Characterisation and in vivo behaviour of a Salmonella typhimurium aroA strain expressing Escherichia coli K1 polysaccharide

Abstract Plasmid pKT274 encoding a determinant for the Escherichia coli K1 polysaccharide was introduced into the Salmonella typhimurium aro A vaccine strain SL3261 and cells harbouring the plasmid were shown to express K1 polysaccharide at their cell surface. SL3261 (pKT274) could be detected in the livers and spleens of BALB/c mice infected by the intravenous route and viable organisms persisted for several weeks. SL3261 (pKT274) was cleared from the livers more rapidly and from the spleens more slowly than SL3261. Unlike mice infected with SL3261 those infected with SL3261 (pKT274) did not exhibit gross splenomegaly during the first three weeks after infection. Mice vaccinated with viable SL3261 (pKT274) were protected against challenge with virulent S. typhimurium but failed to produce detectable levels of humoral anti-K1 polysaccharide antibodies.     

霍乱毒素B亚单位基因在鼠伤寒沙门氏菌中的表达

本工作成功地将霍乱毒素B亚单位(ctx B)基因插入到带有天门冬氨酸β-半醛脱氢酶基困(asd+)的pYAZ48质粒中,并将它转化至天门冬氨酸β-半醛脱氢酶突变(asd-)鼠伤寒沙门氏菌中。实验结果表明,ctx B亚单位基因能在鼠伤寒沙门氏菌中高效表达,并且表达的蛋白能分泌到细胞外。动物实验结果也表明：该疫苗菌株能在肠粘膜细胞定居;口服及全身免疫均能产生较高的抗体,并能增强动物细胞的免疫功能;对伤寒、霍乱有毒株的攻击有良好的保护效果。该系统的应用为疫苗基因工程提供了一个新的途径。     

Efficacy of a DNA vaccine delivered in attenuated Salmonella typhimurium against Eimeria tenella infection in chickens

The efficacy of an oral DNA vaccine carrying the Eimeria tenella 5401 antigen gene delivered by attenuated Salmonella typhimurium was examined in an experimental challenge study. The DNA vaccine preparation was made by transforming the recombinant plasmid pcDNA3-5401 into the attenuated S. typhimurium strain (Dam(-) and PhoP(-)) (designated hereafter as ZJ111/pcDNA3-5401). The chickens were randomly divided into six groups, 50 per group. Group A were given PBS as control. Chickens in group B were fed with 10(8) colony forming units (CFU) of attenuated S. typhimurium carrying pcDNA3. Group C were immunised with 100 microg of the recombinant 5401 protein via intramuscular injection. Groups D to F orally received ZJ111/pcDNA3-5401 at doses of 10(7), 10(8) and 10(9)CFU per chicken, respectively. All immunisations were boosted 2 weeks later. The immunised chickens were challenged with 6x10(4) homologous sporulated oocysts 14 days after the second immunisation. No significant differences in body weight were detected between the groups before immunisation and at week 4 after the booster immunisation. The ZJ111/pcDNA3-5401 was eventually eliminated from the spleen and liver on week 6 post-immunisation. The plasmid pcDNA3-5401 was stably maintained in over 80% of the attenuated S. typhimurium population after 100 generations of growth in antibiotic-free media. Oral immunisation of chickens with ZJ111/pcDNA3-5401 elicited specific humoral responses and stimulated proliferation of peripheral blood lymphocytes. The lymphocyte proliferation response was significantly higher in all vaccinated groups than in the control chickens. Antibody response was significantly lower in group C than in groups immunised with strain ZJ111/pcDNA3-5401. Vaccination with the strain ZJ111/pcDNA3-5401 at 10(8) (group E) and 10(9) (group F) CFU per chicken provided 55.0 and 57.5% protection against E. tenella challenge, respectively. These results have important implications for the development of DNA vaccines against avian coccidiosis by bacteria-vectored oral delivery system.     

