Purification and some properties of a Bacillus cereus mouse lethal toxin.

A mouse lethal toxin (MLT) produced by Bacillus cereus isolated in vomiting-type food poisoning was purified by chromatography on DEAE-Sephadex A-25 followed by gel filtration on Sephadex G-75. Purified MLT possessed a molecular weight of 33,000-34,000. It showed mouse lethality and hemolytic (HL) activity on sheep and rabbit erythrocytes; the latter erythrocytes were more weakly hemolyzed than the former ones. However, fluid accumulation in mouse ligated intestinal loops was not induced by purified MLT at the highest concentration used. Both MLT and HL activities were stable at pH 6-9, during storage at -20 degrees C for 8 weeks, and resistant to papain, cholesterol, lecithin, and dithiothreitol treatments. Most activity was lost during storage at 4 degrees C or 25 degrees C for 2 weeks or upon treatment with trypsin, trypanblue, or ethanol. The activities were resistant to heating at 37 degrees C for 5 min, less resistant at 98 degrees C for 5 min, and sensitive at 60 degrees C for 5 min. It can be concluded from the results that MLT is different from the diarrheagenic toxin produced by B. cereus isolated in diarrheal-type food poisoning, but is similar to, if not identical, hemolysin II.     

Purification and characterization of the vascular permeability factor produced by Bacillus cereus

Purification of an extracellular protein exhibiting the vascular permeability activity produced by Bacillus cereus was performed by ammonium sulfate precipitation followed by chromatography on DE-32 cellulose, Sephadex G-100, and Sephadex G-75. The purified protein was found to be electrophoretically and antigenically almost homogeneous although it contained a trace of contaminant. The molecular weight of the protein was calculated to be 45,000 by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The purified protein showed vascular permeability activity and mouse lethal toxicity, and caused fluid accumulation in ligated mouse intestinal loops, whereas it did not show any hemolytic and lecithinase activities. From these findings, the purified protein is suggested to be an enterotoxin (or a diarrheagenic toxin) responsible for diarrhea caused by B. cereus in a diarrheal-type food poisoning.     

Morphologic and physiologic changes induced by Clostridium perfringens type A alpha toxin in the intestine of sheep

OBJECTIVE: To evaluate the morphologic and physiologic changes induced by Clostridium perfringens type A (alpha toxin in the ileum and colon of sheep. SAMPLE POPULATION: 16 ligated intestinal loops in 4 Merino lambs and 18 explants of ileum and colon from slaughtered lambs. PROCEDURE: alpha Toxin-induced fluid accumulation was evaluated in ligated ileal and colonic loops of sheep. Tissues were evaluated morphologically by use of gross and histologic examination. Effects of toxin on in vitro intestinal net water transport were tested in modified Ussing chambers. RESULTS: Ovine ileal and colonic loops incubated with C perfringens type A alpha toxin retained more fluid than control loops. Histologically, in the ileum of lambs inoculated with 300 LD50 of alpha toxin/mL, there was a mild to moderate multifocal infiltration of neutrophils in the lamina propria and submucosa. The colonic loops of lambs inoculated with 30 or 300 LD50 of alpha toxin/mL had excessive mucus in the lumen, a moderate amount of neutrophils mixed with mucus in the intestinal lumen, and moderate multifocal infiltration of the lamina propria and submucosa with neutrophils; the blood vessels of these layers were engorged with neutrophils. In vitro measurements of water transport also revealed inhibition of net epithelial water absorption in ileum and colon incubated with alpha toxin on the mucosal side. CONCLUSIONS AND CLINICAL RELEVANCE: These results indicate that alpha toxin induces alterations in sheep intestine. Clostridium perfringens type A organisms that produce alpha toxin could be responsible for diseases of intestinal origin in some ruminants.     

