Biological activity of extracts isolated from a virulent strain of Sh. flexneri grown in the presence of calcium ions]

The authors carried out a comparative study of the genetically connected Sh. flexneri cultures (3 virulent strains, 3 clones of an avirulent mutant selected in the flux of an oblique light from the virulent strain, and lac+ Kcp A-hybrids obtained by crossing the initial virulent cultures with the E. coli K12 Hfr strains). The absence of any correlation between the virulence of the strains under study and the lipopolysaccharide (by rhamnose) content in the extracts from them in growing the cultures in the presence of calcium ions was noted. Toxicity of the extracts from the virulent cultures was demonstrated on a model of developing chick embryos. No such property was possessed by the extracts from avirulent strains. The extracts from the virulent cultures in nontoxic doses possessed the capacity to decrease LD50 of shigella strains used for the infection. The biologically active factor determined in the extracts from the virulent cultures apparently was not lipopolysaccharide.     

Comparative characteristics of cell-free filtrates of Shigella flexneri of different virulence]

It was shown for the first time that the virulent Sh. flexneri strain grown on Luria broth differed from the avirulent one by the yield of readily released surface-located complexes--lipopolysaccharide (determined by rhamnose) and protein into the filtrate. There was no distinct correlation between the strain virulence and the content of rhamnose-determined lipopolysaccharide in the filtrate; growing bacteria in the presence of Ca and Mg ions had no significant influence on the lipopolysaccharide release into the filtrate. Protein release into the cell-free filtrate was thrice that in the virulent shigella strain than in the avirulent one. When bacteria were grown in the presence of Ca ions protein release from the virulent strain increased 1.5-fold and changed but little in the avirulent culture. Cell-free filtrates of the virulent strain produced toxic action on L tissue culture cells; in conjunctival infection of guinea pigs they caused some reduction of the LD50 of the virulent strain and sharply aggravated the course of the infectious process. Heating of the filtrate at 100 degrees C for 15 min decreased their toxic action on L cells. The data obtained indicated that the active biological factor revealed in the virulent strain of Sh. flexneri was protein or its derivative.     

Study of the enterotoxic action of the neurotoxin of the Sonne dysentery microbe in a rabbit small intestine loop model]

The enterotoxic action of neurotoxin from Sonne dysentery microbes (obtained by the method of Mesrobeanu et al.), and also of the culture autolysates and homologous Boiven's endotoxin was studied on a model of the isolated loop of the rabbit small intestine. Neurotoxin preparations obtained from virulent strains as well as autolysates of these cultures possessed enterotoxic activity, whereas purifed endotoxin preparations in doses of 1--10 mg failed to cause any dilatation of the isolated intestinal segment. A significant individual rabbit sensitivity to the enterotoxic action of the neurotoxin preparation was revealed. Lyophilization of neurotoxin preparation did not influence its enterotoxicity. However dialysis against distilled water and boiling of the neurotoxin preparations led to the loss of enterotoxic activity.     

Experimental study, using a pulmonary model, of the pathogenic properties of genetically related strains of Sh. flexneri, differing in their ability to cause keratoconjunctivitis]

Complex microbiological and morphological study of the genetically connected Sh. flexneri strains, avirulent by keratoconjunctival test showed that the greatest extent of the virulent loss was revealed in shigellae with mutation in the area glpK-gene: they lost the capacity to penetrate into the cells of the bronchial epithelium, to resist local leukocytic reaction and to produce an injurious action on the pulmonary vessels. Shigellae with the replaced kcpA- gene area mostly lost the capacity to penetrate into the epithelial cells of mouse bronchi and were eliminated from the epithelium by the 9th hour after the administration. Xyl--avir-hybrids obtained from crossing of Sh flexneri with the streptomycin-resistant E. coli K12 donors were characterized by the loss of the capacity to prolonged reproduction in the organism of mice with the retention of the capacity to penetration into the epithelium, of the influence on the development of the leukocytic reaction and of the damaging effect on the pulmonary capillaries.     

Phage conversion of the antigens in Escherichiae and Shigellae. 2. Conversion of the somatic antigen in Shigella flexneri with the moderate E. coli 0129 phage]

The authors studied the converting activity of the moderate EF5 phage isolated from the lysogenic E. coli 0129 strain. It was shown that this phage converted the O-antigen with the detection of the type antigen V in the strains of Sh. flexneri of the serological type la and y-variant. The converted cultures contained the type antigen V and were identical by the antigenic properties to one another and the Sh. flexneri of the serological type 5 and E. coli 0129. A conclusion was drawn that phages converting the antigens of Sh. flexneri could be encountered in escherichia and could modify the antigens in Sh. flexneri and escherichia possessing the antigenic factor 3,4.     

