First evidence that an emerging mammalian alphacoronavirus is able to infect an avian species.

A novel swine enteric alphacoronavirus, swine acute diarrhoea syndrome coronavirus (SADS-CoV), related to Rhinolophus bat CoV HKU2 in the subgenus Rhinacovirus emerged in southern China in 2017, causing diarrhoea in newborn piglets, and critical questions remain about the pathogenicity, cross-species transmission and potential animal reservoirs. Our laboratory's previous research has shown that SADS-CoV can replicate in various cell types from different species, including chickens. Here, we systematically explore the susceptibility of chickens to a cell-adapted SADS-CoV strain both in vitro and in vivo. First, evidence of SADS-CoV replication in primary chicken cells, including cytopathic effects, immunofluorescence staining, growth curves and structural protein expression, was proven. Furthermore, we observed that SADS-CoV replicated in chicken embryos without causing gross lesions and that experimental infection of chicks resulted in mild respiratory symptoms. More importantly, SADS-CoV shedding and viral distribution in the lungs, spleens, small intestines and large intestines of infected chickens were confirmed by quantitative reverse transcription polymerase chain reaction and immunohistochemistry. The genomic sequence of the original SADS-CoV from the pig source sample in 2017 was determined to have nine nucleotide differences compared to the cell-adapted strain used; among these were three nonsynonymous mutations in the spike gene. These results collectively demonstrate that chickens are susceptible to SADS-CoV infection, suggesting that they are a potential animal reservoir. To our knowledge, this study provides the first experimental evidence of cross-species infection in which a mammalian alphacoronavirus is able to infect an avian species.

A novel spike subunit 1-based enzyme-linked immunosorbent assay reveals widespread porcine torovirus infection in eastern China.

Toroviruses (ToVs), closely related but genetically distinct from coronaviruses, are known to infect horses, cows, pigs, goats and humans, mainly causing enteritic disorders. However, due to the lack of an adaptive culture system, porcine ToV (PToV) has received less attention. In this study, we developed a novel serological detection method based on the PToV envelope spike subunit 1 (S1) protein for the first time, and compared it to an existing indirect enzyme-linked immunosorbent assay (ELISA) based on the nucleocapsid protein. By using the S1-based ELISA, we carried out the first seroepidemiological survey of PToV in China, assaying both specific IgG and IgA responses in 1,037 serum samples collected from diarrheic pigs in eastern China. There was a relatively high incidence of seropositivity in pigs of different ages, especially one-week-old piglets and sows (78% and 43%), the former probably reflecting maternal antibodies. Furthermore, 3/20 (15%) of faecal samples collected from one PToV-seropositive swine herd in Zhejiang province tested positive by RT-PCR. The complete PToV genome was sequenced from one of these samples, and its phylogenetic relationship with other full-length PToV sequences available in GenBank was determined. Our data provide the first serological evidence for PToV infection in pigs from China, which will help elucidate the potential pathogenicity of PToV in pigs.

Lactobacillus rhamnosus GG promotes M1 polarization in murine bone marrow-derived macrophages by activating TLR2/MyD88/MAPK signaling pathway.

Lactobacillus rhamnosus GG (LGG) is increasingly applied in functional food products and acts as a probiotic model in nutritious and clinical studies. Increasing evidences have revealed the immune modulation of LGG on macrophages. The aim of this study is to investigate the effect of LGG on macrophage polarization of murine bone marrow-derived macrophages (BMDMs). BMDMs were treated with 108 colony-forming units (CFU)/ml LGG for 1.5, 3, and 6 hr. Results showed that LGG obviously upregulated the mRNA expression of M1-associated cytokines (p < .05), including interleukin-1 beta (IL-1ß), IL-6, tumor necrosis factor-alpha (TNF-α), and inducible nitric oxide synthase (iNOS), whereas had no effect on the expression of M2-associated markers (p > .05), including arginase 1 (Arg1), mannose receptor, and chitinase-like protein 3 (YM1). Furthermore, LGG markedly increased the expression of pro-inflammatory cytokines (IL-12p40, cyclooxygenase-2 [COX-2], and interferon-γ [IFN-γ]) (p < .05) and anti-inflammatory cytokines (IL-10, IL-4, and transforming growth factor-ß [TGF-ß]) (p < .05). In addition, we also found that TLR2/MyD88/MAPK signaling pathway was required for LGG-induced M1 macrophage polarization and M1-related cytokines expression. Together, these findings demonstrate that probiotic LGG facilitates M1 polarization of BMDMs, suggesting that LGG may have an immunotherapeutic potential in regulating the host defense against pathogen invasion.

