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1.
The innate immune system has the basic function of identifying and eradicating microbial invaders and alerting the adaptative immune system to their presence. In this study, the porcine intestinal innate immune response was evaluated by analysing the expression of TLRs, cytokines and chemokines in two porcine epithelial cell lines from different regions: IPEC-J2 (jejunum) and IPI-2I (ileum). Both cells lines were stimulated with 1microg of LPS from Salmonella typhimurium. RNA was collected at 30min, 1, 2, 3 and 4h after treatment. Expression of TLR-1, -2, -3, -4, -6, -8, -9, -10, TNF-alpha, IL-1beta, -8 and MCP-1 was quantified relative to the quantity of Cyclophilin-A mRNA using real-time quantitative PCR (RTQ-PCR). The results obtained show up differences in the gene expression between both cell lines IPEC-J2 and IPI-2I as response to LPS from S. typhimurium during the activation time, which may suggest an in vivo variability in the innate immune response against pathogens in different regions of the host's gut.  相似文献   

2.
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.  相似文献   

3.
Intestinal epithelial cells represent the first line of defence against pathogenic bacteria in the lumen of the gut. Besides acting as a physical barrier, epithelial cells orchestrate the immune response through the production of several innate immune mediator molecules including beta-defensins. Here, we establish the porcine intestinal cell line IPI-2I as a new model system to test the regulation of porcine beta-defensins 1 and 2. Gene expression of both defensins was highly upregulated by foetal calf serum components in normal growth medium. In serum-free medium, baseline expression remained low, but pBD-2 gene expression was increased 10-fold upon infection with Salmonella Typhimurium. Arcobacter cryaerophilus and Salmonella Enteritidis, pathogenic bacteria with comparable adhesion and invasion characteristics, failed to increase pBD-2 mRNA levels. Heat killed or colistin-treated Salmonella Typhimurium had no effect, showing that the upregulation of pBD-2 was dependent on the viability of the Salmonella Typhimurium. Gene expression of pBD-1 was regulated differently since an increase in pBD-1 mRNA was observed by Salmonella Enteritidis infection. We conclude that the IPI-2I cells can serve as a new model to study porcine beta-defensin regulation and that pBD-1 and pBD-2 are differentially regulated in this cell line.  相似文献   

4.
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.  相似文献   

5.
Enterotoxigenic Escherichia coli (ETEC) is an extracellular bacterium that causes post-weaning diarrhoea (PWD) in piglets with different severity of clinical signs. The pathogenesis of ETEC is ascribed to the effect of enterotoxins. ETEC colonizes ileum and probably can penetrate the epithelium and stimulate macrophages. The aim of study was to examine whether there is any difference in cytokine response in vitro produced by two porcine cell lines, intestinal epithelial cell line (IPI-2I) and macrophage cell line (3D4/31) after stimulation with different serotypes of ETEC associated with different clinical course of PWD in piglets. Three serotypes, O149:K88 (F4), O147:F18 and O8:K88, were used. We observed that all the used serotypes were unable to induce IL-8 and TNF-alpha mRNA expression in IPI-2I cell line as measured by the real-time RT-PCR. In 3D4/31 cell line, we detected differences in cytokine response among the used serotypes. The highest IL-8 and TNF-alpha mRNA expression in 3D4/31 was detected after stimulation with serotype O149:K88 frequently associated with hemorrhagic gastroenteritis.  相似文献   

6.
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.  相似文献   

7.
In this study we have compared protein secretion in the wild type of S. Typhimurium and the rfaC mutant. We found out that the rfaC mutant was defective in protein secretion. In addition, the rfaC mutant was defective in its invasion into an IPEC-J2 porcine epithelial cell line and also in motility in semisolid agar. Consistent with this, reduced flagella numbers were observed in the rfaC mutant. In the rfaC mutant, there were no defects in flagellin expression as detected by western blot and immune electron microscopy which demonstrated equal amounts of flagellin in the cytoplasm of both the rfaC mutant and the wild-type S. Typhimurium. However, in the wild-type strain only, the flagellin was assembled to spatially restricted areas on the inner side of cytoplasmic membrane. The oligosaccharide core of LPS is therefore required for the assembly of flagella and T3SS secretion machinery followed by protein secretion.  相似文献   

