Comparison of the Expression Kinetics and Immunostimulatory Activity of Replicating mRNA, Nonreplicating mRNA, and pDNA after Intradermal Electroporation in Pigs.

Synthetic mRNA is becoming increasingly popular as an alternative to pDNA-based gene therapy. Currently, multiple synthetic mRNA platforms have been developed. In this study we investigated the expression kinetics and the changes in mRNA encoding cytokine and chemokine levels following intradermal electroporation in pigs of pDNA, self-replicating mRNA, and modified and unmodified mRNA. The self-replicating mRNA tended to induce the highest protein expression, followed by pDNA, modified mRNA, and unmodified mRNA. Interestingly, the self-replicating mRNA was able to maintain its high expression levels during at least 12 days. In contrast, the expression of pDNA and the nonreplicating mRNAs dropped after respectively one and two days. Six days after intradermal electroporation a dose-dependent expression was observed for all vectors. Again, also at lower doses, the self-replicating mRNA tended to show the highest expression. All the mRNA vectors, including the modified mRNA, induced elevated levels of mRNA encoding cytokines and chemokines in the porcine skin after intradermal electroporation, while no such response was noticed after intradermal electroporation of the pDNA vector.

Evaluation of a xenogeneic vascular endothelial growth factor-2 vaccine in two preclinical metastatic tumor models in mice.

In this study, a xenogeneic DNA vaccine encoding for human vascular endothelial growth factor receptor-2 (hVEGFR-2) was evaluated in two murine tumor models, the B16-F10 melanoma and the EO771 breast carcinoma model. The vaccine was administered by intradermal injection followed by electroporation. The immunogenicity and the biological efficacy of the vaccine were tested in (1) a prophylactic setting, (2) a therapeutic setting, and (3) a therapeutic setting combined with surgical removal of the primary tumor. The tumor growth, survival, and development of an immune response were followed. The cellular immune response was measured by a bioluminescence-based cytotoxicity assay with vascular endothelial growth factor-2 (VEGFR-2)-expressing target cells. Humoral immune responses were quantified by enzyme-linked immunosorbent assay (ELISA). Ex vivo bioluminescence imaging and immunohistological observation of organs were used to detect (micro)metastases. A cellular and humoral immune response was present in prophylactically and therapeutically vaccinated mice, in both tumor models. Nevertheless, survival in prophylactically vaccinated mice was only moderately increased, and no beneficial effect on survival in therapeutically vaccinated mice could be demonstrated. An influx of CD3+ cells and a slight decrease in VEGFR-2 were noticed in the tumors of vaccinated mice. Unexpectedly, the vaccine caused an increased quantity of early micrometastases in the liver. Lung metastases were not increased by the vaccine. These early liver micrometastases did however not grow into macroscopic metastases in either control or vaccinated mice when allowed to develop further after surgical removal of the primary tumor.

Subtherapeutic tetracycline concentrations aggravate Salmonella Typhimurium infection by increasing bacterial virulence.

OBJECTIVES: Antibiotics are among the most frequently prescribed drugs in human and animal medicine. With antibiotic resistance being a serious threat to veterinary and public health, the prudent use of antibiotics receives much attention. Less well known is that incorrect use of antimicrobial agents may also lead to increased bacterial virulence with the potential of a more severe clinical course of infection. Therefore, the aim of this study was to investigate the effect of subtherapeutic doses of tetracyclines on htpG virulence gene expression in Salmonella Typhimurium and on the course of salmonellosis. METHODS: Salmonella strains containing an htpG-luxCDABE transcriptional fusion were constructed. Phenotype microarrays and tetracycline treatment were used to investigate their htpG expression. A Salmonella transposon mutant bank was used to identify genes involved in the induction of htpG gene expression. Finally, the in vitro results were linked to the in vivo situation using a Salmonella mouse model. RESULTS: We demonstrate that subtherapeutic antimicrobial concentrations can exacerbate bacterial infections through direct up-regulation of bacterial virulence factors using Salmonella Typhimurium 112910a phage type 120/ad as a model organism. Phenotype microarrays showed that expression of the Salmonella Typhimurium virulence gene htpG is increased by several tetracycline antimicrobials at values below their MIC, a process that requires intact Salmonella LPS genes. Exposure of experimentally infected DBA/2J mice to subtherapeutic doxycycline concentrations resulted in htpG-mediated exacerbation of Salmonella Typhimurium infection. CONCLUSIONS: These findings show that the Salmonella isolate used in this study can respond to subtherapeutic tetracycline pressure by increasing its virulence and disease severity.

