Salmonella enteritidis temperature-sensitive mutants protect mice against challenge with virulent Salmonella strains of different serotypes

The protection conferred by temperature-sensitive mutants of Salmonella enteritidis against different wild-type Salmonella serotypes was investigated. Oral immunization with the single temperature-sensitive mutant E/1/3 or with a temperature-sensitive thymine-requiring double mutant (E/1/3T) conferred: (i) significant protection against the homologous wild-type Salmonella strains; (ii) significant cross-protection toward high challenge doses of S. typhimurium. Significant antibody levels against homologous lipopolysaccharide and against homologous and heterologous protein antigens were detected in sera from immunized mice. Moreover, a wide range of protein antigens from different Salmonella O serotypes were recognized by sera from immunized animals. Besides, primed lymphocytes from E/1/3 immunized mice recognized Salmonella antigens from different serotypes. Taken together, these results indicate that temperature-sensitive mutants of S. enteritidis are good candidates for the construction of live vaccines against Salmonella.     

Infection of Salmonella typhimurium with coliphage Mu d1 (Apr lac): construction of pyr::lac gene fusions.

A procedure was developed for introducing the coliphage Mu d1 (Apr lac) into Salmonella typhimurium in order to construct gene fusions that place the structural genes of the lac operon under the control of the promoter-regulatory region of other genes. To introduce Mu d1 from Escherichia coli K-12 into S. typhimurium, which is normally not a host for Mu, we first constructed an E. coli double lysogen carrying the defective Mu d1 phage and a Mu-P1 hybrid helper phage (MuhP1) that confers the P1 host range. A lysate prepared from this strain was used to infect a P1-sensitive (i.e., galE), restriction-deficient, modification-proficient strain of S. typhimurium, and a double lysogen carrying Mu d1 and MuhP1 was isolated. Induction of the latter strain produced lysates capable of infecting and generating gene fusions in P1-sensitive strains of S. typhimurium. In this paper we describe the construction of pyr::lac fusions by this technique.     

Genetic mapping of a mutation conferring sensitivity to bacteriophage Mu in Salmonella typhimurium LT2.

Two strains of Salmonella typhimurium LT2, SA1475 and MA411, were fortuitously found to be sensitive to bacteriophage Mu. The Mu-sensitivity allele of SA1475 was called musA1 and shown to be linked to the histidine operon both in conjugation and transduction experiments. The Mus allele of MA411 was unlinked to the his region and was tentatively designated musB2. Strains carrying large deletions of the his operon were also tested for Mu sensitivity; those of which the his-rib region is deleted were also sensitive to Mu. Transduction data led to the order zee-2 hisOGDCBAHFIE gnd musA. An Hfr injecting the his operon early (HfrK9) an carrying hisG9424::Tn10 delta 4 delta 11 and musA1 was isolated; this Hfr made it possible to introduce the Mus character into most derivatives of S. typhimurium LT2. Since strain SA1475 is resistant to bacteriophage P1, it could be used to select a new P1-Mu hybrid which has the host range of Mu and the transduction properties of P1.     

Type 1 fimbriae of Salmonella enteritidis.

Salmonella enteritidis was previously shown to produce fimbriae composed of 14,000-molecular-weight (Mr) fimbrin monomers (J. Feutrier, W. W. Kay, and T. J. Trust, J. Bacteriol. 168:221-227, 1986). Another distinct fimbrial structure, comprising 21,000-Mr fimbrin monomers, has now been identified. These fimbriae are simply designated as SEF 14 and SEF 21, respectively (for S. enteritidis fimbriae and the Mr [in thousands] of the fimbrin monomer). A simple method for the purification of both structures was developed by using the different biochemical properties of these fimbriae. SEF 21 remained intact after being boiled in sodium dodecyl sulfate but readily dissociated into subunits of 21,000 Mr at pH 2.2. The overall amino acid composition and the N-terminal amino acid sequence of the SEF 21 fimbrin were distinct from those of SEF 14 but were virtually identical to the predicted sequence for type 1 fimbrin of Salmonella typhimurium. Immunoelectron microscopy of S. enteritidis clearly revealed fimbrial structures that reacted with immune serum specific to the 21,000-Mr fimbrin. Immune sera raised against this subunit were cross-reactive with type 1 fimbrins found in whole-cell lysates of S. typhimurium, Salmonella illinois, and Salmonella cubana. However, there was no cross-reaction with Escherichia coli type 1 fimbriae or with other fimbrins produced by S. enteritidis. Under certain growth conditions, S. enteritidis produced both SEF 14 and SEF 21. However, when S. enteritidis was grown at 30 degrees C or lower, only the 21,000-Mr SEF 21 fimbrin could be detected. There was a direct correlation between mannose-sensitive hemagglutination and the presence of SEF 21.     