Bacillus amyloliquefaciens SC06 Protects Mice Against High-Fat Diet-Induced Obesity and Liver Injury via Regulating Host Metabolism and Gut Microbiota.

Obesity and the related liver diseases are prevalent around the world. Although probiotics have been shown to prevent obesity through multiple ways, only few researches investigated the lipid-lowering effects of probiotic Bacillus. Moreover, the limited results consistently suggested that Bacillus regulated genes related to lipogenesis and oxidation, but no further exploration was made. Our previous study revealed that Bacillus amyloliquefaciens SC06 has a potent antioxidant capacity in vitro. The aim of this study is to investigate the effects of SC06 on obesity and the associated liver injury of high-fat diet (HFD)-fed-mice and its underlying mechanism. By feeding normal chow (NC), NC+SC06, HFD, and HFD+SC06 to mice, we found that SC06 improved body weight gain, hepatic steatosis, and glucose metabolism of HFD-mice. Furthermore, SC06 also increased the antioxidant capacity of mice through Nrf2/Keap1 signaling pathway. High-throughput sequencing of 16S rRNA gene showed that HFD changed the gut microbiota dramatically, while HFD+SC06 decreased the ratio of Firmicutes/Bacteroidetes and increased TM7 abundance. More differences were also found in lower taxa. Altogether, SC06 is a potential probiotic that decreases HFD-related lipid accumulation and liver injury via regulating the antioxidant capacity and host gut microbiota.

Protocatechuic acid improved growth performance, meat quality, and intestinal health of Chinese yellow-feathered broilers.

The aim of this study was to investigate the effect of protocatechuic acid (PCA) on the growth performance, meat quality, and intestinal health of Chinese yellow-feathered broilers. Growing broilers were fed the basal diet or diets supplemented with 300 or 600 mg/kg PCA, or 200 mg/kg enramycin for 52 D. We found that addition of 300 mg/kg PCA significantly increased body weight, live weight, and carcass weight and decreased the feed to gain ratio of broilers; PCA improved meat quality through reducing shear force, and increasing a* (relative redness) and decreasing b* (relative yellowness) at 24 h after slaughter. The activities of alkaline phosphatase and diamine oxidase in plasma were significantly decreased by administration of 300 mg/kg PCA; PCA also significantly increased total antioxidant capability and decreased malondialdehyde content and activity of xanthine oxidase in liver. Meanwhile, it enhanced activities of total superoxide dismutase, glutathione s-transferase, and glutathione peroxidase in the jejunal mucosa. Interleukin-10 and transforming growth factor-ß were significantly increased in jejunal mucosa and plasma of 300 mg/kg PCA diet group, whereas interluekin-2 and interferon-γ dropped dramatically. Moreover, relative expression of apoptosis-related genes decreased in liver, whereas that of intestinal barrier-related and immunity-related genes increased in jejunum. Furthermore, 300 mg/kg PCA treatment significantly changed α-diversity and structure of the cecal microflora in broilers, with increasing relative abundance of Firmicutes and Actinobacteria while reducing Bacteroidetes and Proteobacteria. These results indicated that PCA improved the feed efficiency, growth performance, meat quality of broilers, and antioxidant capacity. It also enhanced intestinal immune function and improved the structure of intestinal flora to favor improved intestinal health in Chinese yellow-feathered broilers.

