宋内氏痢疾菌无毒株S7表达Vi抗原工程菌的构建

将含有编码Ｖｉ抗原ＶｉａＢ基因片断的质粒转导进入宋内氏痢疾菌无毒株Ｓ７中,组建了重组菌株Ｓ７Ｖｉ。质粒电泳图谱显示重组菌株Ｓ７Ｖｉ中存在被转入的外源质粒带。重组株的生化特性没有改变。菌体凝集及Ｖｉ抗血清标记的ＳＰＡ菌液凝集反应证明在重组株的菌体表面,同时表达了Ｖｉ抗原和宋内氏毒菌的Ｏ抗原。以５×１０~８ＣＦＵ、１０×１０~８ＣＦＵ的重组株免疫近交系的ＬＩＢＰ小鼠,免疫小鼠对宋内氏毒株Ｓ６３攻击的保护率为６０％至９０％,对伤寒毒株Ｔｙ２攻击的保护率为２５％至４０％。     

Effects of buprenorphine on immunogenicity and protective efficacy in the guinea pig keratoconjunctivitis model (Sereny test)

Shigellosis is a disease of global proportions, with an estimated 164.7 million episodes annually throughout the world as well as an estimated 1.1 million associated mortalities in developing countries. Due to increasing incidence, and continued emergence of multi-drug resistant strains, Shigella vaccine development is considered a top public health priority. The guinea pig keratoconjunctivitis model, the basis for the Sereny test, remains the most reliable in vivo indicator of virulence of Shigella strains and immunogenicity and protective efficacy of Shigella vaccine candidates. The model is effective in evaluating the ability of Shigella strains to invade the corneal epithelia of guinea pigs and spread to contiguous cells, with the more virulent strains causing ulcerative keratoconjunctivitis. However, analgesia is not routinely used to relieve this painful condition because of potential immunomodulation and confounding of experimental results. The objective of the study reported here was to evaluate use of buprenorphine hydrochloride as an analgesic during the Sereny test. Local and systemic immune responses were measured in guinea pigs given buprenorphine versus those responses in controls. Results of this study suggest that buprenorphine, administered at an analgesic dose of 0.05 mg/kg of body weight twice daily, can be successfully used with the model without significantly affecting immunologic evaluation of Shigella vaccine candidates. However, in buprenorphine-treated animals, there was a significant increase in the amount of mucopurulent ocular discharge, requiring frequent cleaning of the affected eyes. Additionally, animals treated with buprenorphine had significant reduction in body weight, in comparison with saline controls.     

The pathogenesis of Shigella flexneri infection: lessons from in vitro and in vivo studies

Shigella flexneri is a Gram-negative facultatively intracellular pathogen responsible for bacillary dysentery in humans. More than one million deaths occur yearly due to infections with Shigella spp. and the victims are mostly children of the developing world. The pathogenesis of Shigella centres on the ability of this organism to invade the colonic epithelium where it induces severe mucosal inflammation. Much information that we have gained concerning the pathogenesis of Shigella has been derived from the study of in vitro models of infection. Using these techniques, a number of the molecular mechanisms by which Shigella invades epithelial cells and macrophages have been identified. In vivo models of shigellosis have been hampered since humans are the only natural hosts of Shigella. However, experimental infection of macaques as well as the murine lung and rabbit ligated ileal loop models have been important in defining some of the immune and inflammatory components of the disease. In particular, the murine lung model has shed light on the development of systemic and local immune protection against Shigella infection. It would be naive to believe that any one model of Shigella infection could adequately represent the complexity of the disease in humans, and more sophisticated in vivo models are now necessary. These models require the use of human cells and tissue, but at present such models remain in the developmental stage. Ultimately, however, it is with such studies that novel treatments and vaccine candidates for the treatment and prevention of shigellosis will be designed.     