Conditionally transposition-defective derivative of Mu d1(Amp Lac).

A Mu d1 derivative is described which is useful for genetic manipulation of Mu-lac fusion insertions. A double mutant of the specialized transducing phage Mu d1(Amp Lac c62ts) was isolated which is conditionally defective in transposition ability. The Mu d1 derivative, designated Mu d1-8(Tpn[Am] Amp Lac c62ts), carries mutations which virtually eliminate transposition in strains lacking an amber suppressor. In such strains, the Mu d1-8 prophage behaves like a standard transposon. It can be moved from one strain of Salmonella typhimurium to another by the general transducing phage P22 with almost 100% inheritance of the donor insertion mutation. When introduced into a recipient carrying supD, supE, or supF, 89 to 94% of the Ampr transductants were transpositions of the donor Mu d1-8, from the transduced fragment into new sites. The stability of Mu d1-8 in a wild-type, suppressor-free background was sufficient to permit use of the fusion to select constitutive mutations without prior isolation of deletions to stabilize the fusion. Fusion strains could be grown at elevated temperature without induction of the Mu d prophage. The transposition defect of Mu d1-8 was corrected by a plasmid carrying the Mu A and B genes.     

Modulation of Salmonella infection by the lectins of Canavalia ensiformis (Con A) and Galanthus nivalis (GNA) in a rat model in vivo

The plant lectins, Concanavalin A (Con A) and Galanthus nivalis agglutinin (GNA) have been prefed to rats for 3 d pre- and 6 d postinfection with Salmonella typhimurium S986 or Salm. enteritidis 857. Con A significantly increased numbers of Salm. typhimurium S986 in the large intestine and in faeces, and severely impaired growth of the rats, more severely than is the case of infection with Salmonella typhimurium alone. Con A had much less effect on rats infected with Salm. enteritidis 857 only showing a significant increase in numbers in the colon, accompanied by intermittent increases of Salmonella in the faeces during the study. GNA significantly reduced pathogen numbers in the lower part of the small bowel and the large intestine of rats infected with Salm. typhimurium S986 and significantly improved rat growth. GNA had little effect on infection by Salm. enteritidis 857 with slight decreases in Salmonella numbers in the small intestine and large intestine and transient increases in the faeces.     

Bacteriophage P22 as a vector for Mu mutagenesis in Salmonella typhimurium: isolation of nad-lac and pnc-lac gene fusions.

Salmonella phage P22 was utilized as a vector for phage Mu cts d1(Apr lac) mutagenesis in Salmonella typhimurium. Efficient transposition of phage Mu d1 and the construction of gene fusions were readily accomplished with this procedure. Mutants blocked in the biosynthesis of NAD+ and in pyridine nucleotide cycle metabolism were isolated by this method, resulting in nadB-lac, nadC-lac, and pncB-lac gene fusions.     

Analysis of the host ranges of transposon bacteriophages Mu, MuhP1, and D108 by use of lipopolysaccharide mutants of Salmonella typhimurium LT2.

The lipopolysaccharide receptors for the mutator bacteriophages Mu, MuhP1, and D108 were investigated with lipopolysaccharide mutants of Salmonella typhimurium LT2. Mu adsorbed only to mutants lacking the terminal O antigen but retaining the main chain sugars of the core; the side chain N-acetylglucosamine was not required. MuhP1 and D108 adsorbed partially to cells with the same receptors but adsorbed well only to cells with shorter lipopolysaccharides of the Rc and Rd1 chemotypes.     

