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Objective Meat and eggs from chickens infected with Salmonella Enteritidis, Salmonella Typhimurium and Salmonella Infantis are considered to be an important source of Salmonella infections for humans. In order to control Salmonella infections in chickens, basic biosecurity measures are taken in combination with inactivated or attenuated live vaccines. Apart from an adaptive immune response, some live vaccines also induce innate immune mechanisms that prevent or inhibit systemic invasion with homologous Salmonella serovars. It is unknown whether these invasion inhibition effects are also directed against heterologous Salmonella serovars. Furthermore, it is unclear whether the adaptive immune response after vaccination with a Salmonella Enteritidis phage type 4 live vaccine is also directed against other phage types of Salmonella Enteritidis and Typhimurium. Material and methods Specific pathogen-free day-old chicks were vaccinated orally with a commercially available Salmonella Enteritidis live vaccine. To test the invasion inhibition effect, the animals were challenged orally with a labelled Salmonella Typhimurium or Salmonella Infantis strain 1 day after vaccination. To demonstrate the adaptive immune response against non-phage type 4 Salmonella Enteritidis strains and a monophasic Salmonella Typhimurium strain, the chickens were challenged with Salmonella Enteritidis strains of phage types 1, 8 and 21 and a monophasic Salmonella Typhimurium strain (Definitive Type 193). After challenge, the abundance of the challenge strain in liver and cecal tissue was enumerated and compared with a corresponding control group. Results Findings showed that the live Salmonella Enteritidis vaccine inhibits systemic invasion after early infection with Salmonella Typhimurium and Salmonella Infantis. Furthermore, adaptive immunity against the tested non-phage type 4 Salmonella Enteritidis strains and the monophasic Salmonella Typhimurium strain was demonstrated. Conclusion and clinical relevance The results of this study demonstrate that vaccination with the Salmonella Enteritidis phage type 4 live vaccine significantly inhibits the invasion of Salmonella Typhimurium and Infantis. Furthermore, an adaptive immune response was also detected against non-phage type 4 Salmonella Enteritidis strains and a monophasic Salmonella Typhimurium strain.

Vaccination with the live attenuated vaccine Salmoporc is an effective measure to control Salmonella Typhimurium (STM) in affected swine populations. However, the cellular immune response evoked by the Salmoporc vaccine including differences in vaccinated pigs versus non-vaccinated pigs upon STM infection have not been characterized yet. To investigate this, tissue-derived porcine lymphocytes from different treatment groups (vaccination-only, vaccination and infection, infection-only, untreated controls) were stimulated in vitro with heat-inactivated STM and abundances of IFN-γ, TNF-α and/or IL-17A-producing T-cell subsets were compared across organs and treatment groups. Overall, our results show the induction of a strong CD4+ T-cell response after STM infection, both locally and systemically. Low-level induction of STM-specific cytokine-producing CD4+ T cells, notably for the IFN-γ/TNF-α co-producing phenotype, was detected after vaccination-only. Numerous significant contrasts in cytokine-producing T-cell phenotypes were observed after infection in vaccinated and infected versus infected-only animals. These results suggest that vaccine-induced STM-specific cytokine-producing CD4+ T cells contribute to local immunity in the gut and may limit the spread of STM to lymph nodes and systemic organs. Hence, our study provides insights into the underlying immune mechanisms that account for the efficacy of the Salmoporc vaccine.

Background: Beta-toxin (CPB) is the major virulence factor of Clostridium perfringens type C, causing hemorrhagic enteritis in newborn pigs but also other animals and humans. Vaccines containing inactivated CPB are known to induce protective antibody titers in sow colostrum and neutralization of the CPB activity is thought to be essential for protective immunity in newborn piglets. However, no method is available to quantify the neutralizing effect of vaccine-induced antibody titers in pigs. (2) Methods: We developed a novel assay for the quantification of neutralizing anti-CPB antibodies. Sera and colostrum of sows immunized with a commercial C. perfringens type A and C vaccine was used to determine neutralizing effects on CPB induced cytotoxicity in endothelial cells. Antibody titers of sows and their piglets were determined and compared to results obtained by an ELISA. (3) Results: Vaccinated sows developed neutralizing antibodies against CPB in serum and colostrum. Multiparous sows developed higher serum and colostrum antibody titers after booster vaccinations than uniparous sows. The antibody titers of sows and those of their piglets correlated highly. Piglets from vaccinated sows were protected against intraperitoneal challenge with C. perfringens type C supernatant. (4) Conclusions: The test based on primary porcine endothelial cells quantifies neutralizing antibody activity in serum and colostrum of vaccinated sows and could be used to reduce and refine animal experimentation during vaccine development.

Since 2013 the efficacy of new live Salmonella Enteritidis (SE) vaccines for chickens needs to be demonstrated according to European Pharmacopoeia Monograph 04/2013:2520 to receive approval in the EU. The purpose of this study was to determine whether a vaccine licensed since 1999 could also fulfil the required tests of the current guideline. For this, Salmonella-free chickens (n = 50) were vaccinated on their 2nd, 46th and 84th day of life with the live attenuated S. Enteritidis strain IDT No. 441/014. Non-vaccinated control animals (n = 50) were kept accordingly. To demonstrate the duration of immunity 20 animals of each group were challenge infected 65 weeks after the last vaccination with a virulent SE (PT 4) strain. According to the monograph, cloacal swabs were taken 3, 5, 7, 10 and 14 days post challenge (dpc). Tissue samples of liver, spleen, caeca, ovaries and oviduct were collected during necropsy of 10 animals per group on 7 and 14 dpc, respectively. All samples were analysed bacteriologically regarding the presence of the challenge strain. The number of challenge strain positive tissue samples and cloacal swabs was significantly reduced in vaccinated animals (p < 0.05). Therefore, the vaccine strain complied with the EP guideline. This study is the first that demonstrates the efficacy of this vaccine according to the current regulations. However, efficacy could also be shown during the development of the vaccine but by use of another animal model that comprised fewer animals per group. The use of this model is no longer accepted by EU regulatory authorities. The results need discussion in context with the 3R principle.