Effect of jejunal loop location on the activity of Escherichia coli heat-stable enterotoxin in 4- to 5-week-old pigs

Heat-stable enterotoxin (STa) from Escherichia coli strain 431 was injected into the jejunum of 4 pigs from each of 3 litters, using a ligated intestinal loop assay (with loops beginning 1 m caudad to the pylorus). The jejunum was divided into 4 contiguous areas, with 4 loops in each area. Doses of 0, 10, 100, or 1,000 ng of purified toxin (10 ng/mouse unit) were injected into the loops within an area, using a 4 X 4 Latin square design. Fluid accumulation in the loops increased (P less than 0.05) with increasing concentrations of STa in pigs in all litters, but the magnitude of the response varied across litters. Fluid responses to the STa varied in the different areas of the jejunum, with pigs in 2 litters having a decrease (P less than 0.05) in the response to STa in the caudal areas. These data quantitate the variability within the different areas of the jejunum of the young pig.     

Effect of salicylates on intestinal secretion in calves given (intestinal loops) Escherichia coli heat-stable enterotoxin

The inhibitory effect of salicylates on intestinal secretion in 1- to 5-day-old calves given Escherichia coli heat-stable enterotoxin (ST)-induced intestinal fluid response was investigated. Purified ST was diluted in isotonic saline solution to obtain 1:10, 1:25, 1:50, 1:75, and 1:100 dilutions. Each dilution (1 ml) was inoculated into ligated loops in the distal part of the jejunum of each calf. Acetylsalicylic acid (aspirin) given orally (100 mg/kg) at 4 hours before ST was inoculated did not substantially alter the intestinal fluid response to ST. Sodium salicylate (IV) infusion, begun simultaneously when, or at 1 hour after, ST was inoculated, significantly (P less than 0.05) decreased fluid accumulation in those loops inoculated with ST dilutions of 1:25 or greater. The sodium and potassium concentrations of the accumulated fluid did not differ significantly between or within treatment groups. These results indicate that sodium salicylate infusion may be beneficial in treating enterotoxic colibacillosis in calves. Aspirin given orally at the dose used in the present study, would not have any beneficial effect.     

Fluid accumulation in the ligated intestinal loop and histopathological changes of the intestinal mucosa caused by Clostridium botulinum C2 toxin in the pheasant and chicken

Fluid accumulation was induced in ligated intestinal loops of both pheasants and chickens injected with crude Clostridium botulinum C2 toxin. Necrosis of the epithelium and lamina propria of the duodenum and jejunum occurred in both species of bird after intraduodenal administration of crude C2 toxin. The severity of such reactions depended upon the dose and the period after administration of C2 toxin up to eight hours, and such reactions were suppressed specifically with rabbit anti-C2 toxin. These results confirm that C2 toxin is one of the causes of diarrhoea in avian botulism.     

The effect of cholera toxin and heat labile and heat stable Escherichia coli enterotoxin on cyclic AMP concentrations in small intestinal mucosa of pig and rabbit.

The effect of cholera toxin, heat labile and heat stable Escherichia coli enterotoxin on mucosal cyclic AMP concentrations was determined on the proximal jejunum of weanling pigs and young rabbits. Ligated loops were injected with solutions containing no enterotoxin for control and either cholera toxin, heat labile or heat stable E. coli enterotoxin. The loops were drained after either two, four or six hours incubation at which time accumulated fluid was recorded and mucosal samples removed for determination of cyclic AMP concentration. In the rabbit, cholera toxin and heat labile, but not heat stable E. coli enterotoxin stimulated intestinal secretion while in the pig all three enterotoxins induced net fluid accumulation. Cholera toxin and heat labile, but not heat stable E. coli enterotoxin elevated rabbit mucosal cyclic AMP concentrations. In the pig these enterotoxins had no significant effect on mucosal cyclic AMP concentrations. The results are inconsistent with the hypothesis that the adenyl cyclase system is an essential step for enterotoxin induced intestinal secretion. The activation of intestinal adenyl cyclase by bacterial enterotoxins may only be an associated and not a necessary event for the stimulation of intestinal secretion.     

Prolactin and colibacillus-induced fluid transport in ligated intestinal segments.