Oxygen-regulated stimulons of Salmonella typhimurium identified by Mu d(Ap lac) operon fusions

Using the technique of Mu d1(Ap lac)-directed lacZ operon fusions, several oxygen-regulated genetic loci were identified in Salmonella typhimurium. Thirteen anaerobically inducible and six aerobically inducible operon fusions were identified. Based on control by the oxrA and oxrB regulatory loci, the anti-lacZ fusions were grouped into three classes: class I loci were regulated by both oxr loci, class II genes were regulated by oxrA only, and class III loci were not affected by either regulatory locus. Several of the anti-lacZ fusions required growth in complex medium before they exhibited the inducible phenotype. While the expression of some of these loci was repressed when organisms were grown in nitrate, others were stimulated by nitrate. Fusions into the hyd and phs loci were identified among the isolated anti-lacZ fusions. Six oxygen-inducible (oxi) operon fusions were also identified. Two of the oxi loci mapped near oxygen-regulatory loci: oxiC near oxrA and oxiE near oxyR. However, neither fusion appeared to occur within the regulatory locus. The data presented serve to further define the aerobic and anaerobic stimulons of S. typhimurium but indicate additional regulatory circuits above those already defined.     

Antibodies to lipopolysaccharide and outer membrane proteins of Salmonella enteritidis PT4 are not involved in protection from experimental infection

BALB/c and Schofield mice were inoculated with formalin-killed bacteria prepared from strains of Salmonella enteritidis belonging to phage type (PT) 4 and carrying a 38 MDa plasmid and expressing long-chain lipopolysaccharide, or strains without a 38 MDa plasmid or lacking the ability to express lipopolysaccharide. Vaccinated mice were challenged with viable bacteria belonging to a virulent strain of S. enteritidis (PT4). Mice surviving this viable challenge were examined for a humoral antibody response to membrane antigens of S. enteritidis (PT4) that might relate to the possession of a given virulence property. BALB/c mice immunized with any of the test antigens were found to be immune to S. enteritidis (PT4), and this immunity was protective. Serum antibodies, of the IgG class, were detected to OmpA and a minor outer membrane protein (OMP) of 31 kDa. Schofield mice also raised IgG antibodies to these outer membrane proteins; however, non-immunized mice of this strain were resistant to infection. The virulence of S. enteritidis (PT4) was also tested using mice belonging to strains B10D2 (new), Biozzi (high), Biozzi (low), C3HeJ, B10ITYR and C57/L.     

Monoclonal antibodies demonstrate multiple epitopes on the O antigens of Salmonella typhimurium LPS

To investigate the complexity of the antigenic determinants presented on the surface of Salmonella typhimurium, a panel of murine monoclonal antibodies was generated and characterized. Hybridomas specific for S. typhimurium (strain TML, O antigens 1, 4, 12) were produced by immunization with acetone-killed and dried bacteria and standard fusion procedures. In this report, 15 such monoclonal antibodies, all of which bind lipopolysaccharide (LPS) extracted from S. typhimurium, are described. The fine specificity of these antibodies was assessed by examining the differential binding of each antibody to a panel of Salmonella strains, which selectively express different O antigenic determinants. This analysis defined several distinct categories of monoclonal antibodies of varying isotypes. Four anti-O:4-specific antibodies were identified. Two were specific for O:1. One antibody appears to react with the core polysaccharide of S. typhimurium LPS. Several of the monoclonal antibodies recognized LPS determinants that are presumably created by a combination of O antigens. For instance, one bound only to Salmonella strains that expressed both O:1 and O:12, whereas another bound only to those strains which expressed both O:4 and O:12. A group of three antibodies bound to any strain that simultaneously expressed O:1, O:4, and O:12. A distinct group of three monoclonal antibodies also bound strains that expressed O:1, O:4, and O:12, but only when the O:5 antigenic determinant was not present. The latter are, in that respect, S. typhimurium strain TML LPS-specific. The results of this analysis suggest that the epitopes of the S. typhimurium LPS molecule that are recognized by the host are considerably more complex than has been previously indicated by classical serology.     

Construction of Salmonella strains with both antigen O4 (of group B) and antigen O9 (of group D).

