Sub-lethal levels of carvacrol reduce Salmonella Typhimurium motility and invasion of porcine epithelial cells

The European ban on the use of antibiotic growth promotors has increased the search for new alternatives to prevent pig intestinal microbial diseases and to stimulate growth. The addition of essential oils or components thereof, such as carvacrol, to pig feed is a promising alternative. In this report we determined the effect of sub-lethal concentrations of carvacrol on Salmonella Typhimurium. At concentrations where growth of Salmonella was not inhibited, carvacrol completely inhibited motility of the bacterium. This loss of motility was not due to the loss of the flagellum or to ATP shortage upon carvacrol treatment. Adhesion of Salmonella to IPEC-J2, porcine intestinal epithelial cells, was not affected by carvacrol but invasion was significantly reduced. In addition, the epithelial gene expression of porcine β-defensin 2, an innate immune response to Salmonella infection, was reduced when Salmonella was exposed to carvacrol. This indicates that invasion but not adhesion of Salmonella triggers the porcine β-defensin 2 expression of porcine epithelial cells.     

Early epithelial invasion by Salmonella enterica serovar Typhimurium DT104 in the swine ileum

Salmonella enterica serovar Typhimurium is an important intestinal pathogen in swine. This study was performed to document the early cellular invasion of Salmonella serovar Typhimurium in swine ileum. Ileal gut-loops were surgically prepared in ten 4- to 5-week-old mixed-breed pigs and inoculated for 0-60 minutes. Loops were harvested and prepared for both scanning and transmission electron microscopy (SEM and TEM, respectively). Preferential bacterial adherence to microfold cells (M cells) was seen within 5 minutes, and by 10 minutes bacterial invasion of the apical membrane was seen in M cells, goblet cells, and enterocytes. This multicellular invasion was observed throughout the course of infection. In addition, SEM revealed a specific affinity of Salmonella serovar Typhimurium to sites of cell extrusion. Using TEM, bacteria in these areas were focused in the crevices formed by the extruding cell and the adjacent cells and in the cytoplasm immediately beneath the extruding cell. Our results suggest that early cellular invasion by Salmonella serovar Typhimurium is nonspecific and rapid in swine. Furthermore, the combination of SEM and TEM data suggests that Salmonella serovar Typhimurium may use sites of cell extrusion as an additional mechanism for early invasion.     

In vitro studies of adhesion and invasion by Salmonella strains of bovine epithelial cells

24 Salmonella strains were divided into three groups according to the circumstances of isolation. The adhesion and invasion abilities of the strains were determined using two permanent cell lines (IEC-6, VERO) and an epithelial cell line from the small intestine of a calf fetus (pKD). Strains of different groups showed no differences in their ability to adhere to the cells tested. Significant differences were found for the invasion ability. Strains isolated from organs of calves suffering from salmonellosis showed a significant higher invasiveness for permanent cell lines and a considerable higher invasiveness for pKD cells than strains of the other groups.     

Comparison of early ileal invasion by Salmonella enterica serovars Choleraesuis and Typhimurium

The mechanisms of Salmonella serovar-host specificity are not well defined. Pig ileal loops were used to compare phenotypic differences in early cellular invasion between non-host-adapted Salmonella serovar Typhimurium (SsT) and host-adapted Salmonella serovar Choleraesuis (SsC). By 10 minutes postinoculation, both serovars invaded a small number of M cells, enterocytes, and goblet cells. Multiple SsC organisms (up to 6 per cell) simultaneously invaded M cells, whereas SsT often invaded as one to two organisms per M cell. Internalization of both serovars resulted in vacuoles containing a single bacterium. The follicle-associated epithelium (FAE) of SsC-inoculated loops responded with more filopodia and lamellipodia although exhibiting less cell swelling than SsT. Additionally, SsT showed an enhanced affinity for sites of cell extrusion compared with SsC at 60 minutes. These results suggest: 1) both SsC and SsT exhibit non-cell-specific invasion as early as 10 minutes postinoculation, 2) Salmonella serovars exhibit differences in early invasion of FAE and M cells, and 3) cells undergoing extrusion may provide a site for preferential adherence by SsT and SsC.     

