Biological properties of chloroform/methanol extracts of Coxiella Burnetii.

Biological properties of antigens of whole cells of C. burnetii in phase I (WCI), their residue (CMR) and extract (CME) obtained by extraction of C. burnetti with chloroform/methanol mixture were subjected to a comparative study. It was shown that CME is able to react specifically with sera containing antibodies against WCI. The inoculation of animals with CME, but not with WCI and CMR does not cause changes of humoral and cell immunity to Coxiella and does not strengthen nonspecific antibacterial activity, but it can stimulate the formation of antibodies. Morphological changes of internal organs of mice were most expressed after the CMR inoculation and were practically absent after the CME inoculation.

[Invasiveness characteristics of the causative agent of pseudotuberculosis]. / Kharakteristika invazivnosti vozbuditelia psevdotuberkuleza.

The comparative characteristics of the invasiveness of Y. pseudotuberculosis, the most important element of its pathogenicity, is given on the basis of the results obtained in testing 57 Y. pseudotuberculosis strains and 23 Y. enterocolitica strains, all of them recently isolated, on experimental models (the monolayer cultures of Hep-2 cells, the enteral inoculation of mice and guinea pigs, the keratoconjunctival test). The invasiveness of Y. pseudotuberculosis has been shown to be manifested immediately after the ir adhesion and accompanied by the multiplication of these microbes in the cytoplasma of Hep-2 cells and, in animal experiments, in the cytoplasma of the epithelial cells of mucous membranes and the macrophages of lamina propria mucosae. The intracellular multiplication leads to the destruction of the layer of Hep-2 cells and, in animal experiments, to the disintegration of the infected cells, the development of Hep-2 cells and, in animal experiments, to the disintegration of the infected cells, the development of erosions and ulcers, purulent lymphadenitis of the regional lymph nodes, generalized infection with multiple abscesses in internal organs. Y. enterocolitica strains under investigation induced neither conjunctivitis, nor enterocolitis in the animals, and in experiments on Hep-2 cells, these strains, having less pronounced adhesive properties, either showed sharply limited adhesiveness without the capacity for intracellular multiplication and cytotoxicity, or proved to be absolutely noninvasive.

[Quantitative evaluation of Vibrio cholerae colonization and lymphocyte count in small intestine villi of rabbits immunized with vibrio and cholera toxin]. / Kolichestvennaia otsenka kolonizatsii kholernykh vibrionov i soderzhaniia limfotsitov v vorsinkakh tonkoi kishki krolikov, immunizirovannykh vibriohami i kholerogenom-anatoksinom.

The influence of parenteral immunization with live virulent vibrios and lipopolysaccharide-containing choleragen toxoid and oral immunization with liver avirulent vibrios on the interaction of V. eltor and the mucous membrane was studied on the ligated loops of the small intestine in 164 rabbits. Protection from enterotoxigenicity (the accumulation of fluid) was compared with the suppressive effect on the adhesion and colonization of vibrios. The coefficient of immunization efficacy was determined microbiologically, as the ratio of the adhesion index (i. e. the percentage of the number of vibrios in the media inoculated with the homogenized matter of the washed intestinal wall from the sum of this number and the amount of vibrios in the contents) in the control rabbits to the adhesion index in the immunized ones, and histologically, as the ratio of the average number of vibrios attached to the epithelial number of vibrios attached to the epithelial cells of one intestinal villus in the control rabbits to the corresponding number in the immunized animals. Protection from the enterotoxigenicity of vibrios resulted from the suppression of their adhesion and colonization, lipopolysaccharide being, probably, the factor of adhesiveness. Effective protection was accompanied by an increase in the number of lymphocytes (especially "granular" lymphocytes with lysosome-like granules) between intestinal villi and the number of lymphoid and plasmic cells in the lamina propria of the villi, which indicates the role of lymphocytes in cholera-induced immunity.

