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Salmonella Paratyphi A, the pathogen of paratyphoid A accounts for an obviously growing proportion of cases in many areas. Therefore, development of specific paratyphoid A vaccines is needed. In the present study, the poxA gene of Salmonella Paratyphi A, encoding the aminoacyl-tRNA synthetase, was deleted successfully by the method of lambda Red recombination system, the resulting strain, ΔpoxA was characterized in respect of growth, adhesion and invasion, virulence, immunogenicity and protective efficacy. It was found that the growth of the ΔpoxA strain was significantly delayed compared with the wild type strain, the mutant ΔpoxA was less invasive to Caco-2 BBE epithelioid cells and THP-1 macrophages than the wild type strain, strain ΔpoxA was attenuated at least 1000-fold in mice, significant immune response and efficient protection were provided by the mutant ΔpoxA after oral immunization. It is concluded that the Salmonella Paratyphi A poxA deletion mutant ΔpoxA can be used as a live oral vaccine candidate against paratyphoid A., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Enteric fever remains a major public health problem in South and Southeast Asia. The recent roll-out of the typhoid conjugate vaccine protecting against S. Typhi exhibits great promise for disease reduction in high burden areas. However, some endemic regions remain vulnerable to S. Paratyphi A due to a lack of licensed vaccines and inadequate WASH. Several developmental S. Paratyphi A vaccines exploit O-antigen as the target antigen. It has been hypothesised that O-antigen is under selective and environmental pressure, with mutations in O-antigen biosynthesis genes being reported, but their phenotypic effects are unknown. Here, we aimed to evaluate O-antigen variation in S. Paratyphi A originating from Nepal, and the potential effect of this variation on antibody binding. O-antigen variation was determined by measuring LPS laddering shift following electrophoresis; this analysis was complemented with genomic characterisation of the O-antigen region. We found structural O-antigen variation in <10 % of S. Paratyphi A organisms, but a direct underlying genetic cause could not be identified. High-content imaging was performed to determine antibody binding by commercial O2 monoclonal (mAb) and polyclonal antibodies, as well as polyclonal sera from convalescent patients naturally infected with S. Paratyphi A. Commercial mAbs detected only a fraction of an apparently "clonal" bacterial population, suggesting phase variation and nonuniform O-antigen composition. Notably, and despite visible subpopulation clusters, O-antigen structural changes did not appear to affect the binding ability of polyclonal human antibody considerably, which led to no obvious differences in the functionality of antibodies targeting organisms with different O-antigen conformations. Although these results need to be confirmed in organisms from alternative endemic areas, they are encouraging the use of O-antigen as the target antigen in S. Paratyphi A vaccines., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Typhoid and Paratyphoid fever cause a global health burden, especially for the children of Southern Asia. The impact of the disease is further exacerbated by the dramatic increase of antimicrobial resistance. While vaccines against Salmonella Typhi have been developed and successfully introduced, an effective vaccine targeting S. Paratyphi A is still lacking. Several efforts are currently ongoing to develop vaccines targeting both S. Typhi and S. Paratyphi A. In order to analyze the immune response induced by vaccination and in sero-epidemiological studies, easy to perform and high throughput immunoassays are needed. Here we present the setup and characterization of a customized ELISA assay and of a luminescent-based serum bactericidal assay (L-SBA) to measure the quantity of S. Paratyphi O antigen specific antibodies and their functional activity against S. Paratyphi A. Robust quality control criteria have been put in place both for ELISA and SBA and assays have been fully characterized in terms of quantitation limit, limit of blanks, specificity, linearity and precision. Assays are being employed to analyze samples from clinical trials, enabling the assessment of immunogenicity during clinical vaccine development., Competing Interests: This work was sponsored by GlaxoSmithKline Biologicals SA. GSK Vaccines Institute for Global Health Srl is an affiliate of GlaxoSmithKline Biologicals SA. MC, LR, LM, MI, FBS, SR and OR are employees of the GSK group of companies. EL, BS, PP, EM, SG are employees of Vismederi Srl. FBS, MI, SR, MC, and OR report ownership of GSK shares/share options. AJP is chair of the UK Department of Health and Social Care’s Joint Committee on Vaccination and Immunisation, is chair of WHOs technical advisory group on Salmonella vaccines and was a member of WHO’s SAGE until 2022. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Carducci, Massai, Lari, Semplici, Grappi, Maria, Jones, Conti, Piu, Scorza, Iturriza-Gómara, Montomoli, Pollard, Rondini and Rossi.)

Enteric fever is caused by the infection of Gram-negative bacteria, Salmonella enterica serovar Typhi and Salmonella enterica serovar Paratyphi (S. Paratyphi) A, B and C, through contaminated food and water. The disease almost exclusively affects the populations living in low- and middle-income countries, with the World Health Organization Southeast Asian Region (WHO SEAR) having the highest endemicity. Despite humans being the sole reservoir of infection and antibiotics and vaccines are made available, the disease was not taken up for elimination until recently due to several biological and technical reasons, including the lack of accurate and region-specific disease surveillance data in the real-time diagnostic inaccuracy of acute infections, difficulty in identifying the chronic asymptomatic carriers who are the major reservoirs of infection and the absence of a political will. However, there is now a renewed interest and effort to control the disease in the endemic areas with the help of better surveillance tools to monitor disease burden, wider availability of more accurate blood culture methods for diagnosis, and above all, cost-effective typhoid conjugate vaccines (TCVs) that can provide a high level of durable protection, particularly against the multidrug-resistant strains and to the age group most commonly affected by the disease. However, despite the commercial availability of a few TCVs, they are still in the development stage. Several questions need to be answered before they are taken up for routine immunization in countries like India. Furthermore, typhoid vaccines with a wider coverage, including additional efficacy against Salmonella Paratyphi A and B and preferably the non-typhoidal Salmonella (NTS) serovars, for which no vaccines are currently available would be more desirable. We have developed several subunit vaccine candidates containing the glycoconjugates of the surface polysaccharides of typhoidal and non-typhoidal Salmonellae and an intrinsic Salmonella protein that functions as both antigen and adjuvant. We also developed a novel mouse model of oral Salmonella Typhi infection to test the candidate vaccines, which demonstrated broad protective efficacy against Salmonella spp. through the induction of humoral and cell-mediated immunity as well as memory response.

