Efficient and robust reverse genetics system for bovine rotavirus generation and its application for antiviral screening.

Unveiling the molecular mechanisms underlying rotavirus replication and pathogenesis has been hampered by the lack of a reverse genetics (RG) system in the past. Since 2017, multiple plasmid-based RG systems for simian, human, and murine-like rotaviruses have been established. However, none of the described methods have supported the recovery of bovine rotaviruses (BRVs). Here, we established an optimized plasmid-based RG system for BRV culture-adapted strain (BRV G10P [15] BLR) and clinical isolates (BRV G6P [1] C73, G10P [11] HM26) based on a BHK-T7 cell clone stably expressing T7 polymerase. Furthermore, using this optimized RG system, we successfully rescued the reporter virus BRV rC73/Zs, rHM26/Zs and rBLR/Zs, harboring a genetically modified 1.8-kb segment 7 encoding full-length nonstructural protein 3 (NSP3) fused to ZsGreen, a 232-amino acid green fluorescent protein. Analysis of the stability of genomic insertions showed that the rC73/Zs and rBLR/Zs replicated efficiently and were genetically stable in seven rounds of serial passaging, while rHM26/Zs can be stabilized only up to the third generation, indicating that the BRV segment composition may influence the viral fitness. In addition, we adopted the recombinant reporter viruses for high-throughput screening application and discovered 12 candidates out of 1440 compounds with potential antiviral activities against rotavirus. In summary, this improved RG system of BRVs represents an important tool with great potential for understanding the molecular biology of BRV and facilitates the development of novel therapeutics and vaccines for BRV.

Probiotic potential of Lactobacillus isolated from horses and its therapeutic effect on DSS-induced colitis in mice.

Inflammatory bowel disease (IBD) is a refractory disease that endangers both humans and animals. In recent times, Lactobacillus have been used to treat animal diseases. It may be a good choice to try to isolate Lactobacillus with probiotic potential to treat IBD. Equine, as a kind of hindgut fermentation animal has rich intestinal microflora, but data regarding this is scarce. The isolation of Lactobacillus with probiotic potential from equine may become a new method for the treatment of IBD. Four isolates of Lactobacillus were isolated from fresh feces of healthy male adult horses and analyzed their biological characteristics. According to the phylogenetic analysis, A2.5 and A7.1 were identified as Pediococcus pentosaceus, A3 as Lactobacillus plantarum, and B8.2 as Weissella cibaria. All four isolates showed tolerance to the environment of acid, bile salt concentration and simulated artificial gastrointestinal fluid. The hydrophobic rate and self-aggregation rate of A3 were close to 100%, and the adhesion rate was 28.85 ± 0.74%. Four isolates were negative in hemolysis test and sensitive to common antibiotics and different isolates had different sensitivity to antibiotics. The four isolates had antibacterial and antioxidant activities which can reflect their probiotic potential. Furthermore, they could regulate the LPS (Lipopolysaccharides) stimulated Caco-2 cells. We chose A3 as the treatment strain to intervene Dextran sulfate sodium salt (DSS)-induced mice. The results showed that compared with DSS group, DSS + A3 group exhibited reduced Disease activity index (DAI), increased colon length, reduced pathological score and regulated cytokine secretion at the level of gene expression. In this study, four isolates of Lactobacillus with probiotic potential were isolated, and Lactobacillus plantarum A3 with reduced ulcerative colitis in mice was screened. It might provide a potential treatment for IBD.

Microbiome analysis reveals gut microbiota alteration of early-weaned Yimeng black goats with the effect of milk replacer and age.

BACKGROUND: Colonization of intestinal microbiota in ruminant during the early life is important to host health, metabolism and immunity. Accumulating evidence revealed the ameliorative effect of milk replacer administration in the gut microbial development of early-weaned ruminants. Yimeng black goats (YBGs) inhabiting Shandong, China show a complex intestinal microbial ecosystem, but studies of their gut microbiota are still insufficient to report. Here, this study was performed to investigate how the gut microbiota develops in weaned YBGs with the effect of age and milk replacer. RESULTS: Results indicated that both age and milk replacer were important factors to change the gut microbiota of YBGs. Although the alpha diversity of gut microbiota did not change with the age of YBGs, the taxonomic compositions significantly changed. The relative abundance of some beneficial bacteria such as Lachnospiraceae, Ruminococcaceae, Ruminiclostridium, Eubacterium and Barnesiella significantly decreased and subsequently increase with age, which contributes to maintain the stability of intestinal environment and realize the diversity of intestinal functions. The relative abundance of Porphyromonas, Brevundimonas, Flavobacterium, Stenotrophomonas, Propionibacterium, Acinetobacter, Enterococcus and Clostridium belong to pathogenic bacteria in milk replacer-treated YBGs was significantly decreased. Additionally, some beneficial bacteria such as Ruminococcus, Ruminococcaceae, Christensenellaceae and Ruminiclostridium also display a trend of decreasing first followed by gradually increasing. CONCLUSIONS: This study first revealed the gut bacterial community alterations in YBGs with the effect of age and milk replacer. This study also characterized the gut microbial distribution in YBGs with different ages and provided better insight into microbial population structure and diversity of YBGs. Moreover, milk replacer may serve as a good applicant for improving gut microbial development in early-weaned YBGs.