Behavior of cAMP-deficient mutants of Salmonella typhimurium in macrophage cultures

The behavior of c-AMP-deficient S. typhimurium mutants in the culture of peritoneal macrophages of white mice and the antibacterial activity of immune macrophages obtained from mice previously immunized with c-AMP-deficient S. typhimurium mutants were studied; c-AMP-deficient mutants were shown to have a lesser cytotoxic effect on macrophages than the initial virulent strain, while retaining their capacity for intracellular proliferation. Immune macrophages acquired the ability to withstand the cytotoxic action of the virulent strain.     

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.     

Evaluation of the Lon-deficient Salmonella strain as an oral vaccine candidate

We evaluated the efficacy of CS2022 (the Lon protease-deficient mutant strain of Salmonella enterica serovar Typhimurium) as a candidate live oral vaccine strain against subsequent oral challenge with a virulent strain administered to BALB/c and C57BL/6 mice. CS2022 persistently resided in the spleen, mesenteric lymph nodes, Peyer's patches, and cecum of both strains of mice after a single oral inoculation with 1 x 10(8) colony-forming units. Finally, CS2022 almost disappeared from each tissue sample by week 12 in BALB/c mice, whereas CS2022 still resided in each tissue type at week 12 after inoculation of C57BL/6 mice. A significant increase in the serovar Typhimurium lipopolysaccharide-specific secretory immunoglobulin A (s-IgA), as measured for one of the mucosal immune responses, was detected in bile and intestinal samples of both strains of immunized mice at week 4 after immunization. In addition, the expression of gamma interferon mRNA in the spleens of both strains of immunized mice, especially those of C57BL/6 mice, was significantly increased at week 4 after immunization and was boosted during the following 5 days after the challenge was administered to the mice. Furthermore, peritoneal macrophages isolated from immunized mice at week 4 after immunization exhibited an increase in intracellular killing activity against both virulent and avirulent Salmonella. The present results suggested that salmonellae-specific s-IgA on the mucosal surfaces induced by immunization with CS2022 generally prevented mice from succumbing to an oral challenge with a virulent strain. Simultaneously, CS2022 promoted the protective immunity associated with macrophages in both strains of mice.     

携带新城疫病毒DNA疫苗的鼠伤寒沙门氏菌及其安全性与免疫效力

根据GenBank中发表的新城疫病毒(NDV)融合蛋白(F)基因序列,设计一对引物,通过RTPCR扩增出鹅源新城疫病毒分离株JS5F基因,测序确认后,将其克隆入真核表达载体pVAX1,获得重组真核表达质粒pVAX1F。将pVAX1F转化减毒鼠伤寒沙门氏菌SL7207,构建成携带DNA疫苗的重组沙门氏菌SL7207(pVAX1F)。重组菌以不同剂量口服免疫1日龄雏鸡,结果表明,细菌对雏鸡具有良好的安全性,且不影响鸡的增重。将SL7207(pVAX1F)分别以108CFU和109CFU的剂量3次口服免疫1日龄商品代伊莎褐蛋鸡,抗体检测结果显示,在三免后1周,SL7207(pVAX1F)109CFU剂量组的血清抗体效价与空载体组之间存在显著性差异(p<0.05)。重组菌两个剂量组在首免后2周开始出现粘膜抗体,并于二免后2周和3周达到较高水平。免疫保护试验结果显示,SL7207(pVAX1F)109CFU剂量组的保护率为77.27%,与空载体组之间存在显著性差异(p<0.05)。     

A single dose of recombinant Salmonella typhimurium induces specific humoral immune responses against heterologous Eimeria tenella antigens in chicken

