Search

Materials on the behavior of Y. pseudotuberculosis in the model of marine ecosystem are presented. Y. pseudotuberculosis, both carrying and having lost the virulence plasmid of 45 MD, die in nonsterile sea water, but are capable of prolonged survival in sterile sea water and in the bodies of echinoderms. The death of these bacteria is accelerated in the presence of larvae of echinoderms. In sea water at low temperature Y. pseudotuberculosis maintained their high pathogenicity potential which they use on contact with eukaryotes. Under these conditions Y. pseudotuberculosis preserve their virulence plasmid, thus causing the disease and death of animals.

Heat-stable toxin, lethal for mice when administered parenterally (LD50 was equal to 4.5 micrograms/mouse), was isolated from Y. pseudotuberculosis grown at a temperature of 8-10 degrees C. The toxin was found to be species-specific protein with a molecular weight of 45 kD, resistant to the action of elevated temperature (boiling for 5 minutes), 0.1-1.0% anionic detergent (sodium dodecyl sulfate) and stable at the pH range between 6.0-8.0. The amino acid sequence of the N-end fragment of the toxin was determined: H2N-Ala-Glu-Val-Asp-Ser-Ser-Glu-Phen-Pro. The toxin was inactivated after treatment with trypsin, chemotrypsin, papain. Antibodies to this toxin were detected in the blood sera of pseudotuberculosis patients.

Serum to thermostable Y. pseudotuberculosis toxin is shown to induce agglutination of bacteria belonging to all 8 serologic variants of this species. No cross reactions with other enterobacterial species, including four Yersinia species, were detected. The results permit us recommend the antitoxic serum for identification of Y. pseudotuberculosis.

Virulent strains of Yersinia pseudotuberculosis and Y. enterocolitica cultured at 10 degrees C were studied with respect to their interaction with cultured fish cells. Infection with Yersinia at 25 degrees C resulted in the formation of pseudopodia and the adherence of bacteria to the cell surface. The adherent bacteria were ingested 2 h after infection. Yersinia was found to be cytotoxic for the cells and the intracellular bacteria seemed to reside in phagosomes and were found in stages of division but were never observed in the nucleus.

Y. pseudotuberculosis strains were grown at 6 degrees-8 degrees C and then incubated at 37 degrees C. 3-6 hours later serum resistance appeared in the strains having plasmid virulence and producing outer membrane polypeptide with a molecular weight of 120 kD, known as P1. 10-12 hours later serum resistance appeared in the strain having the virulence plasmid, but not producing P1, as well as in strains in which the plasmid was eliminated.

The protective properties of the species-specific pore-forming polypeptide of Y. pseudotuberculosis outer membrane (porin) were studied. The study showed that 80-90% of mice immunized with porin survived after challenge with 10-30 LD50 of Y. pseudotuberculosis, serovars 1 and 3. The LD50 of the preparation exceeded its ED50 more than 100-fold. Immunization made in two injections was more effective than immunization in one injection. The immunization of the animals with porin led to an increase in the activity of peritoneal exudate macrophages with respect to Y. pseudotuberculosis, serovars 1 and 3.

This work presents data indicating that Y. pseudotuberculosis population, cultivated at lower temperature (6-8 degrees C), has a high potential of cellular and tissue invasiveness. Pseudotuberculosis has been experimentally shown to start as generalized infection due to the rapid (during 10-15 minutes) penetration of the infective agent through the epithelium of the mouth cavity, the small intestine, the urinary bladder, conjunctiva, pulmonary alveoli and vascular walls into the blood and then into internal organs. The data, obtained in this study, on the penetration of Y. pseudotuberculosis through any mucous surfaces in different species of warm-blooded animals are indicative, to a considerable extent, of the fact that this process occurs due to the action of nonspecific mechanisms.

A decrease in the temperature of the cultivation of Yersinia pseudotuberculosis has been shown to lead to the appearance of motility and adhesive properties in these bacteria, to enhance their ability to penetrate the body of the host through mucous membranes, while a rise in the temperature of cultivation has been shown to cause the loss of these properties and, therefore, a decrease in the penetrating capacity of these bacteria. Y. pseudotuberculosis penetrates from the surface of the epithelium into the blood stream in 10 minutes. The capacity of the bacteria penetrating into the blood to induce lethal infection is determined, to a great extent, by the plasmid calcium dependence, and in oral infection, when these bacteria must overcome the barrier formed by the mucous membrane, calcium-dependent bacteria grown at 6-8 degrees C show the highest degree of virulence.