Isolation and Evaluation of Probiotic Potential of Lactic Acid Strains From Healthy Equines for Potential Use in Salmonella Infection.

The objective of the present study was to evaluate the probiotic properties, security and antibacterial ability in vivo of isolated strains from healthy equine. In the present study, two Pediococcus acidilactici (P1 and P2) and two Lactobacillus equi (L1 and L2) were isolated. All isolates were died when exposed to pH 2.0 for 3 hours but survived at pH 3.0 and pH 4.0 with differential survival rate, and there is a higher survival rate at pH 4.0. Similarly, the isolates showed different tolerance to bile. The viable bacteria count was sustained at high levels in a tolerance test with artificial gastrointestinal fluid. The isolates survived and grew at temperatures between 37 and 55°C but died at 65°C. Four strains exhibited inhibitory activity against pathogens, including Salmonella typhimurium (CVCC542), Escherichia coli (C83902), Staphylococcus aureus (BNCC186335), and Pasteurella multocida (clinical isolate). These isolates exhibited differential antibiotic susceptibility. In safety trials, all isolates were γ-hemolytic, and the oral toxicity of strains P1 (gavaged with 1 × 109 CFU/day) and L1 (gavaged with 1 × 109 CFU/day) were analyzed in mice. There were no effects on the overall health status of mice. There were no prominent differences in the incidence of bacteria translocation to blood, liver, and spleen. Mice gavaged with Pediococcus acidilactici P1 (1 × 108 CFU/day) or Lactobacillus equi L1 (1 × 108 CFU/day) as prevention showed lower rates of diarrhea and mortality after being challenged with Salmonella typhimurium (4 × 106 CFU signal dose, 0.1 mL by intragastric gavage). The results indicate that the isolated strains could act as potential probiotics, providing a new way to reduce salmonella infection, which merit future application studies.

Probiotic potential of Weissella strains isolated from horse feces.

In this study, we isolated four Weissella confusa strains from the healthy horse feces to test their potential as equine probiotics. The identification and characteristics of these isolates were determined as per standard methods. Resistance and susceptibility of the isolated strains were tested to low pHs, different heat treatments, commonly used antibiotics and against the pathogenic strains of Salmonella, Pasteurella, Staphylococcus aureus, and Escherichia coli. After 3 h cultural in different pH medium, the 4 strains still had a certain amount of survival above pH 3.0. WH2 and WH4 were still viable at pH2.5. All the isolated strains showed proper growth at 60 °C while no strain survived at 80 °C. The inhibition of α-amylase, the scavenging ability of free radical DPPH· and hydroxyl free radical HO·were also investigated. The results showed that WH4 had highest inhibition rate of α-amylase activity and DPPH· free radical scavenging rate, and the inhibition rate of α-amylase activity was 24.09% and the DPPH· free radical scavenging rate was 35.78%. The inhibition rate ofα-amylase activity and DPPH· scavenging rate of free radicals in the other three strains were about 10%. The clearance rate of hydroxyl radical (HO·) in 4 strains was between 12% and 15%. The antibiotic susceptibilities varied for these four Weisella strains but all of them showed resistance against the frequently used equine antibiotics. All the four strains successfully suppressed the growth of standard strains in in vitro bacteriostasis experiment, which included Salmonella enteritidis (NTNC13349), Escherichia coli (C83902) and Staphylococcus aureus (BNCC186335). they also successfully suppressed the growth of state key laboratory isolating pathogens, which are Pasterurella multocida and Salmonella. Our findings suggest that the isolated strains of Weissella confusa can act as potential equine probiotics and should be explored further.