Effect of heat stable and heat labile Escherichia coli enterotoxins and cholera toxin in combination with theophylline on unidirectional sodium and chloride flux in the small intestine of weanling swine.

The effect of heat stable and heat labile Escherichia coli enterotoxins or cholera toxin in combination with theophylline on net water, sodium and chloride and unidirectional sodium and chloride fluxes was examined in acute isolated loops of jejunum of weanling swine. The effect of heat stable enterotoxin in combination with theophylline was determined in loops located in the proximal jejunum, while combinations of theophylline and either heat labile enterotoxin or cholera toxin were studied in the distal jejunum. In each situation the addition of theophylline resulted in an additive rather than a synergistic increment of intestinal secretory activity. This study implies that the intestinal adenyl cyclase system and enterotoxin induced intestinal secretion may not be directly related in the swine small intestine.     

Effects of intraluminal glucose on intestinal secretion induced by heat stable and heat labile Escherichia coli enterotoxin, cholera toxin and theophylline.

Glucose, l-alanine, l-aspartate, l-methionine and glycine enhanced net fluid and electrolyte absorption in acute isolated loops of the proximal jejunum of weanling swine. The effect of glucose on intestinal secretion induced by heat stable and heat labile Escherichia coli entero-toxin, cholera toxin and theophylline was examined in both the proximal and distal jejunum of weanling swine. In the proximal jejunum glucose enhanced the rate of net fluid and electrolyte absorption. This increase was accompanied by an increase in unidirectional dosium absorption. In loops exposed to either heat stable or heat labile enterotoxins, glucose significantly decreased the rate of net fluid and electrolyte secretion. The magnitude of glucose enhancement in loops exposed to heat stable and heat labile enterotoxins was similar to adjacent control loops. However, glucose enhancement did not occur in loops exposed previously to cholera toxin or concurrently to theophylline. Therefore, cholera toxin and theophylline may inhibit substrate dependent sodium absorption in the proximal jejunum. In the distal jejunum glucose enhancement did occur but the rate of enhancement was less than in the proximal jejunum. In this region glucose enhancement was not evident in loops exposed to either theophylline, heat stable, heat labile or cholera toxin.     

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.     

Effect of Heat Stable and Heat Labile Escherichia coli Enterotoxins, Cholera Toxin and Theophylline on Unidirectional Sodium and Chloride Fluxes in the Proximal and Distal Jejunum of Weanling Swine

Acute, isolated loops of proximal and distal jejunum of weanling swine were exposed to either heat stable porcine Escherichia coli enterotoxin, heat labile porcine Escherichia coli enterotoxin, cholera toxin or theophylline. Unidirectional sodium fluxes in response to heat stable in the proximal jejunum were dependent on the length of time that the intestinal mucosae was exposed to the enterotoxin. Net water, sodium and chloride and unidirectional sodium and chloride flux measurements in the proximal jejunum in response to each agent uniformly indicated that net secretion of fluid and electrolytes was the result of increased unidirectional sodium secretion or blood-to-lumen flux and decreased unidirectional chloride absorption or lumen-to-blood flux. In addition heat stable cholera toxin and theophylline but not heat labile decreased unidirectional chloride secretion a small but significant amount in the proximal jejunum.

Sodium and chloride flux measurements in the distal jejunum demonstrated that all four secretory agents could stimulate net secretion of water, sodium and chloride in that region. The response to these secretory agents as measured by sodium and chloride unidirectional flux rates was not similar to changes observed in the proximal jejunum. In the distal small intestine, whereas heat labile cholera toxin and theophylline induced similar qualitative changes in unidirectional sodium and chloride fluxes, that induced by heat stable differed.

