Specific antibodies bound to secretory IgA in the sera of patients with intestinal infections]

Specific IgA and sIgA antibodies were studied in the sera of patients suffering from various intestinal diseases (dysentery, salmonellosis, typhoid fever, chronic typhoid carrier state) and in the sera of healthy persons immunized by parenteral route with typhoid alcohol vaccine. The nature of antibodies was identified in Coombs' test, using monospecific antisera to alpha-chain and to the secretory component. IgA and sIgA antibodies were revealed most frequently in the sera of dysentery patients and of chronic typhoid carriers. No sIgA antibodies were found in the sera of subcutaneously immunized persons. The presence of specific sIgA antibodies in the serum reflects the participation of local immune mechanisms in the formation of systemic immunity in the intestinal infections.     

Combined rapid (TUBEX) test for typhoid-paratyphoid A fever based on strong anti-O12 response: design and critical assessment of sensitivity

Rapid diagnostics can be accurate but, often, those based on antibody detection for infectious diseases are unwittingly underrated for various reasons. Herein, we described the development of a combined rapid test for two clinically-indistinguishable bacterial diseases, typhoid and paratyphoid A fever, the latter fast emerging as a global threat. By using monoclonal antibodies (mAbs) to bacterial antigens of known chemical structures as probes, we were able to dissect the antibody response in patients at the level of monosaccharides. Thus, a mAb specific for a common lipopolysaccharide antigen (O12) found in both the causative organisms was employed to semi-quantify the amounts of anti-O12 antibodies present in both types of patients in an epitope-inhibition particle-based (TUBEX) immunoassay. This colorimetric assay detected not only anti-O12 antibodies that were abundantly produced, but also, by steric hindrance, antibodies to an adjoining epitope (O9 or O2 in the typhoid or paratyphoid bacillus, respectively). Sensitivity and, particularly, reaction intensities, were significantly better than those obtained using an anti-O9 or anti-O2 mAb-probe in the examination of paired sera from 22 culture-confirmed typhoid patients (sensitivity, 81.8% vs 75.0%) or single sera from 36 culture-confirmed paratyphoid patients (52.8% vs 28.6), respectively. Importantly, sensitivity was better (97.1% for typhoid, 75.0% for paratyphoid) if allowance was made for the absence of relevant antibodies in certain specimens as determined by an independent, objective assay (ELISA)--such specimens might have been storage-denatured (especially the older paratyphoid samples) or procured from non-responders. Benchmarking against ELISA, which revealed high concordance between the two tests, was useful and more appropriate than comparing with culture methods as traditionally done, since antibody tests and culture target slightly different stages of these diseases. Paired sera analysis was insightful, revealing 64% of typhoid patients who had no change in antibody titer over 4-16 days, and 14% with no IgM-IgG class-switching.     

Malnutrition and maternal vaccination against typhoid toxin

Children are particularly susceptible to typhoid fever caused by the bacterial pathogen Salmonella Typhi. Typhoid fever is prevalent in developing countries where diets can be less well-balanced. Here, using a murine model, we investigated the role of the macronutrient composition of the diet in maternal vaccination efficacies of two subunit vaccines targeting typhoid toxin: ToxoidVac and PltBVac. We found that maternal vaccinations protected all offspring against a lethal-dose typhoid toxin challenge in a balanced, normal diet (ND) condition, but the declined protection in a malnourished diet (MD) condition was observed in the PltBVac group. Despite the comparable antibody titers in both MD and ND mothers, MD offspring had a significantly lower level of typhoid toxin neutralizing antibodies than their ND counterparts. We observed a lower expression of the neonatal Fc receptor on the yolk sac of MD mothers than in ND mothers, agreeing with the observed lower antibody titers in MD offspring. Protein supplementation to MD diets, but not fat supplementation, increased FcRn expression and protected all MD offspring from the toxin challenge. Similarly, providing additional typhoid toxin-neutralizing antibodies to MD offspring was sufficient to protect all MD offspring from the toxin challenge. These results emphasize the significance of balanced/normal diets for a more effective maternal vaccination transfer to their offspring.     