Electron microscopic study of the interaction of virulent and avirulent Shigella flexneri strains with the macrophages of murine peritoneal exudate

The electron microscopic dynamic study of the interaction of S. flexneri virulent strain 2a and S. flexneri avirulent strain, genetically related to strain 2a and having the same antigenic structure, with peritoneal macrophages of CBA mice at an early period showed differences in the cytotoxic action of these strains. Even at the early period of the interaction between macrophages and virulent shigellae structural changes were observed in macrophages: ruptures in the membranes of parasitophore phagosomes, the vacuolization of the cytoplasm, the destruction of mitochondria. These changes were indicative of the early cytotoxic action of shigellae belonging to the virulent strain. In macrophages infected with avirulent shigellae the manifestations of this cytotoxicity were less pronounced and appeared later than in macrophages infected with S. flexneri virulent strain.     

The role of delayed hypersensitivity in the development of an experimental dysentery infection and vaccinal process

The results of experiments, indicating the development of cell-mediated delayed hypersensitivity (DH) skin reaction, its dependence on the virulence of Shigella flexneri 2a, the dose and the character of the antigen used for testing the reaction, are presented. One of the immunologically active components of S. flexneri virulent strain 2a No. 516 is a biologically active thermolabile factor detected in filtrate and supernatant fluid. These data suggest that the immunosuppressive action of the virulent strain is probably linked with the function of T-suppressors affecting the reaction at the period of its development (the induction phase). S. flexneri avirulent strain produced no such effect, developing DH being seemingly sufficient for activating T-cell-mediated immune response.     

Study of the role of type-specific antigen in the process of Shigella flexneri interaction with epithelial cell and macrophage cultures]

Epithelial cells of the HEp-2 line infected in parallel by genetically connected strains of Sh. flexneri, differing in the capacity to synthesize the type antigen were studied morphologically. The type antigen proved to show no significant influence on the penetration of dysentery bacilli into the cell. A study of the process of phagocytosis of the mentioned strains by a culture of peritoneal macrophages of guinea pig demonstrated that the presence of a type antigen communicated to the bacteria some selective advantages within the macrophages increasing their resistance to the action of the digestive enzymes.     

Plasmid-mediated bacteriocin production by Shigella flexneri isolated from dysenteric diarrhoea and their transformation into Escherichia coli

AIMS: To determine the production of bacteriocin by Shigella flexneri strains, to relate their production to the presence of dysenteric diarrhoea and to asses the genetic determination of the bacteriocin. METHODS AND RESULTS: One hundred and sixteen strains of Sh. flexneri were isolated from patients with diarrhoea and 49 of them produced bacteriocin active against several Escherichia coli and abacteriocinogenic Sh. flexneri strains. The extrachromosomal DNA isolated from bacteriocinogenic Sh. flexneri strains were used as a substrate to transform E. coli HB-101 cells by means of electroporation. CONCLUSIONS: Only the Sh. flexneri strains isolated from dysenteric diarrhoea produced bacteriocin. It was demonstrated that a plasmid of approx. 3 kb was responsible for the genetic determination of these anti-bacterial substances. Significance and IMPACT OF THE STUDY: A 3-kb plasmid that harboured information for the production of bacteriocin by Sh. flexneri strains was described. The production of this bacteriocin may be related to dysenteric diarrhoea produced by these bacterial strains.     

Functional macrophage activity in infection with virulent and avirulent strains of the dysentery microbe

The effect of S. flexneri virulent and avirulent (vaccine) strains 2a on the cytoplasmic membrane of mouse macrophages has been studied by evaluating the action of these bacteria on the activity of 5-nucleotidase. The dynamics of the activity of 5-nucleotidase after the introduction of both virulent and avirulent strains has a phasic character with alternating rises and falls in the activity of this enzyme in comparison with the control. S. flexneri vaccine strain produces mainly a stimulating effect on the functional activity of peritoneal macrophages in mice, which is confirmed by a decrease in the activity of 5-nucleotidase; on the contrary, S. flexneri virulent strain- has mainly an inhibiting effect on the functional activity of peritoneal macrophages, which is confirmed by an increase in the activity of 5-nucleotidase in these cells. The comparative study of changes in the activity of 5-nucleotidase, following the introduction of S. flexneri, in mice, previously immunized with smallpox vaccine, and in intact mice has shown that the use of animals immunized with smallpox vaccine in the study of metabolic characteristics may lead to distortions in the results of the experiment.     