In vitro inhibition of Eimeria tenella invasion of epithelial cells by phytochemicals

Resistance to coccidiostats and possible future restrictions on their use raise the need for alternative methods of reducing coccidiosis in poultry. The aim of this study was to evaluate the effect of selected phytochemicals on Eimeria tenella sporozoite invasion in vitro. Four phytochemicals were selected on the basis that they reduce the virulence of Eimeria spp. and/or provide immune modulatory benefits to host cells: betaine, carvacrol, curcumin and Echinacea purpurea extract (EP).Madin–Darby bovine kidney (MDBK) cells were covered by medium containing phytochemicals at the highest concentration which was non-toxic to the cells. Salinomycin 50 μg/ml was positive control; negative control was medium only. E. tenella (Houghton strain) sporozoites were added to wells and after incubation for 2, 4 or 20 h at 37 °C, cells were fixed and stained with hematoxylin–eosin. Ten evenly spaced fields per well were photographed and the percentage of cells invaded by sporozoites was calculated and normalized to the control.At 2 h, carvacrol, curcumin and EP showed a significantly lower percentage of sporozoite invasion than the untreated control; in contrast, betaine treatment represented a significantly higher invasion percentage. Combining carvacrol with EP inhibited E. tenella invasion more effectively than applying the compounds individually, but the further addition of curcumin did not reduce invasion further.In conclusion, this study shows that invasion of MDBK epithelial cells by E. tenella sporozoites is inhibited in the presence of carvacrol, curcumin, or EP and enhanced by betaine. There may be potential for developing these phytochemicals as anti-coccidial feed or water additives for poultry.     

Characterization of Salmonella enterica serovar Typhimurium DT104 invasion in an epithelial cell line (IPEC J2) from porcine small intestine

Salmonella Typhimurium DT104 is an emerging enteric pathogen in swine of increasing medical importance. In this study, the time course and the actin-dependent host signaling processes necessary for invasion of a S. Typhimurium DT104 field isolate were investigated in IPEC J2 epithelial cells derived from porcine small intestine. Internalized bacteria were quantified by a gentamicin resistance assay. DT104 internalization into epithelial monolayers increased steadily between 15 and 120 min after apical inoculation. Internalization was reduced by the Rho GTPase inhibitor mevastatin, the N-WASP inhibitor wiskostatin and the actin-disrupting agent cytochalasin D, but not the Rac1 GTPase inhibitor NSC-23766. Early DT104 invasion of porcine enterocytes appears to be mediated by Rac1 GTPase-independent changes in epithelial actin assembly.     

In vitro invasion capacity of Salmonella Typhimurium DT9 isolates sourced from humans and layer hen environments

In Australia, Salmonella Typhimurium definitive type 9 is frequently isolated during foodborne outbreaks of salmonellosis. Multiple‐locus variable number tandem repeat analysis (MLVA) trace back investigations frequently identify isolate distribution patterns that may be epidemiologically linked to disease outbreaks. In this study, the in vitro virulence potential of S. Typhimurium DT9 isolates possessing different MLVA patterns (03 15 07 11 550, 03 24 11 10 523, 03 15 08 11 550 and 03 14 08 11 550) isolated from either humans or layer hens was assessed using a human colon carcinoma cell line. Four strains per MLVA from each host for a total of 32 isolates were included in these experiments. Bacteria were grown to stationary phase and added to cells at a multiplicity of infection of 100. Across all isolates, mean percent recovery ranged from 7.1 ± 1.1 to 33.3 ± 7.1%. The layer hen isolate, KC900 (MLVA profile 03 15 08 11 550), exhibited the greatest invasion with a mean percent recovery of 33.3 ± 7.1%. Overall, layer hen isolates of S. Typhimurium DT9 had significantly higher invasion into Caco2 cells than human isolates (p = .0021). RAPD and enterobacterial repetitive intergenic consensus genomic fingerprinting was also performed. Irrespective of source, the SalmonellaDT9 isolates included in this study exhibited similar fingerprint patterns.     

