Isolation of a highly purified capsular polysaccharide from Salmonella enterica serovar Typhi (Salmonella typhi)--Vi-antigen and its use in serological diagnosis of typhoid fever

For use in differential diagnostics of typhoid fever, samples of the capsular polysaccharide from Salmonella enterica serovar Typhi (usually named Vi-antigen) were isolated and characterized by physicochemical and serological methods. It was shown that only the sample of Vi-antigen with the minimal (0.57%) admixture of the corresponding lipopolysaccharide (LPS) from S. typhi retained a high serological activity in the tests with monoreceptor anti-Vi sera. However, it exhibited a substantially weaker reaction with sera from normal donors and patients with acute nontyphoid salmonelloses, than Vi-antigen preparations with a higher (0.8-1.2%) LPS content. The chromatographically pure Vi-antigen was purified by triple reprecipitation with hexadecyltrimethylammonium bromide. The content of the LPS admixture in the resulting Vi-antigen samples was quantitatively determined by GC. A high purification level of the Vi-antigen from the LPS admixture allows us to hope that this preparation could serve as a basic component of the test system for the diagnostics of typhoid fever. The English version of the paper.     

Isolation of a Highly Purified Capsular Polysaccharide from Salmonella enterica Serovar Typhi (Salmonella typhi) and Its Use in Serological Diagnostics of Typhoid Fever

For use in differential diagnostics of typhoid fever, samples of the capsular polysaccharide from Salmonella enterica serovar Typhi (usually named Vi-antigen) were isolated and characterized by physicochemical and serological methods. It was shown that only the sample of Vi-antigen with the minimal (0.57%) admixture of the corresponding lipopolysaccharide (LPS) from S. typhi retained a high serological activity in the tests with monoreceptor anti-Vi sera. However, it exhibited a substantially weaker reaction with sera from normal donors and patients with acute nontyphoid salmonelloses, than Vi-antigen preparations with a higher (0.8–1.2%) LPS content. The chromatographically pure Vi-antigen was further purified by triple reprecipitation with hexadecyltrimethylammonium bromide. The content of the LPS admixture in the resulting Vi-antigen samples was determined quantitatively by GC. A high purification level of the Vi-antigen from the LPS admixture allows us to hope that this preparation could serve as a basic component of the test system for the diagnostics of typhoid fever.     

Mitogenic action of bacterial T-independent antigens under various conditions of lymphocyte culture

The mitogenic response of lymphocytes in mouse spleen cell culture to the action of Salmonella typhi lipopolysaccharide (LPS) and Vi-antigen under different experimental conditions has been studied. The density of the culture has been shown to influence the activation of lymphocytes with Vi-antigen. Thus, macrophages stimulate mitogenesis at the concentration of lymphocytes equal to 1.0-1.2 X 10(6) cells/ml and suppress it when this concentration increases tenfold. The method used for the purification of cell suspension from adhering cells has been shown to influence the level of the mitogenic response of lymphocytes. The cultures, purified from macrophages by filtration through a column packed with cotton wool, have been found to respond to the mitogenic doses of LPS 7-8 times weaker than those purified by adhesion onto plastic. Background and mitogen-induced inclusion of 3H-thymidine into lymphocytic DNA varies in accordance with the presence of adhering cells. For this reason, in the evaluation of the influence of macrophages on mitogenesis it is expedient to consider not only the stimulation index, but also the absolute inclusion of thymidine into cells.     

The role of adhesin in the polymeric structure of Vi-antigen

Samples of Vi-antigen subjected to hydrolysis of different duration have been studied. As revealed in this study, 12-hour hydrolysis causes the depolymerization of the preparation. The fragments thus obtained have been found to possess Vi-specificity. The polymeric form of Vi-antigen contains adhesin differing in specificity from Vi-determinant. This component is absent in the low-molecular fraction of Vi-antigen. Adhesin is the binding element of the molecule of Vi-antigen and ensures its polymeric structure.     

Determination of O and Vi antigens in typhoid fever in the slide coagglutination reaction

Investigations with a view to the development and trial of a slide coagglutination test system for the detection of specific Salmonella typhi antigens have been made. As a result, diagnostic agents with sensitivity to group D Salmonella lipopolysaccharides and Vi-antigen, equal to 1 X 10(-5) to 1 X 10(-6) g/l, have been obtained. Specimens of saliva, urine and fecal filtrates from 61 adult patients with bacteriologically confirmed typhoid fever and 54 practically healthy persons have been studied. The coagglutination test has been positive with specimens from 90 +/- 4% of typhoid fever patients and, within the first 5 days of the disease, with those from 85 +/- 7% of such patients. The slide coagglutination test with saliva specimens has been found to be more informative than that with urine specimens. The data obtained in these investigations indicate that the slide coagglutination test is highly sensitive and specific, which offers good prospects for its use as a simple, economic and demonstrative method for the early tentative diagnosis of typhoid fever.     

