Chitosan-adjuvanted Salmonella subunit nanoparticle vaccine for poultry delivered through drinking water and feed.

Poor induction of mucosal immunity in the intestines by current Salmonella vaccines is a challenge to the poultry industry. We prepared and tested an oral deliverable Salmonella subunit vaccine containing immunogenic outer membrane proteins (OMPs) and flagellin (F) protein loaded and F-protein surface coated chitosan nanoparticles (CS NPs) (OMPs-F-CS NPs). The OMPs-F-CS NPs had mean particle size distribution of 514 nm, high positive charge and spherical in shape. In vitro and in vivo studies revealed the F-protein surface coated CS NPs were specifically targeted to chicken immune cells. The OMPs-F-CS NPs treatment of chicken immune cells upregulated TLRs, and Th1 and Th2 cytokines mRNA expression. Oral delivery of OMPs-F-CS NPs in birds enhanced the specific systemic IgY and mucosal IgA antibodies responses as well as reduced the challenge Salmonella load in the intestines. Thus, user friendly oral deliverable chitosan-based Salmonella vaccine for poultry is a viable alternative to current vaccines.

Exploratory cohort study to determine if dry cow vaccination with a Salmonella Newport bacterin can protect dairy calves against oral Salmonella challenge.

BACKGROUND: Salmonellosis is a major cause of morbidity and mortality in neonatal calves, often occurring before preventative vaccines can be administered. HYPOTHESIS/OBJECTIVE: To evaluate the protective effect on calves of colostrum from cows vaccinated with a commercially available Salmonella Newport bacterin against a Salmonella Typhimurium challenge. ANIMALS: Twenty Holstein bull calves from a university dairy farm. METHODS: Nonrandomized placebo-controlled trial in which colostrum was harvested from 30 cows that received 2 doses of either Salmonella bacterin or saline before calving. Colostrum collected from each group was pooled and fed to 2 groups of 10 calves at birth. At approximately 2 weeks of age, calves were challenged with Salmonella Typhimurium. Clinical, hematologic, microbiological, and postmortem findings were compared between the 2 groups. RESULTS: No differences in mortality, clinical findings, hematology results, blood and fecal cultures, or necropsy findings between the 2 groups were observed. Vaccinated cows had higher colostral titers, and calves fed this colostrum had higher serum titers (mean difference, 0.429; mean [SE], 0.852 [0.02] for vaccinated versus 0.423 [0.02] for control calves). CONCLUSIONS AND CLINICAL IMPORTANCE: Transfer of colostral immunoglobulins from Salmonella enterica serotype Newport bacterin to neonatal calves was not sufficient to decrease mortality, clinical signs, sepsis, intestinal damage, or fecal shedding when exposed to a highly pathogenic Salmonella isolate. A large-scale randomized controlled clinical trial is needed to evaluate the efficacy of this bacterin when administered in the dry period for prevention of salmonellosis in neonatal calves.

Alginate-coated chitosan microparticles encapsulating an oral plasmid-cured live Salmonella enterica serovar Gallinarum vaccine cause a higher expression of interferon-gamma in chickens compared to the parenteral live vaccine.

Salmonella enterica serovar Gallinarum causes a disease in chickens known as fowl typhoid. Interferon-gamma (IFN-γ) has been shown to be crucial in eliminating salmonellosis infection because of its strong association with T-cell responses. This study was undertaken to compare the expression of IFN-γ in chickens generated by different vaccine formulations. Eighty one-day-old Lohmann layer chicks were divided into four groups of 20 birds each for the experiment. This comprised an unvaccinated negative control group (NEG), a group vaccinated with the live 9R vaccine by the injection route (SC), a group vaccinated with alginate-coated chitosan microparticles encapsulating live plasmid-cured S. Gallinarum strain 9 (PC) by the oral route, and a group vaccinated with a weak attenuated live S. Gallinarum strain 9 encapsulated in alginate-coated chitosan microparticles (VM) given orally. Vaccinations were done at 10 and 14 weeks of age followed by challenge at 16 weeks of age. IgG was measured using ELISA. qRT-PCR was used to compare the mRNA fold expression of IFN-γ in the PC, VM and SC groups using the unvaccinated/unchallenged group as the control. There were significant differences in the IgG levels between each vaccinated group and the unvaccinated group (P < 0.05) after booster vaccination and post-challenge. There was 100% protection of the birds in SC and VM groups, 80% protection in PC group and 0% protection in the NEG group. Using 2-ΔΔCT calculation, IFN-γ was more highly expressed in the PC group than in the SC group or VM group. In conclusion, the IFN-γ was more highly expressed in the PC group (though not significantly higher) compared to the SC and VM groups and this could be attributed to the alginate-coated chitosan microparticles which acted as an adjuvant.