Effects of Probiotic Bacillus as an Alternative of Antibiotics on Digestive Enzymes Activity and Intestinal Integrity of Piglets.

The previous study in our team found that supplementation of probiotic Bacillus amyloliquefaciens (Ba) instead of antibiotics promote growth performance of piglets. Hence, the present study was carried out to further demonstrate the effect of Ba replacement of antibiotics on digestive and absorption enzyme activity and intestinal microbiota population of piglets. A total of 90 piglets were selected and divided into three groups: G1 group was fed with basal diet supplemented with 150 mg/Kg aureomycin, G2 group was fed with 1 × 108 cfu/Kg Ba and half dose of aureomycin, G3 group was used the diet with 2 × 108cfu/Kg Ba replaced aureomycin. Each treatment had three replications of 10 pigs per pen. Results indicated that Ba replacement significantly increased the activities of amylase, disaccharides and Na+/K+-ATPase. And chymotrypsin activity in different section of intestine was dramatically enhanced in half replacement of aureomycin with Ba. Moreover, Ba replacement maintained the intestinal integrity with the significantly decreased activity of DAO compared with aureomycin group. Besides, supplementation with Ba increased the ß-diversity of intestinal microbiota. Taken together, the current study indicated that diet supplementation with Ba instead of aureomycin increased the growth performance of piglets by improving the digestive and absorb enzyme activities, enhancing the intestinal integrity and regulating the population of intestinal micrbiota.

Glycyrrhizin Attenuates Salmonella enterica Serovar Typhimurium Infection: New Insights Into Its Protective Mechanism.

Glycyrrhizin (GL), a triterpenoid glycoside, serves important functions in various biological activities, including antiviral and antitumor immune responses. However, the anti-inflammatory effects of GL on Salmonella enterica serovar Typhimurium (ST)-induced injury in mice and the mechanisms underlying the protection of GL are poorly understood. Here, we investigated the effects of GL on host immune responses against ST infection in mice. A phenotypic analysis using hematoxylin and eosin (H&E) staining and transmission electron microscopy showed that GL relieved ST-induced weight loss and intestinal mucosal injury. A colonization assay showed that GL significantly reduced ST colonization in the ileum and colon and translocation to the liver and spleen. An antibacterial activity assay and real-time PCR revealed that GL had no direct inhibitory impact on ST growth or virulence gene expression. ELISA showed that GL pretreatment significantly decreased proinflammatory cytokine (IFN-γ, TNF-α, IL-6) secretion and increased anti-inflammatory cytokine (IL-10) secretion in the ileum, colon and serum of ST-infected mice. Moreover, flora analysis showed that GL reduced Akkermansia, Sutterella, Prevotella and Coprococcus but enriched Parabacteroides and Anaerotruncus in the cecum of ST-infected mice. These results suggest that GL promotes the secretion of immune factors and modulates intestinal flora to prevent further ST infection. We also analyzed the effect of GL on immunocytes and found that GL promoted the phenotypic and functional maturation of murine bone marrow-derived dendritic cells (BMDCs). Flow cytometry and western blotting demonstrated that NF-κB, ERK, and p38 MAPK were required for GL-induced BMDC maturation. The above findings indicate that GL attenuates ST infection by modulating immune function and intestinal flora. This study enriches our current knowledge of GL-mediated immunological function and provides a new perspective on the prevention of Salmonella infection in animals and humans.

Effects of three probiotic Bacillus on growth performance, digestive enzyme activities, antioxidative capacity, serum immunity, and biochemical parameters in broilers.