Vi antigen and microbial adhesion factors. The detection of adhesins in commercial Vi antigen preparations

Enterobacterial Vi-antigen inhibit the Escherichia coli-induced agglutination of red blood cells of guinea pigs and other animals, which indicates that Vi-antigen contains the admixture of adhesion associated with type I pili. The amount of adhesion contained in Vi-antigen can be determined from the degree to which its hemagglutination-inhibiting effect is manifested.     

Formation of cells secreting antigen-dependent nonspecific immunoglobulins during immunization of mice with 2 T-independent antigens

Immunization of BALB/c and C3H/A mice with T-independent antigens (Vi-antigen of Salmonella typhi and polyvinyl pyrrolidone PVP350) induces the appearance of both antibody-forming cells (AFC) and a sharp increase in the number of cells forming nonspecific immunoglobulins (nIFC). This effect is not related to mitogenic properties of the antigens. The number of nIFC formed after simultaneous injection of both T-independent antigens does not differ from that of nIFC formed during immunization with each of these antigens alone. Thus, there was no summation of nIFC number after injection of two T-dependent or one T-dependent and one T-independent antigens. The mechanisms of nIFC formation under the influence of T-dependent and T-independent antigens are discussed.     

Macrophage participation in the polyclonal and specific response to T-independent antigen in vitro

Effect of adherent cells (macrophages on synthesis of total immunoglobulins (Ig) and specific antibody (Ab) in mice spleen cell culture stimulated by Vi-antigen Salmonella typhi has been studied. Ig and Ab in culture medium were determined by ELISA technique 96 hours after introducing the antigen into cell culture. The adsorption of analysed samples and reference antiserum on antigen coated microplates at pH 3.5 results in reducing nonspecific protein adsorption and promotes quantitative analysis Ab synthesized in vitro. The level of Ab produced in antigen stimulated spleen cell culture during 96 hours after antigen introduction is less than 2% of all synthesized Ig. Removal of most macrophages from spleen cell population results in a considerable decrease of specific and polyclonal immune responses. The basal Ig level in spleen cell culture (without antigen influence) does not decrease after removal of macrophages.     

Determination of the Vi-antigen content of acetone-dried typhoid vaccines

Summary A number of typhoid strains and coli strain 5396/38 are compared for their Vi-antigen content with the aid of complement fixation and erythrocyte sensibilization tests. Acetone-killed and dried germs were used as source material. Parallel batches derived from the same strain showed good agreement. Moreover, results with erythrocyte sensibilization and complement fixation reaction were closely correlated. The conclusion is drawn that serological determination of the Vi-antigen of acetone-dried typhoid vaccine is possible. Further investigations, however, must be carried out to compare the functional capacity to produce antibodies of strains with the same serologically determined content of Vi-antigen. We thank Dr.M. Landy for the revision of this paper.     

Immune response and tolerance to S. typhi Vi-antigen in T-lymphocyte depleted mice]

Comparison of immune response to Vi-antigen in thymetomized letally irradiated and reconstituted with fetal liver cells mice and in control animals revealed no difference between the two groups. The absence of enchancement of antibody formation in T cell depleted mice favours thymic-independent regulation of immune response to optimal dose of Vi-antigen. The induction of cyclophosphamide tolerance to Vi-antigen did not depend on the presence of T cells: tolerogenic treatment was equally effective in T cell depleted mice and in control animals. Therefore cyclophosphamide tolerance was not due to the activation of T suppressors but to direct elimination of immunocompetent clones of B cells.     

Specific blockade of the immune response caused by an excessive dose of S. typhi Vi-antigen

Intravenous injection to adult mice of Vi-antigen (200 microgram) induces in them the state of a short-term (10--12 days) unresponsiveness. This is due to the block of immunocompetent cells, not to masking the antibodies production by the excess of free antigen. Double washing to spleen cells before the test of local passive hemolysis in gel failed to reverse the block of the immune response; besides, there was no free antigen in the spleen that could inhibit the antibodies produced by the cells of the immune animal. The immune response block can be reversed by the injection of heterologous antiserum to Vi-antigen 18 to 24 hours before the Jerne's test. The restoration of immune response by means of the antiserum is prevented by the administration of 6-thioguanine after Vi-antigen (200 microgram). Thus, administration of a massive dose of Vi-antigen failes to block the proliferation and differentiation of the antigen-recognizing cells, but depresses the synthesis or secretion of antibodies.     

