Role of the Salmonella abortusovis virulence plasmid in the infection of BALB/c mice

Following oral inoculation of BALB/c mice, Salmonella abortusovis strain SS44 was recovered in lower numbers from the Peyer's patches and mesenteric lymph nodes compared with S. typhimurium strain SL1344, whereas splenic infections were equivalent between the two serovars. SS44 was cured of its virulence plasmid or subjected to mutagenesis of the spv genes, and the Spv(-) derivatives were tested for virulence in mice. Plasmid-cured S. abortusovis SU40 retained virulence in BALB/c mice when inoculated intraperitoneally. On the other hand, mice infected orally with SU40 had greatly reduced splenic infection compared to those infected with wild-type SS44. Similar results were obtained after Tn5 insertion mutagenesis of the spvR gene or deletion of the spvABCD locus. These results suggest that in the gut-associated lymphoid tissues S. abortusovis may replicate less than S. typhimurium and that the S. abortusovis virulence plasmid primarily affects systemic infection after oral inoculation but not after intraperitoneal administration in the mouse model.     

Detection of Salmonella spp. in milk by using Felix-O1 bacteriophage and high-pressure liquid chromatography

A method is described whereby the presence of less than five salmonellae was detected per milliliter of milk within 24 h of sample collection. Salmonellae were removed from milk by means of electropositive large-pore filters. Eluates from the filters were analyzed for the presence of Salmonella spp. by Felix-O1 bacteriophage and high-pressure liquid chromatographic techniques. The method gave only a positive response when salmonellae were present in the milk. Of the serotypes and strains of Salmonella spp. tested, Salmonella dublin (10 strains), Salmonella typhimurium (5 strains), Salmonella anatum, Salmonella krefeld, and Salmonella saint-paul gave positive responses. One strain of Salmonella agona (three strains tested) and three strains of Salmonella enteritidis (seven strains tested) were not detectable by the method described herein.     

Detection of Salmonella spp. in milk by using Felix-O1 bacteriophage and high-pressure liquid chromatography.

A method is described whereby the presence of less than five salmonellae was detected per milliliter of milk within 24 h of sample collection. Salmonellae were removed from milk by means of electropositive large-pore filters. Eluates from the filters were analyzed for the presence of Salmonella spp. by Felix-O1 bacteriophage and high-pressure liquid chromatographic techniques. The method gave only a positive response when salmonellae were present in the milk. Of the serotypes and strains of Salmonella spp. tested, Salmonella dublin (10 strains), Salmonella typhimurium (5 strains), Salmonella anatum, Salmonella krefeld, and Salmonella saint-paul gave positive responses. One strain of Salmonella agona (three strains tested) and three strains of Salmonella enteritidis (seven strains tested) were not detectable by the method described herein.     

Hepatitis B virus nucleocapsid/pre-S2 fusion proteins expressed in attenuated Salmonella for oral vaccination

Hybrid HBV nucleocapsid-pre-S(2) fusion proteins were stably expressed in several aromatic-dependent attenuated Salmonella typhimurium and Salmonella dublin strains. When these live recombinant bacteria were administered i.p. to BALB/c mice they induced high titer anti-hepatitis B virus core Ag (HBc) and detectable anti-pre-S2 serum antibodies. Upon oral feeding of the recombinant salmonellae to mice, the rate of seroconversion to anti-HBc was dependent on the salmonella strain used. With the best carrier strain high titer anti-HBc antibodies and lower titer anti-pre-S2 serum IgG antibodies were observed two weeks after a single oral immunization. The Ig class and IgG subclass distribution of anti-HBc antibodies after i.p. and oral immunization is consistent with the induction of functional T cell help.     