Shigellosis

Shigellosis is a global human health problem. Four species of Shigella i.e. S. dysenteriae, S. flexneri, S. boydii and S. sonnei are able to cause the disease. These species are subdivided into serotypes on the basis of O-specific polysaccharide of the LPS. Shigella dysenteriae type 1 produces severe disease and may be associated with life-threatening complications. The symptoms of shigellosis include diarrhoea and/or dysentery with frequent mucoid bloody stools, abdominal cramps and tenesmus. Shigella spp. cause dysentery by invading the colonic mucosa. Shigella bacteria multiply within colonic epithelial cells, cause cell death and spread laterally to infect and kill adjacent epithelial cells, causing mucosal ulceration, inflammation and bleeding. Transmission usually occurs via contaminated food and water or through person-to-person contact. Laboratory diagnosis is made by culturing the stool samples using selective/differential agar media. Shigella spp. are highly fragile organism and considerable care must be exercised in collecting faecal specimens, transporting them to the laboratories and in using appropriate media for isolation. Antimicrobial agents are the mainstay of therapy of all cases of shigellosis. Due to the global emergence of drug resistance, the choice of antimicrobial agents for treating shigellosis is limited. Although single dose of norfloxacin and ciprofloxacin has been shown to be effective, they are currently less effective against S. dysenteriae type 1 infection. Newer quinolones, cephalosporin derivatives, and azithromycin are the drug of choice. However, fluoroquinolone-resistant S. dysenteriae type 1 infection have been reported. Currently, no vaccines against Shigella infection exist. Both live and subunit parenteral vaccine candidates are under development. Because immunity to Shigella is serotype-specific, the priority is to develop vaccine against S. dysenteriae type 1 and S. flexneri type 2a. Shigella species are important pathogens responsible for diarrhoeal diseases and dysentery occurring all over the world. The morbidity and mortality due to shigellosis are especially high among children in developing countries. A recent review of literature (Kotloff et al.,1999) concluded that, of the estimated 165 million cases of Shigella diarrhoea that occur annually, 99% occur in developing countries, and in developing countries 69% of episodes occur in children under five years of age. Moreover, of the ca.1.1 million deaths attributed to Shigella infections in developing countries, 60% of deaths occur in the under-five age group. Travellers from developed to developing regions and soldiers serving under field conditions are also at an increased risk to develop shigellosis.     

Evolutionary genetics of a new pathogenic Escherichia species: Escherichia albertii and related Shigella boydii strains

A bacterium originally described as Hafnia alvei induces diarrhea in rabbits and causes epithelial damage similar to the attachment and effacement associated with enteropathogenic Escherichia coli. Subsequent studies identified similar H. alvei-like strains that are positive for an intimin gene (eae) probe and, based on DNA relatedness, are classified as a distinct Escherichia species, Escherichia albertii. We determined sequences for multiple housekeeping genes in five E. albertii strains and compared these sequences to those of strains representing the major groups of pathogenic E. coli and Shigella. A comparison of 2,484 codon positions in 14 genes revealed that E. albertii strains differ, on average, at approximately 7.4% of the nucleotide sites from pathogenic E. coli strains and at 15.7% from Salmonella enterica serotype Typhimurium. Interestingly, E. albertii strains were found to be closely related to strains of Shigella boydii serotype 13 (Shigella B13), a distant relative of E. coli representing a divergent lineage in the genus Escherichia. Analysis of homologues of intimin (eae) revealed that the central conserved domains are similar in E. albertii and Shigella B13 and distinct from those of eae variants found in pathogenic E. coli. Sequence analysis of the cytolethal distending toxin gene cluster (cdt) also disclosed three allelic groups corresponding to E. albertii, Shigella B13, and a nontypeable isolate serologically related to S. boydii serotype 7. Based on the synonymous substitution rate, the E. albertii-Shigella B13 lineage is estimated to have split from an E. coli-like ancestor approximately 28 million years ago and formed a distinct evolutionary branch of enteric pathogens that has radiated into groups with distinct virulence properties.     

Sex and virulence in Escherichia coli: an evolutionary perspective

Pathogenic Escherichia coli cause over 160 million cases of dysentery and one million deaths per year, whereas non-pathogenic E. coli constitute part of the normal intestinal flora of healthy mammals and birds. The evolutionary pathways underlying this dichotomy in bacterial lifestyle were investigated by multilocus sequence typing of a global collection of isolates. Specific pathogen types [enterohaemorrhagic E. coli, enteropathogenic E. coli, enteroinvasive E. coli, K1 and Shigella] have arisen independently and repeatedly in several lineages, whereas other lineages contain only few pathogens. Rates of evolution have accelerated in pathogenic lineages, culminating in highly virulent organisms whose genomic contents are altered frequently by increased rates of homologous recombination; thus, the evolution of virulence is linked to bacterial sex. This long-term pattern of evolution was observed in genes distributed throughout the genome, and thereby is the likely result of episodic selection for strains that can escape the host immune response.     

