Role of SPI-1 in the interactions of Salmonella Typhimurium with porcine macrophages

Salmonella Pathogenicity Island 1 (SPI-1) genes are indispensable for virulence of Salmonella Typhimurium in mice after oral challenge. These genes mediate invasion in intestinal epithelial cells and induce cell death in murine macrophages. The role of SPI-1 in the pathogenesis of Salmonella Typhimurium infections in food producing animals is not known. It was the aim of the present study to characterize the interactions of a porcine Salmonella Typhimurium field strain and its isogenic mutants in the SPI-1 genes hilA, sipA and sipB with porcine macrophages. SPI-1 was found to be important in the invasion of porcine pulmonary alveolar macrophages (PAM) and the induction of the formation of spacious phagosomes. Both early and delayed cytotoxicity were seen in PAM, but only the early cytotoxicity was SPI-1 dependent. Exposure of PAM to Salmonella Typhimurium induced the production of reactive oxygen species (ROS) and interleukin-8, but no differences were noticed between the induction mediated by the wild type strain and its SPI-1 mutant strains. In conclusion, invasion of porcine macrophages and the induction of early, but not delayed, cytotoxicity by Salmonella Typhimurium is SPI-1 dependent. SPI-1 dependent invasion, however, is not a prerequisite to induce a pro-inflammatory response.     

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 limited role for SsrA/B in persistent Salmonella Typhimurium infections in pigs

Virulence genes regulated by the SsrA/B system are indispensable for systemic disease in BALB/c mice. The role of this regulating system in the pathogenesis of Salmonella Typhimurium infections in pigs is not documented. In the present study, the interactions of Salmonella Typhimurium and an ssrA/B mutant were compared in vitro and in vivo. The ssrA/B mutant strain displayed decreased Salmonella Pathogenicity Island 2 (SPI-2) expression levels, showed a replication defect in mouse macrophages and was attenuated in a mouse model after oral inoculation. Using real time qRT-PCR and a porcine ileal loop model, it was shown that the ssrA/B mutant strain was not significantly attenuated in overall virulence and SPI-1 expression in specific. Flowcytometric analysis demonstrated that the ssrA/B mutant strain was defective in intracellular replication in porcine macrophages. After oral inoculation of piglets with the wild type strain or the ssrA/B mutant strain, the animals of both groups excreted Salmonella and were colonized by Salmonella to the same extent. In an intravenous mixed infection model, the ssrA/B mutant strain was defective in the colonization of several internal organs. These results suggest that the ssrA/B gene of Salmonella Typhimurium plays a limited role in the persistent intestinal colonization of pigs.     

SPI-1-encoded type III secretion system of Salmonella enterica is required for the suppression of porcine alveolar macrophage cytokine expression

ABSTRACT: Genes localized at Salmonella pathogenicity island-1 (SPI-1) are involved in Salmonella enterica invasion of host non-professional phagocytes. Interestingly, in macrophages, SPI-1-encoded proteins, in addition to invasion, induce cell death via activation of caspase-1 which also cleaves proIL-1β and proIL-18, precursors of 2 proinflammatory cytokines. In this study we were therefore interested in whether SPI-1-encoded type III secretion system (T3SS) may influence proinflammatory response of macrophages. To test this hypothesis, we infected primary porcine alveolar macrophages with wild-type S. Typhimurium and S. Enteritidis and their isogenic SPI-1 deletion mutants. ΔSPI1 mutants of both serovars invaded approx. 5 times less efficiently than the wild-type strains and despite this, macrophages responded to the infection with ΔSPI1 mutants by increased expression of proinflammatory cytokines IL-1β, IL-8, TNFα, IL-23α and GM-CSF. Identical macrophage responses to that induced by the ΔSPI1 mutants were also observed to the infection with sipB but not the sipA mutant. The hilA mutant exhibited an intermediate phenotype between the ΔSPI1 mutant and the wild-type S. Enteritidis. Our results showed that the SPI-1-encoded T3SS is required not only for cell invasion but in macrophages also for the suppression of early proinflammatory cytokine expression.     

