In Vivo Expression of Chicken Gut Anaerobes Identifies Carbohydrate- or Amino Acid-Utilising, Motile or Type VI Secretion System-Expressing Bacteria.

Complex gut microbiota increases chickens' resistance to enteric pathogens. However, the principles of this phenomenon are not understood in detail. One of the possibilities for how to decipher the role of gut microbiota in chickens' resistance to enteric pathogens is to systematically characterise the gene expression of individual gut microbiota members colonising the chicken caecum. To reach this aim, newly hatched chicks were inoculated with bacterial species whose whole genomic sequence was known. Total protein purified from the chicken caecum was analysed by mass spectrometry, and the obtained spectra were searched against strain-specific protein databases generated from known genomic sequences. Campylobacter jejuni, Phascolarctobacterium sp. and Sutterella massiliensis did not utilise carbohydrates when colonising the chicken caecum. On the other hand, Bacteroides, Mediterranea, Marseilla, Megamonas, Megasphaera, Bifidobacterium, Blautia, Escherichia coli and Succinatimonas fermented carbohydrates. C. jejuni was the only motile bacterium, and Bacteroides mediterraneensis expressed the type VI secretion system. Classification of in vivo expression is key for understanding the role of individual species in complex microbial populations colonising the intestinal tract. Knowledge of the expression of motility, the type VI secretion system, and preference for carbohydrate or amino acid fermentation is important for the selection of bacteria for defined competitive exclusion products.

Repertoire analysis of γδ T cells in the chicken enables functional annotation of the genomic region revealing highly variable pan-tissue TCR gamma V gene usage as well as identifying public and private repertoires.

BACKGROUND: Despite increasing interest in γδ T cells and their non-classical behaviour, most studies focus on animals with low numbers of circulating γδ T cells, such as mice and humans. Arguably, γδ T cell functions might be more prominent in chickens where these cells form a higher proportion of the circulatory T cell compartment. The TCR repertoire defines different subsets of γδ T cells, and such analysis is facilitated by well-annotated TCR loci. γδ T cells are considered at the cusp of innate and adaptive immunity but most functions have been identified in γδ low species. A deeper understanding of TCR repertoire biology in γδ high and γδ low animals is critical for defining the evolution of the function of γδ T cells. Repertoire dynamics will reveal populations that can be classified as innate-like or adaptive-like as well as those that straddle this definition. RESULTS: Here, a recent discrepancy in the structure of the chicken TCR gamma locus is resolved, demonstrating that tandem duplication events have shaped the evolution of this locus. Importantly, repertoire sequencing revealed large differences in the usage of individual TRGV genes, a pattern conserved across multiple tissues, including thymus, spleen and the gut. A single TRGV gene, TRGV3.3, with a highly diverse private CDR3 repertoire dominated every tissue in all birds. TRGV usage patterns were partly explained by the TRGV-associated recombination signal sequences. Public CDR3 clonotypes represented varying proportions of the repertoire of TCRs utilising different TRGVs, with one TRGV dominated by super-public clones present in all birds. CONCLUSIONS: The application of repertoire analysis enabled functional annotation of the TCRG locus in a species with a high circulating γδ phenotype. This revealed variable usage of TCRGV genes across multiple tissues, a pattern quite different to that found in γδ low species (human and mouse). Defining the repertoire biology of avian γδ T cells will be key to understanding the evolution and functional diversity of these enigmatic lymphocytes in an animal that is numerically more reliant on them. Practically, this will reveal novel ways in which these cells can be exploited to improve health in medical and veterinary contexts.

Gene expression in the chicken caecum is dependent on microbiota composition.