This study was conducted to evaluate the effects of three Bacillus strains on growth performance, digestive enzyme activities, antioxidative capacity, serum immunity, and biochemical parameters in broilers. A total of 360 one-day-old Ross 308 chicks were randomly allocated into four groups with three replicates per group (n = 30). The control group was fed a basal diet, whereas the other groups fed basal diet supplemented with either Bacillus subtilis natto or Bacillus licheniformis or Bacillus cereus (108  cfu/kg) for 42 days, respectively. The results revealed that the probiotic-treated groups markedly improved final body weight, daily weight gain, and the activities of trypsin, amylase, lipase and total protease (p < 0.05). Moreover, chicks fed probiotics had higher serum glutathione peroxidase activity and O2 - level, as well as hepatic catalase and superoxide dismutase activities, whereas malondialdehyde levels in serum and liver were reduced (p < 0.05). The significant increased IgA (p < 0.05) was observed in the probiotics groups as compared to the control group. In addition, dietary administration of probiotic strain markedly reduced the levels of serum ammonia, uric acid, total cholesterol, and triglyceride. Taken together, these three probiotic Bacillus showed beneficial effects on chickens with minor strain specificity.

Direct-fed glucose oxidase and its combination with B. amyloliquefaciens SC06 on growth performance, meat quality, intestinal barrier, antioxidative status, and immunity of yellow-feathered broilers.

This experiment investigated the effects of dietary glucose oxidase (GOD) and its combination with B. amyloliquefaciens SC06 (BaSC06) on the growth performance, meat quality, intestinal physical barrier, antioxidative status and immunity of male Lingnan yellow-feathered broilers. A total of 720 1-d-old broilers were assigned into 4 treatments with 6 replicates per treatment (30 birds per replicate): (1) basal diet (Ctr), (2) basal diet with 200 mg/kg enramycin (ER), (3) basal diet with 75 U/kg GOD, and (4) GOD diet (75U/kg) supplemented with 1 × 105 colony-forming units BaSC06/kg feed (GB), for an experimental duration of 52 d. The results showed that there were no significant effects of GOD or GB on growth performance of birds. The shear force and drip loss of breast muscle of birds fed GOD and GB were less than those fed ER, while the shear force in GB significantly decreased compared to Ctr. Also, both GOD and GB treatment increased about 1-fold expression of ZO-1, Claudin-1, Occludin, and MUC-2 genes in jejunal mucosa compared to Ctr, no difference was found between GOD and GB. Compared to Ctr, serum total antioxidant capability and glutathione peroxidase in GOD and GB increased, while the malondialdehyde level and xanthine oxidase activity significantly decreased. Both GOD and GB treatments reduced the relative level of HO-1, p53, and BAX transcripts in liver. It is worth noting that GB decreased transcription of p53 and Bcl-2 by 76.11% and 50.19% compared to GOD, respectively. In addition, compared to Ctr, GOD and GB markedly increased serum IL-2 content by 110% and 182%, while decreased IFN-γ by 43.57% and 57.51%, respectively. The highest sIgA level in GB was found among four groups. In conclusion, dietary treatment with GOD and its combination with BaSC06 both had beneficial effects on shear force and drip loss, expression of intestinal tight junctions, antioxidative capacity and immune function. It is suggested that GB had better effect than GOD on anti-apoptosis.

Antioxidant Properties of Probiotic Bacteria.

Oxidative stress defines a condition in which the prooxidant-antioxidant balance in the cell is disturbed, resulting in DNA hydroxylation, protein denaturation, lipid peroxidation, and apoptosis, ultimately compromising cells' viability. Probiotics have been known for many beneficial health effects, and the consumption of probiotics alone or in food shows that strain-specific probiotics can present antioxidant activity and reduce damages caused by oxidation. However, the oxidation-resistant ability of probiotics, especially the underling mechanisms, is not properly understood. In this view, there is interest to figure out the antioxidant property of probiotics and summarize the mode of action of probiotic bacteria in antioxidation. Therefore, in the present paper, the antioxidant mechanisms of probiotics have been reviewed in terms of their ability to improve the antioxidant system and their ability to decrease radical generation. Since in recent years, oxidative stress has been associated with an altered gut microbiota, the effects of probiotics on intestinal flora composition are also elaborated.