Incidence of R factors in coliform, fecal coliform, and Salmonella populations of the Red River in Canada.

Coliforms, fecal coliforms, and Salmonella were isolated from the Red River, Manitoba, Canada, and identified. These organisms were then examined for resistance to 12 antibiotics. Some fecal coliforms were resistant to all 12 antibiotics, and 18% of the Salmonella isolates were resistant to one or more antibiotics. A total of 52.9% of the fecal coliforms resistant to three or more antibiotics were able to transfer single or multiple resistance (R) determinants to the Salmonella recipient, and 40.7% could transfer R determinants to the Escherichia coli recipient. Of the resistant Salmonella, 57% transferred one or two determinants to the Salmonella recipient, and 39% transferred one or two determinants to the E. coli recipient. It was calculated that populations of fecal coliforms containing R factors were as high as 1,400 per 100 ml and that an accidental intake of a few milliliters of water could lead to transient or permanent colonization of the digestive tract. Consideration of data on bacteria with R factors should be made in future water quality deliberations and in discharge regulations.     

A comparison of the yield of Vi and O antigens during the adaptation of Salmonella typhi strains to cultivation under starvation conditions

S. typhi strains Ty(2)4446 and Vi-1S underwent multiple passages in f synthetic liquid starvation culture medium consisting of water with salts and glucose added. In the process of the adaptation of the cultures to these stress conditions (starvation stress) the increasing yield of biomass from passage to passage was observed. Differences in the accumulation of Vi- and O-antigens were noted in two strains under study. In the cultures of strain Ty(2)4446 an insignificant increase in the antigen content from passage to passage was observed, while in the cultures of strain Vi-1S an increase in the content of Vi- and O-antigens was 4- to 5-fold. With the adaptation of the culture the Vi-antigen to O-antigen ratio changed from 1:57 to 1:20 for strain Ty(2)4446 and from 1:2.7 tp 1:2.2 for strain Vi-1S. Strain Ty(2)4446 had an advantage over strain Vi-1S with respect to the synthesis of Vi-antigen. These data are indicative of the expediency of using not only strain Ty(2)4446, but also strain Vi-1S for the preparation of typhoid vaccine, especially the one based on Vi-antigen.     

M467: a murine IgA myeloma protein that binds a bacterial protein. I. Recognition of common antigenic determinants on Salmonella flagellins.

We have studied the binding of M467, an IgA murine myeloma protein, to flagellin from seven species of Salmonella. It was found that M467 was reacting with antigenic determinants that were common to all the flagellins studied. These determinants were not related to serotypic antigens. Electronmicrographs of unreduced M467 showed a variety of polymeric species bound to flagella in a manner that could produce immobilization as well as agglutination and precipitation through cross-linking of antigenic determinants. Immunodiffusion in agar gel revealed that M467 was recognizing more than one group of peptide determinants on the flagellins studied. Passive hemagglutination inhibition and a solid phase radioimmunoassay provided evidence that there were differences in binding avidities between M467 and the various Salmonella flagellins studied. It was concluded that M467 is binding more than one specific group of antigenic peptide determinants on flagellin molecules. Flagellin from four of the seven species of Salmonella studied were deficient in one or more of these determinants.     

Study of the nature and mechanism of biosynthesis of nonspecific immunoglobulins]

Mice were immunized with Vi-antigen. Spleen cells, removed at different time after immunization, were cultivated in Eagle medium, containing glycine-14C. The biosynthesis of antibodies to Vi-antigens, autoantibodies to mouse IgG and antigen-dependent non-specific immunoglobulins (NigG) were determined by means of specific immunosorbents. Immunization of mice with Vi-antigen resulted in a sharp increase in antigen-dependent NIgG formation. Thus, this protein biosynthesis takes place not only during immunization with thymus-dependent antigens, but also in response to the thymus-independent antigen. It is shown that the synthesized antigen-dependent NIgG were not autoantibodies to self mouse IgG.     

Towards more virulent and antibiotic-resistant Salmonella?