Typhoid vaccine development has been impeded by inability of currently available vaccines to induce cellular immunity along with neutralizing antibodies against all serovars of S. Typhi and S. Paratyphi. Unfortunately, antibiotic treatment has shown to be an ineffective therapy due to development of resistance against multiple antibiotics. In the present study, we have explored the immunogenicity and protective efficacy of in-silico designed multi-epitope DnaK peptides as candidate vaccine molecules against Salmonella. Immunization studies in mouse typhoid model revealed three of these peptides (DP1, DP5 and DP7) are highly efficacious, stimulating both humoral and cell mediated immunity along with long lasting antibody memory response. There was significant increase in antibody titers (IgG, IgG1, IgG2a, IgA and IgM), lymphocyte proliferative responses and cytokine levels. Immunized groups showed marked reduction in organ bacterial load, fecal shedding and pronounced protection (upto 80%) as compared to unimmunized controls after challenge with S. typhimurium. Our results demonstrate the huge potential of DnaK peptide vaccine candidates (DP1, DP5 and DP7) to accord protective immunity with significant increase in survivability against Salmonella infection in mice, thus commending these molecules as promising agents to tackle typhoid., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Several serotypes of non-host-specific or paratyphoid Salmonella have been linked with contamination of poultry meat, and eggs, resulting in foodborne outbreaks in humans. Vaccination of poultry against paratyphoid Salmonella is a frequent strategy used to reduce the levels of infection and transmission, which ultimately can lead to lower rates of human infections. Live vaccines have been developed and used in poultry immediately after hatching as a result of their ability to colonize the gut, stimulate a mucosal immune response, induce a competitive inhibitory effect against homologous wild strains, and reduce colonization and excretion of Salmonella . Furthermore, vaccines can competitively exclude some heterologous strains of Salmonella from colonizing the gastrointestinal tract when young poultry are immunologically immature. In addition, various studies have suggested that booster vaccination with live vaccines a few weeks after initial vaccination is essential to increase the level of protection and achieve better cross-protective immunity. Vaccination of breeders, broilers, layers, and turkeys with modified live Salmonella vaccines is a common intervention that has become an important component in poultry companies' multistep prevention programs to meet increasingly demanding customer and regulatory food safety requirements. Both live and inactivated vaccines play a critical role in a comprehensive control program for chicken and turkey breeders and commercial layers. This review examines the response and protection conferred by live modified vaccines against non-host-specific Salmonella that can be considered for the design and implementation of vaccination strategies in poultry.

Typhoid and paratyphoid fevers have a high incidence worldwide and coexist in many geographical areas, especially in low-middle-income countries (LMIC) in South and Southeast Asia. There is extensive consensus on the urgent need for better and affordable vaccines against systemic Salmonella infections. Generalized modules for membrane antigens (GMMA), outer membrane exosomes shed by Salmonella bacteria genetically manipulated to increase blebbing, resemble the bacterial surface where protective antigens are displayed in their native environment. Here, we engineered S Paratyphi A using the pDC5- viaB plasmid to generate GMMA displaying the heterologous S Typhi Vi antigen together with the homologous O:2 O antigen. The presence of both Vi and O:2 was confirmed by flow cytometry on bacterial cells, and their amount was quantified on the resulting vesicles through a panel of analytical methods. When tested in mice, such GMMA induced a strong antibody response against both Vi and O:2, and these antibodies were functional in a serum bactericidal assay. Our approach yielded a bivalent vaccine candidate able to induce immune responses against different Salmonella serovars, which could benefit LMIC residents and travelers., (Copyright © 2021 American Society for Microbiology.)

Enteric fever is a systemic infection caused by Salmonella Typhi or Paratyphi A. In many endemic areas, these serovars co-circulate and can cause multiple infection-episodes in childhood. Prior exposure is thought to confer partial, but incomplete, protection against subsequent attacks of enteric fever. Empirical data to support this hypothesis are limited, and there are few studies describing the occurrence of heterologous-protection between these closely related serovars. We performed a challenge-re-challenge study using a controlled human infection model (CHIM) to investigate the extent of infection-derived immunity to Salmonella Typhi or Paratyphi A infection. We recruited healthy volunteers into two groups: naïve volunteers with no prior exposure to Salmonella Typhi/Paratyphi A and volunteers previously-exposed to Salmonella Typhi or Paratyphi A in earlier CHIM studies. Within each group, participants were randomised 1:1 to oral challenge with either Salmonella Typhi (104 CFU) or Paratyphi A (103 CFU). The primary objective was to compare the attack rate between naïve and previously challenged individuals, defined as the proportion of participants per group meeting the diagnostic criteria of temperature of ≥38°C persisting for ≥12 hours and/or S. Typhi/Paratyphi bacteraemia up to day 14 post challenge. The attack-rate in participants who underwent homologous re-challenge with Salmonella Typhi was reduced compared with challenged naïve controls, although this reduction was not statistically significant (12/27[44%] vs. 12/19[63%]; Relative risk 0.70; 95% CI 0.41-1.21; p = 0.24). Homologous re-challenge with Salmonella Paratyphi A also resulted in a lower attack-rate than was seen in challenged naïve controls (3/12[25%] vs. 10/18[56%]; RR0.45; 95% CI 0.16-1.30; p = 0.14). Evidence of protection was supported by a post hoc analysis in which previous exposure was associated with an approximately 36% and 57% reduced risk of typhoid or paratyphoid disease respectively on re-challenge. Individuals who did not develop enteric fever on primary exposure were significantly more likely to be protected on re-challenge, compared with individuals who developed disease on primary exposure. Heterologous re-challenge with Salmonella Typhi or Salmonella Paratyphi A was not associated with a reduced attack rate following challenge. Within the context of the model, prior exposure was not associated with reduced disease severity, altered microbiological profile or boosting of humoral immune responses. We conclude that prior Salmonella Typhi and Paratyphi A exposure may confer partial but incomplete protection against subsequent infection, but with a comparable clinical and microbiological phenotype. There is no demonstrable cross-protection between these serovars, consistent with the co-circulation of Salmonella Typhi and Paratyphi A. Collectively, these data are consistent with surveillance and modelling studies that indicate multiple infections can occur in high transmission settings, supporting the need for vaccines to reduce the burden of disease in childhood and achieve disease control. Trial registration NCT02192008; clinicaltrials.gov., Competing Interests: I have read the journal's policy and the authors of this manuscript have the following competing interests: A.J.P chairs the UK Department of Health’s (DH) Joint Committee on Vaccination and Immunisation (JCVI) and the European Medicines Agency Scientific Advisory Group on Vaccines and is a member of the World Health Organization’s (WHO) Strategic Advisory Group of Experts. The views expressed in this manuscript are those of the authors and do not necessarily reflect the views of the JCVI, the DH, or the WHO. Author Vincenzo Cerundulo was unable to confirm his authorship contributions. On his behalf, the corresponding author has reported his contributions to the best of their knowledge.