Laser micro-structured Si scaffold-implantable vaccines against Salmonella Typhimurium.

Salmonella Typhi is responsible for typhoid fever in humans. Despite the efforts, the development of long-lasting vaccines has failed and the available vaccines display only moderate activity, being considered as "international traveler's" vaccines. Taking advantage of the previously described implantable vaccine technology consisting on 3D laser-microstructured Si scaffolds loaded with antigen-seeded macrophages, the present study aimed to apply an antigenic stimulus of whole extracts of S. Typhimurium, which is the mouse analogue of the human Salmonella Typhi, and examine its ability to mount specific antibody response. After defining the experimental conditions for specific anti-S. Typhimurium IgG production in vitro, antigen-seeded macrophages loaded onto the 3D Si-scaffolds were implanted to mice, while parallel experiments used conventional Freund-complete-adjuvant vaccination protocols. The results showed that only the implantable vaccine protocol could mount a specific antibody response 14 days after implantation. The cytokine profile showed increase of IL-10 and IFN-γ in the case of implantable and conventional vaccination respectively, 7 days after implantation. Morphological studies on the excised scaffolds 14 days after implantation, showed the development of a well-structured adherent monolayer, establishing multiple contacts with lymphocytes in favor to immune response development. Based on the hypothesis that both stimulatory and suppressive components in the vaccination preparation, could affect the overall activity, peptidoglycan was applied as an antigen to the vaccination protocols. Surprisingly, peptidoglycan was shown to induce a mitogenic rather than specific immunogenic response. In this case, histological analysis of the excised scaffolds showed a restricted layer of adherent cells with cytoplasmic extensions, but hard to distinguish cell contacts with lymphocytes. Finally, the presented results showed a differential behavior of antigen presenting cells in accordance to the antigenic stimulus and consequently the activation state of the cells. Tailoring the micro/sub-micron 3D structures and chemistry of Si scaffolds, could control cell behavior according to the user's needs.

Effect of a DIVA vaccine with and without in-feed use of coated calcium-butyrate on transmission of Salmonella Typhimurium in pigs.

BACKGROUND: For satisfactory Salmonella control, good biosecurity along the pork production chain is crucial, although additional control measures on-farm need to be considered. This study evaluated the effect of two potential control measures against the spread of Salmonella Typhimurium via a transmission experiment with 56 piglets (3-15 weeks of age): two groups were orally vaccinated with 107 - 108 Colony Forming Units (CFU)/2 mL of a new attenuated Salmonella Typhimurium vaccine 'Salmoporc-∆rfaJ' with DIVA capacities (Differentiation between Infected and Vaccinated Animals) (n = 2x16); the feed of one group was additionally supplemented with coated calcium-butyrate salt. Two weeks post vaccination, four pigs per group were orally challenged with 107 CFU/2 mL of a Salmonella Typhimurium strain 112910a. Both groups were compared with a positive (challenged/untreated; n = 16) and negative (unchallenged/untreated; n = 8) control group. Until six weeks post challenge, blood, individual faecal and finally tissue samples were examined. Adjusted transmission ratios 'Ra' were estimated, based on the challenge strain isolation from faecal and/or tissue samples. RESULTS: In both intervention groups, Ra values were lower compared to the positive control group, although these differences were not significant. In the combination group DIVA vaccine + coated butyrate, less non-challenged contact animals excreted Salmonella and less tissue samples were found Salmonella-positive in all pigs, when compared to the positive control group (P < 0.01). Seroconversion was detected in none of the vaccinated animals before challenge, when using a commercial lipopolysaccharide (LPS) ELISA targeting only Salmonella O-antigens, deleted in this vaccine. This was in contrast with an in-house whole-cell ELISA testing for various Salmonella antigens, in which Salmonella-specific antibodies were found pre-challenge in the serum of the vaccinated pigs. CONCLUSIONS: Both interventions showed a limited, non-significant reduction of Salmonella transmission between piglets. They may have applications towards Salmonella control and surveillance. Firstly, the number of Salmonella excreting contact pigs was significantly lower in the group where vaccination was combined with coated calcium-butyrate salt in the feed; secondly, the new vaccine confirmed its DIVA capacity. Therefore, these interventions merit further research with larger sample sizes, to optimize their use for Salmonella programmes.

A novel adjuvant, mixture of alum and the beta-adrenergic receptor antagonist propranolol, elicits both humoral and cellular immune responses for heat-killed Salmonella typhimurium vaccine.

