Salmonella Enteritidis with double deletion in phoPfliC--a potential live Salmonella vaccine candidate with novel characteristics for use in chickens

Salmonella Enteritidis mutants with deletions in phoP, fliC or phoP fliC were tested for their virulence and their ability to induce parameters of the innate and adaptive immunity in addition to their potential for serological differentiation between vaccinated, non-vaccinated and infected chickens. The double phoP fliC deletion mutant was sufficiently attenuated but not diminished in its capability to inhibit the caecal colonisation and systemic invasion of homologous Salmonella Enteritidis shortly after administration of the vaccine strain to very young chicks. Immunisation with the attenuated ΔphoP fliC mutant resulted in protective effects which were only slightly and insignificantly lower than after “immunisation” with a Salmonella wild-type strain, indicating the capability to induce an intense adaptive immune response and protection against Salmonella exposure in older chickens. The deletion in fliC enabled the effective the differentiation between immunised and infected chickens using a commercially available ELISA kit. The double phoP fliC deletion mutant of Salmonella Enteritidis might be a potential and promising live Salmonella vaccine candidate with novel characteristics for use in poultry.     

Effect of infectious bursal disease (IBD) vaccine on Salmonella Enteritidis infected chickens

BackgroundChickens infected with both infectious bursal disease virus (IBDV) and Salmonella had higher mortality. In this work, we investigated the effect of IBDV vaccine (modified live-virus bursal disease vaccine, Nobilis strain 228E®) on experimentally infected chickens with Salmonella Enteritidis (SE).MethodsFour experimental groups were included in this study, negative control group, 228E®group, 228E® + SE infected group, and SE infected group. Chickens were ocularly administrated 228E® at 12 days of age and orally infected with S. Enteritidis at 13 days of age. Sera, intestinal fluid, blood, cloacal swabs and tissue samples were collected at 1, 2 and 3 weeks post vaccination (PV).ResultsThe recorded mortalities were higher in the 228E® + SE infected group, compared to the SE infected group. The anti-S. Enteritidis serum antibody titer and the intestinal mucosal IgA level were higher in the SE infected group at 2 and 3 weeks PV, compared to 228E® + SE infected group. S. Enteritidis fecal shedding and organ colonization were significantly higher in the 228E® + SE infected group than the SE infected group at 2 and 3 weeks PV. The 228E® + SE group had significantly lower bursa to body weight ratios at 2 and 3 weeks PV, as well as had higher bursal lesion scores than the SE infected group. IBDV vaccine depressed the specific-SE systemic and mucosal antibody responses, but did not affect the specific-SE cellular immune responses.ConclusionChickens administrated IBDV vaccine, followed by S. Enteritidis infection, could cause a significant effect on the bursa of Fabricius, resulting in failure of systemic and mucosal antibody responses to the S. Enteritidis and reduce the elimination and the clearance of S. Enteritidis.     

Zebrafish as a novel model for non-typhoidal Salmonella pathogenesis,transmission and vaccine efficacy

Salmonella-induced gastroenteritis causes massive morbidity and mortality in both adults and children of developing countries. However, it is difficult to study the mode of infection and vaccine efficacy due to inadequacies of current animal models. For this reason, we have explored using zebrafish as an improved model for non-typhoidal Salmonella (NTS) infection, including Salmonella enterica Typhimurium, Salmonella enterica Enteritidis and Salmonella enterica Weltevreden. In this study, we found that after infection of zebrafish with NTS, severe diarrhea like symptoms were observed and NTS significantly colonized the zebrafish intestine without any manipulation of the normal intestinal microbiota of the fish. Furthermore, these strains can colonize for longer than 72 h and induce severe inflammation in the intestine, which may induce fish death. We also found that infected fish can transmit the pathogen into naïve fish. Moreover, we have established that zebrafish is an excellent model for vaccine study. Successive triple bath vaccination with heat-killed single serotype S. Typhimurium and S. Enteritidis immunogen induced protective efficacy against a high dose (10⁸ CFU/ml) of infection with these pathogens. This study provides a natural infection model for the study of NTS infection, transmission and vaccine efficacy.     