Gut microbiota is of considerable importance for each host. Despite this, germ-free animals can be obtained and raised to sexual maturity and consequences of the presence or absence of gut microbiota on gene expression of the host remain uncharacterised. In this study, we performed an unbiased study of protein expression in the caecum of germ-free and colonised chickens. The major difference between these two groups was in the expression of immunoglobulins which were essentially absent in the germ-free chickens. Microbiota also caused a minor decrease in the expression of focal adhesion and extracellular matrix proteins and an increase in the expression of argininosuccinate synthase ASS1, redox potential sensing, fermentative metabolic processes and detoxification systems represented by sulfotransferases SULT1C3 or SULT1E1. Since we also analysed expression in the caecum of E. coli Nissle and E. faecium DSM7134 mono-associated chickens, we concluded that at least immunoglobulin expression and expression of cystathionine synthase (CBS) was dependent on microbiota composition with E. coli Nissle stimulating more immunoglobulin and PIGR expression and E. faecium DSM7134 stimulating more CBS expression. Gut microbiota and its composition therefore affected protein expression in the chicken caecum though except for immunoglobulin production, the remaining differences were unexpectedly low.

Species-specific immunity to Helicobacter suis.

BACKGROUND: Helicobacter (H.) suis is mainly associated with pigs, but is also the most prevalent gastric non-H. pylori Helicobacter species found in humans. Both H. pylori and H. suis may cause persistent infection of the stomach. Several immune evasion mechanisms have been proposed for H. pylori, which focus to a great extent on its major virulence factors, which are absent in H. suis. The aim of this study was to gain more knowledge on immune evasion by H. suis. MATERIALS AND METHODS: Cytokine expression kinetics were monitored in the stomach of BALB/c mice experimentally infected with H. suis. The cytokine expression profile in the stomach of naturally H. suis-infected pigs was also determined. Subsequently, the effect of H. suis on murine and porcine dendritic cell (DC) maturation and their ability to elicit T-cell effector responses was analyzed. RESULTS: Despite a Th17/Th2 response in the murine stomach, the inflammatory cell influx was unable to clear H. suis infection. H. suis-stimulated murine bone marrow-derived dendritic cells induced IL-17 secretion by CD4+ cells in vitro. Natural H. suis infection in pigs evoked increased expression levels of IL-17 mRNA in the antrum and IL-10 mRNA in the fundus. In contrast to mice, H. suis-stimulated porcine monocyte-derived dendritic cells were unable to express MHCII molecules on their cell surface. These semimature DCs induced proliferation of T-cells, which showed an increased expression of TGF-ß and FoxP3 mRNA levels. CONCLUSIONS: Helicobacter suis might evade host immune responses by skewing toward a Treg-biased response.

The response of porcine monocyte derived macrophages and dendritic cells to Salmonella Typhimurium and lipopolysaccharide.

BACKGROUND: Following infection and initial multiplication in the gut lumen, Salmonella Typhimurium crosses the intestinal epithelial barrier and comes into contact with cells of the host immune system. Mononuclear phagocytes which comprise macrophages and dendritic cells (DC) are of key importance for the outcome of Salmonella infection. Although macrophages and DC may differentiate from a common precursor, their capacities to process and present antigen differ significantly. In this study, we therefore compared the response of porcine macrophages and DC differentiated from peripheral blood monocytes to S. Typhimurium and one of the most potent bacterial pathogen associated molecular patterns, bacterial lipopolysaccharide. To avoid any bias, the expression was determined by protein LC-MS/MS and verified at the level of transcription by quantitative RT-PCR. RESULTS: Within 4 days of culture, peripheral blood monocytes differentiated into two populations with distinct morphology and expression of MHC II. Mass spectrometry identified 446 proteins in macrophages and 672 in DC. Out of these, 433 proteins were inducible in macrophages either after infection with S. Typhimurium or LPS exposure and 144 proteins were inducible in DC. The expression of the 46 most inducible proteins was verified at the level of transcription and the differential expression was confirmed in 22 of them. Out of these, 16 genes were induced in both cell types, 3 genes (VCAM1, HMOX1 and Serglycin) were significantly induced in macrophages only and OLDLR1 and CDC42 were induced exclusively in DC. Thirteen out of 22 up-regulated genes contained the NF-kappaB binding site in their promoters and could be considered as either part of the NF-kappaB feedback loop (IkappaBalpha and ISG15) or as NF-kappaB targets (IL1beta, IL1alpha, AMCF2, IL8, SOD2, CD14, CD48, OPN, OLDLR1, HMOX1 and VCAM1). CONCLUSIONS: The difference in the response of monocyte derived macrophages and DC was quantitative rather than qualitative. Despite the similarity of the responses, compared to DC, the macrophages responded in a more pro-inflammatory fashion.