Capsular substance and extracellular proteins of the causative agent of pseudotuberculosis caused in the organism of guinea pigs a specific immunological reconstruction with the accumulation in the blood of antibodies in low titres, chiefly of macroglobulin type. The blood sera of these animals failed to protect the mice from lethal pseudotuberculosis infection in experiments of passive protection. Surface antigens of pseudotuberculosis causative agents produced a sensitizing action on the guinea pig organism; this was shown in vitro with the macrophages of the abdominal cavity of these animals. Extracellular proteins decreased the completeness of phagocytosis by neutrophilic leukocytes (demonstrated in vitro with the donor blood leukocytes).

For the first time the activity of Y. pseudotuberculosis, its extracellular, surface antigen and Westphal's lipolysaccharide has been studied with the use of biological models (the ligated loop of the small intestine of a rabbit, the pulmonary model, the intradermal tests). This microorganism has been found capable of inducing the accumulation of exudate in the ligated loop of the small intestine of a rabbit and interfering with the permeability of the vascular walls. The toxic action of Y. pseudotuberculosis is neutralized by immunization.

Available are data obtained at light and electron microscopy of operative specimens from patients with abdominal pseudotuberculosis and animals challenged orally with Yersinia pseudotuberculosis. The authors are the first to outline detailed characteristics of the intestinal and regional lymph node lesion arising in response to the infection and reflecting growing resistance to it. These features of pathological process involve marked tissue eosinophilia, necrosis due to phagocytes rexis, and granulomatosis suggesting a pronounced role in the pathogenesis of the body allergization in the course of infection. It is proposed to consider pseudotuberculosis-related changes in lymph nodes as lymphoblastic (early affection) and granulomatous-necrotic (advanced infection) lymphadenitis. The evidence obtained can promote differential diagnosis of pseudotuberculosis.

The role of phagocytosis in experimental pseudotuberculosis was demonstrated in vivo and in vitro. Virulent Y. pseudotuberculosis strain caused the death of the majority of the cells, whereas the weakly-virulent ones--degenerative changes and the death of but of few of them. The capacity of the causative agents of pseudotuberculosis to survive and to reproduce within the cells could be regarded as one of the significant factors of virulence of these microbes. The maximal immunizing effect was observed in infection of the animals with sublethal dose of the living Y. pseudotuberculosis culture. A somewhat lesser, but a sufficiently high, immunizing effect was produced by lipopolysaccharide. A method of macrophage culture can be used for the assessment of the immunogenicity of various antigenic complexes of the microbes.

The work presents the data indicating that the temperature of Y. pseudotuberculosis cultivation is very important in regulating the activity of pathogenicity factors, necessary for the initiation of the pathogenic process in the cells of the macroorganism. Low temperature (8-10 degrees C), necessary for the growth of Y. pseudotuberculosis, facilitates the activation of invasive and toxic pathogenicity factors. At a growth temperature of 37 degrees C the inhibition of such necessary attributes of virulence as adhesion and invasion into epithelial cells occurs in Y. pseudotuberculosis, which decreases the capacity of these bacteria for inducing the infectious process. The virulence of Y. pseudotuberculosis population, lost as the result of its cultivation in synthetic culture media at a temperature of 37 degrees C, has been found to be restored at low temperature.

The comparative electron-microscopic study of early stages of the interaction of Y. pseudotuberculosis virulent strain (No. 282) with "professional" (macrophages) and "nonprofessional" (HeLa cells) phagocytes has been carried out. The character of the intimate mechanism of this interaction has been found to be essentially different. The common feature for both systems is the adsorption of bacteria and their penetration into cells due to phagocytosis. But the subsequent fate of Y. pseudotuberculosis is different. In HeLa cells they are isolated from the cytoplasm by multilayer membrane structures, thus remaining morphologically intact. In macrophages the destruction of the microbe in phagolysosomes occurs.

The pore-forming protein, lipopolysaccharide-protein complex and lipopolysaccharide of Y. pseudotuberculosis outer membrane have been shown to participate in the penetration of the bacteria into the cells of the macroorganism, to produce a toxic effect on these cells and to enhance the ingesting activity of macrophages in small doses, while suppressing it in large doses. When introduced parenterally, protein induces a more pronounced clinical picture of specific reactive hepatitis in experimental animals and greater changes in their kidneys than the lipopolysaccharide--protein complex.

