Effects of Salmonella enterica serovar Typhimurium, or serovar Choleraesuis, Lactobacillus reuteri and Bacillus licheniformis on chemokine and cytokine expression in the swine jejunal epithelial cell line, IPEC-J2.

Direct-fed microbials, including Lactobacillus and Bacillus spp., are potential replacements for low dose in-feed antibiotics for swine and other livestock. To understand the function of these microbes in the gut, the current study used pig jejunal epithelial cells (IPEC-J2) to evaluate how Lactobacillus reuteri (LR) and Bacillus licheniformis (BL) differed from Salmonella enterica serovars Typhimurium (ST) or Choleraesuis (SC) in their ability to regulate, stimulate, or modify the proinflammatory mediators, interleukin 8 (IL8), CC chemokine 20 (CCL20), and tumor necrosis factor-alpha (TNFalpha). To optimize the positive control to drive IL8 secretion by IPEC-J2 cells, cells were treated apically with various concentrations of ST (versus control (CTL)) for 1h, followed by a wash. Media containing gentamicin was added and collected at 6h post-treatment. Compared to CTL, 10(8) ST produced maximal IL8 secretion in both the apical and basolateral directions, with significant basolateral polarization (P<0.0001). We next evaluated the time course of IL8 secretion, and IL8, CCL20, and TNFalpha mRNA expression by IPEC-J2 cells treated apically with 10(8) ST, SC, LR, and BL versus CTL. Media and RNA were collected at 1.5, 3.0, and 6.0 h post treatment. Only ST stimulated an increase in IL8 secretion at any time point, with increases in IL8 mRNA at both 3 and 6h (P<0.05). However, BL increased IL8 mRNA at 1.5h (P<0.0001). Neither LR nor SC affected IL8 mRNA expression. CCL20 mRNA was strongly upregulated by ST (P<0.05) and BL (1.5 and 3.0 h; P<0.05), but not LR or SC. Only ST increased TNFalpha mRNA relative to CTL (P<0.05). Two experiments were conducted to determine if pre-exposure of IPEC-J2 cells to LR or BL modified ST induced IL8 secretion. Confluent cells were treated apically overnight with various levels of LR or BL (in separate experiments) followed by ST challenge. Media were collected at 4 (LR experiment) or 5h (BL experiment) post ST. In the LR study, IL8 secretion was increased by ST as compared to CTL (P<0.0001), reduced by LR (P<0.05), and LR+ST co-treatments failed to alter ST stimulated secretion. In the BL experiment, secretion of IL8 was increased by ST (P<0.0001), but blunted basolaterally in BL+ST co-treated wells. The data demonstrate that IPEC-J2 cells increase IL8 secretion in response to ST, and IL8 mRNA in response to ST and BL, but not LR. Furthermore, ST stimulated secretion of IL8 is inhibited basolaterally in the presence of BL.

Expression of Toll-like receptors, interleukin 8, macrophage migration inhibitory factor, and osteopontin in tissues from pigs challenged with Salmonella enterica serovar Typhimurium or serovar Choleraesuis.

Two serovars of Salmonella enterica, namely serovar Typhimurium (ST) and serovar Choleraesuis (SC) account for the vast majority of clinical cases of swine salmonellosis worldwide. These serovars are thought to be transmitted among pigs in production settings mainly through fecal-oral routes. Yet, few studies have evaluated effects of these serovars on expression of innate immune targets when presented to pigs via repeated oral dosing in an attempt to model transmission in production settings. Thus, a primary objective of the current experiments was to evaluate expression of Toll-like receptors (TLR) and selected chemoattractive mediators (interleukin 8, IL8; macrophage migration inhibitory factor, MIF; osteopontin, OPN) in tissues from pigs exposed to ST or SC that had been transformed with kanamycin resistance and green (STG) or red (SCR) fluorescent protein to facilitate isolation from pen fecal samples. In vitro studies confirmed that STG and SCR largely (though not completely) retained their ability to upregulate IL8 and CC chemokine ligand 20 (CCL20) in cultured swine jejunal epithelial cells. Transformed bacteria were then fed to pigs in an in vivo study to determine tissue specific effects on mRNA relative expression. Pigs were fed cookie dough inoculated with bacteria on days 0, 3, 7, and 10 with 10(8)CFU STG (n=8) or SCR (n=8), while control (CTL) pigs (n=8) received dough without bacteria. Animals were sacrificed 14 days from the initial bacterial challenge and samples of tonsil, jejunum, ileum, colon, mesenteric lymph node (MLN), spleen, and liver were removed for subsequent RNA isolation. Expression of mRNA in tissues was determined using real-time quantitative PCR and expressed relative to 18S rRNA. Within CTL pigs, when expressed relative to the content in liver, mRNA for all targets demonstrated substantial tissue effects (P<0.001 for all TLR; MIF, and OPN; P<0.05 for IL8). Feeding STG and SCR resulted in significant (P