     

Evaluation of procholeragenoid against experimental colibacillosis in piglets of vaccinated dams

A toxoid prepared from the toxin of Vibrio cholera was adjuvanted with aluminium hydroxide and used for immunisation of pregnant gilts. Litters of these and of non-vaccinates were experimentally challenged with Escherichia coli producing either heat labile and heat stable (LT and ST) enterotoxins or ST enterotoxin only. Both the challenge strains of E coli produced high rates of mortality (64 and 68 per cent) and morbidity (80 and 100 per cent) in litters of non-vaccinated dams. Statistically highly significant protection against the LT/ST enterotoxin producing strain of E coli was obtained accompanied by the absence of colonisation of the small intestine by the pathogen. No protection against the ST enterotoxin producing strain was found. It is suggested that this vaccine would not confer passive protection to piglets against K99 and 987-positive E coli which usually produce ST enterotoxin only.     

Permeability properties of swine small intestine: effect of a heat stable Escherichia coli enterotoxin.

The permeability of weanling swine small intestine was estimated using measurements of filtration coefficients and equivalent pore size. Hypertonic solutions of mannitol, erythritol and urea were used to calculate reflection coefficients in the duodenum, mid jejunum and distal jejunum. Estimated effective pore radius was 6.4-7.4, 5.6-7.2 and 4.7-4.9A degrees in the three respective regions. Similarly the filtration coefficient induced by hypertonic solutions of mannitol decreased significantly in the distal jejunal segments. The results show an aboral gradient of decreasing permeability along the small intestine of the weanling pig. In situ incubation of loops in the proximal jejunum with a heat stable Escherichia coli enterotoxin for one hour did not significantly change the effective pore size as calculated from reflection coefficients of hypertonic solutions of erythritol and urea. However, the filtration coefficients of loops exposed to the enterotoxin were significantly greater than control loops with hypertonic solutions of erythritol and urea but not mannitol. This suggests the occurrence of a slight reduction in epithelial porosity. The results support the hypothesis that intestinal secretion induced by heat stable E. coli enterotoxin is not the result of an increased mucosal permeability.     

Liposomes targeted to deliver antisecretory agents to jejunal mucosa.

The B subunit of cholera toxin has been covalently attached to the surface of liposomes made from a mixture of phosphatidylethanolamine, phosphatidylcholine and cholesterol. Adenylate cyclase inhibitors and chloride conductance inhibitors were encapsulated within the liposomes. These "targeted" liposomes were used to study the combined effects of this novel delivery system, and a limited number of possible antisecretory agents, on net fluid flux into the pig jejunum. A state of net secretory fluid flux was induced in isolated jejunal loops in weanling pigs by adding theophylline or cholera toxin to the lumen of the isolated loops. There was no reduction in net fluid secretion when liposome suspensions without encapsulated secretory inhibitors were added to fluid in the lumen of loops treated with theophylline. There was also no reduction in net fluid secretion when miconazole, alpha-phenylcinnamate or 5 nitro-2-(3-phenethylamino)benzoate were encapsulated within targeted liposomes added to isolated jejunal loops. The net fluid flux induced by exposure of jejunal loops to theophylline was significantly reduced by adding targeted liposomes containing 2'-deoxy-3'-AMP. The reduction involved a reversal of net secretory fluid flux to an absorptive value. The net fluid secretory response to treatment of loops with cholera toxin was also inhibited by treating loops with targeted liposomes containing 2'-deoxy-3'-AMP. However, the reversal of secretion was less complete for secretion induced by cholera toxin than for secretion induced by theophylline. The reduced antisecretory efficacy versus cholera toxin was not improved by encapsulating higher concentrations of 2'-deoxy-3'-AMP.(ABSTRACT TRUNCATED AT 250 WORDS)     

大肠杆菌热敏毒素研究概况

本文在分析大肠杆菌热敏毒素（LT）生物学特性及其分子学水平研究的基础上，从蛋白质和基因水平阐述了猪源LT与人源LT的亲缘关系，评述了LT在研制人畜大肠杆菌腹泻疫苗中的应用价值，也通过对与LT具有相似蛋白和基因结构的霍乱毒素CT的分析，表明二者具有相同的进化起源，认为通过定点诱变的LT能够取代CT用作幽门螺杆菌疫苗的粘膜免疫佐剂。     

Nine DNA probes for detection of toxin and adhesin genes in Escherichia coli isolated from diarrhoeal disease in animals

