Salmonella enterica serovar Choleraesuis infection of the porcine jejunal Peyer's patch rapidly induces IL-1beta and IL-8 expression

Salmonella enterica serovar Choleraesuis is an enteric pathogen of swine, producing septicemia, enterocolitis, pneumonia, and hepatitis. The initial molecular events at the site of Salmonella infection are hypothesized to be critical in the initiation of innate and adaptive immune responses; however, the acute immune response elicited by porcine intestinal tissues is not well understood. To address this need, we employed explants of jejunal Peyer's patch (JPP) mucosa from pigs to examine Salmonella-induced immune responses under controlled conditions as well as to overcome limitations of whole animal approaches. JPP explants mounted in Ussing chambers maintained normal histological structure for 2 h and stable short-circuit current and electrical conductance for 2.5 h. After ex vivo luminal exposure to Salmonella serovar Choleraesuis, JPP responded with an increase in mRNA expression of IL-1beta and IL-8, but not TNFalpha. Increased IL-1beta and IL-8 expression were dependent on efficient Salmonella adhesion and internalization, whereas mutant Salmonella did not induce inflammatory cytokine expression. Commensal enteric bacteria, present in some experiments, also did not induce inflammatory cytokine expression. These findings indicate that Salmonella uptake by Peyer's patch is important in the induction of an innate response involving expression of IL-1beta and IL-8, and that ex vivo intestinal immune tissue explants provide an intact tissue model that will facilitate investigation of mucosal immunity in swine.     

Use of bioinformatic SNP predictions in differentially expressed genes to find SNPs associated with Salmonella colonization in swine

Asymptomatic Salmonella-carrier pigs present a major problem in preharvest food safety, with a recent survey indicating >50% of swine herds in the United States have Salmonella-positive animals. Salmonella-carrier pigs serve as a reservoir for contamination of neighbouring pigs, abattoir pens and pork products. In addition, fresh produce as well as water can be contaminated with Salmonella from manure used as fertilizer. Control of Salmonella at the farm level could be through genetic improvement of porcine disease resistance, a potentially powerful method of addressing preharvest pork safety. In this research, we integrate gene expression profiling data and sequence alignment-based prediction of single nucleotide polymorphisms (SNPs) to successfully identify SNPs in functional candidate genes to test for the associations with swine response to Salmonella. A list of 2527 genes that were differentially regulated in porcine whole blood in response to infection with Salmonella enterica serovar Typhimurium were selected. In those genes, SNPs were predicted using ANEXdb alignments based on stringent clustering of all publically available porcine cDNA and expressed sequence tag (EST) sequences. A set of 30 mostly non-synonymous SNPs were selected for genotype analysis of four independent populations (n = 750) with Salmonella faecal shedding or tissue colonization phenotypes. Nine SNPs segregated with minor allele frequency ≥15% in at least two populations. Statistical analysis revealed SNPs associated with Salmonella shedding, such as haptoglobin (HP, p = 0.001, q = 0.01), neutrophil cytosolic factor 2 (NCF2 #2, p = 0.04, q = 0.21) and phosphogluconate dehydrogenase (p = 0.066, q = 0.21). These associations may be useful in identifying and selecting pigs with improved resistance to this bacterium.     

Gene expression responses to a Salmonella infection in the chicken intestine differ between lines

Poultry products are an important source of Salmonella enterica. An effective way to reduce food poisoning due to Salmonella would be to breed chickens more resistant to Salmonella. Unfortunately host responses to Salmonella are complex with many factors involved. To learn more about responses to Salmonella in young chickens, a cDNA microarray analysis was performed to compare gene expression profiles between two chicken lines under control and Salmonella infected conditions. Newly hatched chickens were orally infected with S. enterica serovar Enteritidis. Since the intestine is the first barrier the bacteria encounter after oral inoculation, intestinal gene expression was investigated at different timepoints. Differences in gene expression between the two chicken lines were found in control as well as Salmonella infected conditions. In response to the Salmonella infection a fast growing chicken broiler line induced genes that affect T-cell activation, whereas in a slow growing broiler line genes involved in macrophage activation seemed to be more affected at day 1 post-infection. At days 7 and 9 most gene expression differences between the two chicken lines were identified under control conditions, indicating a difference in the intestinal development between the two chicken lines which might be linked to the difference in Salmonella susceptibility. The findings in this study have lead to the identification of novel genes and possible cellular pathways, which are host dependent.     