A mouse model for the human pathogen Salmonella typhi

Salmonella enterica serovar Typhi (S. Typhi) causes typhoid fever, a life-threatening human disease. The lack of animal models due to S. Typhi's strict human host specificity has hindered its study and vaccine development. We find that immunodeficient Rag2(-/-) γc(-/-) mice engrafted with human fetal liver hematopoietic stem and progenitor cells are able to support?S. Typhi replication and persistent infection. A?S. Typhi mutant in a gene required for virulence in humans was unable to replicate in these mice. Another mutant unable to produce typhoid toxin exhibited increased replication, suggesting a role for this toxin in the establishment of persistent infection. Furthermore, infected animals mounted human innate and adaptive immune responses to S. Typhi, resulting in the production of cytokines and pathogen-specific antibodies. We expect that this mouse model will be a useful resource for understanding S.?Typhi pathogenesis and for evaluating potential vaccine candidates against typhoid fever.     

Immunoaffinity-based mass spectrometric characterization of immunoreactive proteins of Salmonella Typhi

Salmonella Typhi, a human-restricted Gram negative enterobacteriaceae, is the causative agent of typhoid fever in human being. The available serodiagnostic tools for the diagnosis of typhoid fever lack sensitivity and/or specificity. This study aimed to identify the immunoreactive proteins of S. Typhi that could help to develop improved diagnostic tools. Here, we performed immunoaffinity-based proteomic approach that uses charged columns to retrieve IgG and IgM antibodies from the plasma of typhoid patients followed by capture of S. Typhi proteins. These proteins were then characterized by mass spectrometry and bioinformatics tools. Using this approach, we identified 28 immunoreactive proteins of S. Typhi, in which 14 proteins were captured by IgG charged column and 4 proteins were captured by IgM column. We also identified 10 proteins (hlyE, rfbH, dapD, argI, glyA, pflB, trxB, groEL, tufA and pepD) captured by both columns. The prediction of antigenicity and immunogenicity resulted that 22 proteins were antigenic while 6 were non-antigenic on the scale of 0.4 threshold value of VaxiJen. These proteins successfully simulated the immune system in silico and in response higher amount of antibodies‘ titers were recorded in C-IMMSIM, confirming the immunogenic nature of these proteins. The identified proteins are of diverse nature and functions including those involved in virulence and pathogenesis, energy metabolism, cell development, biosynthesis of amino acids, regulatory functions and biosynthesis of cofactors. The findings of this study would be helpful in the development of improved vaccines and diagnostic tools for typhoid fever.     

Reaction and response of newborn guinea pigs to experimental Salmonella typhi infection

Newborn guinea pigs, orally infected with Salmonella typhi were examined at various intervals of time in order to determine bacterial distribution in tissues and to establish possible correlation with the clinical aspects manifested. Histopathological examination evidenced typical lesions in jejunum, ileum, caecum and especially in regional lymphatic tissues. Spleen, liver and mesenteric lymph nodes presented granulomatous lesions similar to those observed in in human typhoid fever. After oral administration, the animals reacted with anorexia, febrile reactions, bacteremia, diarrhoea, positive stool cultures, dehydration, lethargy and antibodies too were produced. Our results indicate that typhoid infection may be induced in newborn guinea pigs; the model may be used for an assessment of attenuated live typhoid vaccine control.     