Two experiments, using the ligated intestinal segment technique, were conducted to determine whether the pituitary hormone prolactin (PRL) could reduce Escherichia coli-induced fluid loss into the small intestine of 2- to 3-week-old pigs. Inoculation of 10(6) to 10(8) enteropathogenic E coli organisms into ligated jejunal segments caused a significant accumulation of luminal fluid within 12 hours. In the first experiment, intraluminal inoculation with 0.5 mg of ovine PRL along with the bacteria did not have any effect on fluid accumulation. Systemic IV treatment of the animals with 1.0 mg of ovine PRL at 3-hour intervals, beginning either immediately after or 9 to 10 hours before intestinal ligation, did not significantly (P less than 0.05) reduce fluid accumulation as compared with control animals. In the second experiment, IM administration of 100 microgram of thyrotropin-releasing hormone (TRH) at 3-hour intervals, beginning 6 hours before intestinal ligation, significantly (P less than 0.05) increased circulating PRL concentrations, as measured by radioimmunoassay. However, TRH treatment did not reduce the accumulation of luminal fluid in E coli-inoculated segments.     

Passive haemagglutination and complement fixation as diagnostic tests for contagious caprine pleuropneumonia caused by the F-38 strain of mycoplasma

The protective effects of high levels of O115 antibodies and K88 antibodies in the sera of hyperimmunized rabbits on the intestinal loop dilation elicited by live cultures of Escherichia coli O115: K88:H39 and its enterotoxins were examined. K88 antibodies did not prevent fluid accumulation when live E coli O115:K88:H39 or its enterotoxins were injected into ligated loops in rabbits actively immunised with K12:K88 or when K88 antiserum was mixed directly with the challenge shortly before injection into the loops. In two of six rabbits immunised with heat-killed O115 E coli the live strain failed to elicit a fluid response and in two others the response was reduced. In all cases the O115 antiserum inhibited the fluid response evoked by the live, homologous bacteria, but not by enterotoxin preparations, when the serum was mixed directly with the challenge before injection.     

Enteric pathology and Salmonella-induced cell death in healthy and SIV-infected rhesus macaques

The goal of this study was to morphologically characterize a ligated ileal loop model of Salmonella enterica serotype Typhimurium infection in rhesus macaques (Macaca mulatta) and to verify the occurrence of Salmonella-induced cell death in vivo. Eight adult healthy male rhesus macaques were used for ligated ileal loop surgery. Four macaques had been intravenously inoculated with simian immunodeficiency virus (SIV) mac251. Ileal ligated loops were inoculated with wild-type (WT) S. Typhimurium strain IR715 (ATCC14028 nal (r)), an isogenic noninvasive mutant strain (ATCC14028 nal (r) ΔsipAΔsopABDE2), or sterile Luria Bertani broth. Loops were surgically removed at 2, 5, and 8 hours post-inoculation (hpi). Intestinal samples were processed for histopathology, immunohistochemistry for detecting Salmonella, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL), and transmission electron microscopy. Combined histopathology scores were similar between SIV-infected and control macaques. As expected, the invasion-deficient mutant was less pathogenic than WT S. Typhimurium. Neutrophil infiltrate in the intestinal mucosa correlated with bacterial loads (r = 0.7148; P < .0001) and fluid accumulation (r = 0.6019; P < .0001) in the lumen of the intestinal loops. Immunolabeled WT S. Typhimurium was observed in the epithelium and lamina propria at the tip of the villi at 2 hpi, progressing toward deeper lamina propria at 5-8 hpi. Most TUNEL-positive cells localized to the lamina propria, and some had morphological features of macrophages. Ultrastructurally, bacteria were observed intracellularly in the lamina propria as well as within apoptotic bodies. This study provides morphological evidence of Salmonella-induced cell death in vivo in a relevant nonhuman primate model.     

Effects of chloride conductance inhibitors on fluid secretion into ligated ileal and jejunal loops in pigs

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 greater than 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.     