Host Stress Drives Salmonella Recrudescence.

Host stress is well known to result in flare-ups of many bacterial, viral and parasitic infections. The mechanism by which host stress is exploited to increase pathogen loads, is poorly understood. Here we show that Salmonella enterica subspecies enterica serovar Typhimurium employs a dedicated mechanism, driven by the scsA gene, to respond to the host stress hormone cortisol. Through this mechanism, cortisol increases Salmonella proliferation inside macrophages, resulting in increased intestinal infection loads in DBA/2J mice. ScsA directs overall Salmonella virulence gene expression under conditions that mimic the intramacrophagic environment of Salmonella, and stimulates the host cytoskeletal alterations that are required for increased Salmonella proliferation inside cortisol exposed macrophages. We thus provide evidence that in a stressed host, the complex interplay between a pathogen and its host endocrine and innate immune system increases intestinal pathogen loads to facilitate pathogen dispersal.

Heat-labile enterotoxin of Escherichia coli promotes intestinal colonization of Salmonella enterica.

Enterotoxigenic Escherichia coli (ETEC) is an important cause of infantile and travellers' diarrhoea, which poses a serious health burden, especially in developing countries. In addition, ETEC bacteria are a major cause of illness and death in neonatal and recently weaned pigs. The production of a heat-labile enterotoxin (LT) promotes the colonization and pathogenicity of ETEC and may exacerbate co-infections with other enteric pathogens such as Salmonella enterica. We showed that the intraintestinal presence of LT dramatically increased the intestinal Salmonella Typhimurium load in experimentally inoculated pigs. This could not be explained by direct alteration of the invasion or survival capacity of Salmonella in enterocytes, in vitro. However, we demonstrated that LT affects the enteric mucus layer composition in a mucus-secreting goblet cell line by significantly decreasing the expression of mucin 4. The current results show that LT alters the intestinal mucus composition and aggravates a Salmonella Typhimurium infection, which may result in the exacerbation of the diarrhoeal illness.

HtpG contributes to Salmonella Typhimurium intestinal persistence in pigs.

Salmonella enterica subspecies enterica serovar Typhimurium (Salmonella Typhimurium) contamination of pork, is one of the major sources of human salmonellosis. The bacterium is able to persist and hide in asymptomatic carrier animals, generating a reservoir for Salmonella transmission to other animals and humans. Mechanisms involved in Salmonella persistence in pigs remain poorly understood. In the present study, we demonstrate that the Salmonella htpG gene, encoding a homologue of the eukaryotic heat shock protein 90, contributes to Salmonella Typhimurium persistence in intestine-associated tissues of pigs, but not in the tonsils. HtpG does not seem to play an important role during the acute phase of infection. The contribution to persistence was shown to be associated with htpG-dependent Salmonella invasion and survival in porcine enterocytes and macrophages. These results reveal the role of HtpG as a virulence factor contributing to Salmonella persistence in pigs.

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.

Induction of seroconversion and persistence of Salmonella Typhimurium in pigs are strain dependent.

Foodborne salmonellosis is one of the most important bacterial zoonotic diseases worldwide. Salmonella Typhimurium is the serovar most frequently isolated from persistently infected slaughter pigs in Europe. Salmonella Typhimurium pathogenesis is host species specific. In addition, differences in in vitro behaviour of Salmonella Typhimurium strains have also been described, which may be reflected by a different course of infection within a host species. We compared the course of a Salmonella Typhimurium infection in pigs, using two Salmonella Typhimurium strains that were able to interfere with MHC II expression on porcine macrophages to a different extent in vitro. After experimental inoculation, blood and faecal samples from all pigs were collected at regular time points. At 40 days post inoculation (pi), animals were euthanized and tissue samples were bacteriologically analysed. The proportion of serologically positive piglets at 33 days pi was significantly higher in pigs that were inoculated with the strain that did not downregulate MHC II expression in vitro. Furthermore, this strain was less frequently shed and isolated in lower numbers from tonsils and ileocaecal lymph nodes than the strain that was able to markedly downregulate MHC II expression in vitro. We thus found that the delayed onset of seroconversion after oral inoculation of piglets with a particular Salmonella Typhimurium strain coincided with higher faecal shedding and increased persistence. Strain specific differences in Salmonella pathogenesis might thus have repercussions on the serological detection of Salmonella Typhimurium infections in pigs.