OBJECTIVE: To determine the efficacy of the mixture of propranolol (PRP), a beta-adrenergic receptor antagonist, and alum, as a new adjuvant, in the induction of humoral and cellular immunity in response to heat-killed Salmonella typhimurium (S. typhimurium) (HKST) as a model vaccine. METHODS: BALB/c mice were divided into five groups. Mice in the experimental groups received either the HKST vaccine alone or in combination with the adjuvant alum, PRP or the alum-PRP mixture. Mice in the negative control group received phosphate-buffered saline. All mice were immunized two times on days 0 and 14. Two weeks after the last immunization, immune responses to S. typhimurium were assessed. RESULTS: Administration of the alum-PRP mixture as an adjuvant increased the ability of the HKST vaccine to enhance lymphocyte proliferation, shifted the immune response towards a T-helper (Th) 1 pattern and increased S. typhimurium specific IgG, IgG2a and IgG1. This resulted in improved protective immunity against S. typhimurium. CONCLUSION: Administration of the alum-PRP mixture as an adjuvant in combination with the HKST vaccine, can enhance both humoral and cellular immunity and shift the immune responses to a Th1 pattern.

Feeding a diet containing a fructooligosaccharide mix can enhance Salmonella vaccine efficacy in mice.

Fructooligosaccharides (FOS) are considered prebiotics because of their ability to promote growth of specific beneficial gut bacteria, such as bifidobacteria. Some studies reported potential immune-modulating properties. The aim of this study was to investigate the effect of FOS:inulin mix on murine response to Salmonella vaccine and evaluate the relevance toward protection against Salmonella infection. Balb/c mice were fed a diet containing 5% FOS:inulin mix or a control diet 1 wk before oral immunization with a suboptimal dose of live attenuated Salmonella typhimurium vaccine. Four weeks after vaccination, mice were infected with LD100 of virulent S. typhimurium. Specific blood Salmonella immunoglobulin G and fecal immunoglobulin A significantly increased in mice fed the diet containing prebiotics compared with control mice 4 wk postimmunization. Peritoneal macrophage phagocytic activity also significantly increased in FOS:inulin-fed mice at 1 wk postimmunization compared with control mice. No detectable effects were observed on the percentage of lymphoid cell subsets in the spleen. However, production of cytokines, interferon-gamma, interleukin-12, and tumor necrosis factor alpha, was numerically increased in spleen cell cultures stimulated with mitogens from FOS:inulin-fed mice 1 and 4 wk postimmunization. Salmonella translocation to lymphoid organs was not affected by feeding FOS:inulin. However, the improved response to Salmonella vaccine was concomitant with an increase in the survival rate of FOS:inulin-fed mice upon challenge with virulent Salmonella. No detectable effects were observed on the composition or the metabolic activity of the microbiota. Overall, the data suggest that a diet supplemented with FOS:inulin mix stimulates mucosal immunity and seems to improve efficacy of an oral vaccine.

Salmonella typhimurium grown in iron-rich media, inactivated with ferric chloride and adjuvanted with homologous bacterial DNA is potent and efficacious vaccine in mice.

The present study describes our attempt to construct a novel vaccine formulation that affords full protection against murine typhoid under experimental conditions. Ferric chloride, 100mM, as inactivating agent, bacterial growth under iron-rich conditions and homologous bacterial DNA as adjuvant were used for construction of the experimental Salmonella typhimurium vaccine. The vaccine inoculated twice at 2 weeks interval in Swiss albino mice elicited statistically significant IgG levels when compared with non-adjuvanted and other control groups. All the mice inoculated with the novel vaccine withstood challenge with 50 LD(50) dose of S. typhimurium strain St 585. No significant safety problems were found in mice.

Modulation of human humoral immune response through orally administered bovine colostrum.

Eighteen healthy volunteers were randomized into two treatment groups and consumed liquid prepackaged bovine colostrum whey and placebo for 7 days. On days 1, 3 and 5, an attenuated Salmonella typhi Ty21a oral vaccine was given to all subjects to mimic an enteropathogenic infection. The circulating antibody secreting cells and the expression of phagocytosis receptors of the subjects before and after oral immunization were measured with the ELISPOT assay and flow cytometry. All subjects responded well to the vaccine. No significant differences were observed in ELISPOT values for IgA, IgG, IgM, Fcgamma and CR receptor expression on neutrophils and monocytes between the two groups. There was a trend towards greater increase in specific IgA among the subjects receiving their vaccine with bovine colostrum. These results suggest that bovine colostrum may possess some potential to enhance human special immune responses.