减毒鼠伤寒沙门氏菌全长hpaA基因工程菌的构建

为构建表达HpaA蛋白的重组减毒鼠伤寒沙门氏菌 ,并探讨以减毒鼠伤寒沙门氏菌为载体构建H .pylori疫苗株的意义 ,应用PCR法从H .pylori基因组DNA中扩增 783bp的hpaA基因 ,经酶切 连接反应将其克隆入原核表达质粒pTrc99A的NcoⅠ SalⅠ位点 ,并进行了核苷酸序列测定。重组质粒转化减毒鼠伤寒沙门氏菌SL3 2 6 1 ,提取重组菌质粒 ,PCR和酶切鉴定 ,筛选阳性克隆。用SDS PAGE电泳和Westernblot进行HpaA表达分析和鉴定 ,用薄层扫描分析HpaA含量。重组菌C5 7BL 6小鼠喂灌 ,分批两d和 1 0d后处死小鼠 ,取脾和末段回肠进行细菌培养 ,挑菌落提质粒鉴定。结果表明 ,经PCR和酶切证实 ,构建了含 783bphpaA基因的重组原核表达质粒 ,并将后者成功转化了减毒鼠伤寒沙门氏菌。重组菌能表达约3 0kDHpaA蛋白 ,重组HpaA量约占全菌体蛋白量的 3 8 9% ,Westernblot证实其有免疫反应性。小鼠重组菌喂灌两d或 1 0d后 ,脾和末段回肠均发现携目的基因的菌落。这些结果提示 ,构建了表达H .pyloriHpaA的重组减毒…     

Cytochemical study of different stages in the life cycle of Toxoplasma gondii. I. Amylopectin and lipids in endozoites]

Attempts were made to localize cytochemically acid micopolysaccharides, in addition to sulphate groups of mucosubstances in Toxoplasma endozoites of SS-119 and RH strains with negative results. Amylopectin was found to be the only polysaccharide so far detected in the endozoite stage, using the PAS technique. With RH strain toxoplasmas, the dynamics of amylopectin was followed within 6 days after mouse inoculation. On day 2, toxoplasmas and the host cells of the mouse peritoneal exudate being practically PAS-negative, the amount of amylopectin increased progressively towards day 6. The immune response of the host may be presumably involved in the pattern of this dynamics. Phospholipids were detected in the cytoplasm of intra- and extracellular endozoites of strain SS-119. Little, if any, amount of neutral fat was observed. Of special interest is the increased phospholipid content in the infected host cells.     

A recombinant Salmonella typhimurium vaccine strain is taken up and survives within murine Peyer's patch dendritic cells

The attenuated Salmonella typhimurium PhoP^c strain is avirulent but immunogenic via the oral route in mice and is attenuated in survival in macrophage cell lines. In this study, the fate of PhoP^c bacteria expressing green fluorescent protein was investigated in murine Peyer's patches. The survival of PhoP^c was monitored after orogastric inoculation of BALB/c mice. Bacteria persisted for several weeks in the Peyer's patches and were also recovered from the mesenteric lymph nodes and spleen. Confocal microscopy analysis identified dendritic cells as the Peyer's patch cell type that internalized PhoP^c expressing green fluorescent protein at early time points. In addition, live PhoP^c were found in Peyer's patch dendritic cells and not in B cells 3 days after orogastric inoculation. Taken together, these results provide strong evidence that PhoP^c is internalized and survives within Peyer's patch dendritic cells. As these cells are potent antigen-presenting cells, these data could explain the immunogenicity of S. typhimurium vaccine strains in vivo .     

表达乙型脑炎病毒E蛋白重组减毒沙门菌活载体疫苗株的构建

目的:构建表达乙型脑炎病毒(JEV)E蛋白的口服重组减毒鼠伤寒沙门菌活载体疫苗株。方法:克隆JEV E基因,将其插入表达载体pYA3341中,构建重组质粒pYA3341-E,将重组质粒电转入鼠伤寒沙门菌疫苗株X4550(缺失asd、cya、crp基因),获得重组疫苗菌株X4550(pYA3341-E);鉴定重组菌E蛋白的表达,测定重组菌的稳定性、生长曲线、安全性,以及小鼠的免疫试验和血清中和试验。结果:酶切鉴定和序列测定证实重组质粒构建成功;SDS-PAGE检测有目的蛋白条带;Western印迹证实表达的E蛋白能与猪抗JEV阳性血清特异性结合;重组菌株在体外营养选择压力下,可稳定地携带重组质粒传代繁殖,在体内可较稳定地定居于肠系膜淋巴结和脾脏;小鼠口服试验证实重组菌无毒性作用,安全可靠;小鼠口服重组菌免疫,ELISA检测产生了抗JEV抗体;中和试验表明产生的抗体具有中和活性。结论:构建了能稳定表达JEV E蛋白的口服减毒鼠伤寒沙门菌疫苗株X4550(pYA3341-E),为研究乙型脑炎口服基因工程疫苗奠定基础。     