Zoonotic infections caused by Salmonella enterica serovar Typhimurium pose a constant threat to consumer health, with the pig being a particularly major source of multidrug-resistant isolates. Vaccination, as a promising approach to reduce colonization and shedding, has been scarcely used, as it interferes with current control programs relying on serology as a means of herd classification. In order to overcome this problem, we set out to develop a negative-marker vaccine allowing the differentiation of infected from vaccinated animals (DIVA). Applying an immunoproteomic approach with two-dimensional gel electrophoresis, Western blot, and quadrupole time-of-flight tandem mass spectrometry, we identified the OmpD protein as a suitable negative marker. Using allelic exchange, we generated an isogenic mutant of the licensed live vaccine strain Salmoporc and showed that virulence of Salmoporc and that of the mutant strain, SalmoporcΔ ompD , were indistinguishable in BALB/c mice. In a pig infection experiment including two oral immunizations with SalmoporcΔ ompD and challenge with a multiresistant S. enterica serovar Typhimurium DT104 clinical isolate, we confirmed the protective efficacy of SalmoporcΔ ompD in pigs, showing a significant reduction of both clinical symptoms and colonization of lymph nodes and intestinal tract. OmpD immunogenic epitopes were determined by peptide spot array analyses. Upon testing of several 9-mer peptides, each including an immunogenic epitope, one peptide (positions F 100 to Y 108 ) that facilitated the detection of infected animals independent of their vaccination status (DIVA function) was identified. The approach described overcomes the problems currently limiting the use of bacterial live vaccines and holds considerable potential for future developments in the field.

The aim of this study was to examine the duration of immunity of different vaccination schemes using the S. enteritidis live vaccine Gallivac Se and the S. enteritidis-S. typhimurium inactivated vaccine Gallimune Se+St. Three groups of Lohman Brown chickens were used. Group one was vaccinated three times orally with Gallivac Se at weeks one, seven and 13 of age. Group two was vaccinated twice orally with Gallivac Se in weeks one and seven and once i.m. with Gallimune Se+St in week 14 of age. A third group was not vaccinated and served as the control group. Eight randomly selected chickens from each of the three groups were challenged with a nalidixic acid resistant S. enteritidis PT4 strain in weeks 24, 51 and 71 of age and the same number of animals were challenged with a S. typhimurium DT 104 strain in weeks 26, 54 and 73 (75) of age.The chickens were euthanised seven days post challenge and the number of challenge strain organisms (log10 cfu) in the liver and on caecal mucosa was determined.The quantitative investigation of the challenge strain in the liver and caecal mucosa revealed a statistically significant (p0.05) lower challenge strain burden in the vaccinated groups compared with the non-vaccinated control group up to week 71 (73) of age. The protective effects were demonstrated for both challenge strains.

LPS antibody reaction of S. Typhimurium was detected in a total of 111 two- to ten-week-old pigs, which had been exposed to experimental oral infection with this serovar in 16 separate experiments. The total antibody level was determined according to the meat juice ELISA provided for the Salmonella control programme and the antibody concentrations of the isotopes IgG1, IgG2, IgA and IgM. Although there were significant differences between the individual experiments with respect to the reaction intensity and proportion of the individual immune globulin isotypes, the following general statements on the dynamics of the antibody reaction can be made: Clearly elevated antibody levels could be observed one week after the onset of infection. After one and two weeks, respectively, 22.5 and 52.9% of the infected animals showed antibody levels which were to be evaluated as positive in the sense of the Salmonella control programme (or = 40%). While the concentration of the IgM-related antibodies decreased again during the second week already, the antibody concentration of IgG1 and IgG2 continued to increase until the end of the experiment after 3 weeks. The clear differences in the reactions between the experiments cannot be explained solely by defined conditions of infection such as strain of infection, infective dose or age of the pigs. Obviously, other non-defined or non-recognized factors may have a decisive influence on the dynamics of the antibody reaction in the animal groups.

Hybrid swine (Landrace x Pietrain) aged 3-4 weeks were immunized twice at an interval of 3 weeks solely by the oral route and by the oral/parenteral route to evaluate the efficacy of a live S. Typhimurium vaccine. In each experiment a control group was run without vaccination. The animals were challenged at the age of 8-10 weeks by oral test infection with a labelled S. Typhimurium DT 104 strain. An ELISA was used to establish the presence of antibodies to S. Typhimurium in serum samples, coupled with clinical investigation. The presence of the challenge strain in the ileal and caecal mucosa and in the ileocolic lymph nodes was investigated quantitatively using the Koch plating method to determine the degree of colonization of those organs at the time of slaughter. The clinical course of disease was used to assess the success of vaccination. However, it was not possible to trigger, in a reproducible manner, clinical signs of disease in unvaccinated animals through infection. The vaccinated animals had a significantly lower (p < 0.05) colonization of the ileal and caecal mucosa than the unvaccinated animals. This was also seen to a lesser degree for the ileocolic lymph nodes.