Assessment of microbiological correlates and immunostimulatory potential of electron beam inactivated metabolically active yet non culturable (MAyNC) Salmonella Typhimurium.

This study investigates the microbiological and immunological basis underlying the efficacy of electron beam-inactivated immune modulators. The underlying hypothesis is that exposure to eBeam-based ionization reactions inactivate microorganisms without modifying their antigenic properties and thereby creating immune modulators. The immunological correlates of protection induced by such eBeam based Salmonella Typhimurium (EBST) immune modulators in dendritic cell (DC) (in vitro) and mice (in vivo) models were assessed. The EBST stimulated innate pro inflammatory response (TNFα) and maturation (MHC-II, CD40, CD80 and CD86) of DC. Immuno-stimulatory potential of EBST was on par with both a commercial Salmonella vaccine, and live Salmonella cells. The EBST cells did not multiply under permissive in vitro and in vivo conditions. However, EBST cells remained metabolically active. EBST immunized mice developed Salmonella-specific CD4+ T-cells that produced the Th1 cytokine IFNγ at a level similar to that induced by the live attenuated vaccine (AroA- ST) formulation. The EBST retained stable immunogenic properties for several months at room temperature, 4°C, and -20°C as well as after lyophilization. Therefore, such eBeam-based immune modulators have potential as vaccine candidates since they offer the safety of a "killed" vaccine, while retaining the immunogenicity of an "attenuated" vaccine. The ability to store eBeam based immune modulators at room temperature without loss of potency is also noteworthy.

Introducing a cost-effective method for purification of bioactive flagellin from several flagellated gram-negative bacteria.

The objective of this study was to introduce a simple and cheap method for purification of flagellin. So, flagellin proteins of Salmonella typhimurium (S. typhimurium), Escherichia coli (E. coli), Citrobacter freundii (C. freundii) and Salmonella typhi (S. typhi) were purified by a modified simple method. Bacterial cultures were precipitated by centrifugation. Precipitates were washed twice and flagellin proteins were detached by shaking vigorously (in PBS pH = 2), and then flagellin proteins were precipitated by ammonium sulfate saturation. Evaluation of purification efficiency and concentration were examined by SDS-PAGE and Bradford assay. Polyclonal antibodies were produced against S. typhimurium FliC and cross-reactivity of anti-S. typhimurium was assessed against other flagellins. Bioactivity of flagellins was evaluated by cell proliferation and IL-8 protein expression assay in HEK293 cells, and also, IL-6 and TNF-α genes expression in chicken cells. Results showed a single band for flagellin proteins of all bacteria on %10 SDS-PAGE, which concentration ranged from 150 to 400 µg/mL. All flagellin proteins increased cell proliferation, and IL-8 levels were increased after treatment by flagellins and levels of IL-6 and TNF-α were increased after treatment with S. typhimurium FliC. All flagellin proteins showed cross-reactivity with antibodies. Findings showed that application of our method, not only reduced time and cost, but also, the purified flagellin proteins had acceptable bioactivity.

Assessment of attenuated Salmonella vaccine strains in controlling experimental Salmonella Typhimurium infection in chickens.

Salmonella hold considerable promise as vaccine delivery vectors for heterologous antigens in chickens. Such vaccines have the potential additional benefit of also controlling Salmonella infection in immunized birds. As a way of selecting attenuated strains with optimal immunogenic potential as antigen delivery vectors, this study screened 20 novel Salmonella Typhimurium vaccine strains, differing in mutations associated with delayed antigen synthesis and delayed attenuation, for their efficacy in controlling colonization by virulent Salmonella Typhimurium, as well as for their persistence in the intestine and the spleen. Marked differences were observed between strains in these characteristics, which provide the basis for selection for further study as vaccine vectors.

La bactérie Salmonella est considérée comme un vecteur vaccinal prometteur pour la livraison d'antigènes hétérologues chez les poulets. De tels vaccins ont le potentiel bénéfique supplémentaire de limiter les infections par Salmonella chez les oiseaux immunisés. Comme moyen de sélectionner les souches atténuées avec le potentiel immunogène optimal comme vecteur de livraison d'antigènes, la présente étude a examiné 20 souches vaccinales nouvelles de Salmonella Typhimurium, qui différaient en mutation associées avec une synthèse antigénique retardée et une atténuation retardée, pour leur efficacité à limiter la colonisation par du Salmonella Typhimurium virulent, ainsi que pour leur persistance dans l'intestin et la rate. Des différences marquées furent observées entre les souches pour ces caractéristiques, fournissant ainsi des éléments de sélection pour des études ultérieures comme vecteurs vaccinal.(Traduit par Docteur Serge Messier).

Expansion of Tfh-like cells during chronic Salmonella exposure mediates the generation of autoimmune hypergammaglobulinemia in MyD88-deficient mice.