Salmonella typhimurium vaccine strains were used as antigen delivery system for oral immunisation of chickens against two antigens of the coccidian parasite Eimeria tenella. The cDNAs of the known E. tenella proteins, SO7 and TA4, were isolated from total RNA and subcloned into the expression vectors pQE30 and pTECH2. Subcutaneous immunisation of chickens with Escherichia coli-expressed SO7 and TA4 revealed that both proteins were immunogenic. Both cDNAs were subcloned into plasmids of the pTECH2 vector system, which allows them to be expressed as fusion proteins with the highly immunogenic fragment C of the tetanus toxin under control of the anaerobically inducible nirB promoter. Plasmids were introduced into the S. typhimurium vaccine strains SL3261, C5aroD and C5htrA. SDS-PAGE and Western blot analysis revealed expression of both fusion proteins in all strains under anaerobic culture conditions. Three-week-old white leghorn chickens were orally immunised with 10(9) CFU per animal. The stability of the recombinant bacteria was revealed by recovery of viable Salmonella containing the respective plasmids from the liver of the immunised chickens at day 3 after inoculation. Specific serum IgG antibodies against the SO7-or TA4-antigens were detectable by ELISA 2 weeks after oral immunisation and remained for at least 6 weeks, while specific IgA antibodies were restricted to the bile of the birds. All chickens produced serum IgG and IgA to S. typhimurium lipopolysaccharides. Our data show that a single oral inoculation with recombinant S. typhimurium SL3261, C5aroD and C5htrA can induce specific antibody responses to heterologous Eimeria antigens in chickens, suggesting that recombinant Salmonella are a suitable delivery system for vaccines against Eimeria infections.     

The porin OmpC of Salmonella typhimurium mediates adherence to macrophages.

Macrophages recognize, adhere to, and phagocytose Salmonella typhimurium. The major outer membrane protein OmpC is a candidate ligand for macrophage recognition. To confirm this we used transposon mutagenesis to develop an ompC-deficient mutant in a known virulent strain of S. typhimurium; mutant and wild type were compared in macrophage adherence and association assays. Radiolabeled wild type S. typhimurium bound to macrophages at five-fold higher levels than did the ompC mutant. In association assays, macrophages in monolayers bound and internalized three-fold more wild type than mutant, while macrophages in suspension bound and internalized 40-fold more wild type than mutant. The ompC gene of our test strain of S. typhimurium contains several discrete differences compared with the ompC genes of Salmonella typhi and Escherichia coli. The deduced OmpC amino acid sequence of S. typhimurium shares 77 and 98% identity with OmpC amino acid sequence of E. coli and S. typhi, respectively. Evidence from this study supports a role for the OmpC protein in initial recognition by macrophages and distinguishes regions of this protein that potentially participate in host-cell recognition of bacteria by phagocytic cells.     

Inefficient in vitro killing of virulent or nonvirulent Salmonella typhimurium by murine polymorphonuclear neutrophils

The bactericidal capability of murine peritoneal polymorphonuclear neutrophils against virulent and nonvirulent Salmonella typhimurium was examined in an in vitro system. Although preincubation of the bacteria in specific murine antiserum elicited greater chemiluminescence from phagocytizing neutrophils than did incubation in normal murine serum, antiserum did not enhance ingestion, as less than 5% of the challenge was taken up by neutrophils under any of the conditions studied. Nonvirulent salmonellae showed a transient decrease in viable numbers early during in vitro incubation with or without intact neutrophils. Virulent salmonellae, however, were able to multiply without a lag period except when these bacteria were pretreated with antiserum and incubated in association with intact murine neutrophils. Results of these in vitro studies suggest that the murine polymorphonuclear neutrophil and antisalmonella antibody must act together to effect neutrophil-associated bactericidal activity against virulent salmonellae, and thus, that the neutrophil alone does not play a major role in the protection of unvaccinated, sensitive mice from disease caused by S. typhimurium.     

Oral and intranasal administration of somatostatin DNA vaccine mediated by attenuated Salmonella enterica serovar Typhimurium to promote growth of piglets