DNA gene probes specific for genes encoding heat labile enterotoxin (LTI), heat stable enterotoxins (STIa, STII), vero cytotoxins (VT1, VT2), and adhesins K88 (F4), K99(F5), F41 and 987P(F6) were used to examine 873 isolates of E. coli from cases of diarrhoea (680 from pigs, 187 from cattle and six from sheep). A total of 188 were toxin gene positive and of these 84 belonged to the classical ETEC serogroups. Of the other 104 toxin gene positive strains, 80 hybridized with the VT2 probe of which 34 were from cases of porcine post-weaning diarrhoea belonging to serogroup 0138:K81 and 22 were untypable strains from cattle.     

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.     

Evaluation of heat-labile enterotoxins type IIa and type IIb in the pathogenicity of enterotoxigenic Escherichia coli for neonatal pigs

Type II heat-labile enterotoxins (LT-II) have been reported in Escherichia coli isolates from humans, animals, food and water samples. The goal here was to determine the specific roles of the antigenically distinguishable LT-IIa and LT-IIb subtypes in pathogenesis and virulence of enterotoxigenic E. coli (ETEC) which has not been previously reported. The prevalence of genes encoding for LT-II was determined by colony blot hybridization in a collection of 1648 E. coli isolates from calves and pigs with diarrhea or other diseases and from healthy animals. Only five isolates hybridized with the LT-II probe and none of these isolates contained genes for other enterotoxins or adhesins associated with porcine or bovine ETEC. Ligated intestinal loops in calves, pigs, and rabbits were used to determine the potential of purified LT-IIa and LT-IIb to cause intestinal secretion. LT-IIa and LT-IIb caused significant secretion in the intestinal loops in calves but not in the intestinal loops of rabbits or pigs. In contrast, neonatal pigs inoculated with isogenic adherent E. coli containing the cloned genes for LT-I, LT-IIa or LT-IIb developed severe watery diarrhea with weight loss that was significantly greater than pigs inoculated with the adherent, non-toxigenic parental or vector only control strains. The results demonstrate that the incidence of LT-II appeared to be very low in porcine and bovine E. coli. However, a potential role for these enterotoxins in E. coli-mediated diarrhea in animals was confirmed because purified LT-IIa and LT-IIb caused fluid secretion in bovine intestinal loops and adherent isogenic strains containing cloned genes encoding for LT-IIa or LT-IIb caused severe diarrhea in neonatal pigs.     

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.     

The effect of oral immunisation with heat stable Escherichia coli antigens on the sensitivity of pigs to enterotoxins.

It was found that oral immunisation of pigs with heat stable Escherichia coli antigens resulted in a decrease in the sensitivity of the porcine intestine to both the heat stable (ST) and the heat labile (LT) and the heat labile (LT) form of the enterotoxin produced by enterotoxigenic strains. Antitoxic factors capable of neutralising LT, but not ST, could be passively transferred in the intestinal secretions of the immunised animals.     

Relationships of intestinal enzymes and serum antitoxin to the pig response to Escherichia coli enterotoxin.

The intestinal loop technique was used to evaluate the response of three week old piglets to the heat labile (LT) and the heat stable (ST) enterotoxins produced by Escherichia coli F11(P155). The serum anti-LT activity and the lipase, amylase and trypsin activities in the jejunal lumen of these pigs were determined. Piglets responded independently ti each toxin and no relationship between these responses and serum anti-LT activity or the enzyme activities of the jejunal content could be demonstrated.     

Evaluation of different cell cultures for the detection of heat‐labile enterotoxins of Escherichia coli strains isolated from pigs

The sensitivity of various cell cultures to heat‐labile enterotoxins (LT) and Verocytotoxin (VT) of fifteen E. coli strains isolated from cases of pig colibacillosis in Poland was estimated and compared with the effect of enterotoxins of four standard E. coli strains. Often tested cell cultures, only the following were susceptible: CHO, Vero, GMK, and HeLa.

Eight strains showed CTE in HeLa and CHO cells and five of these reacted in Vero cells. The results appear to suggest that some of the tested E. coli strains isolated from pigs produced VT enterotoxin. Morphological changes caused by the above mentioned E. coli toxins in Vero and GMK cells took the form of cell rounding, followed by cell dissolution.     