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.     

Chromosomal integration and expression of the Escherichia coli K88 gene cluster in Salmonella enterica ser. Choleraesuis strain 54 (SC54)

Attempts to develop live vaccines to protect against enterotoxigenic Escherichia coli (ETEC) infection by induction of both cell-mediated and mucosal immunity, and serum antibody responses have included use of recombinant Salmonella strains that produce K88 fimbrial antigens (Hone et al., 1988; Attridge et al., 1988; Morona et al., 1994). However, none of the recombinant Salmonella vectors has been licensed by the United States Department of Agriculture (USDA) for use as a live vaccine in pigs in the United States. A variant of Salmonella enterica ser. Choleraesuis strain 54 (SC54) is currently used as a safe and effective intranasal attenuated live vaccine in pigs. In order to expand the efficacy of this live vaccine strain, we sought to modify strain SC54 to express the K88 antigens of ETEC. To accomplish this, a plasmid-based system was used to integrate the K88 gene cluster into the chromosome of strain SC54 by site-specific recombination. The K88 antigens were expressed by strain SC54, and the gene cluster was stably maintained in the host.     

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.     

Effects of feeding Salmonella enterica serovar Typhimurium or serovar Choleraesuis on growth performance and circulating insulin-like growth factor-I, tumor necrosis factor-alpha, and interleukin-1beta in weaned pigs

The most common Salmonella serovars causing clinical disease in pigs are Salmonella enterica serovars Typhimurium (Typhimurium) and Choleraesuis. Given that the swine host-adapted serovar Choleraesuis has been reported to cause systemic disease, a different disease outcome from that of Typhimurium, our working hypothesis was that this serovar would likely engage systemic immune-inflammatory mechanisms, resulting in elevated systemic cytokine secretion. Forty-eight weaned pigs were blocked by BW and sex, and randomly allotted to 1 of 3 treatments in a 14-d study. Each treatment had 8 replicates (pens), with 2 pigs/pen. The treatments consisted of a negative control and pigs repeatedly fed 10(8) cfu of Typhimurium or Choleraesuis. On d 0, the pigs were fed Choleraesuis or Typhimurium in dough balls, and the bacteria were refed twice weekly throughout the experiment. Control pigs received dough balls without bacteria. All pigs were housed in temperature-controlled rooms under constant lighting and were fed a standard corn-soybean meal-based nursery diet. Pig BW and feed disappearance were used to determine ADG, ADFI, and G:F. Rectal temperatures were obtained daily from 1 pig/pen beginning 2 d before the first bacterial feeding through d 7 using rapid-response digital thermometers. Serum was collected on d 0, 7, and 14 from a single pig/pen for analysis of IGF-I, tumor necrosis factor-alpha , and IL-1beta. There was no change in the rectal temperature of the control or the Typhimurium-challenged pigs (compared with d 0) or when comparing Typhimurium-challenged pigs with control animals. In contrast, pigs fed Choleraesuis had increased rectal temperatures beginning on d 2 and continuing through d 7 (P < 0.05), with the greatest elevation on d 3 (P < 0.001) compared with the control pigs. Average daily gain and ADFI of pigs challenged with Typhimurium did not differ from those of the control animals. Pigs fed Choleraesuis had a 25% reduction in ADG (P < 0.0001) and ADFI (P < 0.002) compared with the control pigs. On d 7, pigs fed Choleraesuis had reduced serum IGF-I compared with control (P < 0.01) or Typhimurium-challenged pigs (P = 0.01). Bacterial feeding did not affect serum tumor necrosis factor-alpha or IL-1beta compared with control pigs at any time throughout the experiment. We conclude that repeated exposure of weaned pigs to Choleraesuis reduced growth performance in the absence of changes in systemic inflammatory cytokines.     

