A study of 426 rabbits from 3 cities in Jilin province (Changchun City and Jilin City) and Liaoning province (Shenyang City) was conducted between May and June 2015. The overall prevalence of E. bieneusi in rabbits was 0.94% (4/426), with 0% (0/116), 1.72% (3/174), and 0.74% (1/136) in Jilin, Changchun, and Shenyang City, respectively. Only 3 farms (farm 1 and farm 3 in Changchun City, farm 8 in Shenyang City) were PCR-positive for E. bieneusi. Moreover, rabbits of more than 6 months (1.72%) had the highest E. bieneusi prevalence, followed by rabbits of 4-6 months (1.26%), 2-3 months (0.58%), and less than 1 month (0%). Analysis of ITS gene of E. bieneusi suggested that all 4 E. bieneusi isolates were genotype D, and were classified as group 1a. The present results first demonstrated the existence of zoonotic E. bieneusi in domestic rabbits in China. Effective control measures should be implemented to prevent E. bieneusi infection in domestic rabbits, other animals, and humans.

Recently, poly-γ-glutamic acid synthetase A (pgsA) has been applied to display exogenous proteins on the surface of Lactobacillus casei or Lactococcus lactis, which results in a surfacedisplayed component of bacteria. However, the ability of carrying genes encoded by plasmids and the expression efficiency of recombinant bacteria can be somewhat affected by the longer gene length of pgsA (1,143 bp); therefore, a truncated gene, pgsA, was generated based on the characteristics of pgsA by computational analysis. Using murine IL-10 as an exogenous gene, recombinant Lactobacillus plantarum was constructed and the capacity of the surface-displayed protein and functional differences between exogenous proteins expressed by these strains were evaluated. Surface expression of IL-10 on both recombinant bacteria with anchorins and the higher expression levels in L. plantarum-pgsA'-IL-10 were confirmed by western blot assay. Most importantly, up-regulation of IL-1β, IL-6, TNF-α, IFN-γ, and the nuclear transcription factor NF-κB p65 in RAW264.7 cells after stimulation with Poly(I:C) or LPS was exacerbated after co-culture with L. plantarum-pgsA. By contrast, IL-10 expressed by these recombinant strains could reduce these factors, and the expression of these factors was associated with recombinant strains that expressed anchorin (especially in L. plantarum-pgsA'-IL-10) and was significantly lower compared with the anchorin-free strains. These findings indicated that exogenous proteins could be successfully displayed on the surface of L. plantarum by pgsA or pgsA', and the expression of recombinant bacteria with pgsA' was superior compared with bacteria with pgsA.

Transmissible gastroenteritis coronavirus (TGEV) is one of the most severe threats to the swine industry. In this study, we constructed a suite of recombinant Lactobacillus plantarum with surface displaying the spike (S) protein coming from TGEV and fused with DC cells targeting peptides (DCpep) to develop an effective, safe, and convenient vaccine against transmissible gastroenteritis. Our research results found that the recombinant Lactobacillus plantarum (NC8-pSIP409-pgsA-S-DCpep) group expressing S fused with DCpep could not only significantly increase the percentages of MHC-IICD80 B cells and CD3CD4 T cells but also the number of IgA B cells and CD3CD4 T cells of ileum lamina propria, which elevated the specific secretory immunoglobulin A (SIgA) titers in feces and IgG titers in serum. Taken together, these results suggest that NC8-pSIP409-pgsA-S-DCpep expressing the S of TGEV fused with DCpep could effectively induce immune responses and provide a feasible original strategy and approach for the design of TGEV vaccines.

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Cucurbitacin D (CuD), isolated from plants from the Cucurbitaceae family, is a potential antitumour agent since it inhibits proliferation, migration and metastasis of cancer cells. Despite CuD antitumour activity in cancer cells, the effects of CuD on gastric cancer cell lines remain unclear. The present study aimed to investigate the effects of CuD on gastric cancer cell growth and death. Human gastric cancer cell lines (AGS, SNU1 and Hs746T) were cultured and treated with different concentrations of CuD (0, 0.25, 0.5, 1 and 2 µM). Cell proliferation was assessed using Cell Counting Kit-8 assay. Oxidative stress was evaluated by generation of reactive oxygen species (ROS). Cell apoptosis was assessed by terminal deoxynucleotidyl transferase 2′-deoxyuridine-5′-triphosphate nick-end labelling (TUNEL) staining. Levels of intracellular Ca2+ and adenosine triphosphate (ATP) were also assessed. In the present study, CuD effectively inhibited cell proliferation, triggered ROS generation and induced apoptosis in gastric cancer cells (AGS, SNU1 and Hs746T). Treatment with CuD increased intracellular Ca2+ and ATP levels. CuD also stimulated the expression of inducible nitric oxide synthase (iNOS), which augmented nitric oxide production. In addition, CuD activated the mitochondrial apoptosis pathway, which increased the expression of Bax and the release of cleaved caspace-9 (C-caspase-9) and cytochrome c, decreased the expression of B-cell lymphoma 2 (Bcl-2). The mechanism of action of CuD involved the regulation of the protein kinase B/mechanistic target of rapamycin (Akt/mTOR) pathway. We confirmed the effects of CuD on gastric tumours via an in vivo xenograft gastric tumour model. In conclusion, CuD inhibited Akt and activated the iNOS pathway, leading to higher ROS and nitric oxide production, which accelerated gastric cancer cell apoptosis.