A common virulence region on plasmids from eleven serotypes of Salmonella

Cured derivatives of Salmonella dublin and S. typhimurium showed reduced virulence following oral infection of mice (10(4)-10(5)-fold for S. dublin, 10(2)-fold for S. typhimurium). Large plasmids from S. dublin and S. typhimurium independently restored virulence to the cured S. dublin but truncated S. dublin plasmids with deletions in a previously identified virulence region did not. This common virulence region identified in plasmids from S. dublin and S. typhimurium was shown to be carried on plasmids from 11 other serotypes of Salmonella but was absent from 10 plasmid-containing serotypes. TnA and Tn10 were transduced from the virulence region of two TnA-insertion mutants of S. dublin and one Tn10-insertion mutant of S. typhimurium that showed diminished virulence to recipient wild-type strains of S. dublin, S. enteritidis and S. typhimurium. Each transductant showed a decrease in mouse virulence within the range 10(3)-10(5). It is therefore proposed that similar virulence determinants are expressed in different serotypes. It was also shown that integration that occurred during curing was Tn10 dependent.     

The response of foetal sheep to the somatic and flagellar antigens of Salmonella typhimurium.

Following the injection of polymeric flagellin (POL), foetal sheep older than 70 days gestation produced haemagglutinating antibody and synthesized IgM. The maximum titre of antibody in the blood increased with the age at which the foetus was injected. All foetuses synthesized 2-mercapto-ethanol-sensitive antibodies, while older foetuses (approximately 120 days gestation) also produced 2-mercaptoethanol-resistant antibodies and synthesized IgG1. During the primary immune response, there was a poor correlation between the antibody titre and the amount of immunoglobulin synthesized. The majority of IgM synthesized and almost all IgG1 had no demonstrable specificity for POL. During the secondary response to POL, the majority of IgG1 synthesized was specific and in one case appeared to be monoclona. There was no detectable primary antibody response in foetal sheep to the somatic antigens of Salmonella typhimurium, although all foetuses synthesized IgM. Only one of six foetuses receiving a second injection of antigen produced antibody. There was an increase in the numbers of blood lymphocytes following the injection of both POL and S. typhimurium, but only POL induced a rapid increase in the numbers of neutrophils in the blood and produced histological changes in the draining lymph nodes and spleen.     

The serological responses of calves to live Salmonella dublin vaccine--a comparison of different serological tests

The serological responses to live Salmonella dublin vaccine was assessed in three groups of calves; three-day-old colostrum-deprived (3DO C-), three-day-old colostrum-fed (3DO C+) and three-month-old (3MO), by the following tests; serum agglutination test (SAT), indirect haemagglutination test (IHA), complement-fixation test (CFT) and antiglobulin test (AGT). Serological activity was detected by all the tests in the 3MO calves. In the 3DO C+ calves no serological activity was detected by either the somatic SAT or IHA but low levels of CF and somatic AGT antibodies were produced. In 3DO C- calves serological activity, often at low levels, was detected by all the tests except the somatic SAT. High levels of flagellar agglutinins were detected in both groups of 3DO calves. It was concluded that with the exception of the flagellar SAT the tests were affected by the age of the calf and in 3DO calves also by the presence of colostral antibodies. However, the use of the SAT in 3MO calves would provide an indication as to the potency of salmonella vaccines.     

Rapid decay of Salmonella flagella antibodies during human gastroenteritis: a follow up study

An indirect enzyme-linked immunosorbent assay (ELISA) based on Salmonella re-polymerized flagella was employed to measure levels of immunoglobulin (Ig) G, IgM and IgA antibodies in sera from 303 Danish patients diagnosed with either Salmonella enteritidis or Salmonella typhimurium. The antibody-levels were assessed at one, three and six months after onset of salmonellosis, and sera from a control-group of 170 healthy blood donors were additionally analysed in order to establish cut-off values for the analysis. Cross-reactions to other Salmonella serotypes, as well as to Escherichia coli, Yersinia enterocolitica, Campylobacter jejuni, Campylobacter coli and Helicobacter pylori were observed. At one month after onset of symptoms, 70% of the patients recovering from a S. enteritidis infection carried detectable levels of anti-flagella antibodies, as did 77% of the patients recovering from S. typhimurium infection. Three months after onset of symptoms these detection rates had decreased to 46% and 40%; and six months after onset of symptoms the detection rates were 34% and 38%. This rapid decrease in the serum levels of flagella antibodies is in conflict with the "common knowledge" statement of a long-lasting anti-flagella immunoresponse. The present study suggests that such a tenacious statement is (or may be) inaccurate.     