A Salmonella live vaccine causing both O4- and O9-specific immune responses would be of use, but no reported Salmonella serotype has both of these O antigens. Constructed Salmonella typhimurium strains with an rfb (O-antigen-specifying) gene cluster of type D in the chromosome and one of type B in an F'-rfb+ factor, and those with the reverse combination reacted strongly with both anti-O4 (and anti-O5) and anti-O9 sera and, if they carried recA, could be maintained in this state by growth conditions selective for retention of the F' factor. One of the two B.rfb+ gene clusters of a (P22-lysogenic) S. typhimurium strain with a tandem duplication of a chromosomal segment including hisD and B.rfb+ was replaced (by transduction) by a D.rfb+ gene cluster; the resulting strain was O1+ O4+ O5+ O9+ and stable as such after being made recA. A stable O4+ O9+ derivative of a virulent S. enteritidis (O-group D) strain was made by transducing into it first the join point of an appropriate tandem duplication strain, together with the adjacent B.rfb+ gene cluster, and then srl::Tn10 recA.     

禽大肠杆菌、肠炎、鼠伤寒、鸡白痢及鸡伤寒沙门菌多重PCR方法的建立及应用

【背景】大肠杆菌病和沙门菌病是最常见的家禽细菌性疾病,给养禽业造成严重经济损失。另外,禽大肠杆菌和沙门菌也是重要的人畜共患病原菌,可通过禽类及其产品传播给人类,对人类健康造成严重威胁。加强禽大肠杆菌和沙门菌的快速鉴别检测,对养禽业和公共卫生都具有重要意义。【目的】建立禽大肠杆菌、肠炎沙门菌、鼠伤寒沙门菌、鸡白痢沙门菌和鸡伤寒沙门菌的多重PCR检测方法。【方法】通过比较分析确定禽致病性大肠杆菌、肠炎沙门菌、鼠伤寒沙门菌、鸡白痢沙门菌和鸡伤寒沙门菌的特异靶标基因,设计5对特异性引物,通过条件优化建立多重PCR方法,分析该多重PCR方法的特异性、敏感性及可靠性。【结果】该方法能特异性地鉴定禽致病性大肠杆菌、肠炎沙门菌、鼠伤寒沙门菌、鸡白痢沙门菌和鸡伤寒沙门菌,每个PCR反应的最低检出限分别为103 CFU细菌和100 pg基因组DNA。临床分离菌株检测显示,多重PCR与传统血清学方法结果一致。【结论】建立的多重PCR方法能够快速鉴别禽致病性大肠杆菌和不同血清型沙门菌,对禽大肠杆菌病和沙门菌病的流行病学调查及临床检测具有重要意义。     

Monoclonal antibodies of IgA isotype specific for lipopolysaccharide of Salmonella enteritidis: production, purification, characterization and application as serotyping reagents

Hybridomas secreting immunoglobulin A (IgA) monoclonal antibodies (MAbs) against Salmonella enteritidis lipopolysaccharide (LPS) were generated after mucosal immunization of BALB/c mice with heat killed bacteria. Antigen binding properties and specificity of the produced MAbs were studied in ELISA and immunoblotting with purified LPS. Two IgA MAbs agglutinated all Salmonella OD1 strains and all S. enteritidis clinical isolates. MAb 178H11 recognized O:9 antigen of subserogroup OD1 LPS. MAb 177E6/A9 reacted also with OD3 LPS antigen and agglutinated OD3 strains. These data suggest the existence of different O:9 antigen subspecificities, one presented in subgroup OD1 and the other common for OD1 and OD3. Thus the produced IgA MAbs prove to be useful reagents, which could differentiate OD1 and OD3 from OD2 strains.     

High-molecular-weight components in lipopolysaccharides of Salmonella typhimurium, Salmonella minnesota, and Escherichia coli.

Lipopolysaccharide from smooth strains of Salmonella typhimurium, Salmonella minnesota, and Escherichia coli O111:B4, O55:B5, and O127:B8 was fractionated by gel filtration chromatography. All lipopolysaccharide samples separated into three major populations. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the fractions from S. typhimurium and S. minnesota indicated that the three peaks were made up of molecules with average O-antigen lengths of (i) 70 or more repeat units, (ii) 30 and 20 repeats units in the samples from S. typhimurium and S. minnesota, respectively, and (iii) 1 repeat unit. In contrast to the Salmonella samples, peak 1 from the E. coli samples was not detected on polyacrylamide gels and lacked detectable phosphate. This high-molecular-weight material had a sugar composition similar to that of O-antigen and was tentatively identified as capsular polysaccharide. Peaks 2 and 3 of the E. coli samples were analogous to those of the Salmonella isolates, containing lipopolysaccharide molecules with averages of 18 and 1 O-antigen repeat units, respectively. These lipopolysaccharide molecules did not completely dissociate during electrophoresis, and multimers were detected as distinct, anomalous, slow-migrating bands. Increasing the concentration of sodium dodecyl sulfate in the gels resulted in the dissociation of these multimers.     

