RNA-seq profiles of chicken type II pneumocyte in response to Escherichia coli infection.

Avian pathogenic Escherichia coli (APEC) causes great economic loss to the poultry industry worldwide. Chicken type II pneumocytes (CP II cells) secrete surfactants and modulate lung immunity to decrease the infection of the invading pathogen. Nevertheless, the pathogenesis of CP II cells to APEC infection remains poorly understood. Therefore, we conducted global gene expression profiling of CP II cells after APEC-O78 infection to explore the host-pathogen interaction. The differentially expressed genes of CP II cells to APEC infection were characterized by RNA-seq with EB-seq algorithm. In consequence, the mRNA of 18996 genes was identified, and CP II cells responded to APEC infection with marked changes in the expression of 1390 genes. Among them, there are 803 down-regulated mRNAs and 587 up-regulated mRNAs. The KEGG prediction and Gene Ontology terms analysis revealed that the major enriched pathways were related to NF-κB signaling pathway, apoptosis pathway, tight junction, and cytokine-cytokine receptor interaction and other pathways. We adopted qRT-PCR to verify the validity of the selected gene expression. The fold induction of qPCR was similar to the RNA-seq results. These results provide a better understanding of the pathogenesis of APEC, especially apoptosis pathway involved in APEC infection.

Baicalin protects mice against Salmonella typhimurium infection via the modulation of both bacterial virulence and host response.

BACKGROUND: The worsening problems of antibiotic resistance prompt the need for alternative strategies. Baicalin, which is isolated from Scutellaria baicalensisi, has been demonstrated to exhibit anti-inflammatory, anti-virulence and antimicrobial effects. Salmonella typhimurium is an important foodborne pathogenic bacteriaum that causes gastrointestinal disease in humans and many animals. PURPOSE: The aim of this study was to investigate the effects of baicalin on S. typhimurium infection in mice and its possible mechanism in vitro. STUDY DESIGN: To evaluate the effect of baicalin in vivo, mice were orally administered of baicalin, and then were infected by an intragastric administration of S. typhimurium. The minimal inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of baicalin, baicalein, and oroxylin A against S. typhimurium were detected under the guides of the Clinical and Laboratory Standards Institute. In vitro, Caco-2 cells were infected with S. typhimurium in the presence or absence of baicalin, baicalein, and oroxylin A at sub-MICs. METHODS: In the in vivo experiment, the body weight loss, the serum levels of TNFα,  IL-6, and lactic dehydrogenase (LDH), the pathological changes of the caecum and the caecum bacterial burdens were examined. The MICs and MBCs of baicalin, baicalein, and oroxylin A against S. typhimurium were detected by two-fold serial dilutions. In vitro, Caco-2 cells were infected with S. typhimurium, and the invasion capacity, TNFα, nitrate, and LDH were analysed. The transcription levels of Salmonella pathogenicity island 1 virulence associated genes (sopB, sopE, sopE2) of S. typhimurium in the presence of baicalin, baicalein, and oroxylin A were detected by qRT-PCR. RESULTS: Our results showed that baicalin significantly decreased the body weight loss, the serum levels of TNFα,  IL-6, and LDH, and the caecum bacterial burdens of mice challenged with S. typhimurium. Histological examination showed that baicalin decreased the lesion in the caecum of S. typhimurium-infected mice. MICs and MBCs of baicalin, and oroxylin A. against S. typhimurium were > 128 µg/ml. MICs and MBCs of baicalein against S. typhimurium were 64 µg/ml, and > 128 µg/ml, respectively. Pretreatment of Caco-2 cells or S. typhimurium with baicalin, baicalein, and oroxylin A significantly inhibited the invasion of Caco-2 cells by S. typhimurium in a dose-dependent manner. Sub-MICs of baicalin, baicalein, and oroxylin A also significantly decreased the levels of TNFα, nitrate, and LDH from S. typhimurium-infected Caco-2 cells. Moreover, the transcription levels of sopB, sopE, and sopE2 were significantly suppressed by baicalin, baicalein, and oroxylin A. CONCLUSIONS: These results demonstrated that baicalin is a promising agent for the prevention of S. typhimurium infection via the modulation of both bacterial virulence and host response.

Rutin inhibits quorum sensing, biofilm formation and virulence genes in avian pathogenic Escherichia coli.