减毒鼠伤寒沙门菌体内定位分析…

分析减毒鼠伤寒沙门菌口服感染后在小鼠体内定位的情况.将构建的红色荧光蛋白(RFP)原核质粒pYA33-DsRed,以电穿孔法转化减毒鼠伤寒沙门菌X4550,重组菌命名为X4550(33-DsRed).重组菌分别感染巨噬细胞RAW264.7和骨髓源树突状细胞(BMDC),并用流式细胞术检测红色荧光细胞荧光强度.此外,以不同剂量重组菌口服免疫BALB/c小鼠,并于免疫后1d、2d、3d、5d、7d取小鼠脾、肝、肠系膜淋巴结(MLN)、派伊尔氏结(PP)、腹股沟淋巴结(ILN)细胞,检测各组织器官中的红色荧光阳性细胞百分率.重组菌对RAW264.7细胞和BMDC均具有良好的侵袭力.口服小鼠后,第1d,仅在MLN及PP中检测到RFP阳性细胞,其中PP中阳性细胞达到1.4%;第2 d,在ILN中达到0.4%;第3 d,各个组织器官中RFP阳性细胞均有上升趋势,此时在脾、肝中也检测到RFP阳性细胞.第5 d,RFP阳性细胞均减少,第7 d则未检测到任何RFP阳性细胞.减毒鼠伤寒沙门菌具有良好的侵袭力,其黏膜移行方式以及对免疫组织器官靶向定位性,在优化黏膜疫苗以及提高疫苗免疫效力等方面都具有重要作用.     

Role of type 1 and type 3 fimbriae on the adherence and pathogenesis of Salmonella enteritidis in mice.

Through hemagglutination tests two isogenic strains of Salmonella enteritidis were shown to possess type 1 fimbriae (strain V) and type 1 and type 3 fimbriae (strain A). The two strains bound to human buccal and mouse small intestine epithelial cells. Strain A attached to the epithelial cells more readily and in larger numbers in comparison to strain V. Adherence of both strains were sensitive to the presence of D-mannose and pretreatment of the epithelial cells with tannic acid did not promote D-mannose resistant type binding of strain A S. enteritidis to human buccal and mouse small intestine epithelial cells. Furthermore, results from LD50 study indicated that, when the tests were carried out through oral inoculation of the mice the highly fimbriated stain A appeared to be more virulent. However, when the tests were carried out through intraperitoneal inoculation strain V was more virulent. These results indicate that adherence is a major contributing factor to the virulence of S. enteritidis and both type 1 and type 3 fimbriae contribute to this phenomenon.     

Genetic control of the innate resistance of mice to Salmonella typhimurium: expression of the Ity gene in peritoneal and splenic macrophages isolated in vitro

The mouse Chromosome 1 locus Ity regulates the extent to which Salmonella typhimurium replicates within the reticuloendothelial cell system (RES) during the first days of infection. If animals are homozygous for the Itys susceptibility allele, the Gram-negative bacterium undergoes rapid net multiplication, and mice die of a typhoid fever-like disease by day 10 of infection. Animals that are homozygous or heterozygous for the resistance allele, Ityr, control net bacterial replication and survive the first phase of salmonellosis. Indirect studies have implicated the resident macrophage as the effector cell for regulation of early in vivo salmonellae growth. To verify this supposition and to evaluate the phenotypic expression of Ity, we developed an in vitro assay to compare kinetics of S. typhimurium growth within Ityr and Itys macrophages. Resident peritoneal and splenic macrophages were used from inbred Ityr and Itys mice and from Ity congeneic mice. With these mice and through the use of radiolabeled S. typhimurium and an avirulent temperature-sensitive mutant of the bacterium, we found that: phagocytosis of S. typhimurium by Ityr and by Itys macrophages was the same; S. typhimurium grew to a greater extent in Itys peritoneal and splenic macrophages than in Ityr cells; Ityr macrophages killed intracellular salmonellae more efficiently than did Itys macrophages. Thus, we have demonstrated directly that Ity is expressed by the macrophage and have shown for the first time with Ity congeneic mice that the basis for differential net growth of virulent S. typhimurium in Ityr and Itys macrophages is a variation in the degree of bacterial kill.     

