Comparative atherogenic effects of cholesterol and cholesterol oxides.

Previous findings indicating that the oxidation products of cholesterol are associated with atherogenicity have led to a comparative study of the subchronic effects of feeding rabbits purified cholesterol, oxidized cholesterols free of cholesterol and cholesterol esters, or a mixture of cholesterol and oxidized cholesterols. Macroscopically, the cholesterol-fed animals exhibited 6-fold more arterial lesions than the animals fed cholesterol-free oxidized cholesterols. Microscopically, there was no statistically significant difference from the control in the number of histochemically-defined lesions in any of the groups. However, the lesions in the cholesterol-fed group were more severe, as indicated by a statistically significant increase in the magnitude of the lesions. This increased severity was also characterized by greater frequency and intensity of Azure A/Thionin, VonKossa, and Horseradish Peroxidase-Wheat Germ Agglutinin staining. Electron-microscopic studies of normal appearing arterial tissues showed an increased density of viable smooth muscle cells and an increase in vacuolar extracellular debris in the cholesterol-fed group. Oxidized cholesterols in the concentrations and relative compositions administered here are markedly less atherogenic to rabbits than highly purified cholesterol.

Fate of Listeria monocytogenes in orally dosed chicks.

The fate of Listeria monocytogenes in chicks perorally dosed with these bacteria at 2 days of age was determined by bacterial enumeration, immunoperoxidase staining and histological examination of the liver, muscle and gastrointestinal tract. Results revealed listerial egress from the digestive tract and elimination of the organism from the body in most of the chicks within 9 days post-inoculation. L. monocytogenes was isolated from the caecum of only one of 10 chicks examined at 4 weeks post-inoculation. Results indicate that chickens are not likely to be common reservoirs of L. monocytogenes. Intestinal carriage of L. monocytogenes by poultry may frequently be transient, resulting from ingestion of Listeria-contaminated feed and soil.

Indirect immunoperoxidase localization of aflatoxin B1 in rat liver.

Aflatoxin B2a (AFB2a) antibody was used as a histochemical probe in the indirect immunoperoxidase localization of aflatoxin B1 (AFB1) bound to rat liver. The efficacy of the indirect method was initially demonstrated by detecting AFB1 covalently bound to DNA in an enzyme-linked immunosorbent assay. AFB1-modified DNA was attached to a polystyrene microtissue culture plate (solid phase) and then subjected to sequential incubation with AFB2a antiserum followed by goat anti-rabbit peroxidase conjugate. Assays for bound peroxidase revealed that the AFB2a antiserum could be diluted 200,000-fold and still yield a signal-to-noise ratio of 10 when compared to an unmodified DNA control. When the same indirect immunoperoxidase protocol was applied to the light-microscopic localization of AFB1 in liver sections of rats treated in vivo with the mycotoxin, bound toxin could be identified in excellent detail in tissues fixed with periodate-lysine-paraformaldehyde and embedded in glycol methacrylate, but was detectable with only poor resolution in unfixed cryostat sections. Peroxidase-positive reactions in hepatocytes typically exhibited strong nuclear and relatively lighter cytoplasmic staining. Greater concentrations of peroxidase-positive hepatocytes were detected in the periportal area than in the area of the central vein, suggesting a circulatory pattern for AFB1 binding in the liver.

Comparative subchronic oral toxicity of sulphite and acetaldehyde hydroxysulphonate in rats.

A subchronic oral toxicity study of sodium metabisulphite and acetaldehyde hydroxysulphonate was conducted in normal and sulphite oxidase-deficient rats. At the highest dose level (350 mg SO2 equiv./kg body weight/day for 3 wk followed by 175 mg SO2 equiv./kg body weight/day for 5 wk of either compound), gastric lesions were noted histologically in both normal and sulphite oxidase-deficient rats. The lesions were more severe and more frequently encountered in the sulphite oxidase-deficient rats. The no-effect level for Na2S2O5 was 70 mg SO2 equiv./kg body weight/day in both normal and sulphite oxidase-deficient rats. Liver lesions were noted in rats treated with acetaldehyde hydroxysulphonate. These lesions may possibly be attributable to the effects of free acetaldehyde. The no-effect level for acetaldehyde hydroxysulphonate was 7 mg SO2 equiv./kg body weight/day for sulphite oxidase-deficient rats and 70 mg SO2 equiv./kg body weight/day for normal rats. Increased urinary excretion of sulphite was noted in sulphite oxidase-deficient rats whether or not they were given exogenous sulphites. An increase in urinary sulphite levels in sulphite oxidase-deficient rats was observed after dosing with acetaldehyde hydroxy-sulphonate. These findings suggest that acetaldehyde hydroxysulphonate is metabolized to acetaldehyde and free sulphite.