The study aimed to investigate whether rutin affects the quorum sensing (QS) of avian pathogenic Escherichia coli (APEC). In this study, APEC-O78 was selected as the test strain. We mainly examined the effects of rutin on the AI-2 secretion by bioluminescence assay, biofilm formation through a crystal violet staining method, and expression of virulence genes of APEC by qRT-PCR. We found that rutin can significantly interfering with QS through reducing the secretion of AI-2, inhibited the biofilm formation, and reduced the expression of virulence genes of APEC. Moreover, rutin markedly decreased adhesion and damage of APEC to chicken type II pneumocytes. These results suggested rutin reduces cell damage of APEC-infected chicken type II pneumocytes through interfering with QS via decreasing AI-2 production, biofilm formation, and the expression of virulence genes. This paper may provide a new evidence for colibacillosis prevention in chicken.

In vitro antiviral efficacy of caffeic acid against canine distemper virus.

Canine distemper (CD) is a highly contagious disease caused by the canine distemper virus (CDV), and mortality can be as high as 100%. However, there is no specific treatment for CD. In this study, the antiviral activity of the caffeic acid against CDV was evaluated in vitro. The results showed that the IC50 of the caffeic acid against CDV at 1 and 2 h post infection (PI) is 23.3 and 32.3 µg/mL, respectively. Consistently, at 1 and 2 h PI, the caffeic acid exhibited a reduced (23.3-57.0% and 37.2-38.1%) viral inhibitory effect in vero cells. Furthermore, the caffeic acid plus Ribavirin (RBV) has greater antiviral activity against CDV than the caffeic acid or RBV individually. In addition, the caffeic acid reduced the total viral RNA synthesis by 59-86% at 24-72 h. Therefore, our data provided the experimental evidence that the caffeic acid effectively inhibited CDV infection in vero cells, which may potentially be used to treat clinical disease associated with CDV infection.

Cinobufagin enhances the protective efficacy of formalin-inactivated Salmonella typhimurium vaccine through Th1 immune response.

Cinobufagin (CBG), one active ingredient isolated from Venenum Bufonis, has been demonstrated to have immunoregulatory effect. The aim of this study was to investigate whether CBG can enhance the protective efficacy of formalin-inactivated Salmonella typhimurium (FIST) in mice. ICR mice were immunized with FIST (106 CFU/mouse) alone or mixed with CBG (10, 20, and 40 µg) or alum (200 µg) on day 1 and day 15. Two weeks after the second immunization, serum and spleen were sampled for measuring FIST-specific antibody levels, cytokine levels, and splenocyte proliferation. The results showed that CBG enhanced FIST-specific IgG and IgG2a, the levels of interferon-gamma (IFNγ) and nitric oxide (NO), and the splenocyte proliferation response induced by concanavalin A, lipopolysaccharide, and FIST. In vivo protection studies showed that CBG significantly decreased the bacterial burdens in the spleen and prolonged the survival time of FIST-immunized mice challenged with live Salmonella typhimurium. In vivo IFNγ neutralization led to a significant reduction in FIST-specific IgG2a and IFNγ levels, and in the protective efficacy in CBG/FIST-immunized mice. In conclusion, CBG enhances the protective efficacy of formalin-inactivated Salmonella typhimurium vaccine by promoting the Th1 immune response.

Autoinducer-2 of quorum sensing is involved in cell damage caused by avian pathogenic Escherichia coli.

Avian pathogenic Escherichia coli (APEC) infections are responsible for great losses in the poultry industry. Quorum sensing (QS) acts as a global regulatory system that controls genes involved in bacterial pathogenesis, metabolism and protein biosynthesis. However, whether QS of APEC is related to cell damage has not been elucidated. In the present study, we explored the correlation between the damage of chicken type II pneumocytes induced by APEC and the autoinducer-2 (AI-2) activity of APEC. The results showed that when chicken type II pneumocytes were co-cultured with 108 CFU/ml of APEC-O78 for 6 h, the release of LDH reached the highest level (192.5 ± 13.4 U/L) (P < 0.01), and the percentages of dead cells followed the same trend in trypan blue exclusion assay. In addition, the AI-2 activity of cell-free culture fluid (CF) reached the maximum value after 6 h co-culture with 108 CFU/ml of APEC-O78. At the same time, the mRNA expressions of eight virulence genes (papC, fimA, fimC, hlyE, ompA, luxS, pfs, and qseA) of 108 CFU/ml APEC-O78 were significantly increased compared with those of 107 CFU/ml, and the mRNA expressions of four virulence genes (hlyE, tsh, iss, and luxS) of 108 CFU/ml APEC-O78 were higher than those of 109 CFU/ml (p < 0.05) after incubation for 6 h. These results suggested that AI-2-mediated QS is involved in the cell damage induced by APEC-O78, indicating AI-2 may be one new potential target for preventing chicken colibacillosis.