Salmonella are well-known pathogens. Virulence determinants can be present on the chromosome, usually encoded on pathogenicity islands, or on plasmids and bacteriophages. Antibiotic resistance determinants usually are encoded on plasmids, but can also be present on the multidrug resistance region of Salmonella Genomic Island 1 (SGI1). Virulence plasmids show a remarkable diversity in the combination of virulence factors they encode, which appears to adapt them to specific hosts and the ability to cause gastroenteritidis or systemic disease. The appearance of plasmids with two replicons may help to extend the host range of these plasmids and thereby increase the virulence of previously non- or low pathogenic serovars. Antibiotic resistance among Salmonella is also increasing. This increase is not only in the percentage isolates resistant to a particular antibiotic, but also the development of resistance against newer antibiotics. The increased occurrence of integrons is particularly worrying. Integrons can harbour a varying set of antibiotic resistance encoding gene cassettes. Gene cassettes can be exchanged between integrons. Although the gene cassettes currently present in Salmonella integrons encode for older antibiotics (however, some still frequently used) gene cassettes encoding resistance against the newest antibiotics has been documented in Enterobacteriaceae. Furthermore, beta-lactamases with activity against broad-spectrum cephalosporins, which are often used in empiric therapy, have been found associated with integrons. So, empiric treatment of Salmonella infections becomes increasingly more difficult. The most worrisome finding is that virulence and resistance plasmids form cointegrates. These newly formed plasmids can be selected by antibiotic pressure and thereby for virulence factors. Taken together these trends may lead to more virulent and antibiotic-resistant Salmonella.     

The prevalences of Salmonella Genomic Island 1 variants in human and animal Salmonella Typhimurium DT104 are distinguishable using a Bayesian approach

Throughout the 1990 s, there was an epidemic of multidrug resistant Salmonella Typhimurium DT104 in both animals and humans in Scotland. The use of antimicrobials in agriculture is often cited as a major source of antimicrobial resistance in pathogenic bacteria of humans, suggesting that DT104 in animals and humans should demonstrate similar prevalences of resistance determinants. Until very recently, only the application of molecular methods would allow such a comparison and our understanding has been hindered by the fact that surveillance data are primarily phenotypic in nature. Here, using large scale surveillance datasets and a novel Bayesian approach, we infer and compare the prevalence of Salmonella Genomic Island 1 (SGI1), SGI1 variants, and resistance determinants independent of SGI1 in animal and human DT104 isolates from such phenotypic data. We demonstrate differences in the prevalences of SGI1, SGI1-B, SGI1-C, absence of SGI1, and tetracycline resistance determinants independent of SGI1 between these human and animal populations, a finding that challenges established tenets that DT104 in domestic animals and humans are from the same well-mixed microbial population.     

Interaction of chaotropically modified immunoglobulins with protein and glicolipid antigens

The features of interaction of native and chaotropically modified immunoglobulins with proteins (ovalbumin) and glicolipids (lipopolysaccharides, LPS) enterobacteria Escherichia coli K235, Salmonella minnesota and Salmonella enteritidis have been investigated. It has been established, that after processing of native antibodies with 3.5 M KSCN their ability to contact to the specified antigenes repeatedly grows. Besides the intensity of interaction of modified immunoglobulins with the mentioned above antigenes was various, that is determined by the presence of structural distinctions between antigen determinants of proteins and glycolipid antigens, and also between O-polysaccharide chains of LPS in different species of enterobacteria.     

Plasmid-mediated florfenicol and ceftriaxone resistance encoded by the floR and bla(CMY-2) genes in Salmonella enterica serovars Typhimurium and Newport isolated in the United States

Multidrug resistance plasmids carrying the bla(CMY-2) gene have been identified in Salmonella enterica serovars Typhimurium and Newport from the United States. This gene confers decreased susceptibility to ceftriaxone, and is most often found in strains with concomitant resistance to ampicillin, chloramphenicol, streptomycin, sulfamethoxazole and tetracycline. The bla(CMY-2)-carrying plasmids studied here were shown to also carry the florfenicol resistance gene, floR, on a genetic structure previously identified in Escherichia coli plasmids in Europe. These data indicate that the use of different antimicrobial agents, including phenicols, may serve to maintain multidrug resistance plasmids on which extended-spectrum cephalosporin resistance determinants co-exist with other resistance genes in Salmonella.     

Immunologic properties and role of the low-molecular component in a preparation of S. typhi Vi antigen]

The author compared the serological, immunogenic and protective activity of the Vi-antigen and its high- and low-molecular fractions; an interaction between these fractions in administration of their mixture to the animals was studied. The low-molecular antigen (the 2nd fraction), contained in the preparation of the Vi-polysaccharide differed considerably (by properties) from the high-molecular antigen. The 2nd fraction, whose antigenic substance possessed the least immunogenic and protective capacity, failed to induce or to resolve the immunological memory, and also prevented the manifestations of the high immunogenicity of the 1st fraction. Therefore the nonfractional preparation of the Vi-antigen, consisting of 80% of a high-molecular substance of the 1st fraction and having the same serological activity as the 1st fraction, possessed a lesser immunogenic and protective activity.