Pathogenic properties of Yersinia pseudotuberculosis in different biological models

The study of the action of Y. pseudotuberculosis introduced into the cavity of the ligated intestinal loop of a rabbit, into the stomach of 2- to 4-day-old suckling mice or injected intradermally has made it possible to establish the importance of the invasive and toxic activity of this microbe in the pathogenesis of pseudotuberculosis infection. The lesion of the small intestine develops due to the penetration of the bacteria into the mucous membrane and the combined action of the microbial cells inhabiting the stroma, and secreting into the environment of cytotoxins, toxic substances with an enterotropic action or enterotoxins and factors increasing the permeability of the vessels. Y. pseudotuberculosis strains with high and low invasiveness have been isolated.     

Experimental research on cholera vaccines. Local immunity

Parenteral immunization of rabbits with cholera vaccine decreased the number of Vibrio cholerae adhering to the mucous membrane of the small intestine. Cholera toxoid and the complex preparation ensure protection from the local action of cholera toxin on the ligated loop of the rabbit intestine, while cholera vaccine produces no effect under the same conditions. The use of three preparations under study leads to the decrease of exudative reaction to the introduction of live V. cholerae, the effectiveness of these vaccines growing in the following order: cholera vaccine, cholera toxoid, the complex preparation.     

Listerial invasion protein internalin B promotes entry into ileal Peyer's patches in vivo

Listeria monocytogenes (Lm) invades the host intestine using listerial invasion proteins, internalins. The in vivo role of internalin A (InlA) and internalin B (InlB) is reported here. Intragastric (i.g.) administration and ligated loop assays with ΔinlB-Lm demonstrated that a lack of InlB significantly attenuates the invasive ability of Lm into various organs. On the other hand, InlA(m)-Lm expressing a mutant InlA with two substitutions, S192N and Y369S, which has been reported to increase the affinity of InlA to mouse E-cadherin, resulted in little increase in intestinal infection according to both ligated loop and i.g. infection assays. Lm preferentially enters ileal Peyer's patch (PP) via M cells and ΔinlB-Lm showed severely reduced ability to invade though these cells. The present results reveal the importance of InlB, which accelerates listerial invasion into M cells on ileal PPs in vivo.     

The enterotoxigenic capacity of hemolysin-producing strains of Proteus isolated in acute intestinal infections in children

The capacity of P. mirabilis and P. vulgaris strains isolated in acute enteric infections in children for producing enterohemolysin, a new type of hemolysin, has been shown. The relationship between the capacity of Proteus cultures for producing enterohemolysin and their capacity for inducing toxic secretory reaction on a ligated loop on the small intestine of rabbits in the absence of known thermostable and thermolabile antitoxin in bacteria.     

O1/O139新型霍乱疫苗口服免疫动物后的自动免疫保护及被动保护试验研究

O1／O139霍乱口服疫苗是一种新型疫苗。本文通过2种不同途径免疫动物,检测血清抗菌抗体和肠道分泌性IgA抗体以及保护力试验,并探讨了它们之间的关系。通过家兔肠袢试验和细胞四甲基偶氮唑盐(MTT)试验检测免疫后抗血清的被动保护作用。试验提示：在霍乱的保护性免疫应答中,除粘膜免疫可能占主导作用外,血清抗菌抗体也发挥了一定的作用。     

Effect of phosphaden and unithiol on the cyclase system of the small intestine mucosa in rabbits in experimental salmonellosis

The study was made on 59 chinchilla rabbits. S. typhimurium 1847 live culture was introduced into the lumen of an isolated loop of the thin intestine. The activity of adenylate cyclase (AC), guanylate cyclase (GC), the levels of cyclic adenosine 3,5-monophosphate (cAMP), cyclic guanosine 3,5-monophosphate (cGMP), the activity of cAMP- and cGMP-phosphodiesterases were determined in the mucous membrane of the ligated part of the intestine. Considerable fluid accumulation in the loop, activation of AC and cGMP-phosphodiesterase, a rise in the level of cAMP and a drop in the level of cGMP in the mucosa of the ligated part of the intestine were registered. In one group of the animals phosphadene and in the other group unitiol were introduced into the infected intestinal loop; as a result, a decrease in the accumulation of fluid in the loop, on the average, by 40% and a tendency to an increase in the level of cAMP and a drop in the level of cGMP in the mucous membrane of the ligated part of the intestine were observed. Changes in the level of cGMP play, seemingly, a more important role in the development of diarrhea in salmonellosis.     