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.     

Genotypic Prevalence of the Adhesin Involved in Diffuse Adherence in Escherichia coli Isolates in Pre‐weaned Pigs with Diarrhoea in Korea

A total of 1002 Escherichia coli strains isolated from pre‐weaned pigs with diarrhoea on 1114 swine farms were screened for the presence of the adhesin involved in diffuse adherence (AIDA) gene by polymerase chain reaction (PCR). Escherichia coli isolates that carried AIDA genes were also tested by PCR for the detection of five fimbriae (F4, F5, F6, F18 and F41), heat‐stable (STa, STb) and heat‐labile (LT) enterotoxin, enteroaggregative E. coli heat‐stable enterotoxin 1 (EAST1), and Shiga toxin 2 oedema disease (Stx2e) genes. Twenty‐three (2.3%) of the 1002 E. coli isolates carried the gene for AIDA. Among 23 isolates shown to carry genes for AIDA, three carried the AIDA gene as the only shown virulence factor. Other isolates carried other virulence factor genes in addition to AIDA. Four isolates carried genes for at least one of the fimbrial adhesins and enterotoxins. Sixteen isolates carried genes for enterotoxins only. The AIDA may represent an additional virulence determinant in pre‐weaned pigs with diarrhoea.     

Immunity to Escherichia coli in Pigs: Anti-enterotoxins in Colostrum and Serum from Vaccinated and Non-vaccinated Sows

Colostrum from non-vaccinated sows did not contain naturally occurring antibodies to heat labile Escherichia coli enterotoxin. Vaccination of sows by either the intramuscular or intramammary routes with a live formalinized E. coli vaccine resulted in the production of colostrum capable of neutralizing the heat labile toxin. Intramammary vaccination resulted in the production of colostrum which significantly reduced the enterotoxigenic effects of the vaccine strain of E. coli organisms but not that of a heterologous strain.

Vaccination of the sows resulted in the production of serum antibodies to heat labile enterotoxin. Antibodies to heat stable enterotoxin were not demonstrable in the colostrum of either non-vaccinated or vaccinated sows.

     

Enterotoxigenic Escherichia coli in the jejunum of piglets with neonatal diarrhea

Thirty-two Escherichia coli colonies were taken from the primary step of cultivation of the jejunal contents of each of 10 dead piglets which had suffered from diarrhea. The organisms of each colony were examined for the presence of adhesion fimbria (F4 (K88) and F5 (K99)), production of heat-stable and heat-labile enterotoxin and of colicins.The presence of heat-labile enterotoxin in the intestinal content of the necropsied pigs was also tested, and results correlated with enterotoxin production of the isolated E. coli strains. In all but 3 pigs, 50–80 % of the E. coli strains were found to produce one or both of the enterotoxins and to possess the F4 of the F5 antigen. All bacteria producing both heat-labile and heat-stable enterotoxin proved to belong toi O group 149 and to possess the F4 antigen. Strains from 1 pig belonged to O group 64 and possessed the F5 antigen; these bacteria produced heat-stable enterotoxin only. Most of the enterotoxin-producing E. coli also produced colicins.After each subcultivation, the strains produced less heat-labile enterotoxin, some becoming negative when assayed.     

Intestinal responses to enterotoxigenic Escherichia coli heat-stable toxin b in non-porcine species

The Escherichia coli heat-stable enterotoxin (STb) is the most prevalent toxin associated with diarrheagenic E coli isolates of porcine origin. Unequivocal biological activity of this toxin has been observed only in swine intestine. In this study, when endogenous protease activity was blocked with soybean trypsin inhibitor, intestinal secretion was stimulated by STb in jejunal loops of rats, mice, calves, and rabbits. Compared with pigs, rats, mice, and calves, rabbits were relatively insensitive to STb. These data demonstrate that the activity of STb is not a species-specific toxic activity; there is species variation in sensitivity to STb, and some common laboratory animals may have potential to be used to measure biological activity of STb.