Effects of Salmonella enterica serovars Typhimurium (ST) and Choleraesuis (SC) on chemokine and cytokine expression in swine ileum and jejunal epithelial cells.

The gastrointestinal epithelium represents a barrier to potentially invasive enteric pathogens, maintains a role in innate immune surveillance, and is a source of both chemokine and cytokine chemotactic mediators in response to bacterial invasion. In the current study, we evaluated cytokine and chemokine mediators known to regulate movement of macrophages (macrophage migration inhibitory factor; MIF), neutrophils (IL8), dendritic cells (CCL20), and epithelial remodeling (osteopontin; OPN) in response to invasive swine enteropathogens Salmonella enterica serovar Typhimurium (ST) or Choleraesuis (SC). For the in vivo experiment, weaned pigs served as uninfected controls (0 h) or were given 3 x 10(9) CFU ST orally. Pigs were sacrificed at 8, 24, 48, and 144 h after inoculation and total RNA was extracted from defined segments of proximal (PI) and distal (DI) ileum. Relative expression of MIF and OPN were not affected by ST. IL8 expression was increased numerically (P = 0.17 for the interaction term) at 24 and 144 h in the PI and these increases accounted for greater expression in the PI relative to the DI (P < 0.05). Relative expression of CCL20 was increased at 24 h after ST (P < 0.05). Next, we evaluated the time course of MIF, IL8, CCL20, and OPN mRNA expression induced by application of lipopolysaccharide (LPS), ST or SC in vitro using pig jejunal epithelial cells (IPEC-J2). Cells were grown to confluency on permeable membranes, and treated apically with LPS (10 ng/mL), ST or SC (10(8)/well). After 1 h, cells were washed to remove LPS or extracellular bacteria, and media containing gentamicin was added to kill remaining extracellular bacteria. Media and RNA were collected at 1.5, 3, and 6 h after treatment. MIF mRNA was not affected by LPS or bacterial treatment. Similarly, IL8 expression was not affected by LPS, but was increased by ST and SC relative to controls at 1.5 and 3 h post exposure (P < 0.05 for all comparisons). Treatment with SC increased CCL20 mRNA relative to controls at 3 h (P < 0.05), while ST increased CCL20 at 1.5, 3, and 6h with maximal expression at 6 h (P < 0.05 for all comparisons). ST and SC increased polarized IL8 secretion. Our data demonstrate that invasive bacterial pathogens in the pig gastrointestinal tract trigger upregulation of selected cytokine and chemokine mediators, but serovars of Salmonella elicited differing patterns of activation in vitro.

Structural and serological studies of lipopolysaccharides of Citrobacter O35 and O38 antigenically related to Salmonella.

Structural analysis using 13C NMR spectroscopy and methylation showed that lipopolysaccharides (LPSs) of Citrobacter freundii O35 and Salmonella arizonae O59 have structurally identical O-specific polysaccharide chains, and those of C. freundii O38 and Salmonella kentucky differ only in the presence of O-acetyl groups in the former. Serological relationships between the structurally similar LPSs were demonstrated using inhibition of ELISA, rocket immunoelectrophoresis, double gel diffusion, and immunoblotting. The O-acetyl groups present in C. freundii O38 LPS are of little importance for its serological specificity. A cross-reaction was observed in immunoblotting between O-antisera to C. freundii O35 and S. arizonae O59 and a structurally related LPS of Pseudomonas aeruginosa O11a, 11b (Lányi-Bergan classification).