Rabbit Ileal Loop Response to Strains of Clostridium perfringens

The ligated loop of the rabbit intestine was investigated as a possible experimental model for the study of Clostridium perfringens food poisoning. The method of preparation of the challenge inoculum was important in determining whether a given strain would provoke a response. When cultures were grown for 4 hr at 37 C in Skim Milk (Difco), 14 of 29 type A strains isolated from food-poisoning outbreaks consistently produced exudation of fluid and consequent dilation of the ileal segments. In contrast, 15 of the 18 strains derived from other sources failed to elicit a response. By use of different inoculum preparations, nearly all strains could be made to give at least an occasional positive loop reaction. Diarrhea was not obtained in rabbits by intraluminal injection into the normal ileum or by per os administration of the cultures. Lecithinase, purified and in concentrated culture supernatant fractions, failed to produce a response in the isolated ileal loops.     

Factors influencing tone in rabbit large intestine.

Pressure/volume curves obtained in the presence of papaverine have been compared with those obtained under control conditions in rabbit large intestine. Tension/length curves obtained on stimulation of the extrinsic nerve supply to the intestine have been compared with control curves. Evidence is presented for a small contribution of sustained muscular activity to the pressue/volume and tension/length curves. The effect of stretch on peak tension attained by the intestine has also been examined.     

Production and partial purification of a fluid-accumulating factor of non-O1 Vibrio cholerae

A fluid-accumulating factor (FAF in the ligated rabbit ileal loop test) from a strain of non-O1 Vibrio cholerae not producing cholera toxin-like enterotoxin (CTLT) was partially purified by ammonium sulfate precipitation, gel filtration with Sephadex G-100, and DEAE cellulose column chromatography. The preparation thus obtained showed collagenolytic, cytolytic, necrotic, and hemorrhagic activities, but was not lethal to mice nor hemolytic to sheep erythrocytes. Desquamation of epithelial cells, inflammatory edema, and hemorrhage were observed in sections of rabbit intestine after inoculation of partially purified FAF (PPFAF). Biological and enzymatic activities of FAF were completely neutralized with anti-PPFAF rabbit serum. More than 70% of non-O1 V. cholerae strains from human diarrheal feces produced FAF in the shake culture of heart infusion broth (Difco). A fluid-accumulating factor immunologically similar to FAF of non-O1 V. cholerae was also produced by V. mimicus strains isolated from human diarrheal feces. These results indicate that the FAF produced by CTLT-negative non-O1 V. cholerae strains is an entity closely related to a cytolytic and hemorrhagic substance or the like, and that this FAF may play a role in the enteropathogenicity of CTLT-negative strains.     

Experimental data proving the presence of inhibitors of amylase activity in biliary and pancreatic juice

Two parallel studies on albino male rats are performed. In the first study, there is a group which underwent resection of the proximal third of the small intestine. While the other group despite resection of the same segment also has a ligated common biliary and pancreatic duct. In the second study, one group of the experimental animals is only with ligated pancreatic duct and in the other group the same duct is implanted in the initial part of the ileum. On the 15th day after the surgical interventions the amylase activity and the absorption of glucose in the small intestine are studied by the method of turned sacs "in vitro". It is established that the glucose transport does not change after the four surgical interventions. However, the amylase activity increases about twice times after resection of the upper third of the small intestine and more than 4 times after resection of the same segment with simultaneous ligature of the common biliary and pancreas duct. Only at ligating the duct, the amylase activity is decreased in the jejunum and is significantly increased in the ileum, while its implantation in the initial part of the ileum does not change its activity in both studied segments of the small intestine. It is concluded that there are unknown inhibitors for the amylase activity in the biliary and pancreatic juice. The discussed issue is why they inhibit only the enzymatic compensatory processes without influencing the transport systems of the small intestine.     

Methods of selection, preparation and evaluation of the viability of small-intestinal fragments suitable for transplantation

In the experiment performed on cats, using histological, histochemical and impregnative techniques it has been stated that choose of the small intestine area, suitable for transplantation should be based on counting straight vessels ramified in it. In order to ensure viability of the small intestine fragment the method of its preliminary treatment is recommended, based on training blood vessels. For estimating viability of the transplant it is possible to ascertain SDG activity in neurons of the intramural nervous ganglia and estimate amount of secreting gobletlike cells in mucous membrane of the small intestine loop.     