An immunoblotting as a method for the diagnosis of typhoid fever

The patients' sera had been referred to the National Salmonella Centre for routine Widal serology. Sera were predominately from patients suspected of having been infected with Salmonella Typhi, but also included one serum from patient with typhoid fever who was culture positive for Salmonella Typhi. The immunoblotting procedure using Salmonella Typhi somatic (O=9,12 LPS) and flagellar (H=d) antigens was used for preliminary testing of selected patients sera previously evaluated by Widal agglutination assay as containing different levels of antibodies against O and/or H antigens of Salmonella Typhi. Following Chart et al., immunoblotting reactions were graded between 0 and 3, with 0 indicating an absence of antibody binding, and 3 where antibody binding was readily observed. Sera giving reaction of 2 or 3 were considered to be antibody positive for this study. Positive immunoblotting reaction to O=9,12 LPS antigen was obtained only with the serum of patient with typhoid fever. Presence of specific anti-LPS antibodies was also observed in two other patients' sera diluted 1:50, and in case of one of them also in dilution 1:200, but intensity of antigen-antibody reaction was under positive result criterion. The most other sera positive to O=9,12 antigen in law dilutions (1:50, 1:100) by Widal assay, showed the traces of non-specific reaction by immunoblotting. Presence of positive antigen-antibody reaction was indicated for five sera in dilution 1:50 when tested with the >55 kDa H=d flagellar protein subunit, including the serum of patient with typhoid fever. Only in this serum the high level of specific antibodies was detected also in dilution 1:200, what was not observed in case of the other four, which appeared negative. All the other sera were shown not to contain antibodies to flagella antigen. Although the presented results are preliminary and additional study of more sera of people infected with Salmonella Typhi is needed, it can be concluded after Chart et al., that an immunoblotting procedure incorporating O=9,12 LPS and flagellar H=d antigens is a useful method for providing serological evidence of infection with Salmonella Typhi. In our opinion it can serve as a rapid test for the diagnosis of typhoid fever.     

Molecular cloning of a Salmonella typhi LT-like enterotoxin gene

Diarrhoea is a common event during typhoid fever; nevertheless, the possible participation of a diarrhoea-inducing enterotoxin has not been described (Roy et al., 1985). Recombinant bacteriophage lambda FDC1 was isolated from a genomic library of Salmonella typhi, the causal agent of typhoid fever, by screening with a probe for the B subunit gene of the heat-labile, cholera-like, Escherichia coli enterotoxin (LT). Lambda FDC1 codes for an enterotoxin that causes secretion in rat ileal loops, that elongates Chinese hamster ovary (CHO) cells, that is recognized by antibodies against LT, and does not bind in vitro to ganglioside GM1. These results should allow further studies towards elucidating a possible role for the S. typhi enterotoxin in the pathogenesis of typhoid fever.     

Molecular characteristic of antibodies in the blood serum and in the lymph nodes during fractional immunization with small doses of bacterial vaccines.

An experiment on 58 rabbits demonstrated that only macromolecular agglutinins 19S are present in extracts from the lymph nodes after fractional immunization with small doses of typhoid vaccine or some dysentery vaccine. At the same time, antibodies in the blood serum are distributed in fractions of different molecular weight, starting from the 14th day of experiment. It is very probable that this distribution of antibodies is associated with the occurrence in the blood of an inhibitor of the activity of macromolecular antibodies.     

Demonstration of an antigenic protein specific for Salmonella typhi

Current studies were undertaken to determine the presence of a specific antigenic protein on the outer membrane of Salmonella typhi. Immunoblot analysis using sera from patients with fevers revealed that the 50 kD band was specifically recognized only by typhoid sera. The 50 kD band located on the outer membrane is protein by nature and is not a Vi (capsular), dH (flagellar), or O9 (somatic) antigen of S. typhi. These results indicate the usefulness of the specific antigen in the development of a serodiagnostic test for typhoid fever since antibodies of both the IgM and IgG class responses were obtained.     

Effect of aceclidine and fenazepam on the indices of humoral immunity and nonspecific resistance of the body after immunization with a chemical typhoid vaccine

Phenazepam (2.5 mg/kg) and aceclidine (4 mg/kg), when introduced in a single injection at the inductive phase of immune response, enhance the production of antibodies after immunization with chemical typhoid vaccine. The combined use of these preparations has been to produce a potentiating effect.     

Bacterial Vaccine in Multidose Tablet Form for Parenteral Use

Dried typhoid vaccines were prepared by freeze-drying of heat-inactivated phenolized-cell suspensions and by an acetone-killed and dried technique. Portions of the dried powder obtained by each method were compressed into tablets. Tests with the products showed that drying and making of tablets did not affect the ability of the vaccines to protect mice or elicit antibodies in rabbits.     