Intraspecies antagonism of Sh. flexneri in an HEp-2 cell line model]

The authors describe an effect of suppression of invasion of the guinea pig eye conjunctiva and the HEp-2 epithelial cells by virulent Sh. flexneri bacilli, with a simultaneous administration of the same dose of avirulent shigella mutants, genetically connected with them. The data of morphological study and experiments with 3H-glucose labeled shigellae carried out on the cell species model indicated that the bacterial competition for the specific sites for absorption on the epithelial cells underlay the observed phenomenon.     

Safety and immunologic effectiveness of polyantigen from shigellae for oral immunization of monkeys]

Clinical, morphological, and immunological study was carried out in monkeys given shigellae antigen in tablets orally. Each tablet contained 0.5 mg of the antigens obtained by Boiven's method from Sh. flexneri 2a, 3, 4a, 4b, 6 and Sh. sonnei. Daily administration of the preparation for 4 days and then 4 times at the interval of 5 days proved to be harmless for monkeys. A marked immunomorphological reaction in the form of hyperplasia of the lymphoid elements of the wall of the large intestine and the lymph nodes occurred in response. There was found a resistance of the great majority of the immunized animals to the infection with the virulent Sh. flexneri 2a strain, this corresponding to the accumulation of cells secreting IgM and IgA in the wall of the large intestine.     

Nature of the epidemic process in Sonne and Flexner dysenteries and the causes of its induction

A comparative study of the epidemic process in Sh. sonnei and Sh. flexneri dysentery in different regions of the USSR revealed that the morbidity level of Sh. sonnei dysentery changed simultaneously in the regions under study at intervals of 2-3 years. Sh. flexneri dysentery showed morbidity rises occurring at intervals 6-8 years, and their occurrence did not coincide with the periods of elevated morbidity in Sh. sonnei dysentery. The data obtained in the cohort analysis and in the study of recurrent morbidity suggest that Sh. flexneri dysentery produces more pronounced postinfection immunity than Sh. sonnei dysentery, and the immunological factor probably affects the dynamics of the epidemic of these Shigella infections.     

Analysis of merodiploid strains of Sh. flexneri that have lost virulence]

Episome F'13 introduced into the genome of a virulent Sh. flexneri strain brought about changes in a number of properties of the recipient strain. The expression of these properties was not connected with the chromosome area allelic to the plasmid genome. These changes seem to be induced by the mobilization of the chromosome genes of E. coli. The loss of virulence in Sh. flexneri strains carrying episome F'13 seemed to be the consequence of two reasons: the overlapping of kcpA gene by its dominant avirulent allele and abnormal synthesis of cell wall lipopolysaccharide due to the transfer of the mobilized genes from the donor strain F'13. When the preliminary mapping of genes on the chromomome was made with the use of plasmids, it was found necessary to use F-episomes which had no influence on the changes occurring in the phenotypic characteristics of the recipient.     

The modulation of hela cells secretory patterns by invasive Shigella spp. and enteroinvasive E. coli bacterial cells and their soluble components

Considering the important role of cytokines in the initiation and evolution of the inflammatory process induced by Shigella and EIEC strains, the purpose of this study was the characterization of the secretory patterns of HeLa cells induced by Shigella ssp. and EIEC strains and to link the obtained results with the invasiveness level of bacterial strains on this cellular line. During this study there were analyzed two EIEC strains and 12 strains of the following Shigella species: 2 Sh. flexneri 2a, 2 Sh. flexneri 3a, 2 Sh. flexneri 4a, 2 Sh. boydii, 2 Sh. sonnei strains isolated in Romania during 2005 from children with dysentery and diarrhoea and confirmed for their invasive ability by Sereny test. The level of the main pro and anti-inflammatory cytokines, IL-1 beta, IL-6, IL-10, IL-13, IL-17 and TNF-alpha induced by whole bacterial cultures as well as by their soluble mediators was determined by an ELISA test. Our results showed that HeLa cells can be used not only for the qualitative and quantitative assessment of Shigella and EIEC strains invasion ability, but also as a simple work procedure for the investigation of an in vitro complex crosstalk communication mechanisms that involves physical interactions between bacterial cells and epithelial cells (adhesins and complementary receptors) and pro- and anti-inflammatory molecules regulation.The majority of the analyzed Shigella serogroups, with the exception of Shigella sonnei and EIEC strains, inhibited the inflammatory response by reducing the expression of majority of pro-inflammatory cytokines, i.e., IL-1 beta, IL-6, TNF-alpha and IL-17. The reduced cost of the in vitro procedure, the possibility of results interpretation and the strict regulations concerning the use of animals for experimental purposes are the main reasons that support the implementation of such an in vitro test in the research labs.     