Efficacy of Salmonella enteritidis-immune lymphokines on horizontal transmission of S. arizonae in turkeys and S. gallinarum in chickens.

Salmonella arizonae (SA) and S. gallinarum (SG) are of economic importance to international poultry production because of their pathogenesis in young poultry during the first week after hatching. Previous studies from our laboratory have shown immune lymphokines (ILK) produced by S. enteritidis (SE)-immunized chickens provide protection against SE organ invasion in day-old chickens and turkey poults. Previous studies have also demonstrated that SG organ invasion was significantly decreased by administration of ILK to broiler chicks. The objective of the present study was to determine the effect of ILK on the incidence of horizontal transmission of SA in turkey poults and of SG in broiler chicks. The effect of ILK administration on horizontal transmission of SA in poults and SG in chicks was assessed in a seeder/contact model. Seeders were challenged with the appropriate bacterium (SA turkeys, SG chicks), contacts were either untreated or administered ILK. Seeders and contacts cohabited within an experimental group throughout the experiment. Mortality and organ invasion as a result of horizontal transmission were determined. There were no significant differences in mortality between non-treated and ILK-treated contact poults. In contrast, SG was extremely pathogenic to young broiler chicks. Non-treated contact chicks had a mortality rate of approximately 68% whereas significant (P < 0.05) reduction in mortality was demonstrated in the contact chicks treated with ILK (15%). Horizontal transmission, as determined by organ invasion, of SA to contact turkey poults and SG to contact broiler chicks was also significantly (P <0.05) decreased by immunoprophylactic administration of ILK. Bacterial recovery of SA from the liver/spleen and the cecal tonsil in contact poults and SG from contact chicks treated with ILK was dramatically reduced when compared to non-treated contact poults and chicks. Our results strongly suggest the immunoprophylactic administration of SE-immune lymphokines to young turkey poults and broiler chicks significantly reduces the horizontal transmission of Salmonella in poultry. These results suggest the possibilities of using a non-vaccine immunologically-based preventative strategy against Salmonella in poultry.

A computer-assisted structural analysis of regular polysaccharides on the basis of 13C-n.m.r. data.

A computerised approach to the structural analysis of unbranched regular polysaccharides is described, which is based on an evaluation of the 13C-n.m.r. spectra for all possible primary structures within the additive scheme starting from the chemical shifts of the 13C resonances of the constituent monosaccharides and the average values of the glycosylation effects. The analysis reveals a structure (or structures), the evaluated spectrum of which resembles most closely that observed. The approach has been verified by using a series of bacterial polysaccharides of known structure and, in combination with methylation analysis data, for the determination of the presently unknown structures of the O-specific polysaccharides from Salmonella arizonae O59 and O63, and Proteus hauseri O19.

The structure of the O-specific polysaccharide of Salmonella arizonae O62.

The O-specific polysaccharide was liberated by mild acid hydrolysis of the lipopolysaccharide (LPS) isolated from S. arizonae O62 by phenol-water extraction. The branched hexasaccharide repeating-unit of the O-specific chain of the O62 LPS contained L-rhamnose, 2-acetamido-2-deoxy-D-glucose, and 2-acetamido-2-deoxy-D-galacturonic acid in molar ratios of 4:1:1. On the basis of methylation analysis, 1H and 13C NMR spectroscopy, including 2D shift-correlated (COSY) and 1D NOE spectroscopy, the following structure for the repeating unit of the O-specific polysaccharide was established: [formula: see text]

The structure of the O-specific polysaccharide of Salmonella arizonae O21 (Arizona 22) containing N-acetylneuraminic acid.