Reactivity of typhoid patients sera with stress induced 55 kDa phenotype in Salmonella enterica serovar Typhi

Salmonella faces a variety of stresses including acid and heat, in the natural environment whether in the gastrointestinal tract of mammalian host or in the external environment during transmission where survival and multiplication is a priority for the pathogen. In the present study, Salmonella enterica serovar Typhi was grown under acid (inorganic and organic) as well as heat stress and the outermembrane protein (OMP) profiles were compared. A 55 kDa OMP was found to be expressed with high intensity under the selected stress conditions in comparison to normal conditions. The protein expressed under acidic stress reacted with antibodies raised against heat shock protein indicating the similarity of atleast some of the epitopes. In vivo immunogenicity (reactivity with typhoid patient sera) revealed that the 55kDa protein under each stress condition was reactive with 83% of the typhoid sera. In the light of role of the stress induced proteins in pathogenesis of microbial infections and their immunogenic potential, these findings may be relevant for a better understanding of the host-microbe interactions and for future development of diagnostic and preventive strategies.     

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.     

Facility-based disease surveillance and Bayesian hierarchical modeling to estimate endemic typhoid fever incidence,Kilimanjaro Region,Tanzania, 2007–2018

Growing evidence suggests considerable variation in endemic typhoid fever incidence at some locations over time, yet few settings have multi-year incidence estimates to inform typhoid control measures. We sought to describe a decade of typhoid fever incidence in the Kilimanjaro Region of Tanzania. Cases of blood culture confirmed typhoid were identified among febrile patients at two sentinel hospitals during three study periods: 2007–08, 2011–14, and 2016–18. To account for under-ascertainment at sentinel facilities, we derived adjustment multipliers from healthcare utilization surveys done in the hospital catchment area. Incidence estimates and credible intervals (CrI) were derived using a Bayesian hierarchical incidence model that incorporated uncertainty of our observed typhoid fever prevalence, of healthcare seeking adjustment multipliers, and of blood culture diagnostic sensitivity. Among 3,556 total participants, 50 typhoid fever cases were identified. Of typhoid cases, 26 (52%) were male and the median (range) age was 22 (<1–60) years; 4 (8%) were aged <5 years and 10 (20%) were aged 5 to 14 years. Annual typhoid fever incidence was estimated as 61.5 (95% CrI 14.9–181.9), 6.5 (95% CrI 1.4–20.4), and 4.0 (95% CrI 0.6–13.9) per 100,000 persons in 2007–08, 2011–14, and 2016–18, respectively. There were no deaths among typhoid cases. We estimated moderate typhoid incidence (≥10 per 100 000) in 2007–08 and low (<10 per 100 000) incidence during later surveillance periods, but with overlapping credible intervals across study periods. Although consistent with falling typhoid incidence, we interpret this as showing substantial variation over the study periods. Given potential variation, multi-year surveillance may be warranted in locations making decisions about typhoid conjugate vaccine introduction and other control measures.     

vi—PHA试剂的研制及其在检测伤寒带者中的应用

Purified S. typhi Vi antigen is sensitized with equal volume of tannic acid treated formalational sheep erythrocytes (SRBC) at a final concentration of 1 microgram/ml. The Vi-passive hemagglutination assay (Vi-PHA) diagnostic reagent is developed to detect Vi antibodies to S. typhi for the detection of chronic carriers after typhoid fever and the screening S. typhi healthy carriers from food-handlers, which is characterized with high sensitivity, strong specificity and good stability. This Vi-PHA reagent is able to detect 1.16 micrograms/ml of Vi antibodies and doesn't make any cross reaction with healthy sera. For the sera of other diseases, the cross rate is only 0.84%. Using this reagent, 19 positive sera (6.93%) are detected from 274 convalescent sera from typhoid fever, 14 convalescents of which are stool-culture S. typhi positive, that persists a positive rate of 73.68%; 3 positive sera are detected from 106 foodhandlers, one of which is stool-culture S. typhi positive. Therefore, the reagent is simple, convenient, rapid and easy to be applicated in basic unit.     