Paratyphoid fever caused by Salmonella Paratyphi A is a serious public health problem in many countries. In order to and develop a live attenuated candidate vaccine of Salmonella Paratyphi A, a Salmonella pathogenicity island 2 (SPI2, approximate 40 kb) deletion mutant of Salmonella Paratyphi A was constructed by lambda Red recombination, then the biological characteristics and protective ability of the Salmonella Paratyphi A SPI2 mutant were evaluated. Our results showed that the growth and biochemical properties of the SPI2 mutant were consistent with that of its parent strain, and the mutant was stable with the loss of SPI2. The mice lethal test showed that the virulence of the SPI2 mutant was significantly decreased, it can colonize and persistent more than 14 days in the liver and spleen of mice. Vaccination with the SPI2 mutant in mice revealed no significant effect on body weight and clinical symptoms compared to control animals, and specific humoral and cellular immune responses were also significantly induced. Immunization of mice offered efficient protection against Salmonella Paratyphi A strain challenge at 14 days post vaccination based on mortality and clinical symptoms relative to control group. Overall, these findings suggested that SPI2 plays an important role in pathogenicity of Salmonella Paratyphi A, and the SPI2 mutant showed its potential to develop a live attenuated vaccine candidate., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Enteric fever, caused by Salmonella enterica serovars, Typhi (S. Typhi) and Paratyphi (S. Paratyphi) is a major public health challenge for the developing nations. Globally, the disease affects ˜15-30 million individuals every year, resulting in >200,000 deaths. Multidrug-resistant S. Typhi H58 strain has emerged as the dominant circulating strain in a large part of the world and an extensively drug-resistant (XDR) subclade of the strain was recently reported. Many believe that vaccination of the susceptible populations is urgently needed and the best option to control the infection. However, the commercial live attenuated (Ty21a) vaccine is not recommended for children below six years of age while the Vi-polysaccharide-based vaccine has poor long-term efficacy against typhoid fever. Moreover, no vaccines are available against S. Paratyphi infection. Thus, a new formulation capable of providing long term protection against both the pathogens and safe for all age groups is immediately required. We show that recombinant, S. Typhi outer membrane protein STIV (rSTIV) is immunogenic in mice and elicits high serum titers of different immunoglobulin subtypes. STIV antibodies opsonize S. Typhi and S. Paratyphi A to promote antibody-dependent cellular cytotoxicity and complement-mediated lysis. Immunization with rSTIV also induces robust cell-mediated immunity, including antigen-specific T cell proliferation and cytotoxic T lymphocyte response. Finally, mice immunized with rSTIV are significantly protected against S. Typhi and S. Paratyphi A challenge, with reduced visceral bacterial load. Our results underscore the potential of rSTIV as a novel vaccine candidate for enteric fever., (Copyright © 2019 Elsevier GmbH. All rights reserved.)

Mucosal-associated invariant T (MAIT) cells are innate-like T cells that can detect bacteria-derived metabolites presented on MR1. Here we show, using a controlled infection of humans with live Salmonella enterica serovar Paratyphi A, that MAIT cells are activated during infection, an effect maintained even after antibiotic treatment. At the peak of infection MAIT cell T-cell receptor (TCR)β clonotypes that are over-represented prior to infection transiently contract. Select MAIT cell TCRβ clonotypes that expand after infection have stronger TCR-dependent activation than do contracted clonotypes. Our results demonstrate that host exposure to antigen may drive clonal expansion of MAIT cells with increased functional avidity, suggesting a role for specific vaccination strategies to increase the frequency and potency of MAIT cells to optimize effector function.

Introduction: Typhoid and paratyphoid fever remain a global health problem, which - in non-endemic countries - are mainly seen in travelers, particularly in VFRs (visiting friends and relatives), with occasional local outbreaks occurring. A rise in anti-microbial resistance emphasizes the role of preventive measures, especially vaccinations against typhoid and paratyphoid fever for travelers visiting endemic countries. Areas covered: This state-of-the-art review recapitulates the epidemiology and mechanisms of disease of typhoid and paratyphoid fever, depicts the perspective of non-endemic countries and travelers (VFRs), and collectively presents current European recommendations for typhoid fever vaccination. We provide a brief overview of available (and developmental) vaccines in Europe, present current data on cross-protection to S. Paratyphi, and aim to provide a background for typhoid vaccine decision-making in travelers. Expert commentary: European recommendations are not harmonized. Experts must assess vaccination of travelers based on current country-specific recommendations. Travel health practitioners should be aware of the issues surrounding vaccination of travelers and be motivated to increase awareness of typhoid and paratyphoid fever risks.

Previously we have extensively characterized Salmonella enterica serovar Typhi (S. Typhi)-specific cell-mediated immune (CMI) responses in volunteers orally immunized with the licensed Ty21a typhoid vaccine. In this study we measured Salmonella-specific multifunctional (MF) CD8+ T-cell responses to further investigate whether Ty21a elicits crossreactive CMI against S. Paratyphi A and S. Paratyphi B that also cause enteric fever. Ty21a-elicited crossreactive CMI responses against all three Salmonella serotypes were predominantly observed in CD8+ T effector/memory (T(EM)) and, to a lesser extent, in CD8+CD45RA+ T(EM) (T(EMRA)) subsets. These CD8+ T-cell responses were largely mediated by MF cells coproducing interferon-γ and macrophage inflammatory protein-1β and expressing CD107a with or without tumor necrosis factor-α. Significant proportions of Salmonella-specific MF cells expressed the gut-homing molecule integrin α4β7. In most subjects, similar MF responses were observed to S. Typhi and S. Paratyphi B, but not to S. Paratyphi A. These results suggest that Ty21a elicits MF CMI responses against Salmonella that could be critical in clearing the infection. Moreover, because S. Paratyphi A is a major public concern and Ty21a was shown in field studies not to afford cross-protection to S. Paratyphi A, these results will be important in developing a S. Typhi/S. Paratyphi A bivalent vaccine against enteric fevers.

The global epidemic features of enteric fever have changed greatly in recent years. The incidence of enteric fever caused by Salmonella enterica serovar Paratyphi A has progressively increased. In some areas of Asia, infections with S. Paratyphi A have exceeded those with S. Typhi, resulting in S. Paratyphi A becoming the main causative agent of enteric fever. However, two currently licensed typhoid vaccines do not confer adequate cross-protection against S. Paratyphi A infection. Therefore, development of specific vaccines against enteric fever caused by S. Paratyphi A is urgently needed. In the present study, an attenuated strain was constructed by double deletion of the htrA and yncD genes in a wild-type strain of S. Paratyphi A and its safety and immunogenicity assessed. In a mouse model, the 50% lethal dose of the double deletion mutant and the wild-type strain were 3.0 × 10(8) CFU and 1.9 × 10(3) CFU, respectively, suggesting that the double deletion resulted in remarkably decreased bacterial virulence. Bacterial colonization of the double deletion mutant in the livers and spleens of infected mice was strikingly less than that of the wild-type strain. A single nasal administration of the attenuated vaccine candidate elicited high concentrations of anti-LPS and anti-flagellin IgG in a mouse model and protected immunized mice against lethal challenge with the wild-type strain. Thus, our findings suggest that the attenuated vaccine strain is a promising candidate worthy of further evaluation both as a human enteric fever vaccine and as a vaccine delivery vector for heterologous antigens., (© 2015 The Societies and Wiley Publishing Asia Pty Ltd.)