Identification and sequence analysis of lpfABCDE, a putative fimbrial operon of Salmonella typhimurium.

A chromosomal region present in Salmonella typhimurium but absent from related species was identified by hybridization. A DNA probe originating from 78 min on the S. typhimurium chromosome hybridized with DNA from Salmonella enteritidis, Salmonella heidelberg, and Salmonella dublin but not with DNA from Salmonella typhi, Salmonella arizonae, Escherichia coli, and Shigella serotypes. Cloning and sequence analysis revealed that the corresponding region of the S. typhimurium chromosome encodes a fimbrial operon. Long fimbriae inserted at the poles of the bacterium were observed by electron microscopy when this fimbrial operon was introduced into a nonpiliated E. coli strain. The genes encoding these fimbriae were therefore termed lpfABCDE, for long polar fimbriae. Genetically, the lpf operon was found to be most closely related to the fim operon of S. typhimurium, both in gene order and in conservation of the deduced amino acid sequences.     

Genetic characteristics and protective properties of neamin-resistant mutant Salmonella typhimurium and S. dublin of the Nea(r) Str(s) and Nea(r) Str(r) 500 classes

The genetic analysis of attenuated mutants, class Nea(r) Str(s), with the use of bacteriophage P 22 has shown that mutation rendering the mutants resistant to neamine is localized in gene nea A. In experiments with the intraperitoneal infection of mice, the appearance of this mutation in S. typhimurium and S. dublin virulent strains has been found to lead to the decrease of virulence in 100% of clones. On the basis of the data obtained in this investigation, region str-spc in S. typhimurium and S. dublin has been mapped. In contrast to mutation spc A, mutations nea A and str A have been shown to inhibit the action of amber suppressor. The investigation has confirmed the regularity, previously established for Shigella flexneri, concerning the relationship between the influence of mutations, occurring in the genes which determine resistance to neamine and streptomycin and control the synthesis of ribosomal proteins S4, S5, S12 and S17, on the virulence of S. typhimurium and S. dublin and the effect of these mutations on the accuracy of the translation of genetic information in the biosynthesis of protein: mutation spc A has been found to produce no changes in the virulence of salmonellae, while mutations nea A and str A cause its loss. Salmonella strains carrying mutations nea A and nea B have shown pronounced protective properties in experiments on mice.     

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.     

Restricted dietary protein in pregnant beef cows: II. Effect on the immune response

Cows fed 0.37 Kg crude protein per day for four months before calving showed no decrease in colostral IgG, IgA, or IgM from cows fed a normal ration. Similarly, no difference in serum immunoglobulin concentration was detected in their calves. Cows in the low protein group developed significantly lower (P < 0.01) titers to Salmonella pullorum antigen than did the normal group. The data show significant positive linear regressions of calf agglutination titer to S. pullorum on the dam's colostral titers. Serum levels of IgG measured at 24 hours were significantly lower in calves from second-calf heifers than from these of other age groups. The IgA levels of serums from calves from the six-year-old cows were significantly higher than the serum levels of calves from younger dams (P < 0.01).     

Distribution of virulence plasmids within Salmonellae

The virulence region of the Salmonella dublin 50 MDa plasmid shared homology with 678 of 1021 salmonellae tested in colony hybridization experiments. The majority of S. dublin, S. typhimurium and S. enteritidis isolates tested hybridized with the region whereas, with the exception of S. hessarek, S. pullorum and S. gallinarum, other serotypes did not. Homologous virulence regions were plasmid encoded. In S. typhimurium a common 60 MDa plasmid was present in all phage types tested but not in DT4, DT37 and DT170. Smaller plasmids showing partial homology were found in DT12, DT18, DT193 and DT204C. In S. enteritidis a distinct plasmid profile for each of eight phage types was observed. Hybridizing plasmids were found in DT3, DT4, DT8, DT9 and DT11 whereas DT7, which was plasmid free, and DT10 and DT14, which harboured plasmids, did not hybridize. The extent of homology shared between S. dublin, S. typhimurium and S. enteritidis virulence plasmids was about 10 MDa and appeared conserved. Virulence plasmids from S. typhimurium and S. enteritidis did not show homology with a region of the S. dublin 50 MDa plasmid which was not associated with virulence functions whereas plasmids of about 24 MDa and 38 MDa in some S. typhimurium phage types did. The association of conserved virulence regions upon differing plasmids within salmonellae is discussed with reference to possible mechanisms of distribution and evolution of virulence genes.     