The rabbit jejunal 'imino carrier' and the ileal 'imino acid carrier' describe the same epithelial function.

For rabbit jejunal brush border vesicles an 'imino carrier' has been defined as a sodium-dependent, alanine-resistant transporter of cyclic imino acids, while for intact rabbit jejunal and ileal epithelia an 'imino acid carrier' has been defined as a sodium-dependent transporter of both aliphatic and cyclic imino acids. This study on intact rabbit intestine examines whether these two terms describe the same epithelial function. The KPro1/2 and the KProi against JMeAIBmc are identical and so are KMeAIB1/2 and KMeAIBi against JPromc. Likewise, KLeui is the same against the transport of both proline and MeAIB. It is, therefore, concluded that the terms 'imino carrier' and 'imino acid carrier' describe the same epithelial function: A sodium-dependent, relatively high afinity, saturable transporter of both aliphatic and cyclic imino acids. Estimates of the apparent affinity and inhibitory constants for MeAIB, proline and leucine confirm that the jejuno-ileal variation of amino acid transport along the rabbit small intestine is a variation of maximal transport capacity.     

Effect of glutathione on choleragenic diarrhea and disorders of the antioxidant system of rat intestinal and liver cells

The effects of reduced glutathione on the development of choleragenic diarrhea and the activity of glutathione transferase (GT), glutathione peroxidase (GP-GTB and GP-H2O2), superoxide dismutase (SOD), glutathione reductase (GR) in the small intestine and liver of rats with experimentally ligated jejunal loop have been studied. Diarrhea syndrome was found to decrease markedly after glutathione administration in a dose of 1 g/kg bw. GR activity in the jejunum and liver of rats treated with toxin and the following glutathione administration rose by 210 and 186%, respectively, and then reached the control level. Glutathione transferase activity in the jejunum increased by 150% (P less than 0.05), remaining, however, lower than the control values. The activity of other enzymes tested was unchanged. Polyfunctional cellular activity of glutathione suggests that antidiarrhea effect should be considered as an element of pathogenetic therapy.     

Studies on the pathogenicity of Yersinia enterocolitica. III. Comparative studies between Y. enterocolitica and Y. pseudotuberculosis

Comparative studies on pathogenicity between Yersinia enterocolitica and Yersinia pseudotuberculosis were performed using experimental infection systems in vivo and in vitro. All of thestra ins of both species successfully produced experimental enterocolitis in rabbits although the severity varied with the strains challenged. The changes were characterized by granulomatous lesions with necrobiotic centers in reticuloendothelial tissues of the intestine, mesenteric lymph nodes, liver and spleen. These strains uniformly had the ability to penetrate HeLa cells and to survive or multiply within cultured rabbit peritoneal macrophages. In addition, in infections with strain TP-2 or PST-III of Y. pseudotuberculosis, catarrhal inflammation all over the small intestine and/or focal necrosis and parenchymatous degeneration in the liver were observed, along with the granulomatous lesions. These strains, at the same time, exhibited cytotoxic effects on the cultured cells. The pathogenic factors of Y. enterocolitica are discussed in comparison with those of Y. pseudotuberculosis.     

Concanavalin A promotes adherence of Salmonella typhimurium to small intestinal mucosa of rats

A number of dietary lectins have been shown to resist proteolytic digestion. These lectins interact with the small intestinal mucosa causing structural and functional changes. Concomitant to these changes, bacterial overgrowth was reported and a possible interaction between lectins and bacteria in the small intestine was postulated. The aim of this study was to investigate the effect of various lectins on adherence of Salmonella typhimurium to both isolated small intestinal enterocytes and ligated intestinal loops. Isolated intestinal cells or ligated intestinal loops were incubated with [3H] adenine-S. typhimurium in the presence or absence of concanavalin A, phytohemagglutinin, peanut agglutinin, and wheat germ agglutinin. Only concanavalin A promoted the adherence of various strains of nonfimbriated S. typhimurium to isolated viable intestinal cells. Other lectins showed no effect on the adherence. In situ studies showed that bacterial binding was increased in concanavalin A-treated intestinal loops, supporting the significance of the experiments in vitro. These data suggest that lectins may act by promoting bacterial adherence to the small intestine, thereby facilitating colonization and infection, and leading to bacterial overgrowth.     