[Evaluation of the adhesion, colonization and enterotoxigenicity of Vibrio cholerae in the enteral infection of intact and passively immunized newborn animals]. / Ostenka adgezii, kolonizatsii i énterotoksigennosti kholernykh vibrionov pri énteral'nom zarazhenii intaktnykh i passivno immunizirovannykh novorozhdennykh zhivotnykh.

V. eltor were introduced into 323 suckling rabbits through a gastro-duodenal tube and into 629 suckling mice either by the same method, or per annum by a new specially developed technique. Passive immunization was achieved in suckling mice by the preliminary introduction of antibacterial sera into the small intestine or by feeding them with the milk of female mice immunized at the end of pregnancy, and in suckling rabbits by feeding them with the milk of goats immunized by introducing vibrios through the tests of the udder. To evaluate the results, the accumulation of liquid in the intestine and the development of diarrhea were taken into account, quantitative inoculation of homogenized intestinal matter was made; vibrios on the epithelium of the villi in the small intestine were counted and the changes of enterocytes and the lamina propria, observed in light and electron microscopy, were considered. The results of the complex quantitative evaluation in intact suckling animals revealed that the enterotoxigenicity of vibrios was manifested after their adhesion to and later colonization on the intestinal epithelium. Passive antibacterial immunization suppressed their adhesion and colonization, which resulted in protection from the enterotoxigenic effect of vibrios and their elimination from the intestine.

[Choice of models for determining the pathogenic properties of the causative agent of pseudotuberculosis]. / Vybor modelei dlia opredeleniia patogennykh svoistv vozbuditelia psevdotuberkuleza.

The infection of monolayer cultures of HEp-2, HeLa and Vero cells can be used for the quantitative evaluation of the invasiveness of Yersinia, their capacity for intracellular multiplication and cytotoxic action. The oral infection of mice and guinea pigs with Y. pseudotuberculosis permits one to observe initial enteritis and generalized infection accompanied by the multiplication of the microbes and the development of multiple granulomas with abscess formation in the liver and spleen. Administration of the supernatant fluid of the cultures to suckling mice and into the ligated loops of the rabbit small intestine, as well as administration of the native and heated lysates of the cultures into the pad of the mouse paw and into intestinal loops of rabbits have allowed the authors to reveal for the first time that in Y. pseudotuberculosis the production of both thermostable and thermolabile enterotoxins occurs and to confirm that Y. enterocolitica synthesizes only thermostable enterotoxin. For the first time Y. pseudotuberculosis have been found to produce keratoconjunctivitis and generalized infection in guinea pigs as the result of conjunctival infection. The complex of the models used in this study is recommended for the determination of the invasiveness and enterogenicity of Yersinia, and their capacity for intracellular multiplication and generalized infection.

[Use of the keratoconjunctival test for determining the virulence of the causative agent of pseudotuberculosis]. / Primenenie keratokon" iunktival'noi proby dlia opredeleniia virulentnosti vozbuditelia psevdotuberkuleza.

When tested in guinea pigs, Y. pseudotuberculosis strains, serovar I (25 strains) and serovar V (1 strain), were found for the first time to be capable of causing keratoconjunctivitis in the animals; the most virulent of these strains caused progressive conjunctivitis and keratitis with generalized infection. The minimum infective dose was 10(4) for conjunctivitis and 10(6) for keratitis. The studied Y. pseudotuberculosis strains, serovars O3 and O9, were found to be incapable of causing conjunctivitis and keratitis. The authors believe that the keratoconjunctival test allows one to evaluate the invasiveness and toxicity of Y. pseudotuberculosis according to the degree of the manifestation and the time of the development of conjunctivitis and keratitis.

[Protection from virulent Salmonella groups B and D after the peroral immunization of chicks with a hybrid of Salmonella typhimurium and Salmonella dublin]. / Zashchita ot virulentnykh sal'monell grupp B i D posle peroral'noi immunizatsii tsypliat gibridom Salmonella typhimurium i Salmonella dublin.