宋内氏福氏2a双价志贺氏菌芳香族氨基酸营养缺陷型减毒株的研究Ⅱ:生物学特性的研究

进一步对FS54—2、—3、—4、—5、—6、—7及—7b等双价减毒株的侵袭力、毒力、免疫原性与免疫保护性及遗传稳定性等进行了研究,证明双价减毒株仍保持了入侵肠上皮细胞的能力;小动物实验安全低毒;有很好的免疫原性,对小鼠有很好的双重免疫保护性。     

Properties of the R plasmids of Sh. flexneri strains isolated in Bulgaria]

The study of 3,237 Sh. flexneri strains isolated in Bulgaria revealed that 50.72% of them were resistant to antibiotics. 95.88% of the antibiotic-resistant strains were found to have R factor of incompatibility groups F, I or N. The plasmids of incompatibility group F had the property fin+, and the plasmids of groups I and N had the property fin-. R plasmids were divided into 4 groups by their ability for phage restriction.     

Citrate method of extracting antigens from Shigella sonnei. I. Comparison with other methods

The comparative characteristic of extracts obtained from Sh. sonnei by the method of Raynaud and Digeon and neurotoxin obtained by sedimentation with trichloroacetic acid from chloroform autolysates by the method of Mesrobeanu et al. is presented. Both preparations were shown to be similar in their antigenic structure and immunogenicity for mice and guinea-pigs. Nevertheless the extracts obtained by the citrate method contained considerably less protein and endotoxin and had no enterotoxic activity. This allows to recommend the citrate method for extracting protective antigens from Sh. sonnei.     

痢疾杆菌不同群、型间交叉保护性试验研究

本文对痢疾杆菌不同群、型间的交叉保护作用进行了观察。试验模型为恒河猴,将其分为４组,第一、三组分别为感染过福氏１ａ和宋内菌并已康复的猴;第四组为用双价菌苗株ＦＳ（福氏２ａ和宋内）免疫的猴体,剂量依次为４×１０１０、６．５×１０１０、６×１０１０,共１６．５×１０１０活菌,第二组为空白对照。所有４组皆用福氏２ａ２５８００×１０８活菌攻击。从发病率来看,不同菌群与菌型间没有明显的交叉保护作用;从发病程度看则一组显著低于对照组,三组与对照组无显著差异,此结果表明痢疾杆菌Ｂ群内存在交叉保护作用,但较同型保护作用弱。     

Development of a new guinea-pig model of shigellosis

Shigellosis is a major form of bacillary dysentery caused by Shigella spp. To date, there is no suitable animal model to evaluate the protective efficacy of vaccine candidates against this pathogen. Here, we describe a successful experimental shigellosis in the guinea-pig model, which has shown the characteristic features of human shigellosis. This model yielded reproducible results without any preparatory treatment besides cecal ligation. In this study, guinea-pigs were discretely infected with virulent Shigella dysenteriae type 1 and Shigella flexneri type 2a into the cecocolic junction after ligation of the distal cecum. All the experimental animals lost ～10% of their body weight and developed typical dysentery within 24-h postinfection. In the histological analysis, distal colon showed edema, hemorrhage, exudation and inflammatory infiltrations in the lamina propria. Orally immunized animals with heat-killed S. dysenteriae type 1 and S. flexneri type 2a strains showed high levels of serum immunoglobulin G (IgG) and mucosal IgA antibodies and conferred significant homologous protective immunity against subsequent challenges with the live strains. The direct administration of shigellae into the cecocolic junction induces acute inflammation, making this animal model useful for assessing shigellosis and evaluating the protective immunity of Shigella vaccine candidates.