Contact with adult hens affects the composition of skin and respiratory tract microbiota in newly hatched chicks.

Chickens in commercial production are hatched in hatcheries without any contact with their parents and colonization of their skin and respiratory tract is therefore dependent on environmental sources only. However, since chickens evolved to be hatched in nests, in this study we evaluated the importance of contact between hens and chicks for the development of chicken skin and tracheal microbiota. Sequencing of PCR amplified V3/V4 variable regions of the 16S rRNA gene showed that contact with adult hens decreased the abundance of E. coli, Proteus mirabilis and Clostridium perfringens both in skin and the trachea, and Acinetobacter johnsonii and Cutibacterium acnes in skin microbiota only. These species were replaced by Lactobacillus gallinarum, Lactobacillus aviarius, Limosilactobacillus reuteri, and Streptococcus pasterianus in the skin and tracheal microbiota of contact chicks. Lactobacilli can be therefore investigated for their probiotic effect in respiratory tract in the future. Skin and respiratory microbiota of contact chickens was also enriched for Phascolarctobacterium, Succinatimonas, Flavonifractor, Blautia, and [Ruminococcus] torque though, since these are strict anaerobes from the intestinal tract, it is likely that only DNA from nonviable cells was detected for these taxa.

The local immune response of mice after Helicobacter suis infection: strain differences and distinction with Helicobacter pylori.

Helicobacter (H.) suis colonizes the stomach of pigs and is the most prevalent gastric non-H. pylori Helicobacter species in humans. Limited information is available on host immune responses after infection with this agent and it is unknown if variation in virulence exists between different H. suis strains. Therefore, BALB/c and C57BL/6 mice were used to compare colonization ability and gene expression of various inflammatory cytokines, as determined by real-time PCR, after experimental infection with 9 different H. suis strains. All strains were able to persist in the stomach of mice, but the number of colonizing bacteria at 59 days post inoculation was higher in stomachs of C57BL/6 mice compared to BALB/c mice. All H. suis strains caused an upregulation of interleukin (IL)-17, which was more pronounced in BALB/c mice. This upregulation was inversely correlated with the number of colonizing bacteria. Most strains also caused an upregulation of regulatory IL-10, positively correlating with colonization in BALB/c mice. Only in C57BL/6 mice, upregulation of IL-1ß was observed. Increased levels of IFN-γ mRNA were never detected, whereas most H. suis strains caused an upregulation of the Th2 signature cytokine IL-4, mainly in BALB/c mice. In conclusion, the genetic background of the murine strain has a clear impact on the colonization ability of different H. suis strains and the immune response they evoke. A predominant Th17 response was observed, accompanied by a mild Th2 response, which is different from the Th17/Th1 response evoked by H. pylori infection.

SPI-1 encoded genes of Salmonella Typhimurium influence differential polarization of porcine alveolar macrophages in vitro.

BACKGROUND: Within the last decade, macrophages have been shown to be capable of differentiating toward a classically activated phenotype (M1) with a high antimicrobial potential or an alternatively activated phenotype (M2). Some pathogens are capable of interfering with differentiation in order to down-regulate the anti-microbial activity and enhance their survival in the host. RESULTS: To test this ability in Salmonella enterica serovar Typhimurium, we infected porcine alveolar macrophages with wild-type Salmonella Typhimurium and its isogenic mutants devoid of two major pathogenicity islands, SPI-1 and SPI-2. The induction of genes linked with M1 or M2 polarization was determined by quantification of gene expression by RT-qPCR. The ΔSPI-1 mutant induced a high, dose-dependent M1 response but a low M2 response in infected macrophages. On the other hand, wild-type Salmonella Typhimurium induced a low M1 response but a high, dose-dependent M2 response in infected macrophages. The response to ΔSPI-2 mutant infection was virtually the same as the wild-type strain. CONCLUSIONS: We therefore propose that Salmonella Typhimurium DT104 studied here can polarize macrophages towards the less bactericidal M2 phenotype and that this polarization is dependent on the type III secretion system encoded by SPI-1.