A modified glucomannan mycotoxin-adsorbing agent counteracts the reduced weight gain and diminishes cecal colonization of Salmonella Typhimurium in T-2 toxin exposed pigs.

The aim of the study was to investigate the effect of a modified glucomannan binder on the course of a Salmonella Typhimurium infection in pigs. Therefore, four pig diets were provided during 23 days: (1) free of mycotoxins, (2) containing 1g binder per kg feed, (3) containing 83 µg T-2 toxin per kg feed and (4) containing 83 µg T-2 toxin and 1g binder per kg feed. After 18 days, all pigs were inoculated with Salmonella Typhimurium and euthanized five days later. The addition of the binder to T-2 toxin contaminated feed counteracted the reduced weight gain of pigs caused by T-2 toxin and reduced the amount of Salmonella Typhimurium in the cecum and cecal contents. In vitro findings might indicate that the binder captures Salmonella. We thus conclude that the binder counteracts T-2 toxin induced weight loss and possibly binds Salmonella, resulting in a reduced cecal colonization.

Tackling the issue of environmental survival of live Salmonella Typhimurium vaccines: deletion of the lon gene.

Vaccination is an important measure to control Salmonella contamination in the meat production chain. A previous study showed that both the ΔrfaJ and ΔrfaL strains are suitable markers and allow serological differentiation of infected and vaccinated animals. The aim of this study was to verify whether deletion of the lon gene in a Salmonella Typhimurium ΔrfaJ marker strain resulted in decreased environmental survival. Our results indicate that deletion of the lon gene in the ΔrfaJ strain did not affect invasiveness in IPEC-J2 cells and resulted in an increased susceptibility to UV, disinfectants (such as hydrogen peroxide and tosylchloramide sodium) and citric acid. Immunization of pigs with inactivated ΔrfaJ or ΔlonΔrfaJ vaccines allowed differentiation of infected and vaccinated pigs. Furthermore, deletion of the lon gene did not reduce the protection conferred by live wild type or ΔrfaJ vaccines against subsequent challenge with a virulent Salmonella Typhimurium strain in BALB/c mice. Based on our results in mice, we conclude that deletion of lon in ΔrfaJ contributes to environmental safety of the ΔrfaJ DIVA strain.

Salmonella Typhimurium induces SPI-1 and SPI-2 regulated and strain dependent downregulation of MHC II expression on porcine alveolar macrophages.

Foodborne salmonellosis is one of the most important bacterial zoonotic diseases worldwide. Salmonella Typhimurium is the serovar most frequently isolated from persistently infected slaughter pigs in Europe. Circumvention of the host's immune system by Salmonella might contribute to persistent infection of pigs. In the present study, we found that Salmonella Typhimurium strain 112910a specifically downregulated MHC II, but not MHC I, expression on porcine alveolar macrophages in a Salmonella pathogenicity island (SPI)-1 and SPI-2 dependent way. Salmonella induced downregulation of MHC II expression and intracellular proliferation of Salmonella in macrophages were significantly impaired after opsonization with Salmonella specific antibodies prior to inoculation. Furthermore, the capacity to downregulate MHC II expression on macrophages differed significantly among Salmonella strains, independently of strain specific differences in invasion capacity, Salmonella induced cytotoxicity and altered macrophage activation status. The fact that strain specific differences in MHC II downregulation did not correlate with the extent of in vitro SPI-1 or SPI-2 gene expression indicates that other factors are involved in MHC II downregulation as well. Since Salmonella strain dependent interference with the pig's immune response through downregulation of MHC II expression might indicate that certain Salmonella strains are more likely to escape serological detection, our findings are of major interest for Salmonella monitoring programs primarily based on serology.

Vaccination of pigs reduces Salmonella Typhimurium numbers in a model mimicking pre-slaughter stress.