The O-specific polysaccharide of S. arizonae O21 was found to contain 2-acetamido-2-deoxy-D-glucose, 2-acetamidino-2,6-dideoxy-L-galactose, N-acetylneuraminic acid, and O-acetyl groups. On the basis of 1H and 13C NMR studies of the intact and O-deacetylated polysaccharide and oligosaccharide fragments obtained by solvolysis with anhydrous hydrogen fluoride, partial methanolysis and partial hydrolysis, it was concluded that the O-specific polysaccharide has the following structure: [formula: see text]

[Antigenic bacterial polysaccharides. 24. The structure of the O-specific polysaccharide chain of Salmonella arizonae 063 (Arizona 08) lipopolysaccharide]. / Antigennye polisakharidy bakterii. 24. Struktura O-spetsificheskoi polisakharidnoi tsepi lipopolisakharida Salmonella arizonae 063 (Arizona 08).

The O-specific polysaccharide chain of the Salmonella arizonae O63 lipopolysaccharide is composed of D-glucose, D-galactose, N-acetyl-D-galactosamine, and 3-acetamido-3,6-dideoxy-D-galactose (Fuc3NAc) residues in the ratio 1:1:2:1. On the basis of methylation analysis and calculations of 13C-NMR-spectra of the polysaccharide and of the product of its selective cleavage with anhydrous hydrogen fluoride, the linear polymer lacking 3-acetamido-3,6-dideoxygalactose, it was concluded that the polysaccharide has the following structure: (Formula: see text).

[The structure of O-specific polysaccharide chains of lipopolysaccharides from Citrobacter 032 and Salmonella arizonae 064 (Arizona 29)]. / Stroenie O-spetsificheskikh polisakharidnykh tsepei lipopolisakharidov Citrobacter 032 i Salmonella arizonae 064 (Arizona 29).

On the basis of acid hydrolysis, methylation, Smith degradation, selective cleavage with anhydrous hydrogen fluoride, and 13C NMR analysis, the repeating unit of the O-specific polysaccharide of Citrobacter O32 was concluded to have the following structure: (Formula: see text). The repeating unit of the Salmonella arizonae O64 O-specific polysaccharide has the same structure lacking the O-acetyl group.

[Antigenic bacterial polysaccharides. 23. The structure of the O-specific polysaccharide chain of Salmonella arizonae 059 lipopolysaccharide]. / Antigennye polisakharidy bakterii. 23. Stroenie O-spetsificheskoi polisakharidnoi tsepi lipopolisakharida Salmonella arizonae 059.

The O-specific polysaccharide of Salmonella arizonae O59 (Arizona 19) is composed of D-galactose, N-acetyl-D-glucosamine, and N-acetyl-L-fucosamine (FucNAc, 2-acetamido-2,6-dideoxy-L-galactose) in the ratio 1:1:1. The computerized calculation of the 13C NMR spectrum of the polysaccharide, based on the monosaccharide composition, spectra of the free monosaccharides and glycosydation effects, together with the chemical analysis (methylation and Smith degradation) showed that the polysaccharide is built up of trisaccharide repeating units of the following structure: ----3)-alpha-L-FucNAcp(1----3)-beta-D-GlcNAcp-(1----2)-beta- D-Galp-1(----. The molecular basis of serological interrelations between S. arizonae O59 and Pseudomonas aeruginosa O7 (Lányi) is discussed.

[Construction of a dual-promoter expression plasmid delivered by Salmonella choleraesuis C500].

Salmonella choleraesuis C500 strain is an attenuated vaccine preventing piglet from paratyphoid and can also be used as a live vector of other DNA vaccines. Through mucosal immunization, immune response to specific antigens carried by it can be induced. To enhance the immune efficiency of DNA vaccine it carried, promoter Ptrc was inserted into the down stream of the human cytomegalovirus (CMV) immediate early promoter of eukaryotic expression plasmid pEGFP-C1. Then transcription terminator rrnbT1T2 was inserted into down stream of the multiple clone sites of pEGFP-C1, and the dual-promoter expression vector pEGFPPtrcR was constructed. Using 1xTSS method, we transformed the recombinant plasmid into C500, and obtained C500/pEGFPPtrcR. We used SDS-PAGE and Western blotting to detect the expression of report gene EGFP. Strong green fluorescence was observed under fluorescent microscope. The stable passages of this recombinant bacterium were at least 20 generations in vitro. Using liposome we transfected plasmid pEGFPPtrcR into Vero cell. After 24 h, green fluorescent was observed, showing the expression of EGFP in nuclei and endochylema. The construction of dual-promoter expression vector pEGFPPtrcR was successful. The foreign gene was expressed in Salmonella strain C500 and somatocytes, resulting in increased antigen expression. This research provides a foundation for the research of new DNA vaccines which use Salmonella C500 as carrier.