On the toxinogenicity of Pseudomonas aeruginosa strains

Investigation of 367 P. aeruginosa strains primarily isolated from clinical and other biological material as well as from the environment yielded results suggesting a substantial toxinogenic potential. 92.6% of the assayed culture filtrates derived from the strains under investigation proved positive in the early skin tests on rabbits. 49.7% of the assayed material induced cytotoxic alterations on Vero cells, the rates for Y1 and CHO cells being 50.3% and 43.5% respectively. 54.3% culture filtrates caused haemolysis of rabbit RBC and 52.7% lysed horse RBC. Gelatinase activity was found in 96.3% of tested material, protease in 89.8%, lecithinase in 62.4% and elastase in 29.6%. 12.6% of tested material induced fluid accumulation in a ligated intestinal loop. None of the culture filtrates elicited a positive reaction in the suckling mice test suggesting the absence of the thermostable enterotoxin.     

Carbohydrate metabolic disorder in experimental secretory diarrhea induced by cholera toxin

Cholera toxin induced a massive mucus accumulation and a significant enhancement of arteriovenous difference in glucose concentration in the isolated loop of rabbit small intestine. Mucus hexosamine secretion increased threefold, whereas mucous lactate level remained unchanged. It is suggested that intensive mucus secretion is ensured by significant amounts of blood glucose, whose level is 65% increased, possibly due to the observed 1-6-fold liver gluconeogenesis acceleration. Though the mechanism of the latter is not yet known, the data obtained provide evidence that this stimulation is not cAMP-dependent.     

Effects of serotonin on the physiology of the rabbit small intestine

Serotonin has been shown to alter the intestinal transport of ions and intestinal motility. These effects may interfere with each other, modulating the whole physiology of the intestine. We have previously shown that serotonin also alters the transport of nutrients. Thus, the aims of the present work were to determine the possible interference between the secretagogue effect of serotonin and the mechanism by which serotonin inhibits the absorption of nutrients, and to study the effect of serotonin on the digestive activity of nutrients of the brush border membrane jejunum enterocyte in the rabbit. The results show that the secretagogue effect of serotonin neither affects the inhibitory effect of serotonin on the intestinal absorption of the nutrients, nor affects the activity of Na+/K+-ATPase. The activity of sucrase and aminopeptidase N was also not affected by serotonin in the rabbit jejunum. Finally, we also studied different parameters of the motility in the rabbit small intestine. Serotonin seemed to stimulate the motility of the rabbit small intestine by increasing integrated mechanical activity and tone of muscle fibers in duodenum, jejunum, and ileum. In conclusion, serotonin might alter or modulate the whole intestinal physiology.     

The 106-kilobase plasmid of Salmonella braenderup and the 100-kilobase plasmid of Salmonella typhimurium are not necessary for the pathogenicity in experimental models

Among 1.041 clinical isolates (77 serovars) of Salmonella which had been derived from cases with acute enterocolitis, 601 (58%) contained one or more plasmids. Large serovar-specific plasmids were seen in 95 of 307 isolates (31%) of Salmonella typhimurium, in 34 of 34 isolates (100%) of Salmonella enteritidis and in 36 of 38 isolates (94.7%) of Salmonella braenderup: the sizes of which were 100, 60 and 106 kilobases (kb), respectively. In order to determine the role of these plasmids in pathogenicity for enterocolitis, the plasmids were eliminated from some strains of S. braenderup and S. typhimurium and the pathogenicity of the plasmid-less strains was compared with that of the parent strains by invasiveness to HeLa cells, fluid accumulation in the rabbit ligated ileal loop, lesion of mucosal tissue and the Sereny test. The virulence of all the plasmid-less strains was as strong as that of the plasmid-bearing strains in these pathogenicity assay systems. We therefore concluded that the 106-kb plasmid of S. braenderup and the 100-kb plasmid of S. typhimurium are not necessary for their pathogenicity in the experimental models: invasiveness to HeLa cells, fluid accumulation in the rabbit ligated ileal loop, and Sereny test.