Comparison of growth phase on Salmonella enterica serovar Typhimurium invasion in an epithelial cell line (IPEC J2) and mucosal explants from porcine small intestine

Salmonella Typhimurium DT104 is a zoonotic enteropathogen of increasing concern for human health. In this study, the influence of growth phase on invasiveness of a S. Typhimurium DT104 field isolate and two reference strains (SL1344 and ATCC 14028) was compared in IPEC J2 cells and mucosal explants from porcine ileum. Internalized bacteria were quantified by a gentamicin resistance assay. After 90 min of exposure to the apical aspect of epithelial monolayers or luminal surface of explants, internalization of all S. Typhimurium strains in mid-logarithmic phase of bacterial growth was comparable. Internalization of stationary phase bacteria was reduced relative to log phase bacteria, with DT104 exhibiting the greatest decrease. Growth phase-related differences in S. Typhimurium invasion are similar in porcine intestinal epithelial cells and mucosal explants, but may be greater in multidrug-resistant strains.     

Comparison of the in vitro pathogenicity of two Salmonella Typhimurium phage types

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.     

鼠伤寒沙门菌诱导自噬的分子机制研究进展

随着自噬现象的发现以来,有关自噬的研究在很多领域取得了重大突破,而鼠伤寒沙门菌与自噬之间的研究报道较少。文章主要阐述了自噬发生的整个机理过程,标志性蛋白LC3及其上下游蛋白Atg和p62之间的作用机制,自噬信号通路的激活位点和条件,基于鼠伤寒沙门菌刺激TLR系统激活TAK1,导致免疫细胞中的AMPK和ULK1的活化,抑制mTOR的活性,进一步阐述了沙门菌与自噬之间的相互关联性。     

Interactions of butyric acid- and acetic acid-treated Salmonella with chicken primary cecal epithelial cells in vitro

In vitro studies of the interaction between pathogenic bacteria and the chicken intestinal epithelium are hampered by the lack of a host- and tissue-specific in vitro model. Therefore, a reproducible method for isolation and cultivation of chicken primary cecal epithelial cells was developed. Cecal crypts were isolated and cultured in vitro to form a semiconfluent layer of epithelial cells. Incubation of Salmonella enteritidis with these cells resulted in invasion. Pretreatment of the Salmonella bacteria with butyric acid resulted in a significant decrease of invasion of the bacteria in the chicken cecal epithelial cells, whereas pretreatment with acetic acid increased invasiveness. These interactions of S. enteritidis with primary chicken cecal epithelial cells were similar to the interactions with other epithelial cell types.     

Salmonella Typhimurium in chicken manure reduced or eliminated by addition of LT1000

Poultry are normally reared on bedding materials such as wood shavings or rice hulls. Poultry litter reuse for multiple flocks has become economically important in modern broiler production. However, this practice results in the litter serving as a reservoir of numerous microbial organisms, including, yeasts, molds, multiple types of viruses, and bacterial pathogens such as Salmonella, Escherichia, Campylobacter, Clostridium, Staphylococcus, and Pseudomonas. The foodborne pathogens are of particular importance for poultry producers. During the preharvest feed withdrawal period, consumption of contaminated litter and feces by the birds can lead to infection of the upper gastrointestinal tract with Salmonella, which presents substantial problems at processing. The current study was conducted to determine whether the use of a liquid bacterial product (LBP), such as LT1000, could reduce the load of Salmonella Typhimurium in poultry manure. The LBP was added to sterile poultry manure then challenged with 10⁸ cfu/mL of Salmonella Typhimurium. The concentration of Salmonella Typhimurium was measured over 9 d or until the Salmonella Typhimurium was no longer detected. In 91% of the trials, Salmonella Typhimurium was completely eliminated within 9 d. This demonstrates that the LBP used in the current study has the potential to substantially improve the overall microbiological safety of used poultry litter.     

肠炎沙门菌SEF14菌毛与肠上皮细胞IPEC-J2和Caco-2的体外黏附作用研究

为研究肠炎沙门菌SEF14菌毛对肠上皮细胞的黏附作用,本试验利用肠炎沙门菌50336株、突变株50336△sefA、50336△sefD以及互补株50336△sefA (pBRA)、50336△sefD (pACYCD)与肠上皮细胞系细胞(IPEC-J2和Caco-2)进行了黏附作用.结果显示:上述菌株均能与IPEC-J2、Caco-2细胞进行有效黏附,并且随时间延长黏附数量有所增多,相同时间各菌株与IPEC-J2细胞的黏附数量明显多于Caco-2细胞;但细菌和细胞共感染1和4h后,肠炎沙门菌野生株、相应的突变株和互补株与IPEC-J2和Caco-2细胞黏附的数量差别很小,未到达显著差异水平(P＞0.05).结果表明:SEF14菌毛并不特异性介导肠炎沙门菌与肠上皮细胞系IPEC-J2和Caco-2的黏附作用,或者不是介导黏附作用的主要因子.     