Host Species Adaptation of Obligate Gut Anaerobes Is Dependent on Their Environmental Survival.

The gut microbiota of warm-blooded vertebrates consists of bacterial species belonging to two main phyla; Firmicutes and Bacteroidetes. However, does it mean that the same bacterial species are found in humans and chickens? Here we show that the ability to survive in an aerobic environment is central for host species adaptation. Known bacterial species commonly found in humans, pigs, chickens and Antarctic gentoo penguins are those capable of extended survival under aerobic conditions, i.e., either spore-forming, aerotolerant or facultatively anaerobic bacteria. Such bacteria are ubiquitously distributed in the environment, which acts as the source of infection with similar probability in humans, pigs, chickens, penguins and likely any other warm-blooded omnivorous hosts. On the other hand, gut anaerobes with no specific adaptation for survival in an aerobic environment exhibit host adaptation. This is associated with their vertical transmission from mothers to offspring and long-term colonisation after administration of a single dose. This knowledge influences the design of next-generation probiotics. The origin of aerotolerant or spore-forming probiotic strains may not be that important. On the other hand, if Bacteroidetes and other host-adapted species are used as future probiotics, host preference should be considered.

Cytokine expression by CD163+ monocytes in healthy and Actinobacillus pleuropneumoniae-infected pigs.

Distinct monocyte subpopulations have been previously described in healthy pigs and pigs experimentally infected with Actinobacillus pleuropneumoniae (APP). The CD163+ subpopulation of bone marrow (BM), peripheral blood (PB) and lung monocytes was found to play an important role in the inflammatory process. The inflammation is accompanied by elevation of inflammatory cytokines. The aim of the study was to evaluate the contribution of CD163+ monocytes and macrophages to cytokine production during APP-induced lung inflammation. Cytokine production was assessed by flow cytometry (FC) and quantitative PCR (qPCR) in CD163+ monocytes and by qPCR, immunohistochemistry/fluorescence in lungs and tracheobronchial lymph nodes (TBLN). Despite the systemic inflammatory response after APP infection, BM and PB CD163+ monocytes did not express elevated levels of a wide range of cytokines compared to control pigs. In contrast, significant amounts of IL-1ß, IL-6, IL-8 and TNF-α were produced in lung lesions and IL-1ß in the TBLN. At the protein level, TNF-α was expressed by both CD163+ monocytes and macrophages in lung lesions, whereas IL-1ß, IL-6 and IL-8 expression was found only in CD163+ monocytes; no CD163+ macrophages were found to produce these cytokines. Furthermore, the quantification of CD163+ monocytes expressing the two cytokines IL-1ß and IL-8 that were most elevated was performed. In lung lesions, 36.5% IL-1ß positive CD163+ monocytes but only 18.3% IL-8 positive CD163+ monocytes were found. In conclusion, PB and BM CD163+ monocytes do not appear to contribute to the elevated cytokine levels in plasma. On the other hand, CD163+ monocytes contribute to inflammatory cytokine expression, especially IL-1ß at the site of inflammation during the inflammatory process.

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

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-1ß 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-1ß, 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.

Detoxification, Hydrogen Sulphide Metabolism and Wound Healing Are the Main Functions That Differentiate Caecum Protein Expression from Ileum of Week-Old Chicken.