Multifaceted immune responses and protective efficacy elicited by a recombinant autolyzed Salmonella expressing FliC flagellar antigen of F18+ Escherichia coli

Porcine edema disease (ED) caused by F18⁺ Shiga toxin 2e-producing Escherichia coli (STEC) has imposed significant economic losses in the swine industry worldwide, resulting in sudden deaths in post-weaned piglets. The flagellin protein of F18⁺ STEC, a structural component of the flagellar filament, is a known virulence factor that mediates adhesion and invasion to porcine epithelial cells. In this study, Salmonella inactivated by the E lysis gene and expressing the flagellin (fliC) antigen was genetically engineered utilizing a plasmid (pMMP184) carrying an efficient heterologous antigen delivery system. The resulting strain JOL1485 producing FliC was successfully inactivated by the E lysis gene cassette. Following the lysis procedure, FliC secretion and production of JOL1485 was validated by immunoblot analysis. To evaluate protective immunogenicity elicited by the constructed strain, BALB/c mice were injected with 1 × 10⁸ lysed cells via the intramuscular route. The markedly elevated titers of FliC-specific IgG, IgG1 and sIgA antibodies were observed, indicating a robust Th2-associated humoral immune response was raised in the immunized mice. The proportion of CD3⁺ CD4⁺ splenic T cells and proliferative activity were also elevated in in vivo and in vitro stimulated mice splenocytes. Further, JOL1485 successfully elicited upregulated gene expression of cytokines IL-6, IL-8, IL17, IL-21, IFN-γ and TNF-α in naïve porcine peripheral blood mononuclear cells (PBMCs). The overall immune response elicited by JOL1485 conferred a significant rise of protection against a lethal virulent F18⁺ STEC challenge whereas all non-immunized mice died following the challenge. Our results demonstrate that fliC efficiently expressed in the genetically inactivated Salmonella strain has immunostimulatory and protective effects against a F18⁺ STEC lethal challenge, and may be promising as a potential vaccine candidate against ED infection.     

Biofilm formation and genetic diversity of Salmonella isolates recovered from clinical,food, poultry and environmental sources

In the present study, Salmonella isolates (n = 40) recovered from clinical, food, poultry and environmental sources were characterized for serotype identification, genetic diversity and biofilm formation capability. Serotype identification using multiplex PCR assay revealed six isolates to be Salmonella Typhimurium, 14 as Salmonella Enteritidis, 11 as Salmonella Typhi, and the remaining nine isolates unidentified were considered as other Salmonella spp. Most of the Salmonella isolates (85%) produced biofilm on polystyrene surfaces as assessed by microtitre plate assay. About 67.5% isolates were weak biofilm producers and 17.5% were moderate biofilm producers. There was no significant difference in biofilm-forming ability among the Salmonella isolates recovered from different geographical regions or different sources. Among the genetic methods, Enterobacterial Repetitive Intergenic Consensus (ERIC) PCR revealed greater discriminatory power (DI, 0.943) followed by pulsed field gel electrophoresis (PFGE) (DI, 0.899) and random amplification of polymorphic DNA (RAPD) PCR (DI, 0.873). However, composite analysis revealed the highest discrimination index (0.957). Greater discrimination of S. Typhimurium and S. Typhi was achieved using PFGE, while ERIC PCR was better for S. Enteritidis and other Salmonella serotypes. A strong positive correlation (r = 0.992) was observed between biofilm formation trait and clustered Salmonella isolates in composite genetic analysis.     

Profiling the gastrointestinal microbiota in response to Salmonella: Low versus high Salmonella shedding in the natural porcine host

Controlling Salmonella in the food chain is complicated by the ability of Salmonella to colonize livestock without causing clinical symptoms/disease. Salmonella-carrier animals are a significant reservoir for contamination of naïve animals, the environment, and our food supply. Salmonella carriage and shedding in pigs varies greatly both experimentally and on-farm. To investigate the dynamics between the porcine intestinal microbiota and Salmonella shedding, we temporally profiled the microbiota of pigs retrospectively classified as low and high Salmonella-shedders. Fifty-four piglets were collectively housed, fed and challenged with 10⁹ Salmonella enterica serovar Typhimurium. Bacterial quantitation of Salmonella in swine feces was determined, and total fecal DNA was isolated for 16S rRNA gene sequencing from groups of high-shedder, low-shedder, and non-inoculated pigs (n = 5/group; 15 pigs total). Analyses of bacterial community structures revealed significant differences between the microbiota of high-shedder and low-shedder pigs before inoculation and at 2 and 7 days post-inoculation (d.p.i.); microbiota differences were not detected between low-shedder and non-inoculated pigs. Because the microbiota composition prior to Salmonella challenge may influence future shedding status, the “will-be” high and low shedder phylotypes were compared, revealing higher abundance of the Ruminococcaceae family in the “will-be” low shedders. At 2 d.p.i., a significant difference in evenness for the high shedder microbiota compared to the other two groups was driven by decreases in Prevotella abundance and increases in various genera (e.g. Catenibacterium, Xylanibacter). By 21 d.p.i., the microbial communities of high-shedder and low-shedder pigs were no longer significantly different from one another, but were both significantly different from non-inoculated pigs, suggesting a similar Salmonella-induced alteration in maturation of the swine intestinal microbiota regardless of shedding status. Our results correlate microbial shifts with Salmonella shedding status in pigs, further defining the complex interactions among the host, pathogen, and microbiota of this important public health issue and food safety concern.     