8.
The in vitro pathogenicity of Salmonella enterica serovar Typhimurium phage type (pt) 90 and pt 506 (also known as DT 104) isolates from human and porcine origin was studied in adhesion and invasion assays to the human cell line Caco-2 and the porcine cell line IPI-2. Interleukin-8 (IL-8) production by these two cell lines in response to stimulation by the two Salmonella phage types was also measured. Generally, Salmonella Typhimurium pt 506 and pt 90 adhered to and invaded Caco-2 cells and IPI-2 cells equally well. The release of IL-8 by Caco-2 cells or by IPI-2 cells was similar, independent of the Salmonella phage type used for stimulation of the cells. These data suggest that Salmonella Typhimurium pt 90 has a similar ability to cause Salmonella infections as Salmonella Typhimurium DT 104.  相似文献   

9.
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.  相似文献   

10.
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.  相似文献   

11.
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12.
The key process for immune response development is the recognition of bacteria by the immune system of the host based on the sensing of pathogen-associated molecular patterns (PAMP). One of the most important PAMP is the lipopolysaccharide (LPS) molecule, a complex molecule present in the outer membrane of Gram negative bacteria. In this study we were interested in how different parts of the LPS of Salmonella enterica serovar Enteritidis are recognized by porcine neutrophils. To this aim, we constructed S. Enteritidis mutants with rfaL and rfaC genes disabled in the attachment of the O-antigen and in the synthesis of the inner oligosaccharide core of LPS, respectively. We found that in the absence of serum, both the rfa mutants associated with neutrophils and stimulated them for reactive oxygen species (ROS) production significantly more than the wild-type strain. Addition of polymyxin B, which neutralized lipid A, the endotoxic moiety of LPS, effectively decreased the association of the wild-type strain and the rfaC mutant with neutrophils, but not the rfaL mutant. This indicates that the oligosaccharide core newly exposed on the surface in the rfaL mutant, protected from interaction in the wild-type strain by the O-antigen but completely absent in the rfaC mutant, may represent a new ligand for porcine neutrophils that cannot be neutralized by polymyxin B.  相似文献   

13.
Invasion of Salmonella into intestinal epithelial cells is believed to be essential for the pathogenesis of Salmonella infections. Invasion is mediated by genes located on the Salmonella pathogenicity Island I (SPI-1), which are needed for assembling a type three secretion system, that mediates injection of bacterial proteins into the cytosol of epithelial cells, resulting in cytoskeletal rearrangements and as a consequence invasion. HilA is the key regulator of the Salmonella Pathogenicity Island I. To assess the role of hilA in colonization of gut and internal organs in poultry, animals were infected with 10(8) CFU of a delta hilA mutant of S. Enteritidis and its parent strain at day of hatch. Very low numbers of delta hilA mutant strain were able to colonize the internal organs shortly after infection, but they were not eliminated from internal organs at 4 weeks post-infection. At that time, the colonization level of the wild type bacteria in internal organs was decreased to the same low level compared with delta hilA mutant strain bacteria. Shedding of the delta hilA mutant strain and colonization of the caeca was seriously decreased relative to the parent strain starting from Day 5 post-infection. At 4 weeks post-infection, the delta hilA mutant strain was more or less eliminated from the chicken gut, while the parent strain was still shed to a high level and colonized the caeca to a high extent (more than 10(7) CFU/g). It is concluded that hilA is involved in long-term shedding and colonization of S. Enteritidis in the chicken caeca.  相似文献   