Salmonella enterica serovar Typhimurium and Enteritidis infection of pigs and cytokine signalling in palatine tonsils

Pigs are considered as one of the major sources of zoonotic strains of Salmonella enterica for humans. Out of many S. enterica serovars, S. Typhimurium dominates in pigs, however, in several countries in Central Europe, S. Enteritidis is also quite frequent in pig herds. In this study we therefore compared the colonisation of pigs with S. Typhimurium and S. Enteritidis. We found that 3 weeks after infection S. Enteritidis 147 colonised the intestinal tract in higher quantities but was shed in faeces in lower quantities than S. Typhimurium 17C10. In a second experiment we found out that S. Enteritidis 147 and its SPI-1 and SPI-4 mutants increased proinflammatory cytokine (IL-1β and IL-8) signalling in the ileum 5 days post infection. On the other hand, independent of SPI-1 or SPI-4, S. Enteritidis 147 suppressed expression of IL-18, MCP1, TLR2, CD86, IL-7, IL-10 and IL-15 in the palatine tonsils. The suppression of cytokine signalling may facilitate the initial colonisation of the palatine tonsils by Salmonella. Moreover, immune suppression may also influence pig resistance to opportunistic pathogens and Salmonella infection in pigs thus may become an issue not only in terms of pork contamination but also in terms of affecting the immunological status of pig herds.     

Survival of Salmonella serovar Typhimurium inside porcine monocytes is associated with complement binding and suppression of the production of reactive oxygen species

The development of the carrier state in swine after infection with Salmonella serovar Typhimurium (S. Typhimurium) has not been elucidated yet. Possibly, phagocytes like macrophages play a crucial role. It was the aim of the present study to characterize the interaction of a S. Typhimurium strain and its hilA and ssrA mutants with porcine peripheral blood monocytes (PBM). Exposure of porcine PBM to S. Typhimurium induced the production of reactive oxygen species (ROS), requiring bacterial protein synthesis. The numbers of intracellular bacteria sharply decreased over a period of 3h. Monocytes obtained from different pigs differed markedly in their ROS production and in their ability to kill the bacteria. Interestingly, high ROS production did not coincide with increased intracellular killing. Using diphenylene iodonium inhibition of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity, it was shown that bacterial killing was ROS-dependent only within 1h post inoculation, but was ROS-independent from 1h post inoculation onwards. This might be explained by the finding that metabolically active Salmonella bacteria were capable of suppressing the respiratory burst activity in a SPI-1- and SPI-2-independent manner without causing measurable cell damage. Opsonization with complement did not alter the ROS production. Nevertheless, it increased intracellular survival of the bacteria. In conclusion, survival of S. Typhimurium inside porcine PBM is promoted by suppression of respiratory burst activity and complement binding.     

Impact of Salmonella Typhimurium DT104 virulence factors invC and sseD on the onset, clinical course, colonization patterns and immune response of porcine salmonellosis

The present study was conducted to study the impact of the virulence factors invC and sseD of the two type III secretion systems of Salmonella enterica serovar Typhimurium (S. Typhimurium) on the pathogenesis of the porcine S. Typhimurium DT104 infection. For this purpose, two S. Typhimurium mutant strains with a disrupted invC gene of the Salmonella pathogenicity island 1 or with a disrupted sseD gene of the Salmonella pathogenicity island 2 have been studied in experimental infection of pigs. Twenty-two 7-week-old male hybrid pigs were either infected with one of the mutants or the wild-type S. Typhimurium DT104 strain. Each group was examined for clinical signs, Salmonella shedding rate and the specific antibody response. Survival and replication were evaluated by qualitative and quantitative determination of the colonization rate. The humoral and cellular immune responses were examined using isotype-specific ELISA and quantitative real-time PCR of IL-2, IL-4, IL-10, IL-12 and IFN-gamma. The results proved that both mutants had a lower virulence (with marked differences between both mutants) than the wild-type and that both virulence factors have importance in porcine salmonellosis. Only pigs infected with the wild-type S. Typhimurium DT104 exhibited typical clinical symptoms of salmonellosis like anorexia, vomiting, disturbed demeanour, fever and diarrhoea. Deletion of the invC gene resulted in a significantly reduced colonization rate. Interestingly, the mRNA expression of both type-1 and type-2 cytokines were significantly decreased in pigs infected with either the invC-mutant and the sseD-mutant strain.     