Molecular typing and antimicrobial resistance of Salmonella enterica subspecies enterica serovar Choleraesuis isolates from diseased pigs in Japan

Salmonella enterica serovar Choleraesuis (S. Choleraesuis) isolates derived from diseased pigs in Japan during 2001 and 2005 were analyzed for biotype, based on H(2)S production and dulcitol fermentation, pulsed-field gel electrophoresis (PFGE) profile, and antimicrobial resistance profile. S. Choleraesuis biotype Choleraesuis (biotype Choleraesuis) was classified into one genotype, while varietas Kunzendorf (var. Kunzendorf) was classified into two genotypes. The isolates of var. Kunzendorf belonging to one genotype were isolated in a limited area of Japan. Variation in the antimicrobial resistance pattern was observed in isolates of both biotypes Choleraesuis and var. Kunzendorf. We have also shown that the PFGE profile was associated with the biotype and isolation region of each isolate.     

Virulence plasmids of Salmonella enterica--incidence and properties

Salmonella virulence plasmids (SVPs) are large and closely related low-copy plasmids harbored by certain serovars of Salmonella enterica subspecies enterica. These serovars not only comprise those of veterinary significance like Abortusequi, Abortusovis, Choleraesuis, Dublin and Gallinarum/Pullorum, but also Typhimurium and Enteritidis which currently are the most prevalent serotypes in humans and food animals. Experiments with several animal species gave evidence that SVPs increase Salmonella strains' capabilities to replicate in extraintestinal organs of infected hosts thus leading to death of those hosts more frequently and rapidly. The common feature of all SVPs is the "Salmonella plasmid virulence" locus (spv-locus), a highly conserved 7.8 kbp region that is most responsible for the SVP-encoded virulence phenotype of Salmonella. Although functional characterisation of spv gene products has made some progress the molecular mechanism of spv-mediated virulence has not been fully elucidated yet. Some SVPs carry additional gene loci causatively related to Salmonella virulence like the pef-operon of Typhimurium and Enteritidis strains which encodes an adhesive type of fimbria, or genes traT, rsk and rck which are involved in serum resistance. The frequent occurrence of SVPs in host-adapted serovars suggests that SVP-encoded factors represented selective advantages to some Salmonella variants in their effort to colonize certain new niches during Salmonella evolution. This study provides an overview over current knowledge about the virulence plasmids of Salmonella enterica.     

The expression of genes related to adipocyte differentiation in pigs

The purpose of this study was to detect differential expression of genes related to adipocyte differentiation in pigs by suppression subtractive hybridization. Adipocytes and stromal vascular cells (a fraction containing preadipocytes) from pig adipose tissue were isolated for mRNA extraction. The cDNA from preadipocytes was subtracted from the cDNA from adipocytes. The subtracted gene fragments were cloned into pGEM-T Easy TA cloning vector. We selected 384 clones for gene sequence determination and for further analysis. These genes were subjected to a differential screening procedure to confirm the differential expression of genes between the 2 cell types. We found that at least 36 genes were highly expressed in the adipocytes compared with preadipocytes. Among these, 6 genes including 2 novel genes with the greatest differences were selected and confirmed by Northern analysis. We found that angiotensin I-converting enzyme (ACE), ataxia-telangiectasia mutated protein (ATM), calpain 1, and stearoyl coenzyme A desaturase 1 (SCD1) were highly expressed in adipocytes compared with preadipocytes (P < 0.05). The relative mRNA abundance of ACE, ATM, calpain 1, SCD1, and 2 novel genes discovered in the current study was increased at the later stages of adipocyte differentiation (P < 0.05). The results confirmed that the genes involved in lipid metabolism and adipocyte differentiation were highly expressed in porcine adipocytes. However, further investigation is needed to demonstrate specific functions of the novel genes discovered in the current study.     