Regulated expression of virulence gene mviN provides protective immunity and colonization control of Salmonella in poultry

Current live attenuated vaccines for control of Salmonella in poultry persist in the ceca and may persist in the environment. In this paper we report the construction and characterization of the vaccine efficacy of a Salmonella mutant strain with inducible mviN expression and rapid clearance from the host. The mutant was effective in oral immunization of the broiler chicken host against a virulent Salmonella oral challenge strain, having a mean 7 × 10⁶ CFU/g in the ceca of unvaccinated controls compared to a mean 2 × 10³ CFU/g in the ceca of vaccinated chickens at 4 weeks post-challenge (6 weeks of age). The mutant strain also demonstrated immunogenicity, reduced organ colonization, and rapid clearance in broiler chickens within 3 weeks of inoculation.     

Specific and cross-reactive immune response to oral Salmonella Typhi Ty21a and parenteral Vi capsular polysaccharide typhoid vaccines administered concomitantly

BackgroundSince protective efficacy of the current typhoid vaccines—oral whole-cell Salmonella Typhi Ty21a and parenteral Vi-capsular polysaccharide preparation—is not optimal, and no vaccines are available against paratyphoid or non-typhoidal Salmonella (NTS) serotypes, new approaches deserve to be explored. The immunological mechanisms elicited by the two typhoid vaccines are mainly targeted against different structures. We studied whether these vaccines would enhance S. Typhi-specific immune response and cross-reactivity against other Salmonellae, if administered concomitantly.Materials and methodsVolunteers were immunized simultaneously with Ty21a and Vi vaccines (Ty21a + Vi group) or with either of the two singly (Ty21a and Vi groups). All volunteers were investigated for circulating specific and cross-reactive plasmablasts, identified by ELISPOT as IgA, IgG or IgM antibody-secreting cells (ASC) reactive with S. Typhi, S. Paratyphi A/B/C, or selected NTS serotypes (S. Enteritidis, S. Typhimurium).ResultsIn the Ty21a + Vi group, no specific or cross-reactive plasmablasts were detected before vaccination. After vaccination, the number of S. Typhi-specific plasmablasts (878 ASC/10⁶ PBMC, 95%CI 554–1201) proved higher than in the Ty21a (339 ASC/10⁶ PBMC; p < 0.001) and Vi (149 ASC/10⁶ PBMC; p < 0.001) groups. Likewise, cross-reactive responses in the Ty21a + Vi group were higher than in the Ty21a and Vi groups (Ty21a + Vi vs Ty21a: ASC against S. Paratyphi A/B, S. Enteritidis and S. Typhimurium p < 0.05, against S. Paratyphi C p < 0.01; Ty21a + Vi vs Vi: against S. Paratyphi C not significant, others p < 0.0001). A gut-directed homing profile was seen among O antigen-specific and a systemic one among Vi antigen-specific plasmablasts.ConclusionsConcomitant administration of Ty21a and Vi vaccines is well tolerated and induces an additive immune response to the two vaccines. Thus it enhances the magnitude of both typhoid-specific plasmablast responses and those cross-reacting with paratyphoid and most important NTS serotypes. The data encourage concomitant use of Ty21 and Vi vaccines for those at risk.     