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

ABSTRACT: 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.     

Cytokine response of porcine cell lines to Salmonella enterica serovar typhimurium and its hilA and ssrA mutants

Salmonella enterica serovar Typhimurium (S. Typhimurium) is a facultative intracellular bacterium which can infect and colonize pigs. After contact with enterocytes and macrophages, S. Typhimurium induces production of cytokines thus triggering the innate immune response. In this study we evaluated the cytokine response of two porcine cell lines, IPI-2I and 3D4/31, of epithelial or macrophage origins, respectively, to the wild-type S. Typhimurium and its hilA and ssrA mutants. We observed that the 3D4/31 cell line essentially did not respond to S. Typhimurium infection when a medium with foetal calf serum was used. However when the 3D4 cell line was incubated overnight in the presence of porcine serum, it efficiently responded to the wild-type strain and the ssrA mutant but not to the noninvasive hilA mutant as measured by mRNA quantification of TNF-alpha, IL-8 and GM-CSF by the real-time RT-PCR. In IPI-2I, all the cytokines were also induced by the wild-type S. Typhimurium and the ssrA mutant although the induction of TNF-alpha was lower than that induced by the wild-type strain. The hilA mutant was unable to induce any of the cytokines tested. The ssrA mutant can therefore be considered as more suitable for further vaccine development as the stimulation of innate immune response is important for animal protection against a challenge with virulent strains.     

Differences in immune responses of pigs vaccinated with Salmonella Typhimurium and S. Choleraesuis strains and challenged with S. Choleraesuis

S. Choleraesuis (Choleraesuis) and S. Typhimurium (Typhimurium) cause salmonellosis in pigs and humans. The effects of vaccine strains pSV-less Typhimurium OU5048 and Choleraesuis OU7266 and SPI-2-mutant Choleraesuis SC2284 on the immune responses of pigs against Typhimurium, Choleraesuis, and S. Enteritidis (Enteritidis) with or without the virulence plasmid (pSV) were determined. After oral vaccination of three vaccine groups and challenge with Choleraesuis CN36, the level of Salmonella-specific IgG in sera and the bactericidal effects and superoxide generation of peripheral blood mononuclear cells (PBMCs) and polymorphonuclear leukocytes (PMNs) against the above strains were determined using ELISA and NBT assay, respectively. Among three vaccine strains tested, OU7266 stimulated the highest Salmonella-specific IgG levels. Complement inactivation increased IgG concentration, while E. coli absorption reduced IgG levels. The pSV-containing strains were less resistant to serum killing than the pSV-less strains, and Enteritidis exhibited the lowest resistance to serum killing. Serovars tested, vaccine strains, and timeline periods postvaccination and challenge were important factors affecting superoxide production. The two Choleraesuis vaccine strains stimulated greater levels of superoxide from PMNs and PBMCs than the Typhimurium strains. The PMNs and PBMCs in challenged and vaccinated pigs reduced more superoxide than those in challenged hosts. In vaccinated hosts, pSV-less Salmonella strains triggered lower levels of PMN/PBMC-generated superoxide upon challenge than strains with pSV against Enteritidis and Choleraesuis. Overall, Choleraesuis OU7266 may be better than the other vaccine strains in generating the greatest IgG levels, serum bactericidal activity and superoxide levels. The pSV likely influences the immune responses.     

Oxygenated drinking water enhances immune activity in pigs and increases immune responses of pigs during Salmonella typhimurium infection

It has been considered that drinking oxygenated water improves oxygen availability, which may increase vitality and improve immune functions. The present study evaluated the effects of oxygenated drinking water on immune function in pigs. Continuous drinking of oxygenated water markedly increased peripheral blood mononuclear cell proliferation, interleukin-1β expression level and the CD4(+):CD8(+) cell ratio in pigs. During Salmonella Typhimurium infection, total leukocytes and relative cytokines expression levels were significantly increased in pigs consuming oxygenated water compared with pigs consuming tap water. These findings suggest that oxygenated drinking water enhances immune activity in pigs and increases immune responses of pigs during S. Typhimurium Infection.     