In pigs, infection with Salmonella Typhimurium often results in the development of carriers that re-excrete the organism during periods of stress. Previous studies have shown that cortisol plays a significant role in the recrudescence of Salmonella Typhimurium and that re-excretion can be induced by injections of dexamethasone. This study evaluated whether a commercially available Salmonella Typhimurium vaccine was able to reduce Salmonella excretion in a model mimicking pre-slaughter stress. Pigs were randomly assigned to either vaccination or a control group and, 5 weeks later, were infected with Salmonella Typhimurium. Twenty-three days post infection, pigs were injected with dexamethasone to induce recrudescence and Salmonella Typhimurium numbers were determined. Salmonella loads were significantly lower in the ileum and colon and in the contents of the ileum and caecum in vaccinated pigs than in non-vaccinated pigs. In addition, significantly more Salmonella positive tonsil and colon samples were found in non-vaccinated pigs. Vaccination with an attenuated vaccine reduced but did not eliminate Salmonella Typhimurium in pigs in conditions mimicking pre-slaughter stress.

T-2 toxin induced Salmonella Typhimurium intoxication results in decreased Salmonella numbers in the cecum contents of pigs, despite marked effects on Salmonella-host cell interactions.

The mycotoxin T-2 toxin and Salmonella Typhimurium infections pose a significant threat to human and animal health. Interactions between both agents may result in a different outcome of the infection. Therefore, the aim of the presented study was to investigate the effects of low and relevant concentrations of T-2 toxin on the course of a Salmonella Typhimurium infection in pigs. We showed that the presence of 15 and 83 µg T-2 toxin per kg feed significantly decreased the amount of Salmonella Typhimurium bacteria present in the cecum contents, and a tendency to a reduced colonization of the jejunum, ileum, cecum, colon and colon contents was noticed. In vitro, proteomic analysis of porcine enterocytes revealed that a very low concentration of T-2 toxin (5 ng/mL) affects the protein expression of mitochondrial, endoplasmatic reticulum and cytoskeleton associated proteins, proteins involved in protein synthesis and folding, RNA synthesis, mitogen-activated protein kinase signaling and regulatory processes. Similarly low concentrations (1-100 ng/mL) promoted the susceptibility of porcine macrophages and intestinal epithelial cells to Salmonella Typhimurium invasion, in a SPI-1 independent manner. Furthermore, T-2 toxin (1-5 ng/mL) promoted the translocation of Salmonella Typhimurium over an intestinal porcine epithelial cell monolayer. Although these findings may seem in favour of Salmonella Typhimurium, microarray analysis showed that T-2 toxin (5 ng/mL) causes an intoxication of Salmonella Typhimurium, represented by a reduced motility and a downregulation of metabolic and Salmonella Pathogenicity Island 1 genes. This study demonstrates marked interactions of T-2 toxin with Salmonella Typhimurium pathogenesis, resulting in bacterial intoxication.

The complex interplay between stress and bacterial infections in animals.

Over the past decade, an increasing awareness has arisen of the role of neuroendocrine hormones in the susceptibility of mammalian hosts to a bacterial infection. During a stress response, glucocorticoids, catecholamines and neuroendocrine factors are released into the circulation of the host. For a long time the effects of stress on the course of an infection have been exclusively ascribed to the direct effect of stress-related hormones on the immune system and the intestinal barrier function. Chronic stress is known to cause a shift from T helper 1-mediated cellular immunity toward T helper 2-mediated humoral immunity, which can influence the course of an infection and/or the susceptibility to a microorganism. Bacteria can however also respond directly to stress-related host signals. Catecholamines can alter growth, motility, biofilm formation and/or virulence of pathogens and commensal bacteria, and as a consequence influence the outcome of infections by these bacteria in many hosts. For some bacteria, such as Salmonella, Escherichia coli and Pseudomonas aeruginosa it was shown that this influence is regulated by quorum sensing mechanisms. In this manuscript an overview of how and when stress influences the outcome of bacterial infections in animals is provided.

Stress induced Salmonella Typhimurium recrudescence in pigs coincides with cortisol induced increased intracellular proliferation in macrophages.