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.     

In vitro and in vivo efficacy of a live attenuated Salmonella Typhimurium vaccine at preventing intestinal colonization in chicks

Vaccination of chicks with Salmonella (S .) Typhimurium aroA deletion mutants has previously been shown to inhibit intestinal colonization of wild‐type S.  Typhimurium strains. In Australia, Bioproperties VaxSafe? STM1 strain is the only licensed and commercially available S . Typhimurium vaccine. This vaccine is a live attenuated aroA deletion mutant. Currently, it is recommended that the first dose of the STM1 vaccine is administered through coarse spray. It is unclear whether this mode of administration effectively permits intestinal colonization. Furthermore, it is not known whether the STM1 strain prevents or inhibits Salmonella colonization of chicks following this first dose. This study investigated both in vitro and in vivo colonization parameters. Invasiveness was assessed using an in vitro invasion assay into sections of ileum and caecum collected from day‐old chicks. The S.  Typhimurium definitive types (DT) 9 and 44 exhibited the greatest invasion into both intestinal segments. STM1 was invasive but was significantly less so than both isolates of S.  Typhimurium. In dual and triple infections, no competitive microbial interactions between STM1 and wild‐type Salmonella were observed. In vivo colonization inhibition was also tested. Vaccinated and nonvaccinated day‐old chicks were challenged with S.  Typhimurium DT9. Both STM1 and S.  Typhimurium DT9 were found in spleen, liver, ileum, caecum and caecal contents from day 2 postinfection. No significant exclusion effect was observed in vaccinated and challenged chicks.     

The influence of viscosity on adhesion and invasion of Salmonella strains in an in vitro model

The influence of viscosity on the adhesion and invasion behaviour of Salmonella strains of different origin was investigated using an in-vitro-model. These processes seem to be strain-dependent. Compared to the controls, the number of internalized Salmonella was elevated. This increase was a result of the greater number of Salmonella which adhered to Caco-2-cells and was detected only for strains from organs of calves having died from salmonellosis. The average motility of these strains was determined to be 1.6 +/- 0.5 mm/h. A possible association between adhesion ability and motility was discussed.     

Growth of differentiated ovine tracheal epithelial cells in vitro

Culture of ovine tracheal epithelial cells is a useful tool for conducting various in vitro studies. We describe herein an in vitro technique and the conditions for culturing primary epithelial cells derived from tracheas of adult sheep. Ovine tracheas were surgically removed from 2- to 3-month-old healthy sheep and tracheal epithelial cells were isolated by 0.15% pronase digestion. After epithelial cells isolation, a Millicell insert with porous membrane was coated with 0.05% human placental collagen and the epithelial cells were added to the membrane. To create an air-liquid interface environment for the cells, the apical compartment of the membrane containing the tracheal epithelial cells was left exposed to 5% CO(2) at 37 degrees C for 2 days then increased to 9% CO(2) while cells in the basolateral compartment underneath the membrane contained the growth medium necessary for cells nourishment. Pepsin digestion was more effective in reducing the number of fibroblasts than other procedures. Cells were allowed to grow for 6-7 days to form a confluent monolayer and nearly 21 days for cilia formation on the apical surface as determined by light microscopy of haematoxylin and eosin-stained sections of membranes. In order to further confirm the epithelial origin of cells, cells were stained for cytokeratin antigen by immunohistochemistry. Most ciliated epithelial cells were immunoreactive for cytokeratin. This is the first report of differentiated ovine tracheal epithelial cells growth and isolation. This technique can be used in numerous in vitro investigative studies in ovine species as an animal model for human disease.     

体外培养奶山羊乳腺上皮细胞的形态学观察

采用胶原酶和透明质酸酶组合消化法分离培养奶山羊乳腺上皮原代细胞,并通过胰蛋白酶和EDTA选择性消化法纯化奶山羊乳腺上皮细胞.建立了奶山羊乳腺上皮细胞系.显微镜下观察,培养的细胞具有典型的上皮细胞形态特征;角蛋白免疫组化染色后,呈现阳性反应.