Enterotoxin activity of a Salmonella typhimurium of equine origin in vivo in rabbits and the effect of Salmonella culture lysates and cholera toxin on equine colonic mucosa in vitro

To determine whether a strain of Salmonella typhimurium (UCD 1755) of equine origin had enterotoxin activity, 2 ml of a cell-free culture lysate of strain UCD 1755 and approximately 10(9) viable strain UCD 1755 organisms were inoculated into ligated small intestinal segments of rabbits. Intestinal segments inoculated with viable strain UCD 1755 organisms and those inoculated with a cell-free culture lysate of strain UCD 1755 had significant (P less than 0.05) accumulations of fluid 10 hours after inoculation when compared with ligated intestinal segments either inoculated with sterile brain-heart infusion broth or left empty. There was not a statistically significant difference between fluid accumulation of intestinal segments inoculated with viable strain UCD 1755 and that of segments inoculated with a cell-free culture lysate of strain UCD 1755. The responses of equine colonic mucosa to culture filtrates of 2 strains of salmonella typhimurium (UCD 1755 and SL 1027) and purified cholera toxin were studied in vitro. Isolated smaples of colonic mucosa were incubated for 4 hours at 37 C in Krebs-Henseleit bicarbonate buffer (KHB) alone, KHB plus culture lysate of strain UCD 1755, KHB plus culture lysate of strain SL 1027, and KHB plus 1 microgram of cholera toxin/ml. Cyclic adenosine monophosphate (cAMP) content of each sample was determined.(ABSTRACT TRUNCATED AT 250 WORDS)     

Uptake of ferritin by follicle-associated epithelium in the colon of calves.

Uptake of macromolecules (e.g., ferritin) by M cells in follicle-associated epithelium in small and large intestine was investigated in three healthy, conventionally raised, 2- to 3-week-old, female Holstein Frisian calves. A 2.5% solution of ferritin was injected into the ligated loops in mid-jejunum, in terminal ileum, in the ascending colon adjacent to the ileocecal junction, and in the proximal loop of the ascending colon containing gut-associated lymphoid tissue. After exposure times that ranged from 82 to 165 minutes, ferritin was detected in M cells of domes in the small intestine, as well as in cells in follicle-associated epithelium of proprial lymphoid nodules and lymphoglandular complexes of colon that morphologically resembled M cells of small intestine. Ferritin was found in apical invaginations, apical vesicles, multivesicular bodies, basal vesicles, and adjacent intercellular spaces. In addition to ferritin, apical vesicles, multivesicular bodies, and intercellular spaces contained 50-nm membrane-bound particles. More ferritin was endocytosed by M cells of the small intestine than by M cells of the large intestine. In the large intestine, higher amounts of ferritin were found in M cells of follicle-associated epithelium overlying proprial lymphoid nodules than in M cells of follicle-associated epithelium in the depth of lymphoglandular complexes. Based on these results, we concluded that M cells of follicle-associated epithelium in the colon of calves provide a route for antigen uptake into the intestinal lymphoid system.     

Nicotinic acid inhibits enterotoxin-induced jejunal secretion in the pig.

The use of nicotinic acid for preventing intestinal secretion caused by cholera toxin and by the heat-stable enterotoxin of Escherichia coli has been investigated in the weanling pig. Secretory effects were measured in ligated jejunal loops of halothane-anesthetized pigs by dilution of a nonabsorbable marker added to the loop fluid. Different routes of administration and different initial pH values for nicotinate solutions were studied to determine optimal conditions for secretory inhibition. The neutral sodium salt of nicotinic acid had no significant antisecretory activity under any conditions used in these trials. Inhibition of secretion was most effective with partly neutralized nicotinic acid at pH 4.5 added directly to loops containing enterotoxin. Net fluid secretion induced by cholera toxin or heat-stable enterotoxin of E. coli was prevented by this treatment. Reversal of secretion was not accompanied by any measurable changes in cyclic nucleotide concentration in intestinal mucosa. Nicotinic acid antagonism of a secretory step common to cholera toxin and heat-stable enterotoxin of E. coli but subsequent to cyclic nucleotide involvement is indicated by these data.     