Salmonella Typhimurium infections in pigs often result in the development of carriers that intermittently excrete Salmonella in very low numbers. During periods of stress, for example transport to the slaughterhouse, recrudescence of Salmonella may occur, but the mechanism of this stress related recrudescence is poorly understood. Therefore, the aim of the present study was to determine the role of the stress hormone cortisol in Salmonella recrudescence by pigs. We showed that a 24 h feed withdrawal increases the intestinal Salmonella Typhimurium load in pigs, which is correlated with increased serum cortisol levels. A second in vivo trial demonstrated that stress related recrudescence of Salmonella Typhimurium in pigs can be induced by intramuscular injection of dexamethasone. Furthermore, we found that cortisol, but not epinephrine, norepinephrine and dopamine, promotes intracellular proliferation of Salmonella Typhimurium in primary porcine alveolar macrophages, but not in intestinal epithelial cells and a transformed cell line of porcine alveolar macrophages. A microarray based transcriptomic analysis revealed that cortisol did not directly affect the growth or the gene expression or Salmonella Typhimurium in a rich medium, which implies that the enhanced intracellular proliferation of the bacterium is probably caused by an indirect effect through the cell. These results highlight the role of cortisol in the recrudescence of Salmonella Typhimurium by pigs and they provide new evidence for the role of microbial endocrinology in host-pathogen interactions.

Tissue-specific Salmonella Typhimurium gene expression during persistence in pigs.

Salmonellosis caused by Salmonella Typhimurium is one of the most important bacterial zoonotic diseases. The bacterium persists in pigs resulting in asymptomatic 'carrier pigs', generating a major source for Salmonella contamination of pork. Until now, very little is known concerning the mechanisms used by Salmonella Typhimurium during persistence in pigs. Using in vivo expression technology (IVET), a promoter-trap method based on ΔpurA attenuation of the parent strain, we identified 37 Salmonella Typhimurium genes that were expressed 3 weeks post oral inoculation in the tonsils, ileum and ileocaecal lymph nodes of pigs. Several genes were expressed in all three analyzed organs, while other genes were only expressed in one or two organs. Subsequently, the identified IVET transformants were pooled and reintroduced in pigs to detect tissue-specific gene expression patterns. We found that efp and rpoZ were specifically expressed in the ileocaecal lymph nodes during Salmonella peristence in pigs. Furthermore, we compared the persistence ability of substitution mutants for the IVET-identified genes sifB and STM4067 to that of the wild type in a mixed infection model. The ΔSTM4067::kanR was significantly attenuated in the ileum contents, caecum and caecum contents and faeces of pigs 3 weeks post inoculation, while deletion of the SPI-2 effector gene sifB did not affect Salmonella Typhimurium persistence. Although our list of identified genes is not exhaustive, we found that efp and rpoZ were specifically expressed in the ileocaecal lymph nodes of pigs and we identified STM4067 as a factor involved in Salmonella persistence in pigs. To our knowledge, our study is the first to identify Salmonella Typhimurium genes expressed during persistence in pigs.

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.

Salmonella Typhimurium resides largely as an extracellular pathogen in porcine tonsils, independently of biofilm-associated genes csgA, csgD and adrA.

Persistent Salmonella Typhimurium infections in pigs are a major concern for food safety and human health. Tonsils play a key role in the persistence of Salmonella Typhimurium in pigs. Previous studies indicated that Salmonella virulence genes involved in invasion and intracellular survival are of little importance for the colonization of porcine tonsils, suggesting a predominantly extracellular location of the Salmonella bacteria. Biofilm formation might promote extracellular persistence of Salmonella Typhimurium. The aim of this study was to determine whether the bacterium resides predominantly intra- or extracellularly in tonsils of pigs and to examine the contribution of biofilm-associated genes csgA, csgD and adrA in Salmonella persistence in porcine tonsils. Single cell suspensions were prepared from tonsils of orally inoculated pigs (2 x 10(7)colony forming units (CFU) wild type Salmonella Typhimurium) to determine the ratio of extracellular versus intracellular bacteria. Both at 5 and 28 days post-inoculation (pi), the majority of Salmonella bacteria was found extracellularly in porcine tonsils. To determine the contribution of biofilm formation in extracellular persistence, pigs were orally inoculated with a mixture of 2 x 10(7)CFU of the Salmonella Typhimurium wild type strain and 2 x 10(7)CFU of one of the Salmonella Typhimurium csgA, csgD or adrA mutants. At 10 days pi, equal numbers of both wild type and mutant Salmonella bacteria were found not only in tonsils, but also in ileum, ileum contents, ileocecal lymph nodes and faeces. In conclusion, we showed that Salmonella Typhimurium resides extracellularly in porcine tonsils, using a biofilm independent mechanism.