Sections of chicken gut differ in many aspects, e.g., the passage of digesta (continuous vs. discontinuous), the concentration of oxygen, and the density of colonising microbiota. Using an unbiased LC-MS/MS protocol, we compared protein expression in 18 ileal and 57 caecal tissue samples that originated from 7-day old ISA brown chickens. We found that proteins specific to the ileum were either structural (e.g., 3 actin isoforms, villin, or myosin 1A), or those required for nutrient digestion (e.g., sucrose isomaltase, maltase-glucoamylase, peptidase D) and absorption (e.g., fatty acid-binding protein 2 and 6 or bile acid-CoA:amino acid N-acyltransferase). On the other hand, proteins characteristic of the caecum were involved in sensing and limiting the consequences of oxidative stress (e.g., thioredoxin, peroxiredoxin 6), cell adhesion, and motility associated with wound healing (e.g., fibronectin 1, desmoyokin). These mechanisms are coupled with the activation of mechanisms suppressing the inflammatory response (galectin 1). Rather prominent were also expressions of proteins linked to hydrogen sulphide metabolism in caecum represented by cystathionin beta synthase, selenium-binding protein 1, mercaptopyruvate sulphurtransferase, and thiosulphate sulphurtransferase. Higher mRNA expression of nuclear factor, erythroid 2-like 2, the main oxidative stress transcriptional factor in caecum, further supported our observations.

Eggshell and Feed Microbiota Do Not Represent Major Sources of Gut Anaerobes for Chickens in Commercial Production.

In this study, we addressed the origin of chicken gut microbiota in commercial production by a comparison of eggshell and feed microbiota with caecal microbiota of 7-day-old chickens, using microbiota analysis by 16S rRNA sequencing. In addition, we tested at which timepoint during prenatal or neonatal development it is possible to successfully administer probiotics. We found that eggshell microbiota was a combination of environmental and adult hen gut microbiota but was completely different from caecal microbiota of 7-day-old chicks. Similarly, we observed that the composition of feed microbiota was different from caecal microbiota. Neither eggshell nor feed acted as an important source of gut microbiota for the chickens in commercial production. Following the experimental administration of potential probiotics, we found that chickens can be colonised only when already hatched and active. Spraying of eggs with gut anaerobes during egg incubation or hatching itself did not result in effective chicken colonisation. Such conclusions should be considered when selecting and administering probiotics to chickens in hatcheries. Eggshells, feed or drinking water do not act as major sources of gut microbiota. Newly hatched chickens must be colonised from additional sources, such as air dust with spores of Clostridiales. The natural colonisation starts only when chickens are already hatched, as spraying of eggs or even chickens at the very beginning of the hatching process did not result in efficient colonisation.

Development and characterization of recombinant tick-borne encephalitis virus expressing mCherry reporter protein: A new tool for high-throughput screening of antiviral compounds, and neutralizing antibody assays.

The flavivirus, tick-borne encephalitis virus (TBEV) is transmitted by Ixodes spp. ticks and may cause severe and potentially lethal neurological tick-borne encephalitis (TBE) in humans. Studying TBEV requires the use of secondary methodologies to detect the virus in infected cells. To overcome this problem, we rationally designed and constructed a recombinant reporter TBEV that stably expressed the mCherry reporter protein. The resulting TBEV reporter virus (named mCherry-TBEV) and wild-type parental TBEV exhibited similar growth kinetics in cultured cells; however, the mCherry-TBEV virus produced smaller plaques. The magnitude of mCherry expression correlated well with progeny virus production but remained stable over <4 passages in cell culture. Using well-characterized antiviral compounds known to inhibit TBEV, 2'-C-methyladenosine and 2'-deoxy-2'-ß-hydroxy-4'-azidocytidine (RO-9187), we demonstrated that mCherry-TBEV is suitable for high-throughput screening of antiviral drugs. Serum samples from a TBEV-vaccinated human and a TBEV-infected dog were used to evaluate the mCherry-based neutralization test. Collectively, recombinant mCherry-TBEV reporter virus described here provides a powerful tool to facilitate the identification of potential antiviral agents, and to measure levels of neutralizing antibodies in human and animal sera.