Bivalent non-typhoidal Salmonella outer membrane vesicles immunized mice sera confer passive protection against gastroenteritis in a suckling mice model

Invasive non-typhoidal Salmonella (iNTS) serovars, especially Salmonella Typhimurium (ST) and Salmonella Enteritidis (SE), cause gastroenteritis worldwide. Due to the emergence of multi-drug resistance in iNTS, a broad-spectrum vaccine is urgently needed for the prevention of iNTS infection. Currently, there is no effective licensed vaccine against iNTS available in the market. We have formulated an outer membrane vesicles (OMVs) based bivalent immunogen as a vaccine candidate to generate broad-spectrum protective immunity against both recently circulating prevalent ST and SE. We have isolated OMVs from ST and SE and formulated the immunogen by mixing both OMVs (1:1 ratio). Three doses of bivalent immunogen significantly induced humoral immune responses against lipopolysaccharides (LPSs) and outer membrane proteins (OMPs) as well as a cell-mediated immune response in adult mice. We also observed that proteins of OMVs act as an adjuvant for generation of high levels of anti-LPS antibodies through T cell activation. We then characterized the one-day old suckling mice model for both ST and SE mediated gastroenteritis and used the model for a passive protection study. In the passive protection study, we found the passive transfer of bivalent OMVs immunized sera significantly reduced ST and SE mediated colonization and gastroenteritis symptoms in the colon of suckling mice compared to non-immunized sera recipients. The overall study demonstrated that OMVs based bivalent vaccine could generate broad-spectrum immunity against prevalent iNTS mediated gastroenteritis. This study also established the suckling mice model as a suitable animal model for vaccine study against iNTS mediated gastroenteritis.     

Salmonella Typhimurium LPS mutations for use in vaccines allowing differentiation of infected and vaccinated pigs

Contaminated pork is a major source of human salmonellosis and the serovar most frequently isolated from pigs is Salmonella Typhimurium. Vaccination could contribute greatly to controlling Salmonella infections in pigs. However, pigs vaccinated with the current vaccines cannot be discriminated from infected pigs with the LPS-based serological tests used in European Salmonella serosurveillance programmes. We therefore examined which LPS encoding genes of Salmonella Typhimurium can be deleted to allow differentiation of infected and vaccinated pigs (DIVA), without affecting the vaccine strain's protective capacity. For this purpose, deletion mutants in Salmonella strain 112910a, used as vaccine strain, were constructed in the LPS encoding genes: ΔrfbA, ΔrfaL, ΔrfaJ, ΔrfaI, ΔrfaG and ΔrfaF. Primary inoculation of BALB/c mice with the parent strain, ΔrfaL, ΔrfbA or ΔrfaJ strain but not the ΔrfaG, ΔrfaF or ΔrfaI strain protected significantly against subsequent infection with the virulent Salmonella Typhimurium strain NCTC12023. Immunization of piglets with the ΔrfaJ or ΔrfaL mutants resulted in the induction of a serological response lacking detectable antibodies against LPS. This allowed a clear differentiation between sera from pigs immunized with the ΔrfaJ or ΔrfaL strains and sera from pigs infected with their isogenic wild type strain. In conclusion, applying deletions in the rfaJ or the rfaL gene in Salmonella Typhimurium strain 112910a allows differentiation of infected and vaccinated pigs in an LPS based ELISA without reducing the strain's protective capacities in mice.     

A colonisation-inhibition culture consisting of Salmonella Enteritidis and Typhimurium ΔhilAssrAfliG strains protects against infection by strains of both serotypes in broilers

Consumption of contaminated poultry meat is still an important cause of Salmonella infections in humans and there is a need for control methods that protect broilers from day-of-hatch until slaughter age against infection with Salmonella. Colonisation-inhibition, a concept in which a live Salmonella strain is orally administered to day-old chickens and protects against subsequent challenge, can potentially be used as control method. In this study, the efficacy of a Salmonella Typhimurium ΔhilAssrAfliG strain as a colonisation-inhibition strain for protection of broilers against Salmonella Typhimurium was evaluated. Administration of a Salmonella Typhimurium ΔhilAssrAfliG strain to day-old broiler chickens decreased faecal shedding and strongly reduced caecal and internal organ colonisation of a Salmonella Typhimurium challenge strain administered one day later using a seeder bird model. In addition, it was verified whether a colonisation-inhibition culture could be developed that protects against both Salmonella Enteritidis and Typhimurium. Therefore, the Salmonella Typhimurium ΔhilAssrAfliG strain was orally administered simultaneously with a Salmonella Enteritidis ΔhilAssrAfliG strain to day-old broiler chickens, which resulted in a decreased caecal and internal organ colonisation for both a Salmonella Enteritidis and a Salmonella Typhimurium challenge strain short after hatching, using a seeder bird model. The combined culture was not protective against Salmonella Paratyphi B varietas Java challenge, indicating serotype-specific protection mechanisms. The data suggest that colonisation-inhibition can potentially be used as a versatile control method to protect poultry against several Salmonella serotypes.     