Net replication of Salmonella enterica serovars Typhimurium and Choleraesuis in porcine intestinal mucosa and nodes is associated with their differential virulence.

Salmonella enterica is a facultative intracellular pathogen of worldwide importance and causes a spectrum of diseases depending on serovar- and host-specific factors. Oral infection of pigs with S. enterica serovar Typhimurium strain 4/74 produces acute enteritis but is rarely fatal, whereas serovar Choleraesuis strain A50 causes systemic disease with a high mortality rate. With a porcine ligated ileal loop model, we observed that systemic virulence of serovar Choleraesuis A50 is not associated with enhanced intestinal invasion, secretory responses, or neutrophil recruitment compared to serovar Typhimurium 4/74. The net growth in vivo of serovar Choleraesuis A50 and serovar Typhimurium 4/74 was monitored following oral inoculation of pigs with strains harboring pHSG422, which exhibits temperature-sensitive replication. Analysis of plasmid partitioning revealed that the enteric virulence of serovar Typhimurium 4/74 relative to that of serovar Choleraesuis A50 is associated with rapid replication in the intestinal wall, whereas systemic virulence of serovar Choleraesuis A50 is associated with enhanced persistence in intestinal mesenteric lymph nodes. Faster replication of serovar Typhimurium, compared to that of serovar Choleraesuis, in the intestinal mucosa was associated with greater induction of the proinflammatory cytokines tumor necrosis factor alpha, interleukin-8 (IL-8), and IL-18 as detected by reverse transcriptase PCR analysis of transcripts from infected mucosa. During replication in batch culture and porcine alveolar macrophages, transcription of genes encoding components of type III secretion systems 1 (sipC) and 2 (sseC) was observed to be significantly higher in serovar Typhimurium 4/74 than in serovar Choleraesuis A50, and this may contribute to the differences in epithelial invasion and intracellular proliferation. The rapid induction of proinflammatory responses by strain 4/74 may explain why pigs confine serovar Typhimurium infection to the intestines, whereas slow replication of serovar Choleraesuis may enable it to evade host innate immunity and thus disseminate by stealth.

[Serological integration of all known Arizona-species into the Kauffmann-White-scheme (author's transl)]. / Serologische Integration aller bekannten Arizona-Species in das Kauffmann-White-Schema.

This paper reports on the integration of the Arizona group as sub-genus III into the K. W.-Schema. For many years comparative serological analyses of all serotypes belonging to sub-genus III have been carried out and detailed antigenic formulae have been established which now, for the first time, are published in full. These detailed antigenic formulae are not only of the utmost significance for the precise definition of serotypes but are also of immense importance to reference laboratories, in particular for the production of specific diagnostic sera, their correct absorption and specificity control.

A new Salmonella serovar S.III b 58:z10:z53:Rz50.

A new Salmonella serovar S.III b 58:z10:z53:Rz50 was isolated from the water samples of Ashida river, Fukuyama city, Japan. Its antigenic structure is described.

[Further studies of salmonella findings in reptiles. IV. Communication (author's transl)]. / Weitere Untersuchungen über Salmonellabefunde bei Reptilien. IV. Mitteilung.