Genetically engineered Bifidobacterium animalis expressing the Salmonella flagellin gene for the mucosal immunization in a mouse model

BACKGROUND: A critical component of the host defense against enteric infections is the immunological response of the mucosal membrane, a major starting point of infectious disease, such as typhoid fever. The mucosal immune system consists of an integrated network of lymphoid tissues, mucous membrane-associated cells, and effector molecules. In the present study, we developed a recombinant Bifidobacterium animalis (B. animalis) genetically modified with the Salmonella flagellin gene for mucosal immunization as an oral typhoid vaccine. METHODS: We constructed an oral vaccine against Salmonella typhimurium, consisting of recombinant B. animalis containing the flagellin gene of Salmonella. The recombinant B. animalis was administered orally to mice every other day for 6 weeks. Anti-flagellin antibodies in the serum and stools were measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: We detected significantly higher levels of flagellin-specific IgA in the serum and stools of the mice treated with the recombinant B. animalis containing the flagellin gene than was seen in those treated with parental B. animalis. CONCLUSIONS: Our findings suggest that an oral vaccination using recombinant B. animalis genetically modified with the flagellin gene of Salmonella may be effective against Salmonella infections.     

Peptides representing the specific variable regions but not the full porin proteins can be useful for antibody based diagnosis of typhoid fever

Antibody response to major porin proteins of S. Typhi (OmpC and OmpF) was evaluated in sera of typhoid patients (culture positive, n = 28; Widal positive, n = 16). Sera from fever patients (n = 6) having etiology other than Salmonella, and normal healthy human controls (n = 18) were also included. No significant difference between the anti-OmpC and anti-OmpF antibodies (Ab) of typhoid patients and controls was observed. The amino acid sequences of OmpC (and OmpF) porin of enterobacteria was aligned and searched for the variable regions specific to S. Typhi. Two regions, each representing one specific variable region of OmpC and OmpF, were selected (peptides for these regions were custom synthesized). The peptides were evaluated for Ab response of sera. A significantly higher level of Ab to both the peptides was observed in the sera of typhoid patients. The findings suggest that porins of S. Typhi are cross reactive and are not good markers for Ab-based diagnosis of typhoid fever, however, peptides representing the variable regions specific to S. Typhi may have greater diagnostic potential.     

Multi‐isotype antibody responses against the multimeric Salmonella Typhi recombinant hemolysin E antigen

The detection and measurement of different antibody isotypes in the serum provide valuable indicators of the different stages of typhoid infection. Here, the ability of S. Typhi recombinant hemolysin E (HlyE) to detect multi‐isotype antibody responses in sera of patients with typhoid and paratyphoid A was investigated using an indirect antibody immunoassay. Nanogram amounts of HlyE were found to be sufficient for detection of IgG and IgA isotypes and, in a study of individuals' sera (n = 100), the immunoassay was able to distinguish between typhoid and non‐typhoid sera. The overall sensitivity, specificity and efficiency of the ELISA were 70% (39/56), 100% (44/44) and 83% respectively.     

Typhoid fever survey in two localities in Vietnam

As a part of multipurpose health survey of the population in Vietnam the antibodies against S. typhi were determined by the micromethod using haemagglutination test (O-antigen 9, 12) and agglutination test using standard H-diagnostic antigen (d). Totally 292 sera were examined, 139 from Duyen Thai village and 154 from Mai Chau. The data on vaccination against typhoid fever are recorded only in 102 persons. The positivity on Vi antibodies is very high--70% in Duyen Thai and 47% in Mai Chau. This finding is significant according to the high titres in the carriers of S. typhi. The titres of all antibodies are lower in Mai Chau area situated in mountains then in crowded lowlands of Duen Thai. The level of antibodies is decreasing with age. The frequency distribution of antibodies by age proves endemicity of the disease in area, where a large part of population is infected already before reaching 20 years of age. The effectivities of vaccination is discussed.