Nose-to-nose transmission of Salmonella Typhimurium between weaned pigs

Little attention has been paid to the possibility of transmission of Salmonella in intensive pig production systems through alternate methods, such as airborne or direct nose-to-nose contact. This experimental study tested the hypothesis of nose-to-nose transmission of Salmonella enterica serovars Typhimurium (Trial I) and Agona (Trial II) in weaned pigs using stainless steel/glass isolation cabinets. In each trial, cabinet 1 (control pigs) and cabinet 2 (sentinel pigs) were connected directly to the fan unit. Cabinet 3 (seeded pigs) was not directly linked to the fan, but was arranged to receive a constant unidirectional airflow from cabinet 2 (sentinel pigs) through a 10cm diameter hole, which also allowed nose-to-nose contact between pigs housed in these two cabinets. Air was taken out of the system through ducts connecting cabinets 1 and 3 to the exhauster. Therefore, direct contact among seeded and sentinel pigs was allowed but possible aerial transference of contaminated particles between those cabinets was prevented. The system was opened 21 days post-inoculation and tissue samples were collected for bacteriological analysis. The recovery of nalidixic acid-resistant Salmonella Typhimurium from sentinel pigs corroborates the hypothesis of nose-to-nose transmission of that pathogen in pigs. However, serovar-related differences might exist regarding the nose-to-nose transmissibility of Salmonella in pigs, since Salmonella Agona was not detected in sentinel pigs (Trial II).     

Host specific differences alter the requirement for certain Salmonella genes during swine colonization

The pathogenic potential of Salmonella is determined during the complex interaction between pathogen and host, requiring optimal regulation of multiple bacterial genetic systems within variable in vivo environments. The mouse model of systemic disease has been an extremely productive model to investigate the pathogenesis of Salmonella enterica serovar Typhimurium (S. Typhimurium). Although the mouse model is a widely used paradigm for studying the pathogenesis of systemic disease caused by Salmonella, investigations concerning food safety interventions should employ natural hosts to examine gastrointestinal colonization by Salmonella. Recent research has demonstrated specific differences in the attenuation of certain S. Typhimurium mutants in mice compared to swine. This variation in pathogenesis between the mouse model and pigs for the S. Typhimurium mutants is presumably dependent upon either the requirements for specific gene products during systemic disease (mouse) versus gastrointestinal colonization (pig) or host specific differences. In addition, host specific diversity in Salmonella colonization of swine has also been described in comparison to other food-producing animals, including cattle and chickens. Differences in Salmonella colonization and pathogenesis across diverse animal species highlight the importance of species-specific studies of gastrointestinal colonization for the development of Salmonella interventions to enhance pork safety.     

Effects of microcin 24-producing Escherichia coli on shedding and multiple-antimicrobial resistance of Salmonella enterica serotype typhimurium in pigs

OBJECTIVE: To investigate the effect of an Escherichia that produced microcin 24 (Mcc24) on shedding of of Salmonella enterica serotypeTyphimurium in swine and evaluate evidence of in vivo activation of the Mcc24-mediated, multiple-antibiotic resistance (mar) operon. ANIMALS: 36 crossbred weaned pigs. PROCEDURE: 24 pigs were allocated to 2 groups (12 pigs/group). Pigs in 1 group received daily oral administration of an Mcc24-producing E coli, whereas the other group received a non-Mcc24-producing E coli. All pigs were challenge exposed with Salmonella Typhimurium chi4232. A third group of 6 pigs received Mcc24-producing E coli and was challenge exposed with an Mcc24-sensitive, marA-deleted strain of Salmonella Typhimurium 4232. After challenge exposure, fecal samples from all pigs were cultured to detect shedding of Salmonella Typhimurium and Salmonella Typhimurium isolates were screened for resistance to ciprofloxacin. Fecal samples were collected throughout the study, and tissue samples were collected during necropsy. RESULTS: Differences in shedding of Salmonella Typhimurium were not detected between groups receiving Mcc24-producing or non-Mcc24-producing E coli. No significant differences were found in quantitative analysis between groups receiving Mcc24-producing and non-Mcc24-producing E coli. Evidence of mar activation was not detected. CONCLUSIONS AND CLINICAL RELEVANCE: Microcin-producing E coli did not exert an effect on shedding of SalmonellaTyphimurium or mar activation in pigs. It may be difficult or impractical to create the conditions required for Mcc24 to be an effective part of a food safety intervention to reduce shedding of Salmonella Typhimurium.     