There are no vaccines in clinical use against paratyphoid fever, caused by Salmonella Paratyphi A and B or, rarely, C. Oral Salmonella Typhi Ty21a typhoid vaccine elicits a significant cross-reactive immune response against S. Paratyphi A and B, and some reports suggest cross-protective efficacy against the disease. These findings are ascribed to the O-12 antigen shared between the strains. The Vi capsular polysaccharide vaccine has been shown to elicit antibodies reactive with O-9,12. Twenty-five volunteers immunized with the parenteral Vi vaccine (Typherix(®) ) were explored for plasmablasts cross-reactive with paratyphoid strains; the responses were compared to those in 25 age- and gender-matched volunteers immunized with Ty21a (Vivotif(®) ). Before vaccination, 48/50 vaccinees had no plasmablasts reactive with the antigens. Seven days after vaccination, 15/25 and 22/25 Vi- and Ty21a-vaccinated volunteers had circulating plasmablasts producing antibodies cross-reactive with S. Paratyphi A, 18/25 and 23/25 with S. Paratyphi B and 16/25 and 9/25 with Paratyphi C, respectively. Compared to the Ty21a group, the Vi group showed significantly lower responses to S. Paratyphi A and B and higher to S. Paratyphi C. To conclude, the Vi vaccine elicited a cross-reactive plasmablast response to S. Paratyphi C (Vi antigen in common) and less marked responses to S. Paratyphi A and B than the Ty21a preparation. S. Paratyphi A and B both being Vi-negative, the result can be explained by trace amounts of bacterial cell wall O-12 antigen in the Vi preparation, despite purification. The clinical significance of this finding remains to be determined., (© 2014 John Wiley & Sons Ltd.)

Salmonella enterica serotype Paratyphi A is a human-restricted pathogen and the cause of paratyphoid A fever. Using a high-throughput immunoscreening technique, in vivo-induced antigen technology (IVIAT), we identified 20 immunogenic bacterial proteins expressed in humans who were bacteremic with S. Paratyphi A but not those expressed in S. Paratyphi A grown under standard laboratory conditions. The majority of these proteins have known or potential roles in the pathogenesis of S. enterica. These include proteins implicated in cell adhesion, fimbrial structure, adaptation to atypical conditions, oxidoreductase activity, proteolysis, antimicrobial resistance, and ion transport. Of particular interest among these in vivo-expressed proteins were S. Paratyphi A (SPA)2397, SPA2612, and SPA1604. SPA2397 and SPA2612 are prophage related, and SPA1604 is in Salmonella pathogenicity island 11 (SPI-11). Using real-time quantitative PCR (RT-qPCR), we confirmed increased levels of mRNA expressed by genes identified by IVIAT in a comparison of mRNA levels in organisms in the blood of bacteremic patients to those in in vitro cultures. Comparing convalescent- to acute-phase samples, we also detected a significant increase in the reaction of convalescent-phase antibodies with two proteins identified by IVIAT: SPA2397 and SPA0489. SPA2397 is a phage-related lysozyme, Gp19, and SPA0489 encodes a protein containing NlpC/P60 and cysteine, histidine-dependent amidohydrolase/peptidase (CHAP) domains. In a previous study utilizing a different approach, we found that transcripts for 11 and 7 of the genes identified by IVIAT were detectable in organisms in the blood of humans in Bangladesh who were bacteremic with S. Paratyphi A and Salmonella enterica serovar Typhi, respectively. S. Paratyphi A antigens identified by IVIAT warrant further evaluation for their contributions to pathogenesis and might have diagnostic, therapeutic, or preventive relevance.

Growing evidences suggest that Saccharomyces boulardii (SB) is efficacious against bacterial infections and inflammatory bowel diseases. This study investigated the effects of treatment with SB provided in a murine model of typhoid fever. Mice were divided into two groups: (1) control animals challenged with Salmonella Typhimurium (ST), and (2) animals receiving SB, and then challenged with ST. At days 0, 1, 5, 10 and 15 post-challenge, animals were euthanized and tissues collected to analyze bacterial translocation, cytokines, signaling pathways and histological analysis. Survival rate and animal weight were also evaluated. Treatment with SB increased survival rate and inhibited translocation of bacteria after ST challenge. Histological data showed that SB also protected mice against liver damage induced by ST. SB decreased levels of inflammatory cytokines and activation of mitogen-activated protein kinases (p38, JNK and ERK1/2), phospho-IκB, p65-RelA, phospho-jun and c-fos in the colon, signal pathways involved in the activation of inflammation induced by ST. Further experiments revealed that probiotic effects were due, at least in part, to the binding of ST to the yeast. Such binding diminishes ST translocation, resulting in decreased activation of signaling pathways which lead to intestinal inflammation in a murine model of typhoid fever., (Copyright © 2013 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Background: There are no vaccines against paratyphoid fever in clinical use. The disease has become more wide-spread and there is a growing problem of antibiotic resistance among the strains. Previous reports suggest that the oral live Salmonella Typhi Ty21a-vaccine confers protection against paratyphoid B fever. Data on efficacy against paratyphoid A fever are somewhat contentious. The present study investigated the immunological basis for such efficacy reports at a single-cell level: plasmablasts (identified as antibody-secreting cells, ASC) were studied for secretion of antibodies cross-reactive with Salmonella Paratyphi in the circulation of patients with enteric fever and of volunteers vaccinated with Ty21a., Materials and Methods: Thirty volunteers immunized with Ty21a and five patients with enteric fever were investigated for Salmonella Typhi and Salmonella Paratyphi A/B/C-specific circulating plasmablasts. PBMC were sorted by their expression of homing receptors (HR) for the intestine (α4β7), peripheral lymph node (l-selectin) and skin (CLA) and typhoid- and paratyphoid-specific plasmablasts were enumerated with ELISPOT., Results: Before vaccination, no cross-reactive ASC were found in the volunteers. In addition to the Salmonella Typhi-specific response, a significant cross-reactive immune response was mounted against Salmonella Paratyphi A and B both in the patients and the vaccinees. The magnitude of the response increased in the order Salmonella Paratyphi A (median 30 ASC/10(6) PBMC)→Salmonella Paratyphi B (median 81)→Salmonella Typhi (median 301) in the vaccinees. Both in patients and in vaccinees, the homing receptor (HR) selection favored homing to the gut, indicating a humoral intestinal immune response., Conclusions: These immunological data provide evidence consistent with previous reports describing certain levels of cross-protective efficacy of Ty21a against paratyphoid fever. Controlled studies are needed to evaluate cross-protective efficacy. In the current situation where paratyphoid fever is emerging and no vaccines are available, any level of cross-protective capacity is valuable., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Enteric fever is an invasive life-threatening systemic disease caused by the Salmonella enterica human-adapted serovars Typhi and Paratyphi. Increasing incidence of infections with Salmonella enterica serovar Paratyphi A and the spreading of its antibiotic-resistant derivates pose a significant health concern in some areas of the world. Herein, we describe a molecular and phenotypic characterization of an S. Paratyphi A strain accounted for a recent paratyphoid outbreak in Nepal that affected at least 37 travelers. Pulsed-field gel electrophoresis analysis of the outbreak isolates revealed one genetic clone (pulsotype), confirming a single infecting source. Genetic profiling of the outbreak strain demonstrated the contribution of specific bacteriophages as a prime source of genetic diversity among clinical isolates of S. Paratyphi A. Phenotypic characterization in comparison with the S. Paratyphi A ATCC 9150 reference sequenced strain showed differences in flagellar morphology and increased abilities of the outbreak strain with respect to its motility, invasion into nonphagocytic cells, intracellular multiplication, survival within macrophages, and higher induction of interleukin-8 (IL-8) secreted by host cells. Collectively, these differences suggest an enhanced virulence potential of this strain and demonstrate an interesting phenotypic variation among S. Paratyphi A isolates. In vivo profiling of 16 inflammatory cytokines in patients infected with the outbreak strain revealed a common profile of a remarkable gamma interferon (IFN-γ) induction together with elevated concentrations of tumor necrosis factor alpha (TNF-α), IL-6, IL-8, IL-10, and IL-15, but not IL-12, which was previously demonstrated as elevated in nontyphoidal Salmonella infections. This apparent profile implies a distinct immune response to paratyphoid infections.