Bacteriophage P22 H5 transfection and infection of plasmid Salmonella strains

The results of the Ca2+-dependent transfection of the DNA of bacteriophage P22 H5 to constructed Salmonella typhimurium F'- and R+-strains LT2 WT-R and SA118 demonstrated that in these salmonellae the effectiveness of transfection depended on the specificity of the interrelation of plasmids with host strains. Plasmids RA1, R538-1 and RP1 stimulated the transfection of S. typhimurium strain LT2 WT-R, but suppressed the transfection ability of S. typhimurium strain SA118. At the same time the expression of the function of plasmids R446b and R64-11 did not depend on the host strain, as the former did not affect and the latter suppressed the release of transfectants in both Salmonella strains. The presence of plasmids R124, RA1, R64-11 and R724 in strain SA118, heat-sensitive in respect to the synthesis of cell-wall lipopolysaccharide, not only led to a decrease in the effectiveness of transfection; the effectiveness of the inoculation of bacteriophage P22 H5 was also suppressed 10(4) times in the presence of plasmid R124 and at least 10(10) times in the presence of 3 other plasmids. The development of resistance to S-specific bacteriophage P22 H5 was not linked with disturbances in the adsorption of this bacteriophage. Besides, the addition of CaCl2 into the medium completely removed the limitation of infection with bacteriophage P22 H5, determined by plasmid R124.     

Comparative study of the effectiveness of HBcAg presentation to the immune system using attenuated Salmonella strains, serovars S.enteritidis and S.typhimurium

In this work the results of obtaining HBcAg-producing attenuated Salmonella strains, serovars S. enteritidis and S. typhimurium, and their comparative study is presented. As revealed in this study, attenuated S. enteritidis strain E-23 and S. typhimurium strain T-10, producing HBcAg, induce cell-mediated and humoral immune response to HBcAg after injected into anovals. After injection S. typhimurium strain T-10 induces a much higher titer of specific antibodies than S. enteritidis strain E-23. The level of specific antibodies induced by recombinant HBcAg seems to correlate with the capacity of salmonellae for survival inside macroorganisms.     

Effects of Food Intake on Numbers of Salmonellae and Escherichia coli in Rumen and Faeces of Sheep

When 10⁷–10⁸ Salmonella anatum or Salm. typhimurium were inoculated into the rumen of sheep consuming 1·3 kg of lucerne chaff daily, salmonellae were eliminated from the rumen in 2 days, and could not be detected in the faeces after c. 1 week. During starvation, both Escherichia coli and salmonellae grew in the rumen. Resumption of feeding after starvation for 3 days caused further multiplication of E. coli and salmonellae in the rumen. The organisms were subsequently eliminated with further feeding. Inoculation with as few as 400 salmonellae cells into a starved sheep led to large numbers of salmonellae appearing in the faeces and being excreted in varying numbers for at least 5 weeks after resumption of feeding.     

Host cell responses of Salmonella typhimurium infected human dendritic cells

Live attenuated Salmonella are attractive vaccine candidates for mucosal application because they induce both mucosal immune responses and systematic immune responses. After breaking the epithelium barrier, Salmonella typhimurium is found within dendritic cells (DC) in the Peyer's patches. Although there are abundant data on the interaction of S. typhimurium with murine epithelial cells, macrophages and DC, little is known about its interaction with human DC. Live attenuated S. typhimurium have recently been shown to efficiently infect human DC in vitro and induce production of cytokines. In this study, we have analysed the morphological consequences of infection of human DC by the attenuated S. typhimurium mutant strains designated PhoPc, AroA and SipB and the wild-type strains of the American Type Culture Collection (Manassas, VA, USA), ATCC 14028 and ATCC C53, by electron microscopy at 30 min, 3 h and 24 h after exposure. Our results show that genetic background of the strains profoundly influence DC morphology following infection. The changes included (i) membrane ruffling; (ii) formation of tight or spacious phagosomes; (iii) apoptosis; and (iv) spherical, pedunculated membrane-bound microvesicles that project from the plasma membrane. Despite the fact that membrane ruffling was much more pronounced with the two virulent strains, all mutants were taken up by the DC. The microvesicles were induced by all the attenuated strains, including SipB, which did not induce apoptosis in the host cell. These results suggest that Salmonella is internalized by human DC, inducing morphological changes in the DC that could explain immunogenicity of the attenuated strains.     