Chickens over 10 days old, infected orally with virulent salmonellae, were found to remain alive. Histologic investigation showed the development of mild enteritis and more pronounced, lasting for more than two weeks, inflammation of the cecum, dissemination and focal lesions in the liver (granulomas, necrosis). In experiments on the oral immunization of 3-day old chickens the bivalent hybrid of S. typhimurium vaccine strain 274 and S. dublin induced only pronounced blast transformation in lymphatic follicles of the cecum, hyperplasia of activated macrophages and formation of granulomas from these macrophages and lymphocytes. After oral challenge of the immunized chickens with virulent salmonellae of group B (S. typhimurium) and group D (S. enteritidis, S. gallinarum-pullorum) the chickens exhibited sharply pronounced protection against adhesion, colonization and invasion, and a few penetrating bacteria were rapidly destroyed by immune macrophages. Hybrid strain 274/O9 proved to be suitable for use as oral bivalent vaccine against salmonellosis in chickens.

[The use of a live Salmonella bivalent vaccine for decreasing the circulation of salmonellae in a poultry plant]. / Primenenie zhivoi sal'monelleznoi bivalentnoi vaktsiny dlia snizheniia tsirkuliatsii sal'monell na ptitsefabrike.

S. typhimurium (expressing antigen 09 of S.dublin) recombinant vaccine strain was tested at a large poultry-breeding farm under the conditions of permanent circulation of salmonellae. The oral immunization of laying hens in three administrations with doses of 5 x 10(8), 5 x 10(9) and 5 x 10(9) microbial cells induced an increase in titers of antibodies only to group D salmonellae, while in immunized chickens (each receiving a dose of 3 x 10(8) microbial cells) antibodies to both group B and group D salmonellae were detected. The vaccination of laying hens and chickens produced a protective effect due to the development of immunity and the decrease of Salmonella contamination of eggs laid by immunized hens, thus reducing the risk of infection at incubator stations. Total mortality during the first 3 months of life was essentially lower (p < 0.001) among immunized chickens (4.41 +/- 0.09%) than in the control group of nonimmunized chickens (5.91 +/- 0.10%).

[The host reaction to the administration of different components of Coxiella burnetii]. / Reaktsiia khoziaina na vvedenie razlichnykh komponentov Coxiella burnetii.

After the intraperitoneal injection of corpuscles of C. burnetii antigen (Ag), phospholipid (PL), and sediment obtained after the extraction of PL from Ag with chloroform-methanol (CM) slight leukocytic reaction developed in the peritoneum on day 1, and on day 2 it could be observed in the liver and in the spleen. Ag induced the most pronounced morphological changes. In the spleen they were manifested by the activation of T- and B-dependent zones of white pulp from day 2 and by the pronounced hyperplasia of reticular cells and macrophages, leading to splenomegaly, by days 7-14. Simultaneously lymphoid-macrophagal granulomas and hepatomegaly developed in the liver. By days 7-14 the foci of necrosis in the liver were caused by the thrombosis of portal veins and were not registered after the injection of PL and CM (and earlier also in experiments with Ag in doses of 0.1-0.3 mg).

[A morphological evaluation of the protective properties of noninvasive recombinant strains of Shigella]. / Morfologicheskaia otsenka protektivnosti neinvazivnykh rekombinantnykh shtammov shigell.

In 2-3 weeks after the oral immunization of rabbits, made in one or two administrations, with attenuated two-marker S. dysenteriae 1 strain VS-12 and recombinant S. dysenteriae VS-12/S. sonnei NR-18 and S. flexneri y433/S. sonnei NR-18 pronounced immunological reaction developed in the mucous membrane of the small intestine: blast transformation follicles of Peyer's patches, an increase in the number of lymphoblasts and plasmocytes in the cupolae of follicles and in intestinal villi, and an increase in the number of lymphocytes and macrophages in the intestinal epithelium with their release into the lumen of the intestine after challenge with virulent shigellae. The protective potency of these recombinants after challenge with massive doses of virulent shigellae was found to be high, which was shown by quantitative evaluation of the decrease of adhesion, invasiveness and cytotoxicity, suppression of epithelial lesions and development of inflammation in the intestinal mucosa.