Recall of Immunity in Mice Vaccinated with Salmonella enteritidis or Salmonella typhimurium

Mice were immunized with living Salmonella enteritidis or S. typhimurium and then were reinfected 8, 30, 60, and 150 days later with streptomycin-resistant strains of S. enteritidis, S. typhimurium, or a mixture of the two organisms. The level of resistance at the time of challenge and the rate at which resistance was recalled in late convalescence was determined by daily liver and spleen counts of the challenge population. An immediately effective specific antibacterial immunity was maintained only while the vaccinating strain could still be detected in the liver and spleen. Reinfection of vaccine-free mice with the homologous organism caused a rapid recall of antibacterial immunity (within 3 days), but the response to the heterologous organism was much slower (5 to 8 days). Simultaneous injection of both pathogens into the vaccinated mice resulted in liver and spleen growth curves which resembled those obtained when the two organisms were administered separately. The implications of these growth studies in the development of specific cellular immunity to Salmonella infections are discussed.     

Structural and immunochemical studies of the lipopolysaccharides of Salmonella strains with both antigen O4 and antigen O9.

Two Salmonella hybrid strains, SL5313 (Salmonella typhimurium with a D.rfb+ gene cluster) and SL5396 (S. enteritidis with a B.rfb+ gene cluster), each expressing both O-antigen 4 (of serogroup B) and O-antigen 9 (of serogroup D) were studied by immunofluorescence using a mixture of O4-specific mouse monoclonal and O9-specific rabbit polyclonal antibodies. Bound antibodies, detected by anti-mouse antibody labelled with fluorescein isothiocyanate and anti-rabbit antibody labelled with tetramethylrhodamine isothiocyanate showed that more than 98% of the bacteria expressed both the O4 and O9 epitopes. Phenol-water-extracted lipopolysaccharide from batch-grown cultures subjected to sugar and methylation analyses by gas-liquid chromatography and mass spectrometry were shown to contain abequose (of the O4 epitope) and tyvelose (of the O9 epitope) in ratios of 1:1.5 and 1:2.5 for SL5313 and SL5396, respectively. Isolated polysaccharide chains, obtained by weak-acid hydrolysis of the lipopolysaccharides, were found to contain both O4 and O9 specificities in the same molecule, since polysaccharide bound to O4 antibody attached to a solid-phase-adsorbed O9-specific antibody and vice versa. This demonstrates that in strains SL5313 and SL5396 O chains containing both O4 repeating units (from S. typhimurium) and O9 units (from S. enteritidis) are present.     

Identification of contemporary plasmid virulence genes in ancestral isolates of Salmonella enteritidis and Salmonella typhimurium

Six epidemiologically distinct ancestral strains of Salmonella enteritidis and 5 of S. typhimurium from the pre-antibiotic era were examined for plasmid content, and for presence of plasmid genes implicated in mouse-virulence. Five sizes of plasmid were detected in S. enteritidis varying from 1 to 60 MDa. Two sizes of plasmid were found in S. typhimurium, 28 and 60 MDa. Plasmids of the same size were not common to both serovars. The HindIII restriction patterns of 3 of the ancestral S. enteritidis plasmids were identical to the modern 38 MDa plasmid, while all contained identical bands of 3.5, 2.7 and 1.9 kb. All the 60-MDa S. typhimurium plasmids, ancestral and contemporary, had an identical restriction pattern. Three different sized S. enteritidis plasmids and one size S. typhimurium plasmid contained a 3.5-kb DNA fragment carrying the virulence locus VirA. The VirB virulence locus was located on a 2.7-kb DNA fragment in S. enteritidis and on a 2.5-kb fragment in S. typhimurium. Both loci were precisely conserved between the ancestral strains and the modern representatives of both serovars.     

Bacteriophage P1 as a vehicle for Mu mutagenesis of Salmonella typhimurium.

We developed a procedure using bacteriophage P1 as a vector for transferring Mu phage deoxyribonucleic acid into Salmonella typhimurium. Mu phage transferred in this manner yielded lysogenic auxotrophs, and we demonstrated that specific deletions and lac gene fusions can be selected.