Colonization of gastrointestinal tracts of chicks by Campylobacter jejuni.

Bacterial enumeration and histologic examination of organs and tissues of 8-day-old chicks 7 days after peroral inoculation with Campylobacter jejuni revealed that the organism colonized primarily the lower gastrointestinal tract. The principal sites of localization were the ceca, large intestine, and cloaca, where densely packed cells of C. jejuni were observed in mucus within crypts. Examination of C. jejuni-colonized crypts by transmission electron microscopy revealed that the campylobacters freely pervaded the lumina of crypts without attachment to crypt microvilli. Understanding the mechanism of colonization may lead to approaches that will reduce the incidence of C. jejuni carriage by poultry.

Chemotactic behavior of Campylobacter jejuni.

The chemotactic behavior of Campylobacter jejuni was determined in the presence of different amino acids, carbohydrates, organic acids, and preparations and constituents of mucin and bile. L-Fucose was the only carbohydrate and L-aspartate, L-cysteine, L-glutamate, and L-serine were the only amino acids producing a chemotactic (positive) response. Several salts of organic acids, including pyruvate, succinate, fumarate, citrate, malate, and alpha-ketoglutarate, were also chemoattractants, as were bile (beef, chicken, and oxgall) and mucin (bovine gallbladder and hog gastric). Most constituents of bile tested individually were chemorepellents, but the mucin component was chemoattractant. The chemotactic behavior of C. jejuni toward L-fucose, a constituent of both bile and mucin, may be an important factor in the affinity of the organism for the gallbladder and intestinal tract.

Colonization of chicken cecae by Escherichia coli associated with hemorrhagic colitis.

Bacterial enumeration, histologic examination, and immunoperoxidase staining demonstrated the ability of an Escherichia coli strain associated with hemorrhagic colitis (serotype O157:H7) to colonize chicken cecae for up to 90 days postinoculation after a peroral challenge at 1 day of age. The bacteria induced mild, transient, mucous membrane damage confined to the proximal cecae of healthy, normal-appearing chickens, principally at 14 to 28 days postinoculation. Attachment, effacement, and penetration of the cecal surface epithelium by E. coli O157:H7 were observed. With the exception of splenic, hepatic, and cecal tonsil immune-related changes and cecal damage and colonization, no other organ systems or portions of the gastrointestinal tract were affected by the bacteria. Bacterial counts indicated that E. coli O157:H7 was predominantly present in the cecae (often at levels greater than 10(6) CFU/g of tissue and contents) and to a lesser extent in the colon. Our results suggest that E. coli O157:H7 colonizes chicken cecae and is passed through the colon with fecal excrement. The ability of this organism to colonize chicken cecae indicates that chickens may serve as hosts and possibly as reservoirs for E. coli O157:H7.

Immunohistochemical fate of ochratoxin A in mice.