Reversible synthesis of colanic acid and O-antigen polysaccharides in Salmonella Typhimurium enhances induction of cross-immune responses and provides protection against heterologous Salmonella challenge

Colanic Acid (CA) and lipopolysaccharide (LPS) are two major mannose-containing extracellular polysaccharides of Salmonella. Their presence on the bacterial surface can mask conserved protective outer membrane proteins (OMPs) from the host immune system. The mannose moiety in these molecules is derived from GDP-mannose, which is synthesized in several steps. The first two steps require the action of phosphomannose isomerase, encoded by pmi (manA), followed by phosphomannomutase, encoded by manB. There are two copies of manB present in the Salmonella chromosome, one located in the cps gene cluster (cpsG) responsible for CA synthesis, and the other in the rfb gene cluster (rfbK) involved in LPS O-antigen synthesis. In this study, it was demonstrated that the products of cpsG and rfbK are isozymes. To evaluate the impact of these genes on O-antigen synthesis, virulence and immunogenicity, single mutations (Δpmi, ΔrfbK or ΔcpsG) and a double mutation (ΔrfbK ΔcpsG) were introduced into both wild-type Salmonella enterica and an attenuated Δcya Δcrp vaccine strain. The Δpmi, ΔrfbK and ΔcpsG ΔrfbK mutants were defective in LPS synthesis and attenuated for virulence. In orally inoculated mice, strain S122 (Δcrp Δcya ΔcpsG ΔrfbK) and its parent S738 (Δcrp Δcya) were both avirulent and colonized internal tissues. Strain S122 elicited higher levels of anti-S. Typhimurium OMP serum IgG than its parent strain. Mice immunized with S122 were completely protected against challenge with wild-type virulent S. Typhimurium and partially protected against challenge with either wild-type virulent S. Choleraesuis or S. Enteritidis. These data indicate that deletions in rfbK and cpsG are useful mutations for inclusion in future attenuated Salmonella vaccine strains to induce cross-protective immunity.     

rOmpF and OMVs as efficient subunit vaccines against Salmonella enterica serovar Enteritidis infections in poultry farms

Salmonella enterica serovar Enteritidis remains the most prevalent serotype causing human salmonellosis through the consumption of contaminated foods, especially poultry products. The development of a subunit vaccine against S. Enteritidis can not only protect chickens against Salmonella infection in the poultry industry but also cut the transmission sources. In this study, both the expressed recombinant outer membrane protein F (rOmpF) and extracted outer membrane vesicles (OMVs) were developed as subunit vaccines against S. Enteritidis challenge in chickens. Immunization with the subunit vaccine could induce not only antibody production but also strong cell-mediated immune response. Both rOmpF plus QuilA adjuvant and OMVs alone had highly protective efficacy against S. Enteritidis challenge and rapidly decreased the colonization of bacteria in chicken. These findings revealed the potential application of rOmpF and OMVs as subunit vaccines in the poultry industry.     

Immunisation of chickens with live Salmonella vaccines – Role of booster vaccination

It is accepted that booster vaccinations of chickens with live Salmonella vaccines are essential part of vaccinations schemes to induce an effective adaptive immune response. As manufacturer of registered live Salmonella vaccines recommend different times of booster the question raises whether the duration between the first and second immunisation might influence the protective effect against Salmonella exposure. Chickens were immunised with a live Salmonella Enteritidis vaccine on day 1 of age followed by a booster vaccination at different intervals (day 28, 35 or 42 of age) to study the effects on the colonisation and invasion of the Salmonella vaccine strain, the humoral immune response and the efficacy against infection with Salmonella Enteritidis on day 56 of age. Immunisation of all groups resulted in a very effective adaptive immune response and a high degree of protection against severe Salmonella exposure, however, the time of booster had only an unverifiable influence on either the colonisation of the vaccine strain, the development of the humoral immune response or the colonisation of the Salmonella challenge strain. Therefore, the first oral immunisation of the chicks on day 1 of age seems to be of special importance and prerequisite for the development of the effective immune response. A booster immunisation should be carried out, however, the time of booster may vary between week 3 and week 7 of age of the chickens without adversely impact on the efficacy of the adaptive immune response or the protective effects.     