Influence of 5 major Salmonella pathogenicity islands on NK cell depletion in mice infected with Salmonella enterica serovar Enteritidis.

BACKGROUND: In this study we were interested in the colonisation and early immune response of Balb/C mice to infection with Salmonella Enteritidis and isogenic pathogenicity island free mutants. RESULTS: The virulence of S. Enteritidis for Balb/C mice was exclusively dependent on intact SPI-2. Infections with any of the mutants harbouring SPI-2 (including the mutant in which we left only SPI-2 but removed SPI-1, SPI-3, SPI-4 and SPI-5) resulted in fatalities, liver injures and NK cell depletion from the spleen. The infection was of minimal influence on counts of splenic CD4 CD8 T lymphocytes and gammadelta T-lymphocytes although a reduced ability of splenic lymphocytes to respond to non-specific mitogens indicated general immunosuppression in mice infected with SPI-2 positive S. Enteritidis mutants. Further investigations showed that NK cells were depleted also in blood but not in the caecal lamina propria. However, NK cell depletion was not directly associated with the presence of SPI-2 and was rather an indicator of virulence or avirulence of a particular mutant because the depletion was not observed in mice infected with other attenuated mutants such as lon and rfaL. CONCLUSIONS: The virulence of S. Enteritidis for Balb/C mice is exclusively dependent on the presence of SPI-2 in its genome, and a major hallmark of the infection in terms of early changes in lymphocyte populations is the depletion of NK cells in spleen and blood. The decrease of NK cells in circulation can be used as a marker of attenuation of S. Enteritidis mutants for Balb/C mice.

Virulence potential of five major pathogenicity islands (SPI-1 to SPI-5) of Salmonella enterica serovar Enteritidis for chickens.

BACKGROUND: Salmonella is a highly successful parasite of reptiles, birds and mammals. Its ability to infect and colonise such a broad range of hosts coincided with the introduction of new genetic determinants, among them 5 major pathogenicity islands (SPI1-5), into the Salmonella genome. However, only limited information is available on how each of these pathogenicity islands influences the ability of Salmonella to infect chickens. In this study, we therefore constructed Salmonella Enteritidis mutants with each SPI deleted separately, with single individual SPIs (i.e. with the remaining four deleted) and a mutant with all 5 SPIs deleted, and assessed their virulence in one-day-old chickens, together with the innate immune response of this host. RESULTS: The mutant lacking all 5 major SPIs was still capable of colonising the caecum while colonisation of the liver and spleen was dependent on the presence of both SPI-1 and SPI-2. In contrast, the absence of SPI-3, SPI-4 or SPI-5 individually did not influence virulence of S. Enteritidis for chickens, but collectively they contributed to the colonisation of the spleen. Proinflammatory signalling and heterophil infiltration was dependent on intact SPI-1 only and not on other SPIs. CONCLUSIONS: SPI-1 and SPI-2 are the two most important pathogenicity islands of Salmonella Enteritidis required for the colonisation of systemic sites in chickens.

Influence of the microbiota-gut-brain axis on behavior and welfare in farm animals: A review.

There is increasing evidence of a pivotal role of the gut microbiota (GUT-M) in key physiological functions in vertebrates. Many studies discuss functional implications of the GUT-M not only on immunity, growth, metabolism, but also on brain development and behavior. However, while the influence of the microbiota-gut-brain axis (MGBA) on behavior is documented in rodents and humans, data on farm animals are scarce. This review will first report the well-known influence of the MGBA on behavior in rodent and human and then describe its influence on emotion, memory, social and feeding behaviors in farm animals. This corpus of experiments suggests that a better understanding of the effects of the MGBA on behavior could have large implications in various fields of animal production. Specifically, animal welfare and health could be improved by selection, nutrition and management processes that take into account the role of the GUT-M in behavior.

Cytokine mRNA expression in porcine cell lines stimulated by enterotoxigenic Escherichia coli.

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.

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.