By a highly specific antibody against ochratoxin A (OA), the immunohistochemical fate of po administered OA in Swiss mice given a single dose of 25 mg/kg was examined by light microscopy with two immunoperoxidase methods, an indirect method and a peroxidase-antiperoxidase method. Immunohistochemical staining for OA was demonstrated in the esophagus, stomach, small intestine, kidneys, and liver. OA was most intense in the gastrointestinal tract, intermediate in the kidneys, and least in the liver. In the gastrointestinal tract, OA was demonstrable: (i) in the squamous layer of the esophagus, (ii) in the surface mucus and within macrophages and neutrophils of the stomach lamina propria, (iii) within the cytoplasm of duodenal and jejunal surface epithelial cells as well as within macrophages and neutrophils of the duodenal and jejunal lamina propria. Maximal gastrointestinal tract staining was evident from 5 min to 3 hr, while the esophagus remained stained for 24 hr postdosing. No evidence of OA was obtained in the ileum. In the kidneys, the vast majority of OA was localized within the cytoplasm of the proximal convoluted tubular cells as brown-stained vacuoles, whose size became largest at 3 hr postdosing. Staining for OA was also demonstrated within the epithelium of distal convoluted tubules, the macula densa, the loop of Henle, and sometimes within the epithelium of Bowman's capsule and glomerulus. OA in hepatocytes was most intense between 40 min and 3 hr postdosing. Stained hepatocytes were slightly more concentrated in the periportal area than in the area of the central vein and typically exhibited strong cytoplasmic but weak and infrequent nuclear staining. Biliary excretion of OA was demonstrated, since OA was localized in the lumina of biliary ducts but not within biliary cells.

Immunoperoxidase localization of T-2 toxin.

Antibody against T-2 toxin was used for monitoring the fate of T-2 toxin in mice given a single po dose of 11 mg/kg by the peroxidase-antiperoxidase (PAP) method. T-2 toxin was demonstrable in the esophagus from 5 min to about 24 hr postdosing. In the stomach, T-2 toxin was detected within the cytoplasm of intact and injured epithelial cells. In the duodenum, T-2 toxin was primarily localized within the surface epithelium and phagocytic elements (macrophages and neutrophils) of the duodenal lamina propria, especially toward the tips of the villi. Following sloughing of duodenal villous tips, the recovering villous tip epithelial cells frequently showed both cytoplasmic and nuclear T-2 toxin. The jejunum showed weak T-2 toxin within the cytoplasm of villous tip epithelial cells only. The ileum never demonstrated T-2 toxin. Tissue response in the gastrointestinal (GI) tract was characterized by transient edema, marked cytolysis and sloughing, and a subsequent leukocytic invasion of the stomach and proximal small intestine. Evidence of severe gastric and less severe duodenal bleeding was apparent and associated with a marked loss of gastric epithelium and intestinal villous tips. The kidney medulla contained the majority of T-2 toxin stain. T-2 toxin was noted within the distal tubular cells, the cells of the collecting tubules, and the epithelium covering the papilla. T-2 toxin was never demonstrated in any of the hepatic tissue examined.

Cadaverine and aminoguanidine potentiate the uptake of histamine in vitro in perfused intestinal segments of rats.

Examination of the serosal fluid following in vitro luminal perfusion of rat intestinal segments with 1 mg/ml [3H]histamine for 2 hr showed that histamine constituted only 22.1% of the total serosal radioactivity. The remainder of the radioactivity was comprised of histamine metabolites. When equimolar amounts of either aminoguanidine and cadaverine were added to the luminal perfusate, the percentage of the serosal radioactivity as histamine increased to 67.0 and 60.4%, respectively. However, when equal amounts of histamine and anserine were added to the luminal perfusate, only 30.6% of the 3H translocated within 2 hr was [3H]histamine. In all cases, the gross translocation rate based on the percentage of total serosal radioactivity for total radioisotope [( 3H]histamine plus [3H]histamine metabolites) was unchanged by the addition of these substances to the luminal perfusate. The results indicate that the potentiation of histamine toxicity by putrefactive amines, such as cadaverine, results from the inhibition of histamine metabolism which leads to increased uptake of unmetabolized histamine. The results do not support the hypothesis that potentiation occurs via an overall increase in the absorption of histamine and its metabolites due to some disruption in the barrier function of the intestine.

Colonic hemorrhage produced in mice by a unique vero cell cytotoxin from an Escherichia coli strain that causes hemorrhagic colitis.

A Vero cell cytotoxin that produces colonic lesions and subsequent colonic hemorrhage in mice has been purified from a strain of Escherichia coli O157:H7 that causes hemorrhagic colitis in humans. This toxin is different in physicochemical properties from the Shiga-like toxin previously associated with this organism and may be responsible for the unique diffuse mucosal hemorrhage in the colon of individuals with E. coli O157:H7 infections.

Serological relatedness of mouse-virulent Yersinia enterocolitica.