Pathogenesis of Brucella ovis in pregnant mice and protection induced by the candidate vaccine strain B. Ovis ΔabcBA

Ovine brucellosis caused by Brucella ovis is a major cause of reproductive failure in sheep. This study aimed to evaluate transplacental infection and pathogenicity of B. ovis wild type strain ATCC 25,840 (WT B. ovis) and the candidate vaccine strain B. ovis ΔabcBA in pregnant mice. A total of 40 BALB/c mice were equally divided into 4 groups: (i) non immunized and uninfected control mice (3/10 mice became pregnant); (ii) non immunized and challenged with WT B. ovis (5/10 pregnant); (iii) inoculated only with B. ovis ΔabcBA (6/10 pregnant); (iv) immunized with B. ovis ΔabcBA and challenged with WT B. ovis (5/10 pregnant). Female mice bred, and five days after visualization of the vaginal plug, they were inoculated intraperitoneally (ip) with 100 µL of sterile PBS, 100 µL of 1 × 10⁶ CFU of B. ovis ΔabcBA, or 100 µL of 1 × 10⁶ CFU of B. ovis WT, according to each group. At the 17th day of gestation, samples of spleen, liver, uterus, placenta, fetus and mammary gland were obtained for bacteriology, histopathology and immunohistochemistry. Non immunized mice challenged with B. ovis WT developed necrotizing placentitis as well as microgranulomas in the liver and spleen. These findings support the notion that B. ovis infection in pregnant mice induces lesions that are similar to those caused by B. abortus in the same animal model. B. ovis ΔabcBA was not recovered from any of the sampled organs, and it did not cause any gross or microscopic lesions, indicating that it is a safe and attenuated strain in this experimental model. In addition, B. ovis ΔabcBA was induced protective immunity as demonstrated by decreased numbers of B. ovis WT in the liver, uterus and fetuses of immunized mice after the challenge with B. ovis WT.     

Induction of a homologous and heterologous invasion–inhibition effect after administration of Salmonella strains to newly hatched chicks

Administration of live Salmonella strains to day-old chicks provides protection against infection within hours by intestinal colonisation–inhibition. However, the extent to which the oral application of live Salmonella wild-type or vaccine strains may induce an early invasion–inhibition effect is unknown. Potentially protective pre-treatment strains of Salmonella Enteritidis and Infantis were examined for their ability (i) to colonise the caeca, to invade the liver, to induce an influx of granulocytes in caecal mucosa and, (ii) for their capacity to inhibit the systemic invasion of homologous and heterologous Salmonella challenge organisms. The highly invasive strain Salmonella Enteritidis induced a strong influx of heterophils in the caecal mucosa followed by a complete invasion–inhibition of both homologous and heterologous Salmonella challenge organisms administered 24 h later. Pre-treatment with a less invasive Salmonella Infantis resulted in a lower influx of granulocytes in the caecal tissue followed by a complete invasion–inhibition of the homologous serovar Infantis but only an incomplete invasion–inhibition of heterologous serovars. This invasion–inhibition effect has not been described previously in chickens and should be considered in the development of novel live Salmonella vaccines to prevent an early invasion of extra-intestinal organs by Salmonella challenge organisms in young chicks.     

Development of a dual vaccine for prevention of Brucella abortus infection and Escherichia coli O157:H7 intestinal colonization

Zoonoses that affect human and animal health have an important economic impact. In the study now presented, a bivalent vaccine has been developed that has the potential for preventing the transmission from cattle to humans of two bacterial pathogens: Brucella abortus and Shiga toxin-producing Escherichia coli (STEC). A 66 kDa chimeric antigen, composed by EspA, Intimin, Tir, and H7 flagellin (EITH₇) from STEC, was constructed and expressed in B. abortus Δpgm vaccine strain (BabΔpgm). Mice orally immunized with BabΔpgm(EITH₇) elicited an immune response with the induction of anti-EITH₇ antibodies (IgA) that clears an intestinal infection of E. coli O157:H7 three times faster (t = 4 days) than mice immunized with BabΔpgm carrier strain (t = 12 days). As expected, mice immunized with BabΔpgm(EITH₇) strain also elicited a protective immune response against B. abortus infection. A Brucella-based vaccine platform is described capable of eliciting a combined protective immune response against two bacterial pathogens with diverse lifestyles—the intracellular pathogen B. abortus and the intestinal extracellular pathogen STEC.     