Biological properties of plasmid-free strains of Salmonella typhimurium and S. dublin

The study of Salmonella virulent strains has revealed that the characteristic feature of such strains is the presence of plasmids with a molecular weight of 90.2-91.5 kb for S. typhimurium and 77.2-78.5 kb for S. dublin. From Salmonella strains harboring only a single plasmid, variants with no plasmid at all have been obtained. These variants possess lower virulence for mice infected through enteral and intraperitoneal routes; besides, they lose their capacity for penetration into epithelial cells of HeLa line. S. typhimurium and S. dublin have shown decreased multiplication rate in vivo in comparison with the parent strains, while the multiplication rates in vitro were similar. These results suggest that the products of plasmid genes are either responsible for the virulent properties of salmonellae, or they have regulatory functions, thus controlling the work of chromosomal genes.     

Changes in the immunoglobulin levels of the mouse gut and serum during conventionalisation and following administration of Salmonella typhimurium.

Increasess in all immunoglobulin classes, except IgM, were observed in the sera of specific pathogen-free (SPF) mice beginning 10 days after their removal from barrier conditions. Concentrations of serum immunoglobulins, comparable with those of conventional mice, were obtained in these animals between 21 and 35 days. Following the removal of germ-free mice from their sterile isolaters, their intestinal IgA levels increased over 35 days to attain levels found in conventional animals. A marked increase in serum immunoglobulin occurred within one day following intravenous administration of live Salmonella typhimurium organisms to SPF animals, and this persisted for longer than 7 weeks (the duration of the study), This rapid elevation in serum immunoglobulin was not elicited by nonbacterial antigens, killed Salmonellae, or viable Vibrio cholerae. Negligible amounts of this immunoglobulin increase could be attributed to specific antibody.     

The characteristics of the O-specific humoral immune response of mice vaccinated with Salmonella choleraesuis antigens]

The work deals with the results of the comparative enzyme immunoassay (EIA) of serum samples taken from (CBA X C57BL/6) F1 mice immunized with O-specific polysaccharides, O-antigens (O-Ag) obtained by Boivin's method and antigenic preparations isolated with hydroxylamine (HA) from S. choleraesuis and S. typhimurium. O-Ag and lipopolysaccharide (LPS) of the corresponding bacterial species were used as antigens for the sensitization of polystyrene plates. The primary and secondary humoral immune response was studied by means of EIA. As revealed in this investigation, the immunization of mice with HA-isolated antigenic preparations and O-Ag, obtained from S. typhimurium, in a single injection (in doses of 1-100 micrograms) led to the development of weak specific immune response to O-Ag. Response to LPS was absent. After the second immunization of the animals pronounced immune response to O-Ag and LPS was observed. It developed as a response of both IgM and IgG type. The immunization of mice, made in a single injection, with HA-isolated antigenic preparations and O-Ag, obtained from S. choleraesuis, did not lead to the development of O-specific immune response. After the immunization of mice with these antigens in two injections sharply pronounced nonspecific activity of IgM and IgG serum antibodies with respect to O-Ag and LPS of homologous and heterologous bacterial species was noted in EIA. Neither S. typhimurium O-polysaccharide, nor S. choleraesuis O-polysaccharide did not induce O-specific immune response even after the second immunization.     

Immunogenic dialyzable factor derived from a ribosomal fraction of Salmonella typhimurium III. Analysis of resistance induced by dialyzable factors

Dialyzable factors (DF) were prepared from ribosomal fractions of several organisms including rough mutants of Salmonella typhimurium LT2, salmonella species of different serogroups, other enteric bacteria and gram-positive organisms, and tested for their immunogenicity against S. typhimurium infection in mice. All of them conferred local resistance on mice challenged intramuscularly with S. typhimurium LT2 in the early stage of immunization before the establishment of delayed-type hypersensitivity (DTH) to salmonella antigens. Although DFs of enteric bacteria including rough mutants of S. typhimurium induced DTH to salmonella antigens, only DF of a two-heptose mutant of S. typhimurium LT2 afforded significant mouse protection but others only prolonged the mean time to death. DF of Listeria monocytogenes induced the cross-reacting immunity which afforded the low level of mouse protection as well as an increase in mean time to death without inducing DTH. Passive transfer of anti-O antibody did not enhance the mouse protection provided by each DF. Resistance conferred by DF of S. typhimurium LT2 consisted of two phases: (i) nonspecific macrophage activation resulting in reduction of organisms at the infected site, which became active in the early stage of immunization and (ii) salmonella-specific immunity capable of preventing systemic infection, which became active in the late stage of immunization.     