An antiserum (WA-SAA) was produced which agglutinated specifically with mouse-virulent but not with avirulent strains of Yersinia enterocolitica. Expression of the antigenic determinant(s) reaction with WA-SAA was temperature dependent; for growth temperatures of 20 to 40 degrees C, agglutination titers were lowest for cultures grown at 20 degrees C and highest for cultures grown at 35 to 40 degrees C. Addition of Ca2+ (2.5 to 10 mM) to the growth medium had little effect on the agglutination titer, and gel diffusion studies with monospecific anti-V serum indicated that V antigen was not likely to be the determinant reacting with WA-SAA. Immunohistological studies of Peyer's patches of mice infected with Y. enterocolitica WA revealed that the antigenic determinant(s) reacting with WA-SAA was expressed in vivo. The strong correlation of agglutination titer with mouse virulence and the expression in vivo of the antigenic determinant(s) reacting with WA-SAA suggest that the antigen(s) may be associated with the pathogenicity of Y. enterocolitica.

Emetic action of staphylococcal enterotoxin A on weanling pigs.

Peroral and intraduodenal administration of staphylococcal enterotoxin A (SEA) to weanling pigs elicited an emetic response. Peroral administration of an emetic dose of SEA resulted in a single emetic episode occurring 90 to 180 min after dosing. Intraduodenal administration via a surgically implanted catheter of 100 or 150 micrograms of SEA resulted in multiple emetic episodes occurring 150 to 210 min after dosing, suggesting an intestinal site of action for SEA. The 50% emetic dose for perorally administered SEA was between 40 and 50 micrograms for 4.1- to 9.1-kg weanling pigs and 20 micrograms in 0.9- to 2.3-kg weanling pigs. Neurobehavioral responses, including alternating periods of drowsiness and restlessness, staggering, temporary loss of the righting reflex, and refusal to feed were also observed in pigs given an oral dose of SEA. Based on the demonstrated responsiveness of pigs to SEA, pigs should be considered suitable animal models for studies on the sites and mode of action of the toxin.

Effects of staphylococcal enterotoxin A on the rat gastrointestinal tract.

Staphylococcal enterotoxin A (SEA) was administered orally (15 micrograms) to two groups of rats. A marked immune reaction was evoked in the stomach and proximal small intestine of the first group. The second group of rats was used to study the absorptive fate and sites of action of orally administered SEA, utilizing immunoperoxidase staining. After oral dosing of the second group of rats. SEA-related immunoperoxidase staining was confined to: (i) neutrophils and macrophages, principally in the duodenum, and (ii) glomerular neutrophils and cells of the proximal convoluted tubules. Peroxidase staining of the kidney was noted within 15 min of exposure, indicating that SEA or some major postabsorption antigenic product can promptly pass through an intact gastrointestinal mucous membrane and become renally localized. Intestinal and renal detoxification and removal was indicated by an absence of detectable antigen in rats 180 min postexposure. Neuronal binding of SEA in the gastrointestinal tract was not demonstrable.

Survival of Listeria monocytogenes in milk during high-temperature, short-time pasteurization.

Milk from cows inoculated with Listeria monocytogenes was pooled for 2 to 4 days and then heated at 71.7 to 73.9 degrees C for 16.4 s or at 76.4 to 77.8 degrees C for 15.4 s in a high-temperature, short-time plate heat exchanger pasteurization unit. L. monocytogenes was isolated from milk after heat treatment in six of nine pasteurization trials done at 71.7 to 73.9 degrees C and in none of three trials done at 76.4 to 77.8 degrees C. An average of 1.5 to 9.2 L. monocytogenes cells was seen in each milk polymorphonuclear leukocyte before heat treatment in 11 of 12 pasteurization trials. Noticeable degradation of leukocytes with intracellular listeria was detected in unpasteurized milk after 3 days of storage at 4 degrees C, and by 4 days of storage leukocytes had deteriorated to cellular debris, suggesting that holding unpasteurized milk refrigerated for 4 or more days would eliminate a protective effect leukocytes may provide for increasing heat resistance of L. monocytogenes. Results indicate that under the conditions of this study, L. monocytogenes can survive the minimum high-temperature, short-time treatment (71.7 degrees C, 15 s) required by the U.S. Food and Drug Administration for pasteurizing milk.