Enteric fevers remain a common and serious disease, affecting mainly children and adolescents in developing countries. Salmonella enterica serovar Typhi was believed to cause most enteric fever episodes, but several recent reports have shown an increasing incidence of S. Paratyphi A, encouraging the development of a bivalent vaccine to protect against both serovars, especially considering that at present there is no vaccine against S. Paratyphi A. The O-specific polysaccharide (O:2) of S. Paratyphi A is a protective antigen and clinical data have previously demonstrated the potential of using O:2 conjugate vaccines. Here we describe a new conjugation chemistry to link O:2 and the carrier protein CRM(197), using the terminus 3-deoxy-D-manno-octulosonic acid (KDO), thus leaving the O:2 chain unmodified. The new conjugates were tested in mice and compared with other O:2-antigen conjugates, synthesized adopting previously described methods that use CRM(197) as carrier protein. The newly developed conjugation chemistry yielded immunogenic conjugates with strong serum bactericidal activity against S. Paratyphi A.

Background: Antibiotic-resistant Salmonella enterica serovar Paratyphi A, the agent of paratyphoid A fever, poses an emerging public health dilemma in endemic areas of Asia and among travelers, as there is no licensed vaccine. Integral to our efforts to develop a S. Paratyphi A vaccine, we addressed the role of flagella as a potential protective antigen by comparing cell-associated flagella with exported flagellin subunits expressed by attenuated strains., Methodology: S. Paratyphi A strain ATCC 9150 was first deleted for the chromosomal guaBA locus, creating CVD 1901. Further chromosomal deletions in fliD (CVD 1901D) or flgK (CVD 1901K) were then engineered, resulting in the export of unpolymerized FliC, without impairing its overall expression. The virulence of the resulting isogenic strains was examined using a novel mouse LD(50) model to accommodate the human-host restricted S. Paratyphi A. The immunogenicity of the attenuated strains was then tested using a mouse intranasal model, followed by intraperitoneal challenge with wildtype ATCC 9150., Results: Mucosal (intranasal) immunization of mice with strain CVD 1901 expressing cell-associated flagella conferred superior protection (vaccine efficacy [VE], 90%) against a lethal intraperitoneal challenge, compared with the flagellin monomer-exporting mutants CVD 1901K (30% VE) or CVD 1901D (47% VE). The superior protection induced by CVD 1901 with its cell-attached flagella was associated with an increased IgG2a:IgG1 ratio of FliC-specific antibodies with enhanced opsonophagocytic capacity., Conclusions: Our results clearly suggest that enhanced anti-FliC antibody-mediated clearance of S. Paratyphi A by phagocytic cells, induced by vaccines expressing cell-associated rather than exported FliC, might be contributing to the vaccine-induced protection from S. Paratyphi A challenge in vivo. We speculate that an excess of IgG1 anti-FliC antibodies induced by the exported FliC may compete with the IgG2a subtype and block binding to specific phagocyte Fc receptors that are critical for clearing an S. Paratyphi A infection.

We aimed to elucidate the immunological (cytokines), biochemical (antioxidative), and patho-morphological responses in the gut and liver evoked by the addition of Lactobacillus fermentum ME-3 to ofloxacin (OFX) treatment in an experimental infection model of Salmonella enterica serovar Typhimurium. After challenge with S. Typhimurium and treatment according to different schemes, either with OFX and/or addition of L. fermentum ME-3, the mice were killed. Blood, liver, spleen, and small intestine samples were plated to detect S. Typhimurium and lactobacilli. Histological slides were prepared from the liver and ileum. The cytokines (IL-10, IFN-γ, and TNF-α), the glutathione peroxidase and reductase, the glutathione ratio, and the lipid peroxides (LPO) in mucosa of the small intestine and liver were estimated. The addition of L. fermentum ME-3 to OFX increased the eradication of S. Typhimurium from tested sites because of antagonistic and antioxidative properties, reduced the presence of typhoid nodules in the liver, and decreased the values of LPO. The immunological response included the reduction of pro-inflammatory cytokines interferon-γ and tumour necrosis factor-α and the increase in anti-inflammatory cytokine interleukin-10 in the livers of mice without typhoid nodules., (© 2010 The Authors. Journal Compilation © 2010 APMIS.)

Eight Salmonella enterica serovar Paratyphi A strains were screened as candidates to create a live attenuated paratyphoid vaccine. Based on biochemical and phenotypic criteria, four strains, RKS2900, MGN9772, MGN9773 and MGN9779, were selected as progenitors for the construction of DeltaphoPQ mutant derivatives. All strains were evaluated in vitro for auxotrophic phenotypes and sensitivity to deoxycholate and polymyxin B. All DeltaphoPQ mutants were more sensitive to deoxycholate and polymyxin B than their wild-type progenitors, however MGN10028, MGN10044 and MGN10048, required exogenous purine for optimal growth. Purine requiring strains had acquired point mutations in purB during strain construction. All four mutants were evaluated for reactogenicity and immunogenicity in an oral rabbit model. Three strains were reactogenic in a dose-dependent manner, while one strain, MGN10028, was well-tolerated at all doses administered. All DeltaphoPQ strains were immunogenic following a single oral dose. The in vitro profile coupled with the favorable reactogenicity and immunogenicity profiles render MGN10028 a suitable live attenuated Paratyphi A vaccine candidate.