Effects of Escherichia coli heat-stable enterotoxin b on small intestinal villi in pigs, rabbits, and lambs

Culture supernates from two strains of E. coli were placed into different ligated intestinal sections (loops) of each animal. The two bacterial strains were identical except that one contained a plasmid carrying the heat-stable toxin b (STb) gene, while the other did not. Morphometric techniques were used to assess villous epithelial surface areas and mucosal volumes in both intestinal segments exposed to STb-positive (test) and to STb-negative (control) supernates. In pigs whose intestines were exposed to STb-positive supernatants for 2 hours, both villous epithelial surface area and mucosal volume were significantly smaller in test loops than in control loops (P less than 0.02). In test loops of pigs incubated for 1 hour, and in test loops of lambs incubated for 2 hours, there was a decrease in villous epithelial surface area which approached the test for significance but did not meet it (0.05 less than P less than 0.10). Rabbit test loops did not differ from rabbit control loops in either villous epithelial surface area or mucosal volume. Histological examination of the tissues from all three species revealed epithelial changes in porcine and ovine tissues only. In porcine and ovine tissues, epithelium at villous tips was seen to be cuboidal or squamous, or even to be absent. Villi with similarly altered epithelium were seen in control loops, but were seen much more frequently in test loops. These epithelial changes were seen as early as 30 minutes of incubation in pigs. Intestinal tissues from these pigs were examined by transmission electron microscopy, but no difference between test and control tissues was seen.(ABSTRACT TRUNCATED AT 250 WORDS)     

THE SONOGRAPHIC APPEARANCE OF INTESTINAL MUCOSAL FIBROSIS IN CATS

The medical records of 11 cats with full‐thickness intestinal biopsies and histopathologic confirmation of segmental mucosal fibrosis were reviewed. All cats received an abdominal ultrasonographic evaluation. The sonographic feature of a small intestinal mucosal hyperechoic band paralleling the submucosa was present in all cats. Other intestinal sonographic findings included wall thickening, and altered wall layering (increased mucosal echogenicity, thickened submucosa, and/or muscularis layer). None of the cats had complete loss of wall stratification. All cats had clinical signs related to the gastrointestinal (GI) tract at the time of presentation. Three of the 11 cats had palpably thickened small intestinal loops, 3/11 abdominal pain, and 2/11 abdominal fluid. Histopathologically, mucosal fibrosis was associated with inflammatory cell infiltrates in all cats. In those cats with histopathologic evidence of mural fibrosis, all cats had a visible hyperechoic band through several intestinal segments. We speculate that the hyperechoic mucosal band represents the zone of mucosal fibrosis. Independently and prospectively, we reviewed the clinical presentation of 35 cats having this visible hyperechoic mucosal band on ultrasound. Twenty‐four of these 35 cats had clinical signs related to the digestive system at the time of record. Our study suggests that the hyperechoic mucosal band represents fibrosis, and in presence of concurrent GI signs, further diagnostic tests may be warranted.     

A Comparative Study of the Rabbit and Pig Gut Loop Systems for the Assay of Escherichia coli Enterotoxin

A comparison was made between segments of pig and rabbit small intestine in their response to heat-labile (LT) and heat-stable (ST) preparations from porcine enteropathogenic Escherichia coli. Either whole cell lysates or dialysed broth culture supernatants were used as sources of LT and soft agar culture fluids as a source of ST. Whole cell lysates of all thirteen LT-producing E. coli strains tested regularly elicited fluid accumulation in rabbit gut loops. Whole cell lysates of certain E. coli strains considered to be nonenteropathogenic in pigs could also elicit a positive response in rabbit gut loops. When graded doses of LT were tested in pig and rabbit gut loops, the rabbit was more sensitive and is therefore considered preferable to the pig for quantitation of LT. In the rabbit, upper (jejunal) and lower (ileal) small intestine were compared for their response to LT and it was found that ileal loops were twice as sensitive but more prone to false positive reactions. When soft agar culture fluids of several enteropathogenic E. coli strains were tested in the rabbit, the response was inconsistent, and it was concluded that the rabbit is unsuitable for the assay of the heat-stable enterotoxin.     

