of reduced capsule production and that encapsulation is an important virulence determinant of A pleuropneumoniae, serotype 1.

Colony hybridizations with DNA probes for 3 heat-stable (STaP, STaH, and STb) enterotoxins and 1 heat-labile (LT) enterotoxin and for 4 adhesins (K99, F41, K88, 987P) were performed on 870 Escherichia coli isolates to determine pathotypes prevalent among enterotoxigenic E coli (ETEC) isolated from cattle in Belgium. One hundred thirty-two E coli isolates (15.2%) hybridized with probes STaP, K99, and/or F41. The 5 other probes were not hybridized by E coli isolates. Therefore, only STaP enterotoxin and K99 and F41 adhesins were virulence factors of ETEC isolated from cattle. Two major pathotypes accounted for 95% of the ETEC: STaP+K99+F41+ (67.4%) and STaP+K99+ (27.3%). The last 5% of probe-positive isolates had STaP+, STaP+F41+, or K99+F41+ minor pathotypes. Of 12 American ETEC isolates also assayed, 7 were positive with STb and/or 987P probes (pathotypes STaP+STb+,STaP+ 987P+, or STaP+STb+987P+) and may be porcine- rather than bovine-specific enteropathogens. The remaining 5 American ETEC isolates belonged to 3 minor pathotypes (STaP+,STaP+F41+, and K99+F41+) also found among Belgian E coli isolates. Such isolates may be derivatives of STaP+K99+F41+ or STaP+K99+ ETEC after in vivo or in vitro loss of virulence genes and/or non-ETEC isolates, which have acquired virulence genes by in vivo transfer.

Serum tumor necrosis factor (TNF) activity wasquantitated in 8 horses given an IV infusion of endotoxin (0.03 microgram of lipopolysaccharide/kg of body weight, from Escherichia coli 55:B5) in 0.9% NaCl solution over 1 hour. Serum TNF activity was likewise measured in 6 horses given only 0.9% sterile NaCl solution at the same rate. The duration of serum TNF activity was determined, and serum TNF activity was correlated with clinical and laboratory changes during the induced endotoxemia. Horses had no serum TNF activity prior to endotoxin administration, but geometric mean serum TNF activity was significantly higher from 1 to 4 hours after the start of the infusion. In response to endotoxin, horses seemed depressed, had signs of mild to moderate abdominal pain, developed tachycardia and fever, and had leukopenia followed by leukocytosis. Association between serum TNF activity and temperature, heart rate, attitude abnormality score, and WBC count of horses given endotoxin was significant. Serum TNF activity had a significant positive linear correlation with attitude abnormality and heart rate and a negative linear correlation with the WBC count during endotoxemia. Geometric mean serum TNF activity peaked approximately 1.5 hours prior to mean peak fever, and these data were significantly correlated. Results of this study suggest that TNF is an important mediator of endotoxemia in horses.

Alterations in the various leukocyte populations in milk, blood, and mammary lymph were studied by use of the flow cytometric method during acute mastitis episodes induced by endotoxin infusion (50 microgram of lipopolysaccharide of Salmonella typhimurium SH 4809) via the teat canal. Lymph samples were collected via a semipermanent catheter from an afferent duct to the supramammary lymph node. Milk somatic cell count increased at 4 hours after infusion of endotoxin. Neutrophils were the predominant cell population for up to 59 hours after infusion. Numbers of lymphocytes and monocytes-macrophages in milk also increased after the endotoxin infusion. The total cell count in milk started to decrease during the third postinfusion day and returned to preinfusion values during the fourth day. Lymphocyte numbers remained high for about 1 week after the infusion, and lymphocytes were the predominant cell population between postinfusion days 4 and 8. Total blood leukocyte count decreased during the first 6 hours after infusion, followed by an increase until postinfusion hour 31. The proportion of neutrophils in blood increased during the first day, whereas that of lymphocytes decreased. Lymph flow rate and leukocyte numbers in lymph increased after endotoxin infusion. The proportion of neutrophils in the lymph increased during the first 6 hours, whereas that of lymphocytes decreased. After postinfusion hour 6, the inverse course of events was seen.