The Vi capsular polysaccharide vaccine is one of two vaccines against typhoid recommended worldwide and is the vaccine generally used in China. However, in recent years a Salmonella paratyphi A strain that is naturally devoid of capsule has caused frequent outbreaks of typhoid fever in Southern China, leading to the need for identification of additional antigens that could be incorporated into new vaccines. SpaO acts as a major invasion factor of Salmonella enterica spp. and H1a is the unique flagellin subunit ofS. paratyphi A. In this study, the two prokaryotic recombinant antigens, rSpaO and rH1a, were expressed and their immunogenicity was demonstrated by the slide agglutination test and Western blot assays. Using PCR and sequencing analysis as well as ELISA, we find that the spaO and h1a genes are widely distributed in 196 S. paratyphi A isolates (97.5 and 100%, respectively), with high expression frequencies for the SpaO (98.0%) and H1a (100%) antigens. The two genes also show high sequence conservation (similarities from 99.31 to 99.88% for both genes). In sera from 172 paratyphoid A patients, anti-SpaO and anti-H1a IgGs were detectable by ELISA, in 94.8 and 98.8% of patients, respectively. Furthermore, 41.7-66.7% of mice immunized with rSpaO or rH1a alone were protected against subsequent infection, and the protection rate rose to 75.0-91.7% in mice co-immunized with the two antigens. As the spaO and h1a genes of S. paratyphi A are sequence conserved, extensively distributed and highly expressed, the rSpaO and rH1a immunogens should be considered in the development of novel vaccines to prevent S. paratyphi A-caused typhoid fever.

Objective: To determine the baseline Widal titres in apparently healthy children in Davangere., Methods: Cross-sectional study was done on 250 children. Widal titers were found using tube agglutination test., Results: Out of 250,64.2% had a titre of less than 1:20,22.4% had a titre equal to 1:20,9.6% had a titre of 1:40 and 3.6% had a titre of 1:80 to 'O' antigen and 67.2% had a titre of less than 1:20, 21.2% had a titre equal to 1:20, 8% had a titre of 1:40 and 3.6% had a titre of 1:80 to 'H' antigen of S. enterica subsp. enterica ser. Typhi. No children in age group 6 months-2 years had a titre of 1:80 to either antigen. All children in this age group had a titre of less than 1:20 to AH antigen and older children had a titre upto 1:40 dilution., Conclusion: Baseline titres for either S. enterica subsp. enterica ser. Typhi antigen in 6 month-2 year was 1:40 and older children was 1:80. Baseline titres for H antigen of S. enterica subsp. enterica ser. Paratyphi A in 6 month-2 year was less than 1:20 and for older children was 1:40 dilution.

In randomized, controlled field trials in Area Norte and Area Occidente of Santiago, Chile, 2 (Norte) or 3 (Occidente) doses of live oral typhoid vaccine Ty21a in enteric-coated capsules conferred protection against confirmed Salmonella enterica serovar Typhi disease (53% efficacy in Norte; 67% efficacy in Occidente) during 3 years of follow-up. There was also a trend in each trial showing protection against S. enterica serovar Paratyphi B disease (56% efficacy in Norte; 42% efficacy in Occidente). To enhance statistical power, an analysis was performed using pooled data from the 2 trials; this pooling of data was justified by the following facts: epidemiologic surveillance and microbiological methods were identical, the trials overlapped during 22 of the 36 months of follow-up in each trial, the estimates of efficacy against paratyphoid B fever in the 2 trials were roughly similar, and the ratio of follow-up of vaccine recipients to control subjects in both trials was ~1 : 1. In the pooled analysis, Ty21a conferred significant protection against paratyphoid B fever (efficacy, 49%; 95% confidence interval, 8%-73%; P=.019).

ClyASTy and ClyASPaA are closely related pore-forming cytolysins of Salmonella enterica serovars Typhi and Paratyphi A whose expression is strongly repressed under standard in vitro growth conditions. We show here that human infections by these pathogens cause a specific antibody response to ClyA, indicating effective toxin production during infection.

Objective: To study the regulating effects of a novel CpG oligodeoxynuleotide and the synergistic effect of chitosan-nanoparticles (CNP) with CpG on immune responses of mice, which were used to develop a novel immunoadjuvant to boost immune response to conventional vaccines., Methods: A novel CpG ODN containing 11 CpG motifs was synthesized and its bioactivities to stimulate the proliferation of lymphocytes of pig in vitro were detected. Then it was entrapped with CNP prepared in our laboratory by the method of ionic cross linkage, and immunized Kunming mice were co-inoculated with paratyphoid vaccine. The peripheral blood was collected weekly from the tail vein of inoculated mice to detect the contents of IgG, IgA, IgM, and specific antibody against salmonella as well as the levels of interleukin-2 (IL2), IL-4, and IL-6 by SABC-ELISA assay. The numbers of leucocytes, monocytes, granuloytes, and lymphocytes were calculated separately using the routine method. The experimental mice were orally challenged with virulent salmonella 35 days after inoculation., Results: This CpG ODN could remarkably provoke the proliferation of lymphocytes of pig in vitro in contrast with the control (P < 0.05). Compared with those of the control, immunoglobulins, including IgG, IgA, IgM, and specific antibodies to paratyphoid vaccine, increased significantly in sera from the CpG or CpG-CNP-vaccinated mice (P < 0.05). IL-2, IL-4, and IL-6 increased remarkably in sera from immunized mice (P < 0.05). The leucocytes, monocytes, granuloytes, and lymphocytes of the mice immunized with CpG or CpG-CNP were also increased in number (P < 0.05). After the challenge, these immunity values were elevated in the mice vaccinated with CpG or CpG-CNP. The immunized mice all survived, while the control mice fell ill with evident lesions with diffuse hemorrhage in stomach, small intestine, and peritoneum., Conclusions: CpG ODN entrapped with CNP is a promising effective immunoadjuvant for vaccination, which promotes humoral and cellular immune responses, enhances immunity and resistance against salmonella by co-administration with paratyphoid vaccine.