The role of TLR signaling in linking the innate and adaptive immune systems has been a controversial issue that remains to be solved. Here, we determined whether MyD88-dependent TLR signals are required for the generation of B-cell responses during chronic Salmonella infection. Oral administration of recombinant attenuated Salmonella enterica serovar Typhimurium vaccine (RASV) strain in MyD88(-/-) mice resulted in chronic infection. Infection was accompanied by enlarged germinal centers and hypergammaglobulinemia with anti-double-stranded DNA (dsDNA)-specific Ab in sera, and the deposition of immune complexes in the kidneys, suggesting onset of autoimmunity. CD4(+) T cells expressing PD-1, CXCR5, ICOS, and IL-21 were dramatically increased in chronically infected mice, indicating the expansion of follicular helper T (Tfh)-like cells. Of note, the depletion of CD4(+) T cells completely blocked the generation of polyclonal IgG Ab in sera after oral RASV challenge. Inflammatory myeloid cells expressing CD11b and Gr-1 accumulated in high numbers in the spleen of MyD88(-/-) mice. Interestingly, the blockade of PD-1 or ICOS significantly reduced the hypergammaglobulinemia and dsDNA-specific autoantibody production. Overall, these results suggest that Tfh-like cells in chronic bacterial infection trigger autoimmune hypergammaglobulinemia in a PD-1- and ICOS-dependent manner.

Development of a liposome-based immunochromatographic strip assay for the detection of Salmonella.

Salmonella species are ubiquitous human pathogens which pose a dangerous threat to the elderly and children worldwide. In this study, to develop a more efficient assay procedure for the rapid detection of Salmonella Typhimurium, an immunochromatographic strip assay was developed using immunoliposome (anti-Salmonella IgG-tagged) encapsulated with sulforhodamine B (SRB). The detection sensitivity of the developed immunochromatographic assay was compared with a commercial immunochromatographic test strip using colloidal gold nanoparticles. The liposomes were prepared through a reverse-phase evaporation method by using a lipid and phospholipid mixture and SRB, a fluorescence dye, which was encapsulated in the phospholipid bilayer. Furthermore, the outer surface of the SRB-encapsulated liposome was conjugated with antibody (affinity-purified polyclonal goat anti-Salmonella IgG) to form an immunoliposome (size, 223 nm), used as the analytical reagent in the developed immunoassay. For this strip assay, a plastic-backed nitrocellulose strip was immobilized with two antibody zones. The lower zone of the strip referred to Salmonella antigen capture zone (test line), while the other zone served as a positive control (control line). The lower zone was coated with affinity-purified polyclonal goat anti-Salmonella IgG, while the upper zone was coated with rabbit anti-goat IgG. During capillary migration of the wicking solution (diluted liposome and Salmonella culture, each 50 µl), Salmonella was captured with surface-bound immunoliposomes at the antigen capture zone, while the unbound liposomes migrated upward and bound to another zone. The color density of the antigen capture zone was directly proportional to the amount of S. Typhimurium in the test sample. As a result, the detection limit of the immunochromatographic strip assay developed in this study against S. Typhimurium was found to be 10(2) CFU/ml, which was significantly higher than the detection limit (10(7) CFU/ml) of the commercial immunochromatographic test strip assay.

Assessment of 2 Salmonella enterica serovar Typhimurium-based vaccines against necrotic enteritis in reducing colonization of chickens by Salmonella serovars of different serogroups.

This study assessed the protective efficacy of oral vaccination with 2 experimental attenuated Salmonella Typhimurium-vectored vaccines for necrotic enteritis in protecting chickens against intestinal colonization by common serovars of Salmonella belonging to the 4 major serogroups affecting chickens. Birds were vaccinated orally with 1 × 108 colony-forming units (CFU) of 1 of the vaccine strains χ9241 and χ9352, which express a plasmid-encoded partial recombinant hypothetical protein gene (tHP) of Clostridium perfringens, at days 1 and 7 of age, and then were challenged at 14 d of age with 106 CFU of Salmonella serovars Anatum, Enteritidis, Heidelberg, Kentucky, or Typhimurium (representative serovars of serogroups B, C, D, and E). Birds were necropsied at 4 wk of age, and samples were collected to determine reduction in tissue and intestinal colonization. The chickens vaccinated with χ9241-tHP showed reduced colonization by Salmonella Enteritidis (serogroup D) and by Salmonella Heidelberg and Salmonella Typhimurium (serogroup B) compared with the control birds. No reduction in colonization was observed in the chickens vaccinated with χ9352-tHP. There was an association between the efficacy of these vaccine strains in protecting against necrotic enteritis, assessed on an earlier occasion, and their efficacy in protecting against Salmonella colonization. Thus, the choice of an attenuated Salmonella Typhimurium vaccine vector for delivery of heterologous antigens to chickens should be based partly on the vaccine's value in protecting against colonization by serovars within serogroups B and D. Such vectors would have the additional benefit of reducing colonization of important Salmonella serovars.

[Assessment of protective effect of heat shock protein-lypopolysaccharide of Salmonella typhimurium recombinant construction in mice].