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 enterica serovar Choleraesuis derivatives harbouring deletions in rpoS and phoP regulatory genes are attenuated in pigs, and survive and multiply in porcine intestinal macrophages and fibroblasts, respectively

Live attenuated Salmonella enterica strains have been extensively studied as potential vectors for the oral delivery of heterologous antigens. Due to its ability to target immune cells, its specific mechanism for crossing the intestinal barrier, and its swine-restricted tropism, S. enterica subspecies enterica serovar Choleraesuis (S. Choleraesuis) has attracted a great deal of interest for the production of bacterial-based oral carriers specifically adapted to swine. In this study, two mutants of S. Choleraesuis were constructed and their attenuation and intracellular fate analysed with the purpose of engineering new attenuated live strains with improved properties as oral vaccine carriers. Those strains harboured a specific deletion either within the phoP or rpoS genes, which encode virulence-related regulators in S. Typhimurium. In comparison to the wild-type parental S. Choleraesuis, the mutant strains, especially DeltaphoP, were extremely low in virulence in the murine model and in the natural host, the pig. Moreover, when compared with a commercial live vaccine strain, SC-54, the two mutants showed a higher level of attenuation in mice and DeltaphoP also in pigs. In addition, DeltarpoS and DeltaphoP presented a proliferation and survival phenotype within swine intestinal primary fibroblast and macrophage cell cultures, respectively. Collectively, the present results indicate that the DeltarpoS and DeltaphoP strains of S. Choleraesuis gather adequate features to be potential candidates for vaccine vectors for the specific delivery of heterologous antigens adapted to pigs.     

Coated fatty acids alter virulence properties of Salmonella Typhimurium and decrease intestinal colonization of pigs

Salmonella Typhimurium infections in pigs are a major source of human foodborne salmonellosis. To reduce the number of infected pigs, acidification of feed or drinking water is a common practice. The aim of the present study was to determine whether some frequently used short- (SCFA) and medium-chain fatty acids (MCFA) are able to alter virulence gene expression and to decrease Salmonella Typhimurium colonization and shedding in pigs using well established and controlled in vitro and in vivo assays. Minimal inhibitory concentrations (MIC) of 4 SCFA (formic acid, acetic acid, propionic acid and butyric acid) and 2 MCFA (caproic and caprylic acid) were determined using 54 porcine Salmonella Typhimurium field strains. MIC values increased at increasing pH-values and were two to eight times lower for MCFA than for SCFA. Expression of virulence gene fimA was significantly lower when bacteria were grown in LB-broth supplemented with sub-MIC concentrations of caproic or caprylic acid (2 mM). Expression of hilA and invasion in porcine intestinal epithelial cells was significantly lower when bacteria were grown in LB-broth containing sub-MIC concentrations of butyric acid or propionic acid (10 mM) and caproic or caprylic acid (2 mM). When given as feed supplement to pigs experimentally infected with Salmonella Typhimurium, coated butyric acid decreased the levels of faecal shedding and intestinal colonization, but had no influence on the colonization of tonsils, spleen and liver. Uncoated fatty acids, however, did not influence fecal shedding, intestinal or tonsillar colonization in pigs. In conclusion, supplementing feed with certain coated fatty acids, such as butyric acid, may help to reduce the Salmonella load in pigs.