Some data showing specific ways of interaction of Yersinia pseudotuberculosis with the epithelium of the small intestinal mucosa have been received for the first time by using light microscopy of semithin sections. Comparative study of the lesion of the epithelium of different parts of rabbit small intestine by oral inoculation with Yersinia tuberculosis has expanded the ideas of the infection entry. It has been established for the first time that the most intensive penetration of the microorganisms into the epithelium of the mucous membrane takes place in the duodenum and jejunum. As the infected contents is evacuated via the intestinal tract, the Yersinia pathogenicity decreases, and in the ileum, the invasion of the microorganisms becomes less pronounced. It has been revealed that in pseudotuberculosis, there takes place a repeated dissemination of the infection in the intestinal tract because of decay of the involved enterocytes, which is of pathogenetic importance for the progress and outcome of the infection.

Y. pseudotuberculosis cells grown at biologically low temperature have been shown capable of chemotaxis with respect to carbohydrates and amino acids. During cultivation at 36-37 degrees C Y. pseudotuberculosis cells retain this property for 10-15 hours and then lose it. The mechanism of chemotaxis makes it possible for Y. pseudotuberculosis to "find" human and animal tissues and can facilitate the realization of the pathogenicity potential of these bacteria. When administered orally to mice motile bacteria, i. e. those grown at 6-8 degrees C, have been more virulent for the animals than nonmotile ones cultivated at 36-37 degrees C.

The results of the study on the immunostimulant effect of mytilan, a polysaccharide isolated from Crenomytilus grayanus inhabiting the Sea of Japan are presented. It was shown that a single dose of the drug administered parenterally stimulated the natural protective reactions of the macroorganism evident from an increase in the leukocyte count in the peripheral blood of the experimental animals and the phagocytic activity of polymorphonuclear nuclear leukocytes. Mytilan stimulated humoral immunity. Its use brought about an increase in the hemolysin titers in response to administration of the sheep red blood cells card a significant increase in the number of antibody-forming cells as compared to the control. Mytiland had a preventive protective effect with regard to generalized pseudotuberculous and Salmonella infections. The drug increased the macroorganism resistance to both the psedotuberculous microbe and its toxins.

A total of 145 patients having Far Eastern scarlatiniform fever, 99 healthy persons and 54 patients with diseases of non-pseudotuberculous etiology have been examined by means of Fradkin's neutrophil damage test, the capillary cell migration inhibition test and the intradermal test. The specific transformation of the cellular immunity system has been shown to occur in scarlatiniform fever. This transformation takes the form of delayed type hypersensitivity reaction. The immunological tests used in this study have been found to be specific, highly sensitive, simple and suitable for diagnosing Far Eastern scarlatiniform fever.

Experimental pseudotuberculosis arthritis was reproduced by means of antigenic irritation of the knee joint in the presence of specific sensibilization. Pathomorphology is characteristic of the acute exudative-proliferative synovitis and periarthritis. The results obtained allow one to draw the conclusion on the infectious-allergic genesis of the joint involvement in pseudotuberculosis. The stages in the allergic responses of the synovial membrane with subsequent development of early and late immune inflammation are revealed.

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.

Pseudotuberculous microbe produced (in natural medium, at 10-14 degrees) toxins and exoenzymes, pathogenic effect of which was studied in experimental conditions. After invasion of pseudotuberculous microbe into macroorganism its populations appear to change, as a result of this activities of neuraminidase and hyaluronidase were increased. Cultivation of pseudotuberculosis bacteria in vivo may serve as a promising model for studies of enzymatic mechanisms of pathogenesis.

When cultivated in the presence of glucose, irrespective of temperature and the degree of aeration, Y. pseudotuberculosis cells have the ovoid form, constant size and low hydrophobic properties of their surface. Meanwhile the characteristics of the bacteria grown in the medium, carbohydrate-free or with galactose added, essentially depend on the conditions of medium aeration. Under the conditions of intensive stirring at both temperatures these bacteria acquire the coccoid form, not typical for Yersinia, they have a smaller area (approximately 2 times) and more hydrophobic surface in comparison with the cells grown in the presence of glucose. Under stationary conditions the differences between the cells, cultivated in the presence of galactose and glucose, in form and area disappear, but the differences in the hydrophobic properties of the surface are retained. As revealed in this study, the cells grown in the presence of galactose and under the conditions of intensive medium stirring, in contrast to those grown with glucose, have 1.5-fold greater invasive activity, irrespective of aeration conditions, eightfold greater resistance to ampicillin and twofold greater resistance to streptomycin and erythromycin.