AIM: To study protective activity of recombinant construction of heat-shock protein with lypopolysaccharide (rcHSP-LPS) as well as its variants (with destroyed protein or bounded LPS) against Salmonella typhimurium. It was also planned to study the ability of rcHSP-LPS to interact with toll-like receptors (TLRs) expressed on continuous cell lines. MATERIALS AND METHODS: One of the following preparations was administered to outbred mice: rcHSP-LPS; rcHSP-LPS treated by polymyxin B (PMB) for bounding of LPS - rc(HSP-LPS)PMB; rcHSP-LPS in which protein was treated by boiling during 30 min--rc (HSP-LPS)B; LPS (E. coli K-235); polymyxin B (PMB). Twenty-four hours after single or last administration of rcHSP-LPS, each mice was intraperitoneally inoculated with 63 LD50 of S. typhimurium 415 contained in 0.5 ml of physiologic solution. Antibody titer to LPS of Salmonella typhimurium was measured by immunoenzyme assay. RESULTS: It was demonstrated that rcHSP-LPS administered 24 hours before inoculation induced resistance to S. typhimurium infection. Protection formed after 3 injections of rcHSP-LPS with 10 mcg in each or single injection with 100 mcg/mouse. Forty to eighty percent of immunized mice survived after challenge while 90% of control animals died. Destroy of the HSP by boiling of the construction led to loss of protective effect. Bounding of LPS by PMB did not lead to loss of protective properties of the construction but they expressed only after its multiple administration with 10 mcg per mouse. LPS of E. coli in dose 0.0266 mcg per mouse as well as PMB did not influence the course of S. typhimurium infection in mice. CONCLUSION: It was shown that rcHSP-LPS effectively protects mice from S. typhimurium infection by activating innate immunity; one of the possible mechanisms for such protection determined by interaction with TLRs 2 and 4 was considered. Other studies are needed in order to elucidate other mechanisms of innate immunity, which can be activated by rcHSP-LPS.

Construction and immunological assessment of Salmonella typhimurium expressing fox sperm LDH-C4.

This study examined immune responses of foxes to oral doses of recombinant Salmonella typhimurium expressing fox sperm-specific lactate dehydrogenase (fLDH). The cDNA for fLDH was cloned into the expression plasmid pKK233.2 (pKKfLDH). Salmonella typhimurium aroA- (SL3261) was transformed with either the pKK233.2 plasmid alone (SpKK) or the pKKfLDH construct (SpKfLDH). The fLDH expressed by SpKfLDH retained enzymatic activity and was recognized by human LDH-C4-specific antibody. Male European red foxes (Vulpes vulpes) were given an initial oral dose of 1 x 10(11) cfu of either SpKK (control, n = 3) or SpKfLDH (test, n = 6), followed four weeks later with a further dose of 1 x 10(11) cfu. Antibodies to Salmonella lipopolysaccharide (LPS-04) and fLDH were measured in plasma and saliva for eight consecutive weeks after the initial doses. Both LPS-04 IgG- and IgA-specific antibodies as well as fLDH-specific IgG antibodies were detected in plasma and saliva. However, there was a marked fLDH-specific IgA response in saliva consistent with induction of the common mucosal immune system. The antibody measurements demonstrated the feasibility of using recombinant Salmonella as an oral vaccine to elicit gamete antigen-specific mucosal immune responses in foxes.

Assessment of attenuated Salmonella typhimurium strains designed as potential carriers of foreign antigens for mucosal localization and immuno-genicity in a rabbit model.

Neither wild type nor attenuated S. typhimurium strains induced diarrheal illness in rabbits. All strains localized to the Peyer's patch at higher concentrations than in lumenal contents or adjacent ileum. Wild type S. typhimurium C5 induced a typhoid-like illness in rabbits with severe weight loss, bacteremia, persistent splenic colonization, and serum IgG response. Both attenuated strains were disseminated to spleen (day 3) but produced minimal systemic illness. They induced biliary IgA responses greater than the wild type (day 7), but minimal serum IgG responses. Both mutants of S. typhimurium are suitable for further development as live enteric vaccines to carry foreign antigens since they localize to Peyer's patch after oral inoculation, induce biliary antibody, and produce minimal systemic disease. The attenuated strains tested are systemically disseminated. It remains to be determined whether dissemination (determined by a large virulence plasmid) is necessary for the desired mucosal immune response or acceptable for an oral vaccine strain.

T-2 toxin impairment of murine response to Salmonella typhimurium: a histopathologic assessment.

T-2 toxin and other trichothecene mycotoxins experimentally impair normal immune function and may predispose humans and animals to infectious disease. In this study, the histopathologic effects of Salmonella typhimurium challenge concurrently with sublethal T-2 toxin exposure were examined in the Salmonella-resistant C3H/HeN mouse. Oral administration of T-2 toxin (1 mg/kg) every other day for 10 d had little effect on the tissues examined when compared to control animals. Mice challenged with S. typhimurium and then treated with T-2 toxin every other day for 10 d had markedly larger and more bacterial-related lesions in the spleens, kidneys, and livers than animals challenged with S. typhimurium alone. Differences in bone marrow, Peyer's patches and ileal tissues were less discernable between S. typhimurium and S. typhimurium plus T-2 toxin treated groups. These results were consistent with previous findings that T-2 toxin compromised murine resistance to S. typhimurium infection and ultimately caused death in animals challenged with a sublethal dose of the organism.