Background & Objective: Typhoid fever still continues to be a major public health problem around the world. A simple, reliable and affordable rapid diagnostic test has been a long felt need of the clinicians. We therefore prospectively evaluated the sensitivity and specificity of Typhidot test., Methods: The study was carried out between January 2002 and December 2003, on a total of 563 samples from patients clinically suspected to have typhoid fever; blood culture as well as serum for Typhidot test were received., Results: Of the 563 samples, Typhidot test and blood culture were positive in 36 patients, both the tests were negative for 503 patients. Typhidot test was positive for 9 patients with S. Paratyphi A infection. The sensitivity and specificity of the test using blood culture as gold standard were 92.3 and 98.8 per cent respectively for the typhoid fever., Interpretation & Conclusion: Typhidot test is rapid, easy to perform and reliable test for diagnosing typhoid fever, useful for small less equipped laboratories as well as for those with better facilities.

Background: Salmonella antibodies are known to cross-react with other antigens including those from Escherichia coli. Malaria antigens may also have similar characteristics., Objectives: To establish antibody levels in malaria patients and controls and to decipher (if any), relationship(s) between significant agglutinin responses to the various antigens as outcome factor and malaria parasitaemia as exposure factor., Study Setting: University of Benin Teaching Hospital and Egor Local Government Area of Edo state, Nigeria, between July and August, 1999., Study Design: Prospective and a case control study., Patients and Methods: Agglutinins against H and O antigens of Salmonella typhi, Salmonella paratyphi A, B and C were determined using the Widal test in 189 school aged children(5-16 years) with malaria (males, 54.0%; females, 46.0%) and 175 apparently healthy children,(52.0% males, and 48.0 %) of comparable age and gender distributions. Agglutination reaction at >1.80 were significantly more in malaria patients (52/189 or 27.5%) than in controls (28/175 or 16.0%) (x2 = 7.07; p<0.05). Agglutination reaction to various antigens were significantly higher in controls with malaria parasitaemia (10/36 or 27.8%) than those without parasitaemia (18/139 or 12.9%) (x2 = 4.57; p<0.02) (OR=2.59). Significant titres were also more in subjects and controls with malaria parasitaemia (62/235 or 26.38%) than in those without (18/139 or 12.94%) (x2=9.32; p< 0.05) (OR=2.41). At dilution of > or =1.80 children aged 9 -14 years (as compared to those < or =8 years of age) and more males (subjects and controls) had demonstrable agglutinins. At most serum dilutions, antibodies to H antigens were more commonly found in subjects and controls than agglutinins against O antigen of Salmonella typhi. Fifty percent of subjects and controls had demonstrable agglutination at approximate dilutions of 1:60 and 1:40 respectively. The number of subjects and controls that showed demonstrable agglutinins against Salmonella paratyphi antigens were less than 10.0%, Conclusion: Enhanced agglutinin to DO and DH may be a phenomenon associated with childhood malaria.

Three hundred and seven healthy food handlers and 34 blood-culture positive enteric fever patients were screened for Salmonellae agglutinins using the Widal test. Of the 307 healthy food handlers, only 3 (1.0%) had an anti-O titre of > or = 1/160 and 8 (2.6%) an anti-H titre of > or = 1/320 for Salmonella typhi, but the majority, 214 (69.7%) and 149 (48.5%) had titres of < 1/20 for O and H agglutinins respectively. Similar agglutinin titres were also seen for S. Paratyphi A, B, and C. In the 34 enteric fever patients, for S typhi, based on anti-O titre of > or = 1/160, 25 persons showed a significant titre, a sensitivity of 73.5%, and a specificity of 99.0%. And 21 persons showed a significant titre of > or = 1/320 for anti-H, a sensitivity of 61.8% and a specificity of 97.4%. Based on these findings, titres of > or = 1/160 and > or = 1/320 for anti-0 and anti-H respectively, were considered diagnostic for enteric fever in Kumasi, Ghana.

Objective: To determine the antibody titre levels to typhoid/paratyphoid fever organisms among apparently healthy volunteers., Design: Cross sectional study., Setting: General community and University Teaching Hospital., Participants: Volunteer sample of 323 apparently healthy individuals with body temperatures < or = 37.8 degrees C., Main Outcome Measures: Questionnaire administration to classify volunteers into three socio-economic status (SES)., Results: There were 35.29% of the apparently healthy population in Jos community with antibodies to typhoid/paratyphoid fever organisms. The presence of these antibodies were neither sex nor SES related. Normal antibody titres were up to 1:40 and 1:80 for O and H Salmonella antigens respectively. Contrary to the general belief, typhoid/paratyphoid fevers have not affected virtually everybody in Nigeria. The difference between those without previous history and those with previous history was significant (p < 0.05) with those in the former category having a higher percentage., Conclusion: For a single sero-diagnosis to have any diagnostic value in Jos community and its environs, only a four-fold rise to what has been found to be normal should be significant. This means that only titres of 1:160 and 1:320 and above for O and H antigens should be considered significant.

Salmonella paratyphi A is a pathogenic bacterium that causes paratyphoid fever. The current laboratory diagnostic techniques are unsatisfactory. To improve diagnosis, a plasmid (pSK-8E) encoding phase 1 flagellin gene nucleotide position 452-890 from S. paratyphi A has been constructed. The recombinant protein expressed from the plasmid has been used to develop an indirect ELISA for IgM antibody detection. Sera from patients with hemoculture positive for S. paratyphi A, S. typhi, other gram-positive and gram-negative bacteria, and dengue hemorrhagic fever as well as from healthy control subjects were tested. Sensitivity, specificity, positive and negative predictive values of the test were 56.9%, 98.8%, 90.6% and 92.1%, respectively. Since the sensitivity was low, the explanation for this result was investigated. It was found that the sensitivity of the test could be increased to 83.3% if the sera were obtained 9-12 days after onset of fever. The sera obtained earlier or later gave only 33.3% and 66.6% sensitivity, respectively. This result suggests that the IgM antibody detection assay which we have developed is a valuable tool for diagnosis of S. paratyphi A infection when the serum samples are taken at the appropriate time.

Salmonella paratyphi A, the second most common cause of enteric fever in Southeast Asia, is a habitant of and a pathogen for humans only. Lipopolysaccharides (LPS) are both essential virulence factors and protective antigens for systemic infections caused by groups A, B, C, and D nontyphoidal salmonellae. The O-specific polysaccharide of S. paratyphi A is composed of a trisaccharide, -->2-alpha-D)-Manp-(1-->4)-alpha-L-Rhap-(1-->3)-alpha-D-Galp -(1-->, with a branch of D-paratose from the C-3 of alpha-D-mannose, and the C-3 of beta-L-rhamnose is partially O acetylated (C. G. Hellerqvist, B. Lindberg, K. Samuelsson, and A. A. Lindberg, Acta Chem. Scand. 25:955-961, 1971). On the basis of data from our investigational vaccines for enteric bacterial pathogens, including group B salmonellae (D. C. Watson, J. B. Robbins, and S. C. Szu, Infect. Immun. 60:4679-4686, 1992), conjugates composed of the detoxified LPS of S. paratyphi A bound to tetanus toxoid (TT) were prepared by several schemes. LPS was detoxified with acetic acid or with hydrazine; the latter removed O acetyls from the O-specific polysaccharide. The detoxified polysaccharides were activated with cyanogen bromide (CNBr) or with 1-cyano-4-dimethylaminopyridinium tetratfluoroborate (CDAP) and bound to TT with or without a spacer. Solutions of 2.5 microgram of saccharide, alone or as a conjugate, were injected subcutaneously into young mice, and LPS and TT antibodies were measured by enzyme-linked immunosorbent assaying. A conjugate synthesized with higher-molecular-weight O-SP elicited the highest anti-LPS levels. Only conjugates with O acetyls elicited serum immunoglobulin G anti-LPS with bactericidal activity. There were no statistically significant differences between LPS antibody levels elicited by conjugates synthesized with or without a spacer. The conjugate with O-specific polysaccharide activated by CDAP and bound to TT without a spacer elicited the highest level of TT antibodies. Clinical evaluation (if S. paratyphi A conjugates is planned.