[A morphological evaluation of the harmlessness of noninvasive recombinant Shigella strains]. / Morfologicheskaia otsenka bezvrednosti neinvazivnykh rekombinantnykh shtammov shigell.

The interaction of Shigella recombinant strains (with attenuating chromosomal mutations, with transposon-neutralized plasmid gene mutations, the hybrids of both strains), incapable of inducing keratoconjunctivitis in guinea pigs, with cells Hep-2 and enterocytes in the ligated loop of the small intestine of rabbits was studied. These strains retained, to varying extent, pronounced adhesiveness, but practically lost their invasiveness (though in Peyer's patches the translocation of bacteria by M-cells was observed) and cytotoxicity, as well as their capacity to multiply in epithelial cells and to cause destructive inflammation in the intestine. According to these criteria of evaluation, morphological investigations confirm the safety of the recombinant shigellae under study.

[The vaccinal process after the intramuscular immunization of sheep with Salmonella typhimurium strain 274]. / Vaktsinal'nyi protsess posle vnutrimyshechnoi immunizatsii ovets shtammom Salmonella typhimurium 274.

After the immunization of sheep by the intramuscular injection of S. typhimurium vaccine strain 274 the bacteria did not proliferate, but were phagocytosed and destroyed by leukocytes and macrophages; in some sheep these bacteria persisted up to day 13, inducing inflammation neither at the site of injection, nor in lymphatic nodes and internal organs. The vaccinal process in lymphoid tissue was characterized by an initial decrease in the number of lymphocytes, then a sharp rise in blast transformation, mitosis rate and the number plasmatic cells in the centers of multiplication of follicules in lymph nodes and the spleen. After day 13 gradual normalization began and by day 32 only residual manifestations of immunomorphological changes remained. In the lamina propria of the intestinal mucosa individual small macrophagal granulomas developed. The number of lymphocytes also increased in the epithelium and the lamina propria of the uterine mucosa. In the liver variations in the number of lymphocytes were observed in portal interlayers, around central veins and in the region of sinuses.

[The vaccinal process after the peroral immunization of lambs with the Salmonella typhimurium 274 strain]. / Vaktsinal'nyi protsess posle peroral'noi immunizatsii iagniat shtammom Salmonella typhimurium 274.

After oral immunization of lambs with S. typhimurium vaccine strain 274 these bacteria were characterized by low adhesion to the epithelial surface of the ileum, cecum, and colon, by very insignificant invasion into intestinal epithelium and then into lamina propria of the intestinal mucosa, mesenteric lymph nodes and the blood; the blood stream carried these bacteria further to the liver, spleen and other organs. Bacteria of strain 274 produced no damages, did not multiply and only persisted for 7-13 days in some lambs. No inflammation developed in response to oral immunization of the animals. The developing immunomorphological reaction presented as just a negligible increase in lymphocyte count in the intestinal epithelium, blast transformation and plasmatization in lymphatic follicles of Peyer's patches, hyperplastic mesenteric lymph nodes, blast and lymphocyte recirculation, moderate hyperplasia of white splenic pulp and insignificant lymphoid infiltration of portal interlayers of the liver.

Interaction of vibrio cholerae El Tor and gut mucosa in ligated rabbit ileal loop experiment.

Light and electron microscope studies in ligated rabbit gut loops showed that cholera vibrios El Tor attached themselves to the enterocytes and multiplied, most probably on the intestinal epithelium surface. But they did not penetrate deeper and were unable to cause destruction of the epithelium or marked inflammation. Ultrastructural manifestations of both apocrine and merocrine secretion of the enterocytes were observed. In addition, the enterocytes showed signs of generally enhanced activity. Some other changes and alterations in the lamina propria, presumably induced by excessive transport of water, are also described. The enterotoxin-induced epithelial hypersecretion with fluid accumulation in the gut loops resulted in the attached vibrios being cleared off the epithelium lining into the gut lumen. NAG vibrios were mostly incapable of inducing the above changes. We discuss the importance of cholera vibrio attachment to and its multiplication on the intestinal epithelium in the pathogenesis of cholera.