14.
Cytokine response against Salmonella Typhimurium is traditionally studied in conventional animals. Germ-free animals, however, enable to study response against infection without background effect of other microorganisms. Plasma and ileal inflammatory cytokines in germ-free piglets orally infected with virulent LT2 strain or, with a non-virulent SF1591 rough mutant were quantified by ELISA. In plasma and ileal washes, IFN-gamma levels significantly increased in both infected groups. TNF-alpha and IL-18 were mostly missing in plasma 24 h after infection. In the ileum, IFN-gamma, TNF-alpha, and IL-1beta were induced mainly by the virulent strain, whereas IL-18 was induced in highest quantity by non-virulent Salmonella. These data confirmed an important role of IFN-gamma, as well as other inflammatory cytokines in early stage of salmonellosis.  相似文献   

15.
Adherence of Streptococcus suis to porcine endothelial cells   总被引：3，自引：0，他引：3  
Streptococcus suis can cause invasive diseases in pigs and humans, such as meningitis or arthritis. Adherence to and invasion of endothelial cells might represent important steps in survival and spread of S. suis within the host. We tested in vitro adherence and invasion of S. suis strains using a porcine brain microvascular and aortal endothelial cell line. Four S. suis strains were tested with and without prior treatment with porcine serum containing anti-S. suis antibodies. Strains included a capsular serotype 2 strain and its non-encapsulated isogenic mutant strain, as well as two non-typeable (NT) strains, which expressed no capsule under our experimental conditions. Strains adhered to both cell lines to different extents depending on encapsulation and pre-treatment with porcine immune serum. The serotype 2 strain showed almost no adherence, whereas the non-encapsulated mutant strain adhered strongly. Similarly, both NT strains adhered substantially better than the serotype 2 strain. Pre-treatment of bacteria with porcine serum increased adherence of the encapsulated serotype 2 strain and decreased adherence of the non-encapsulated strains. None of the strains was able to efficiently invade either of the two cell lines, except for one NT strain, which showed a very low extend of invasion. Our results suggest that S. suis can adhere to but not invade porcine endothelial cells, and that this interaction may involve different bacterial surface structures, such as capsular polysaccharides and/or binding sites for serum components.  相似文献   

16.
Enhancing and/or modulating innate and adaptive immunity by cytokines appears to be greatly useful to provide effective protective immunity against infectious diseases. However, an effective delivery system for mass administration in livestock industry is needed because of limitations such as cost, labor, time, and protein stability. Here the immunomodulatory functions of swine interleukine-18 (swIL-18), known as IFN-γ-inducing factor (IGIF), were evaluated in a vaccination model of pseudorabies virus (PrV) using attenuated Salmonella enterica serovar Typhimurium as the oral delivery system. The oral administration of S. enterica serovar Typhimurium expressing swIL-18 prior to vaccination with inactivated PrV vaccine induced enhanced levels of serum PrV-specific IgG and its IgG2 isotype, compared to administration of S. enterica serovar Typhimurium harboring the empty vector. Furthermore, S. enterica serovar Typhimurium expressing swIL-18 mounted Th1-biased cellular immune responses against PrV antigen, as evaluated by the production of IFN-γ and IL-4 from peripheral blood mononuclear cells of piglets. Subsequently, Th1-biased immunity induced by S. enterica serovar Typhimurium expressing swIL-18 showed rapid response and rendered piglets displayed more alleviated clinical signs following the virulent PrV challenge. Also, this alleviation of clinical signs was further confirmed by the reduction of nasal excretion of PrV after challenge. The present study demonstrates the extended use of immunomodulatory functions of swIL-18 orally delivered by attenuated S. enterica serovar Typhimurium.  相似文献   