The distribution of Salmonella typhi, S. paratyphi A. and S. paratyphi C. agglutinins among 364 sera from a randomly selected group of healthy individuals in Nairobi and Naivasha (Kenya) were analysed in relation to bacteriologically confirmed cases of typhoid fever at the Kenyatta National Hospital (KNH). Out of these, 30% of the healthy individuals had no detectable Widal titres. Overall agglutinin titres obtained for the healthy population showed that 96% of the individuals had low level reactivity with titres < 1:80 for both H and O antigens of S. typhi, while 4% had titres of 1:160 or above. Agglutinins for S. paratyphi A and C were rarely present in the sera tested. Age and sex were found to have no effect on antibody titre distribution in the two populations but differences in the water source may have affected the percentage of positive tests recorded. In this typhoid endemic region titres upto 1:80 are not uncommon but both H and O titres of 1:160 and above found in conjunction with the clinical picture may be taken to be suggestive of typhoid fever.

Hybrid clones producing monoclonal antibodies (MAbs) specific for Salmonella paratyphi A (72 clones), S. paratyphi B (9 clones) and S. paratyphi C (8 clones) were produced by using the affinity purified Salmonella protein (Bp) as immunogens. MAbs to S. paratyphi A and S. paratyphi B reacted specifically with the 52 kDa homologous flagellin protein components while those to S. paratyphi C reacted with a 61 kDa flagellin protein component. The MAbs against S. paratyphi A and S. paratyphi B were used to establish a double antibody sandwich ELISA for detection of the 52 kDa flagellin antigens in serum samples from patients with acute paratyphoid A and paratyphoid B fever. With this assay system, 6.25 ng per ml of flagellin antigens of S. paratyphi A and S. paratyphi B could be detected. However, the assay system could not detect the flagellin antigens in patients' sera. The presence of IgM antibodies to the 52 kDa antigens of S. paratyphi A and S. paratyphi B in the acute sera from paratyphoid A or paratyphoid B patients suggested that the 52 kDa protein components of both salmonellae are good immunogens for human and might be used as antigens for early diagnosis of paratyphoid A and paratyphoid B fever.

Polysaccharide of O:9,12 specificity purified from Salmonella typhi was conjugated to tetanus toxoid or bovine serum albumin in order to obtain defined antigenic material that would contain O chain free of other S. typhi antigens and that would be suitable for characterizing host humoral response to only S. typhi O-chain antigens. These artificial conjugates were strongly reactive in immunodots with 18 pooled and 3 individual serum samples from patients with typhoid fever and with rabbit anti-Salmonella O antiserum (group D, factors 1, 9, and 12). They reacted weakly with one serum sample from one human with paratyphoid A. These results suggest that the periodate oxidation and the reductive amination used in the conjugation conserved the immunogenicity of the O chain and allowed its absorption to nitrocellulose. They also suggest that the bovine serum albumin conjugate could be used in the diagnosis of S. typhi infections as normal sera may react with the protein molecule of the tetanus toxoid conjugate.

The Widal test was performed in 114 normal individuals from the Gezira area in Sudan. Salmonella typhi O agglutinins were found at a titre of 1.320 in 12 (10.5%) of them. Salmonella paratyphi A agglutinins were found at 1.160 in 5 (4.3%) and Salmonella paratyphi B "O" agglutinins were found in 6 (5.3%) at a titre of 1.160. None of these individuals had a history of typhoid fever or vaccination with TAB vaccine. The following points emerged: (i) normal healthy people in the Sudan have high antibody titres of Salmonella typhi; (ii) the Widal test in the Sudan should be interpreted against this background; (iii) previous diagnostic titre of 1.160 for S. typhi results in high false positive results; (iv) a titre above 1.320 is suggested as diagnostic for S. typhi; 1.160 for both S. partyphi B and S. paratyphi A.

Cross-reactivity in a delayed-type hypersensitivity (DH) response was studied in mice immunised with live Salmonella typhi, S. paratyphi A and S. paratyphi B. Extensive cross-reactions outside the serogroup limits were observed. The ability of DH cross-reacting and non-cross-reacting sonicates to generate activated macrophages was studied in mice immunised 3 months earlier with S. paratyphi B. Whereas DH cross-reacting S. poona sonicate generated activated macrophages the non-cross-reacting S. typhi sonicate did not. To determine whether infections due to diarrhoea-causing salmonellae generated cross-reactive cell-mediated immune responses against enteric fever-causing organisms, similar reverse experiments were performed in mice immunised with S. enteritidis. S. paratyphi A sonicate generated both effector responses, i.e., DH and activated macrophages.

The development of cell-mediated immune response to lipopolysaccharide and Barber protein from Salmonella typhi was investigated in patients suffering from typhoid fever. The cell-mediated immunity as measured by the leukocyte adherence inhibition test, was demonstrable in 77% of patients with typhoid fever but only in 5.6% of healthy controls. It was found that cell-mediated immune response appeared after the first week of illness and persisted for at least 4 weeks. The time course development of cell-mediated immune response and humoral immune response was correlated but the magnitude of each response was independent on one another.

Protein A-containing Staphylococcus aureus was coupled to Salmonella C1, D and Vi monovalent antisera to produce C1-, D- and Vi-COAG reagents. The reagents were used to detect their homologous Salmonella antigens in blood cultures (BC). The D and Vi antigens were detected in 79 of 239 BC from patients with suspected typhoid fever and Salmonella typhi was later isolated from the same 79 BC. The C1 antigen was detected in 8 BC from which only S. oranienburg was later isolated. The COAG test was generally positive at the same time the BC became culture positive. However, because of subculture and biochemical identification requirements the COAG test could be interpreted 1-2 days before culture results were available. The COAG test can be used to presumptively identify Salmonella typhi and Salmonella group C1 in blood cultures before the culture results are available.