Shigella flexneri infection: pathogenesis and vaccine development

Shigella flexneri is a gram-negative bacterium which causes the most communicable of bacterial dysenteries, shigellosis. Shigellosis causes 1.1 million deaths and over 164 million cases each year, with the majority of cases occurring in the children of developing nations. The pathogenesis of S. flexneri is based on the bacteria's ability to invade and replicate within the colonic epithelium, which results in severe inflammation and epithelial destruction. The molecular mechanisms used by S. flexneri to cross the epithelial barrier, evade the host's immune response and enter epithelial cells have been studied extensively in both in vitro and in vivo models. Consequently, numerous virulence factors essential to bacterial invasion, intercellular spread and the induction of inflammation have been identified in S. flexneri. The inflammation produced by the host has been implicated in both the destruction of the colonic epithelium and in controlling and containing the Shigella infection. The host's humoral response to S. flexneri also appears to be important in protecting the host, whilst the role of the cellular immune response remains unclear. The host's immune response to shigellosis is serotype-specific and protective against reinfection by the same serotype, making vaccination a possibility. Since the 1940s vaccines for S. flexneri have been developed with little success, however, the growing understanding of S. flexneri's pathogenesis and the host's immune response is assisting in the generation of more refined vaccine strategies. Current research encompasses a variety of vaccine types, which despite disparity in their efficacy and safety in humans represent promising progress in S. flexneri vaccine development.     

Dynamics of the change in sensitivity of shigellae to gentamycin and cephaloridine in vitro]

The dynamics of the changes in the Shigella sensitivity to gentamicin and cephaloridin was studied in vitro using liquid nutrient media with gradually increasing concentrations of the drugs. 50 passages were performed. It was found that Shigella flexner and sonnei decreased their sensitivity to gentamicin to a little extent and remained middle sensitive. Sensitivity of Shigella flexner to cephaloridin also changed to a little extent, while Shigella sonnei became moderately resistant.     

核桃楸树皮提取物对志贺氏菌抑菌效果研究

目的：观察核桃楸树皮提取物对志贺氏茵的抑菌效果。方法：采用两倍稀释法和纸片琼脂扩散法考察桃楸树皮提取物对志贺氏茵的抑茵作用。结果：核桃楸树皮提取物对痢疾志贺氏Ⅰ型茵、痢疾志贺氏Ⅱ型菌、鲍氏志贺Ⅰ型茵、宋内氏志贺茵均为0．0313g／mL,福氏志贺Ⅱ型菌为O．0625g／mL。结论：核桃楸树皮提取物对志贺氏茵均有良好的抑菌作用,各菌对药物的敏感强弱顺序为：宋内氏志贺菌〉鲍氏志贺Ⅰ型菌〉痢疾志贺Ⅰ型菌〉痢疾志贺菌Ⅱ型菌〉福氏志贺Ⅱ型茵。     

Specific antigens of the causative agents and their antibodies in the circulating immune complexes in acute dysentery

The level of circulating immune complexes has been determined in 53 patients in the dynamics of the disease. For the first time circulating immune complexes have been found to contain Shigella sonnei K-antigen and Shigella flexneri O-antigen, as well as IgA, IgG and IgM to Shigella. Shigella antigens can be detected from the first week of the disease, and their occurrence does not depend on the level of circulating complexes in patients blood serum.     

食蟹猴肠道志贺氏菌感染情况的调查

本文对 337只食蟹猴肠道志贺氏菌进行调查 ,其感染率为 1 1 %。对所分离到的 2 7株志贺氏菌进行生化、血清学鉴定 ,分属三个群 ,八种血清型 ,痢疾志贺氏菌、福氏志贺氏菌、宋内氏志贺氏菌所占比例分别为 1 0 8%、86 5%、2 7% ,以福氏志贺氏 2a型居多 ,占 59 5%。对八种志贺氏菌血清型菌株进行药敏试验 ,结果表明 ,不同血清型菌株对同一种抗菌药物的敏感性、耐药性均有差异。提示不同血清型的菌株均可自然感染食蟹猴 ,在治疗时 ,对感染不同血清型志贺氏菌的食蟹猴区别用药 ,以提高治愈率 ,避免因用药不当所造成的经济损失。     