Campylobacteriosis in an aborted equine fetus

Abortion caused by Campylobacter fetus subsp fetus was diagnosed in a 7-month-old equine fetus. The fetus was small for its gestational age. Macroscopically, the proximal portion of the small intestine was hemorrhagic and its wall was thick. Histologically, the Brunner glands were distended with neutrophils, and the submucosa was thick, owing to fluid accumulation and/or cellular infiltrates. Curved bacteria were observed in the Brunner glands and intestinal glands. Campylobacter fetus subsp fetus was isolated from stomach contents, liver, and lungs, and was detected by dark-field microscopic examination of ocular fluid and stomach contents. Placenta was not available for examination.     

Effects of indomethacin on Salmonella typhimurium- and cholera toxin-induced fluid accumulation in the porcine small intestine

The effect of the cyclooxygenase and prostaglandin E2 (PGE2) synthesis inhibitor, indomethacin, on the secretory responses induced by Salmonella serotype Typhimurium (ST) and cholera toxin (CT), in the porcine small intestine was investigated. ST (10(10) colony-forming units) and CT (56 micrograms) were instilled in tied-off intestinal loops in young anaesthetized pigs receiving intravenous indomethacin in a total dose of 7.5 mg/kg, or saline. The accumulated fluid in the loops and the luminal content of endogenous secretagogues PGE2 and 5-hydroxytryptamine (5-HT) were measured. ST induced fluid accumulation in the jejunum, whereas CT induced fluid accumulation in the jejunum and ileum. Indomethacin had no effect on the secretory responses. Indomethacin had a significant effect on the luminal content of PGE2 in jejunal ST and CT loops, whereas no effect of indomethacin was observed on the luminal content of 5-HT in ST and CT loops. In ST and CT loops, an increased content of PGE2 and 5-HT compared with test loops infused with Ringer's solution was observed. These results indicate that the porcine jejunal secretory response to ST and CT does not involve prostaglandins although indomethacin has an influence on the luminal release of PGE2 but not of 5-HT.     

The Effect of Adsorbant and Anti-inflammatory Drugs on Secretion in Ligated Segments of Pig Intestine Infected with Escherichia coli

Four adsorbant drug preparations, Kaopectate, colloidal Attapulgite, noncolloidal Attapulgite and Pepto-bismol were investigated for their effects on fluid accumulation in ligated segments of pig intestine inoculated with enteropathogenic Escherichia coli. Two anti-inflammatory drugs. aspirin and methylprednisolone, and two antibiotics, lincomycin and polymyxin B, were also tested. All the drugs except the two anti-inflammatory products were given by injection into the lumen of the intestine. Aspirin was given orally and methylprednisolone was given intramuscularly. The antibiotics were tested at levels at which they had no significant antibacterial effect in in vitro tests. The adsorbant drugs colloidal Attapulgite and Pepto-bismol were shown to be effective in reducing fluid accumulation in ligated segments of pig intestine infected with enteropathogenic E. coli. In the case of Peptobismol this effect was associated with an antibacterial effect as well as an antitoxic effect, probably due to its adsorbant properties. It is possible that an aspirin-like effect in the gut due to the active ingredient bismuth subsalicylate may have contributed to the effectiveness of Pepto-bismol. Colloidal Attapulgite was demonstrated to have an antitoxic effect but did not have an antibacterial effect.

In high doses, the anti-inflammatory drugs acetylsalicylic acid and methylprednisolone were marginally effective in reduction of fluid accumulation in the same test system. Lincomycin was shown to reduce intestinal fluid secretion, whereas polymyxin B had no effect.