An attenuated strain of Salmonella typhimurium has been used as a carrier for oral and intranasal genetic immunization. Here, we evaluate the efficacy of a vaccine strain of S. typhimurium. CSO22 (pGM-CSF/SS, plasmid granulocyte-macrophage colony-stimulating factor/somatostatin) expressing two copies of SS genes. A total of 115 piglets, aged 2 months old, were either orally or intranasally immunized against the vaccine strain CSO22 (pGM-CSF/SS) with three dosages (5 × 10(10) colony forming units (CFU), 5 × 10(9) CFU and 5 × 10(8) CFU). For oral immunization, the specific anti-SS antibodies were detected in the immunized piglets. The levels of SS antibodies in the high-dose immunized group (5 × 10(10) CFU) were significantly higher than that in the phosphate buffered saline immunized group (P < 0.01) and 40% of animals were positive in SS antibodies in the high-dose immunized group. Moreover, the weight gain of the high-dose group was increased by 20.86%, 10.26% and 15.30% during 4, 8 and 12 weeks, respectively, after immunization in comparison to the control. For intranasal immunization, the growth of the low-dose group was increased by 10.23% in the whole test period (12 weeks). In conclusion, our results suggest that the recombinant strain could elicit anti-SS antibodies and improve the growth performance of immunized piglets, and that the oral immunization program is better than the intranasal program.     

鸡新城疫口服DNA疫苗的安全性、稳定性与免疫效力

将含新城疫病毒（NDV）F₄₈E₉株融合蛋白（F）基因的真核表达质粒pcDNA3-F的减毒鼠伤寒沙门氏菌ZJ111株（ZJ111/pcDAN3-F）口服接种小鼠和雏鸡。结果表明：利用该减毒株作为载体传递DNA疫苗具有相对安全性。用酶切和PCR鉴定法证实在体内外无抗生素存在的条件下重组质粒在受体菌ZJ111菌株内比较稳定。ZJ111/pcDAN3-F以每羽10⁸cfu免疫雏鸡,2周后二免,二免后4周攻击致死剂量的强毒株F₄₈E₉,观察其免疫效力。结果表明：重组ZJ111/pcDNA3-F菌株不仅能诱导雏鸡产生抗NDV抗体,而且能诱导法氏囊B淋巴细胞和胸腺T淋巴细胞的增殖反应,对强毒株攻击的保护率为66.7%,高于pcDNA3-F裸质粒DNA疫苗注射免疫组（50%）。上述结果初步提示减毒沙门氏菌为载体传递DNA疫苗具有良好的安全性、稳定性和免疫原性,为研制低成本、实用化的口服禽类DNA疫苗奠定了基础。     

The Salmonella typhimurium flgM gene, which encodes a negative regulator of flagella synthesis and is involved in virulence, is present and functional in other Salmonella species

Abstract FlgM inhibits the flagella-specific sigma factor FliA and is involved in the mouse-virulence of Salmonella typhimurium . In recent experiments, we observed that: (i) a flgM gene that could function to negatively regulate flagella synthesis was present in a variety of salmonellae; and (ii) the flgM gene derived from Salmonella species that are not normally virulent in mice could complement the S. typhimurium flgM mutant for virulence. Our results suggest that a functional flgM , has been retained in most, and perhaps all, Salmonella species, regardless of the motility or virulence phenotype of the strain.     

Efficiency of various bacterial suspensions derived from cecal floras of conventional chickens in reducing the population level of Salmonella typhimurium in gnotobiotic mice and chicken intestines

The antagonistic effect exerted towards Salmonella typhimurium by the flora issued from conventional chickens was studied in gnotobiotic animals. In germfree chickens and mice inoculated with S. typhimurium, the highest bacterial counts were observed in ceca, and were not significantly different in either host. The protection afforded by the inoculation of cecal flora issued from a conventional chicken was more effective when this flora was inoculated first into germfree chickens than when it was given only after inoculation with S. typhimurium. Administration of a cecal flora from a 15-day-old chick to gnotobiotic mice and chicken resulted in the inhibition of a further intestinal colonization by S. typhimurium in both hosts. Sixteen strains were isolated among the predominant populations of the fecal flora from chicken flora recipient mice. Association of 14 strains of strictly anaerobic bacteria with 2 strains of Escherichia coli and Streptococcus faecium only decreased the number of S. typhimurium in the ileum of gnotobiotic mice, but not in their cecum. Anaerobe cultures were obtained from 10(-6) and 10(-8) dilutions prepared from the fecal flora of gnotobiotic recipient mice. Antagonistic bacteria were present only in cultures from the 10(-6) dilution. Cecal concentrations of volatile fatty acids were shown not to be the sole factor implicated in the antagonistic effect against S. typhimurium.     