Shigella interactions with the actin cytoskeleton in the absence of Ena/VASP family proteins

Shigella move through the cytosol of infected cells by assembly of a propulsive actin tail at one end of the bacterium. Vasodilator-stimulated phosphoprotein (VASP), a member of the Ena/VASP family of proteins, is important in cellular actin dynamics and is present on intracellular Shigella. VASP binds both profilin, an actin monomer-binding protein, and vinculin, a component of intercellular contacts that also binds the Shigella actin assembly protein IcsA. It has been postulated that VASP might serve as a linker between vinculin and profilin on intracellular Shigella, thereby delivering profilin to the Shigella actin assembly machinery. We show that Shigella actin-based motility is unaltered in cells that are deficient for the Ena/VASP family of proteins. In these cells, Shigella form normal-appearing actin tails and move at rates that are comparable to the rates of bacterial movement in Ena/VASP-deficient cells complemented with the Ena/VASP family member Mena. Finally, whereas vinculin can bind the Arp2/3 complex, we show that Arp2/3 recruitment to Shigella is not correlated with vinculin recruitment, indicating that the role of vinculin in Shigella motility is not recruitment of Arp2/3. Thus, although VASP is recruited to the surface of intracellular Shigella, it is not essential for Shigella actin-based motility.     

Further Evaluation of Immunofluorescence Techniques for Detection of Shigellae in Fecal Specimens

The fluorescent antibody (FA) tests for group B and group D Shigella were reevaluated. Duplicate swab specimens from patients suspected of having shigellosis were cultured shortly after collection and after transport in a soft-agar holding medium. Smears for FA examination were made at the same time. Results obtained for the group D Shigella agree closely with those obtained in previous studies. The percentage of isolations of group B Shigella from transported specimens which were positive by FA was 59.6% as compared to 39.3 and 53.3% in previous studies. S. flexneri was isolated from 76.7% of the FA-positive specimens when they were examined shortly after collection. More isolates of Shigella were obtained when specimens were examined by both methods than when either method was used alone. The results indicated that FA tests for both group B and group D Shigella are practical and may be useful to laboratories engaged in Shigella isolation.     

Shigella toxicity in immunological tolerance

The toxic effect of killed and live Shigella sonnei cultures on normal mice and on mice, tolerant to Shigella O-antigen and to human erythrocytes of different blood groups (in the ABO system) was under study. The toxicity of shigellae, introduced intraperitoneally, has been found to depend on their viability, on their capacity for penetration into the blood, and on the split character of immunological tolerance to Shigella antigens.     

Cathepsin D activity in splenocytes in relation to Shigella virulence in an experiment

The overtime study of changes in the activity of cathepsin D, a lysosomal enzyme, in the splenocytes of CBA mice after their infection with virulent and avirulent Shigella strains of the same origin and with the same antigenic structure has been made. As the result of two months of observations, changes in the activity of this enzyme in the cytoplasmic and lysosomal cell fractions have been found to occur in phases. The activity of cathepsin D has been shown to depend on the virulence of Shigella strains used for inoculation. Virulent Shigella strains induce the pronounced and prolonged activation of the enzyme in the lysosomes, as well as in the cytoplasm. The latter phenomenon is probably indicative of the pathological labilization of the lysosomal membranes, induced by the virulent culture. Avirulent Shigella strains induce only the transient activity of the enzyme in the lysosomes without any essential changes in the permeability of their membranes. These data point to the possibility of differentiating virulent and avirulent Shigella strains by the determination of the enzymatic activity of splenocytes in infected animals.     

Calpain activation by the Shigella flexneri effector VirA regulates key steps in the formation and life of the bacterium's epithelial niche

The enteropathogen Shigella flexneri invades epithelial cells, leading to inflammation and tissue destruction. We report that Shigella infection of epithelial cells induces an early genotoxic stress, but the resulting p53 response and cell death are impaired due to the bacterium's ability to promote p53 degradation, mainly through calpain protease activation. Calpain activation is promoted by the Shigella virulence effector VirA and dependent on calcium flux and the depletion of the endogenous calpain inhibitor calpastatin. Further, although VirA-induced calpain activity is critical for regulating cytoskeletal events driving bacterial uptake, calpain activation ultimately leads to necrotic cell death, thereby restricting Shigella intracellular growth. Therefore, calpains work at multiple steps in regulating Shigella pathogenesis by disrupting the p53-dependent DNA repair response early during infection and regulating both formation and ultimate death of the Shigella epithelial replicative niche.     