17.
CBA/J mice were used in the present study to establish differences between the immune response to three chlamydial strains: AB7 (Chlamydia psittaci wild-type strain), 1B (C. psittaci vaccinal strain) and iB1 (C. pecorum). The evolution of chlamydial infection was evaluated in each strain by studying the clinical signs, the number of bacteria isolated from the spleen and the pathology of the liver. Three aspects of the immune response were then studied: the characterization of the infiltrate of leukocytes in the liver, the percentages of T- and B-cells, macrophages and neutrophils in the spleen, and the presence of cytokines in the serum. Infection followed a different course in the C. psittaci-infected mice; 1B-infected mice showed milder levels in all the parameters analysed than their AB7-infected counterparts. The resolution of infection was earlier in 1B-infected mice and, although the immune response to both strains was Th1-like, a more intense CD8+ T-cell response and an earlier presence of TNF-alpha in serum were observed in this group. C. pecorum infection was controlled mainly by a non-specific immune response, since these mice showed no signs of a systemic specific immune response. Neutrophil depletion experiments showed that these cells play a very limited role in the non-specific response against C. pecorum.  相似文献   

18.
In contrast to other mastitis pathogens, the host response evoked during Staphylococcus aureus intramammary infection is marked by the absence of the induction of critical cytokines, including IL-8 and TNF-alpha, which have established roles in mediating host innate immunity. The elucidation of changes in the expression of other mediators with the potential to regulate mammary inflammatory responses to S. aureus remains lacking. Transforming growth factor (TGF)-alpha, TGF-beta1, and TGF-beta2 are cytokines that regulate mammary gland development. Because these cytokines also have a demonstrated role in mediating inflammation, the objective of the current study was to determine whether S. aureus intramammary infection influences their expression. Ten cows were challenged with S. aureus and milk samples collected. Increases in milk levels of TGF-alpha were evident within 32h of infection and persisted for 16h. Increases in TGF-beta1 and TGF-beta2 levels were detected within 40h of S. aureus infection and persisted through the end of the study. Thus, in contrast to IL-8 and TNF-alpha, S. aureus elicits host production of TGF-alpha, TGF-beta1, and TGF-beta2. This finding may suggest a role for these cytokines in mediating mammary gland host innate immune responses to S. aureus.  相似文献   

19.
Lactococcus lactis is an immunomodulator and candidate live mucosal delivery vehicle for vaccine antigens and for biologically active molecules, including immunoregulatory cytokines such as interferon-gamma (IFN-gamma). To provide a tool for investigating downregulation of allergic predisposition of pigs to experimental food allergy, porcine IFN-gamma was cloned and expressed as a fusion protein with the usp45 secretion signal. Immunoblot analysis with polyclonal anti-pIFN-gamma-antibody demonstrated that the recombinant porcine IFN-gamma (rpIFN-gamma) protein was expressed in the L. lactis transformants as a secreted product. Activity of rpIFN-gamma was confirmed by ability to upregulate class II major histocompatibility complex (MHC) on cells of the porcine monocytic cell line 3D4/31. The L. lactis producing biologically active rpIFN-gamma will be used to investigate its possible ability to modulate the allergic immune response phenotype of pigs.  相似文献   

20.
We have demonstrated that severe systemic disease caused by virulent LT2 strain Salmonella enterica serotype Typhimurium in gnotobiotic piglets can be alleviated by oral inoculation with an avirulent rough (R) mutant of the same serotype 24 h before challenge with the virulent strain. Protected piglets had no signs of enteritis. The concentrations of TNF-alpha, IL-1beta, IL-8 and IL-10 were measured by ELISA in ileal washings and plasma of uninfected and infected pigs. The cytokines were not detected in plasma of germ-free piglets, and low concentrations of IL-1beta and IL-8 were found in their ileal washings. The pre-inoculation of the rough mutant induced an increase in IL-8 and decrease in IL-1beta and IL-10 in plasma. The virulent LT2 strain induced very high TNF-alpha concentrations in the ileum which were reduced in the pigs pre-inoculated with the R mutant.  相似文献   

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