Changes in the cAMP level in macrophages in Salmonella typhimurium phagocytosis

The effect of a virulent S. typhimurium strain and its cAMP-deficients mutant on the level of cAMP in macrophages in the process of phagocytosis has been studied. The virulent strain has been shown to induce the 3-fold increase of the level of cAMP in macrophages, while the mutant renders no such effect.     

Role of peritoneal macrophages in the development of an experimental Salmonella infection

The effect produced on the course of Salmonella infection in mice by the removal of peritoneal macrophages with agarose has been studied. Peritoneal macrophages have been shown to control the multiplication of faintly virulent and virulent S. typhimurium strains in the spleen of mice. In immune mice the elimination of the virulent strain of the causative agent of superinfection may occur without the control of peritoneal macrophages.     

Salmonella typhimurium harboring plasmid expressing interleukin-12 induced attenuation of infection and protective immune responses

IL-12 is known to be an essential cytokine which appears to provide protective immunity against intracellular bacteria, such as Salmonella. In this study, we investigated the possibility of developing a vaccine using IL-12 against virulent Salmonella. We used the host defense system activated by cytokine IL-12. The highly virulent Salmonella strain (Salmonella typhimurium UK-1) was transformed with cytokine-expressing plasmids. These live, wild-type pathogens were used as vaccine strains without undergoing any other biological or genetic attenuating processes. The newly developed strains induced partial protection from infections (30-40%). Of note, the interleukin-12-transformed pathogen was safe upon immunization with low doses (10(3) cfu), induced IgG responses, and stimulated protective immune responses against Salmonella typhimurium in mice (80-100%). These results suggest that IL-12 induced attenuation of wild-type Salmonella in the host infection stage and vaccine development using the wild-type strain harboring plasmid-secreting IL-12 may be considered as an alternative process for intracellular bacterial vaccine development without the inconvenience of time-consuming attenuation processes.     

Effect of constitutively expressed phoP gene on the localization of Salmonella typhimurium within Mac-1 positive phagocytes

Intracellular localization of the wild-type (Spv+), the phoP-constitutively expressed strain (PhoPc), and the spv-deleted strain (Spv-) of Salmonella typhimurium was examined by the use of confocal laser scanning microscopy analysis of immunostained sections of mouse spleens after oral or subcutaneous inoculation. Only 40% of salmonellae of both the PhoPc and the Spv- strains were detected intracellularly within Mac-1 positive cells at day five after oral or day four after subcutaneous inoculation. In contrast, over 85% of salmonellae of the Spv+ strain were detected inside Mac-1 positive cells. In both inoculation trials, the splenic colony-forming unit values for the PhoPc and Spv- strains were significantly lower than the corresponding value for the Spv+ strain. These findings suggest that the constitutively expressed phoP gene of S. typhimurium attenuated virulence by limiting intracellular proliferation within mouse spleen phagocytes, and that the lack of spv genes had the same effect.     

Cutting edge: role of B lymphocytes in protective immunity against Salmonella typhimurium infection

Infection of mice with Salmonella typhimurium gives rise to a disease similar to human typhoid fever caused by S. typhi. Since S. typhimurium is a facultative intracellular bacterium, the requirement of B cells in the immune response against S. typhimurium is a longstanding matter of debate. By infecting mice on a susceptible background and deficient in B cells (Igmu-/- mice) with different strains of S. typhimurium, we could for the first time formally clarify the role of B cells in the response against S. typhimurium. Compared with Igmu+/+ mice, LD50 values in Igmu-/- mice were reduced during primary, and particularly secondary, oral infection with virulent S. typhimurium. After systemic infection, Igmu-/- mice cleared attenuated aroA- S. typhimurium, but vaccine-induced protection against systemic infection with virulent S. typhimurium involved both B cell-dependent and -independent effector mechanisms. Thus, B cell-mediated immunity plays a distinct role in control of S. typhimurium in susceptible mice.