Long-term survival and infectivity of Salmonella choleraesuis

It is believed that Salmonella Choleraesuis, the host-adapted serotype of swine, does not survive well outside the host. We examined the survival capability as well as the presence of latent DNA of S. Choleraesuis in swine feces. Pigs were infected with S. Choleraesuis and feces was collected and pooled on days 2, 4, 7, and 10 post inoculation (PI). Feces was stored in a wet and a dry form and survival was measured over 13 months. Salmonella Choleraesuis was recovered from wet feces through 3 months of storage. In a desiccated (dry) form, S. Choleraesuis was recovered from at least 13 months. Direct PCR analysis did not detect S. Choleraesuis subsequent to the final culture recovery for any stored sample. We also examined the infectivity of S. Choleraesuis resident in dry feces. Six or 13 week old pigs were inoculated with dry feces that had been stored either 2 months or 4 months, respectively. Pigs were inoculated either intranasally or by mixing dry feces with the swine ration. Although clinical signs were mild, S. Choleraesuis was widely disseminated among the tissues of all the pigs inoculated. This study demonstrates that S. Choleraesuis remains viable and infective in the environment. Therefore, contaminated fecal matter can serve as a reservoir for S. Choleraesuis as well as other Salmonella spp. Control measures must consider this environmental reservoir as a source of new infections.     

Porcine circovirus type 2 (PCV2) vaccination reduces PCV2 in a PCV2 and Salmonella enterica serovar Choleraesuis coinfection model

We previously reported that prior porcine circovirus type 2 (PCV2) infection potentiates the severity of clinical signs, lung lesions, and fecal shedding and tissue dissemination of Salmonella enterica serovar Choleraesuis in infected pigs. Here, we evaluated whether PCV2 vaccination is effective in reducing fecal shedding and tissue dissemination of S. Choleraesuis and improving clinical signs associated with PCV2 and S. Choleraesuis infection in 15 Cesarean-derived, colostrum-deprived pigs randomly assigned to 3 groups (n = 5/group). The vaccinated and co-infected (VAC-COINF) group received 2 ml of a commercial PCV2 vaccine at age 3 weeks. The VAC-COINF and co-infected (COINF) groups were inoculated intranasally with PCV2 and S. Choleraesuis at 5 and 7 weeks of age, respectively. The CONTROL group pigs received a similar volume of PBS for sham-vaccination and sham-inoculation. PCV2 vaccination clearly reduced PCV2 DNA load in the serum and postmortem tissue samples and decreased PCV2 antigen levels in tissue samples of the VAC-COINF group. After S. Choleraesuis infection, the incidence of several clinical signs increased in the VAC-COINF group compared to that in the COINF group. The microscopic lung lesions and weight gain, fecal shedding and tissue dissemination of S. Choleraesuis except in the spleen were not significantly different in the VAC-COINF and COINF groups. Thus, PCV2 vaccination reduced PCV2 in the S. Choleraesuis and PCV2 coinfection model and the effects on S. Choleraesuis were minimal.     

An outbreak of Salmonella enterica serotype Choleraesuis in goitered gazelle (Gazella subgutrosa subgutrosa) and a Malayan tapir (Tapirus indicus)

An outbreak of Salmonella enterica serotype Choleraesuis enteritis occurred in two juvenile goitered gazelles and an adult Malayan tapir over a period of 5 wk at the Minnesota Zoo. Diagnosis was made postmortem on one gazelle and one tapir, and a second gazelle was diagnosed via fecal culture. The death of the tapir was attributed to S. enterica serovar Choleraesuis septicemia, while salmonellosis was considered to be a contributing factor besides ostertagiasis for the death of one goitered gazelle and for the diarrhea of another goitered gazelle. A third gazelle became ill in the same time period, but Salmonella infection was not confirmed by culture. All exhibited the clinical signs of profuse, watery diarrhea. The gazelles developed a protein-losing enteropathy, and the tapir showed signs of sepsis and endotoxemia. Serotyping and pulsed-field gel electrophoresis revealed the Salmonella isolates to be indistinguishable from each other. One year prior to this outbreak, Salmonella sp. was cultured from a Visayan warty pig (Sus cebifrons) housed in the same building as the tapir. After further investigation into the outbreak, spread of this pathogen was speculated to be associated with human movement across animal areas.     