Immunization with Salmonella Enteritidis secreting mucosal adjuvant labile toxin confers protection against wild type challenge via augmentation of CD3+CD4+ T-cell proliferation and enhancement of IFN-γ, IL-6 and IL-10 expressions in chicken

The protective efficacy and immunological profiles of chickens immunized with an attenuated Salmonella Enteritidis (SE) constitutively secreting double mutant heat labile enterotoxin (dmLT) were investigated. The dmLT is a detoxified variant of Escherichia coli heat labile toxin and is a potent mucosal adjuvant capable of inducing both humoral and cell-mediated immunity. In this study, four-week-old chickens were inoculated with SE-dmLT strain JOL1641, parental SE strain JOL1087 or phosphate buffered saline control. Peripheral blood mononuclear cells of SE-dmLT inoculated birds showed significant proliferation upon stimulation with SE antigens as compared to the control and JOL1087 groups (P ⩽ 0.05). One week post-challenge, the ratio of CD3⁺CD4⁺ to CD3⁺CD8⁺ T-cells showed a significant increase in the immunized groups. Significant increases in IFN-γ levels were observed in JOL1641 birds immunized via oral and intramuscular routes. While immunizations with the JOL1087 strain via the intramuscular route also induced significant increases in IFN-γ, immunization via the oral route did not trigger significant changes. Pro-inflammatory cytokine IL-6 was also elevated significantly in immunized birds; a significant elevation of IL-10 was observed only in oral immunization with JOL1641 (P ⩽ 0.05). JOL1641 immunized birds showed significant reduction of challenge bacterial-organ recovery as compared to JOL1087 and non-immunized birds. Collectively, our results revealed that immunization with the adjuvant-secreting S. Enteritidis confers protection against wild type SE challenge via induction of strong cell proliferative response, augmentation of CD3⁺CD4⁺: CD3⁺CD8⁺ T-cells ratio and enhancement of IFN-γ, IL-6 and IL-10 cytokine secretion.     

Preclinical identification of vaccine induced protective correlates in human leukocyte antigen expressing transgenic mice infected with Coccidioides posadasii

There is an emerging interest to develop human vaccines against medically-important fungal pathogens and a need for a preclinical animal model to assess vaccine efficacies and protective correlates. HLA-DR4 (DRB110401 allele) transgenic mice express a human major histocompatibility complex class II (MHC II) receptor in such a way that CD4⁺ T-cell response is solely restricted by this human molecule. In this study HLA-DR4 transgenic mice were immunized with a live-attenuated vaccine (ΔT) and challenged by the intranasal route with 50–70 Coccidioides posadasii spores, a potentially lethal dose. The same vaccination regimen offers 100% survival for C57BL/6 mice. Conversely, ΔT-vaccinated HLA-DR4 mice displayed 3 distinct manifestations of Coccidioides infection including 40% fatal acute (FAD), 30% disseminated (DD) and 30% pulmonary disease (PD). The latter 2 groups of mice had reduced loss of body weight and survived to at least 50 days postchallenge (dpc). These results suggest that ΔT vaccinated HLA-DR4 mice activated heterogeneous immunity against pulmonary Coccidioides infection. Vaccinated HLA-DR4 mice displayed early expansion of Th1 and Th17 cells and recruitment of inflammatory innate cells into Coccidioides-infected lungs during the first 9 dpc. While contraction rates of Th cells and the inflammatory response during 14–35 dpc significantly differed among the 3 groups of vaccinated HLA-DR4 mice. The FAD group displayed a sharply reduced Th1 and Th17 response, while overwhelmingly recruiting neutrophils into lungs during 9–14 days. The FAD group approached moribund by 14 dpc. In contrast, vaccinated HLA-DR4 survivors gradually contracted Th cells and inflammatory response with the greatest rate in the PD group. While vaccinated HLA-DR4 mice are susceptible to Coccidioides infection, they are useful for evaluation of vaccine efficacy and identification of immunological correlates against this mycosis.