All together 139 snakes - 134 Coluber jugularis, 3 Natrix natrix and 2 Natrix tessellata were examined for the presence of Salmonella and Arizona bacteriae. From the 139 specimens examined 112 proved to be Salmonella positive. 30 Salmonella respectively Arizona serotypes were isolated, there from 3 new Arizona types were found: Ar. 10a, 10c:27-21, Ar. 10a, 10c:23-31 and Ar. 16:23-31. 41 specimens contained single serotypes, while 51 were double infected, 12 specimens harbored 3 different serotypes of Salmonella, 6 specimens contained four and 2 contained five different serotypes. The result of these examinations present once more the variety of Salmonella species in reptiles.

Unusual antigenic composition of an Arizona strain.

The immunochemical analysis of Arizona strain 23:23:30:42 displayed a peculiar antigenic composition. Hyperimmune sera prepared in rabbits with formaldehyde-treated bacteria precipitated, in agar-gel, the proteins from numerous Salmonellae and E. coli by homogenous lines of apparent serological identity with the Arizona proteins. The reactions were unilateral. The proteins from Arizona did not react against the heterologous antibacterial sera tested; they precipitated, however against sera, rich in common antibodies, that were prepared with proteins from Salmonellae and E. coli. The genetic architecture of this Arizona's chromosome is, apparently, responsible for the unusual antigenic composition of the strain as exhibited by the immunochemical analysis of its hyperimmune serum.

Functional characterization of mouse lymphocyte subpopulations identified by their natural binding of bacteria. I. Identification of the Ig-secreting cell subpopulation.

Three mouse B cell subpopulations (B1, B2 and B3) can be identified by their natural binding of bacteria. To determine whether these subpopulations have unique functions, we assayed the number of anti-SRBC-secreting cells and the number of Ig-secreting cells in unseparated populations and in populations in which the B2 and B3 cells were removed by immobilized monolayers of Escherichia coli-2, a bacteria that binds B2 and B3 cells. Essentially all of the plaque forming cells present in the unseparated population were found in the B1-enriched population, suggesting that most of the antibody-secreting and Ig-secreting cells were in the B1 subpopulation. To show conclusively that the anti-SRBC-secreting cells resided in the B1 subpopulation, the Jerne plaque assay was performed on slides by using lymphocytes prelabeled with various bacteria and the cells that gave rise to the plaques were directly examined. Essentially all of the secreting cells were labeled with Corynebacterium xerosis, which binds to the B1 and B2 cells, whereas very few of the secreting cells were labelled with Arizona hinshawii, which binds to the B2 cells, or with Escherichia coli-2, which binds to the B2 and B3 cells. Thus, the B1 subpopulation contained essentially all of the antibody-secreting cells, which indicates that the B cell subpopulations identified by bacteria are functionally different.

Fluorescent bacterial rosetting of lymphocyte subpopulations. I. Methodology.

Fluorochrome-labelled bacteria were tested for their rosette-forming properties with human lymphocytes in suspension. Acridine orange stained human buffy coat cells or isolated mononuclear cells are rosetted with tetramethyl-rhodamine-isothiocyanate (TRITC)-labelled bacterial strains alone (mono-bacterial rosetting test) or simultaneously with a TRITC-labelled strain and a mutant or taxonomically different strain, labelled with fluorescein-isothiocyanate (double bacterial rosetting test [D-BRT]). Suspensions are centrifuged, washed, finally counterstained with ethidium bromide and examined by fluorescence microscopy. The bacterial binding properties of B cells may be studied by using anti-Ig pretreated mononuclear cells and TRITC bacteria (anti-Ig/BRT). In this study the methodology for bacterial staining, mono-, double- and anti-Ig/BRT is given. Estimation of rosette-forming cells is very accurate, easy and quick due to the bright fluorescence of the bacterial 'beads'. Furthermore, broad applicability of the bacterial rosetting phenomenon to study lymphocyte heterogeneity is gained with the fluorescent assay system.

Fluorescent bacterial rosetting of lymphocyte subpopulations. II. Identification of human lymphocyte subpopulations.

In a previous study, fluorochrome-labelled bacteria were found to be a very objective tool to investigate human lymphocyte heterogeneity with regard to bacterial rosette formation. The application of these assay systems (mono-, double- and anti-Ig/bacterial rosetting test) to identify lymphocyte subpopulations is given.