Salmonella Enteritidis universal stress protein (usp) gene expression is stimulated by egg white and supports oviduct colonization and egg contamination in laying hens

Salmonella enterica subspecies enterica serovar Enteritidis has caused a worldwide egg-associated pandemic since the mid 1980s. The exact mechanisms causing this egg tropism are still largely unknown, and only a few Salmonella genes have been implicated in the interaction with the oviduct or eggs. A in vivo expression technology screening performed previously, identified the uspA and uspB genes as being highly expressed in the chicken oviduct and in eggs. Here, we demonstrate that uspA and uspB gene expression is indeed induced after contact with egg white. Intra-oviduct inoculation of Salmonella Enteritidis uspB and uspBA mutant strains showed that the mutants had a decreased ability to colonize the magnum and isthmus of the oviduct, the organs that produce the egg white and eggshell membranes, respectively, at 7 days post-inoculation. Intravenous challenge showed that a Salmonella Enteritidis uspBA mutant strain had a decreased ability to contaminate eggs. Analogous to the function of universal stress proteins A and B in other bacterial species, we hypothesize that the Salmonella uspA and uspB genes are involved in long term persistence of Salmonella Enteritidis in harmful environments, such as in the oviduct and eggs, by conferring resistance against compounds that damage the bacterial cell membrane and DNA.     

Enrichment for isolating Salmonella Choleraesuis and other Salmonella spp. from pigs

The growth of Salmonella Choleraesuis was examined in Rappaport Vassiliadis broth (RV) and Hajna-tetrathionate broth (HTT) at 37 and 42 degrees C. As the enrichment in RV at 37 degrees C was satisfactory for isolating S. Choleraesuis, we used this enrichment for isolation from the samples collected from 15 asymptomatic pigs reared on a S. Choleraesuis contaminated farm. S. Choleraesuis was frequently isolated from six pigs (40.0%) under field conditions. The isolation of other Salmonella serovars than S. Choleraesuis was attempted by using both RV enrichment at 37 degrees C and HTT enrichment at 42 degrees C. Salmonella organisms were isolated from 156 (44.8%) of 348 fecal samples and more frequently with HTT at 42 degrees C (43.4%) than with RV at 37 degrees C (20.9%). If other serovars in addition to S. Choleraesuis are to be surveyed, HTT enrichment should be used in combination with RV enrichment.     

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.     

A clinical field trial to evaluate the efficacy of vaccination in controlling Salmonella infection and the association of Salmonella-shedding and weight gain in pigs

A clinical field trial was performed to determine the effectiveness of an autogenous Salmonella Typhimurium bacterin compared with a commercial live S. Choleraesuis vaccine in pigs. The association between Salmonella shedding and weight gain was also investigated. Nine cohorts of weaned pigs, (330 to 350 pigs per cohort), were randomly assigned to 1 of 3 treatment groups (injection with S. Typhimurium bacterin, vaccination via water with S. Choleraesuis vaccine, or a control group receiving no vaccine). In each cohort, the average daily gain was calculated for a selected pen throughout the production stage. Pen (pooled) fecal samples were collected bi-weekly and cultured. The odds of Salmonella shedding in both vaccinated groups was higher than in the control group (P < 0.05). The prevalence of Salmonella shedding declined overall as pigs aged (P = 0.04). However, the control pigs showed the smallest decrease in Salmonella shedding over the entire production stage, while prevalence of Salmonella shedding in the vaccinated groups decreased twice as much as the control group over the entire production stage. Salmonella Typhimurium var. Copenhagen DT104, S. Cerro, and S. Agona, which had been isolated on the study farm previously, were recovered from pigs in this study. Shedding of S. Typhimurium var. Copenhagen decreased over time in both vaccine treatment groups. On the other hand, S. Cerro shedding rate was lower in the control pigs compared with vaccinated pigs and S. Agona could be recovered only from the samples collected from S. Choleraesuis vaccinated pigs. The pigs from pens with a higher Salmonella recovery rate experienced slower growth compared with pigs from pens where Salmonella was not isolated. This latter finding indicates that there might be an economic incentive for producers to try to control endemic salmonellosis if effective programs could be developed.