The virulence plasmid of Salmonella dublin: detailed restriction map and analysis by transposon mutagenesis

A detailed restriction map of the virulence plasmid of Salmonella dublin has been determined and used for comparison with the virulence plasmid from S. typhimurium. Two regions were identified which appeared to be similar based on blotting and restriction data. One, of about 22 kb, encompassed the virulence region; the other, of about 8 kb, was outside it. The locations of 259 transposon insertions on the S. dublin plasmid were determined and related to their effect on virulence. One gene involved in virulence but outside the essential virulence region was shown to affect citrate metabolism.     

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.     

The quantitative analysis of lipopolysaccharide antigen in the blood serum of patients with salmonellosis by using complement-bound liposome lysis]

Titration of group B Salmonella O-antigen in the blood sera of patients and donors was carried out by means of the complement-dependent lysis of liposomes sensitized with S. typhimurium LPS. Good correlation (r = 0.95) of the levels of S. typhimurium somatic O-antigen in the patients' sera determined by liposomal immunoassay and aggregate hemagglutination test was established. The concentration of the antigens in the tested samples was within 0.5-50 micrograms/ml. Statistical analysis of the results obtained by liposomal immunoassay techniques demonstrated differences in the distribution functions for the blood sera of patients with different diseases and of donors.     

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.     

Evidence for related virulence sequences in plasmids of Salmonella dublin and Salmonella typhimurium

Transposon-insertion mutants were prepared from virulent field isolates of Salmonella dublin and Salmonella typhimurium. Detailed restriction-enzyme mapping of the single sites of TnA insertion in two mutants (M51 and M173) of S. dublin that showed diminished virulence in a mouse assay indicated that these sites were about 5 kbp apart on the approximately 70 kbp plasmid harboured by the isolate. A Tn10-insertion mutant (M242) of S. typhimurium that showed diminished virulence was also identified. A single copy of Tn10 was inserted into the approximately 90 kbp plasmid harboured by this isolate. Hybridization studies indicated that homology existed between the region encompassing the sites of TnA insertion in M51 and M173 and that encompassing the site of Tn10 insertion in M242. Restriction mapping indicated that the two regions were very similar and could even be identical and, if so, the Tn10 insertion in M242 could be mapped to a point 1.5 kbp from the TnA insertion in M51 and 6.5 kbp from that in M173. It appeared that the maximal extent of the putative similarity/identity was between 13 and 23 kbp. It is proposed that this stretch of high homology could represent a virulence sequence that has been conserved during the evolutionary divergence of the two Salmonella serotypes.     

Bacterial copper- and zinc-cofactored superoxide dismutase contributes to the pathogenesis of systemic salmonellosis

Copper/zinc-cofactored superoxide dismutase ([Cu,Zn]-SOD) has been found in the periplasm of many bacterial species but its biological function is unknown. Here we report the cloning and characterization of sodC , encoding [Cu,Zn]-SOD, from Salmonella typhimurium . The predicted protein sequence shows only 58% identity to Escherichia coli SodC, and from this its chromosomal location and its immediate proximity to a phage gene, sodC , in Salmonella is speculated to have been acquired by bacteriophage-mediated horizontal transfer from an unknown donor. A sodC mutant of S . typhimurium was unimpaired on aerobic growth in rich medium but showed enhanced sensitivity in vitro to the microbicidal action of superoxide. S . typhimurium , S . choleraesuis and S . dublin sodC mutants showed reduced lethality in a mouse model of oral infection and persisted in significantly lower numbers in livers and spleens after intraperitoneal infection, suggesting that [Cu,Zn]-SOD plays a role in pathogenicity, protecting Salmonella against oxygen radical-mediated host defences. There was, however, no observable difference compared with wild type in the interaction of sodC mutants with porcine pleural, mouse peritoneal or J774 macrophages in vitro , perhaps reflecting the hierarchical capacity of different macrophage lines to kill Salmonella , the most efficient overwhelming the proposed protective effect of periplasmic SOD.