Genetic control of the innate resistance of mice to Salmonella typhimurium: Ity gene is expressed in vivo by 24 hours after infection

The early response of inbred mice to infection with S. typhimurium is controlled by the mouse Chromosome 1 locus, Ity. To better understand the expression of this gene, the initial interactions between the reticuloendothelial system (RES) and i.v. injected salmonellae were compared in resistant (Ityr) and susceptible (Itys) mice. In both mouse strains 99% of the bacteria was cleared from the blood within 2 hr, and uptake of S. typhimurium by splenic and hepatic macrophages was similar regardless of Ity genotype. In vivo phagocytosis of bacteria was followed by a 30 to 60% decline in viable bacteria, which was attributed to the bactericidal activity of RES macrophages. Experiments with radiolabeled S. typhimurium strains TML and TML/TS27 (a temperature-sensitive mutant) confirmed that the efficiency of this early phase killing was not under Ity control. Despite the equivalent uptake and initial bactericidal activity by resident macrophages, bacterial numbers in the RES organs of Itys mice were significantly greater than in Ityr mice by approximately 24 hr after infection. These data suggest that Ity regulates the level of surviving intracellular bacteria that accumulate within resident macrophages of the liver and spleen.     

An immunofluorescence analysis of the ontogeny of myeloid, T, and B lineage cells in mouse hemopoietic tissues

The population dynamics of granulopoietic cells, B-lineage cells, and T lymphocytes were analyzed by immunofluorescence in mouse hemopoietic tissues as a function of age. Mac-1+ myeloid cells were present on day 11 of gestation in the liver, where they peaked shortly after birth and declined subsequently. Waves of myeloid population growth began in spleen and bone marrow by days 15 and 19, respectively. Mac-1+ cells increased in number to relatively low plateau levels in spleen by the 3rd wk after birth, whereas in the bone marrow higher plateau levels were reached around 3 mo of age. The 14.8 monoclonal antibody was utilized as one marker of B-lineage precursor cells. 14.8+ cells were detected in the liver on day 11 of gestation, reached peak numbers during the first week after birth and decreased thereafter. On day 15 and 19, 14.8+ cells were found in spleen and bone marrow, respectively, and progressively increased in numbers to reach plateau levels in both sites by 3 mo of age. Mu+ pre-B cells appeared in significant numbers in the 13-day fetal liver, reached a peak shortly after birth, and disappeared from the liver by the end of the second postnatal week. Pre-B cells were found in the spleen and bone marrow on days 15 and 19, respectively. In the spleen pre-B cells reached peak values at birth and disappeared 2 wk later. In spite of the sequential appearance of mu+ pre-B cells in fetal liver, spleen, and bone marrow, their sIgM+ B cell progeny appeared in all these hemopoietic tissues on day 17 of gestation. In the liver, sIgM+ B cells reached their peak at birth and declined thereafter. In the spleen and bone marrow, B cells increased to plateau levels between 1 and 4 mo of age. Thy-1.2+ T cells were relatively late acquisitions in all three hemopoietic tissues. Finally, the expression of the 14.8 antigen by mu+ cells was examined as a function of gestational age. While pre-B cells from day-13 fetuses had no detectable 14.8 antigen, the antigen was weakly expressed on the vast majority of the mu+ pre-B cells by day 17 of gestation. Newborn liver cells expressing 14.8 antigen were found to include a small proportion of cells with peroxidase+ granules. Thus, demonstration of rearrangement and expression of immunoglobulin genes may be required for precise identification of cells of B lineage early in ontogeny.     

Protection from virulent Salmonella groups B and D after the peroral immunization of chicks with a hybrid of Salmonella typhimurium and 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.