Development of drug resistance and the cross resistance in Shigella to certain chemotherapeutic preparations in vitro]

A total of 700 Shigella cultures isolated in different regions of the Ukraine in 1974 and occasionally in 1971-1973 were studied. No cultures resistant to furazolidone or enteroseptol were found. Under the experimental conditions resistance to nitrofuran and especially oxycholine preparations in Shigella developed slowly when the cultures were passaged in broths with increasing concentrations of the drugs. Cross resistance in Shigella was observed with respect to (a) various nitrofuran preparations, less pronounced with respect to furacryline and (b) enteroseptol and 5-NOK. No cross resistance was noted between the above groups of the chemotherapeutics and levomycetin and tetracycline. 5-NOK inhibited the resistance development to streptomycin, while enteroseptol inhibited the development of resistance to furazolidone in Shigella. The studies showed that in the near future there will be observed no mass development of resistance to furazolidone and enteroseptol in Shigella.     

Enteric Pathogens in Monkeys

From 1964 to 1967, 6,646 monkeys, representing 10 primate species, were examined for Shigella and Salmonella infections upon arrival at the National Center for Primate Biology. Of these animals, 12% were infected with Shigella, and 75% of the Shigella isolates were S. flexneri 4. The incidence of Salmonella infections decreased from 12 to 3% during the period of study. Epidemiological studies of animals in the colony for 90 days or more indicated no seasonal variation in the occurrence of Shigella and Salmonella. Many of the isolates from incoming monkeys as well as from laboratory-conditioned animals were resistant to chloramphenicol, dihydrostreptomycin, and tetracycline. The possible operation of drug-resistance factors in these infections is discussed.     

Antibiotic resistance of Shigella in different regions of the USSR]

Shigella were most sensitive to polymyxin ceporin, ampicillin, neomycin and furazolidone and resistant to chloramphenicol, tetracycline and streptomycin. Shigella resistant simultaneously to two or three drugs mainly to tetracycline + chloramphenicol, tetracycline + streptomycin and tetracycline + chloramphenicol + streptomycin were most frequent. The frequency of the Shigella strains carrying R-plasmids increased from 28 per cent in 1969--1970 to 72.6 per cent in 1977. The Shigella strains isolated during the dysentery outbreak in 1973--1977 carried the R-factor controlling resistance to tetracycline + chloramphenicol, tetracycline + chloramphenicol + streptomycin, tetracycline + chloramphenicol + streptomycin + neomycin. Interaction between separate biochemical types, colicinogenicity and drug resistance classes was found in the Shigella isolates. The data on the effect of antibiotic (tetracyclines) intensive use in stock-raising defining wide spread of the R-plasmids controlling resistance to these drugs were obtained.     

Cdc42 facilitates invasion but not the actin-based motility of Shigella

The enteric pathogen Shigella utilizes host-encoded proteins to invade the gastrointestinal tract. Efficient invasion of host cells requires the stimulation of Rho-family GTPases and cytoskeletal alterations by Shigella-encoded IpaC. Following invasion and lysis of the phagosome, Shigella exploits the host's actin-based polymerization machinery to assemble an actin tail that serves as the propulsive force required for spreading within and between cells. The Shigella surface protein IcsA stimulates actin-tail formation by recruiting host-encoded N-WASP to drive Arp2/3-mediated actin assembly. N-WASP is absolutely required for Shigella motility, but not for Shigella invasion. Although Rho-family GTPases have been implicated in both the invasion and motility of Shigella, the role of Cdc42, an N-WASP activator, in this process has been controversial. In these studies, we have examined the role of Cdc42 in Shigella invasion and actin-based motility using Cdc42-deficient cells. We demonstrate that Cdc42 is required for efficient Shigella invasion but reveal a minor Cdc42-independent pathway that can permit Shigella invasion. However, the actin-based motility of Shigella, as well as vaccinia, proceeds unperturbed in the absence of Cdc42. These data further support the involvement of distinct host-encoded proteins in the steps regulating invasion and intercellular spread of Shigella.