Isolation of Limosilactobacillus reuteri Strain with Anti-porcine Epidemic Diarrhea Virus from Swine Feces.

Porcine epidemic diarrhea virus (PEDV) causes severe diarrhea diseases in piglets, which has brought huge economic losses to the pig industry. As the dominant Lactobacillus species in the piglet intestine, the antiviral effect of Limosilactobacillus reuteri (L. reuteri) has been reported. Nine L. reuteri strains were isolated and identified from swine feces in this study. The CCK-8 assay examined the anti-PEDV potential of their cell-free supernatant (CFS). Among the nine L. reuteri isolates examined, LRC8 had a higher inhibition rate to PEDV than the other strains. Thus, the biological properties of the LRC8 strain, such as growth ability, acid production ability, acid and bile salt tolerance, and adhesion to IPEC-J2 cells, were evaluated. Besides, the anti-PEDV activity of LRC8-CFS (LRC8 metabolites, LRM) was assessed using plaque reduction assays, indirect immunofluorescence assays, RT-qPCR, and western blotting. The mRNA relative expression levels of inflammatory factors including IL-1ß, IL-6, IL-8, MCP1, and TNF-α were determined by RT-qPCR. The results showed that the LRC8 strain grew well, was resistant to acid, tolerated bile salts, and adhered strongly to IPEC-J2 cells. In addition, treatment with its CFS (LRM) dramatically downregulated the mRNA expression levels of inflammatory cytokines, and in the Vero cell culture, prophylactic, therapeutic, competitive, and direct-inhibitory actions were seen against PEDV. Finally, we explored the anti-PEDV effects of the LRC8 strain in piglets and found that the LRC8 strain effectively relieved the clinical symptoms and intestinal damage of piglets infected by PEDV. To sum up, we found a L. reuteri strain with an anti-PEDV effect.

An ultrasensitive electrochemical sensor for detecting porcine epidemic diarrhea virus based on a Prussian blue-reduced graphene oxide modified glassy carbon electrode.

This study developed a novel, ultrasensitive sandwich-type electrochemical immunosensor for detecting the porcine epidemic diarrhea virus (PEDV). By electrochemical co-deposition of graphene and Prussian blue, a Prussian blue-reduced graphene oxide-modified glassy carbon electrode was made, further modified with PEDV-monoclonal antibodies (mAbs) to create a new PEDV immunosensor using the double antibody sandwich technique. The electrochemical characteristics of several modified electrodes were investigated using cyclic voltammetry (CV). We optimized the pH levels and scan rate. Additionally, we examined specificity, reproducibility, repeatability, accuracy, and stability. The study indicates that the immunosensor has good performance in the concentration range of 1 × 101.88 to 1 × 105.38 TCID50/mL of PEDV, with a detection limit of 1 × 101.93 TCID50/mL at a signal-to-noise ratio of 3σ. The composite membranes produced via co-deposition of graphene and Prussian blue effectively increased electron transport to the glassy carbon electrode, boosted response signals, and increased the sensitivity, specificity, and stability of the immunosensor. The immunosensor could accurately detect PEDV, with results comparable to real-time quantitative PCR. This technique was applied to PEDV detection and served as a model for developing additional immunosensors for detecting hazardous chemicals and pathogenic microbes.

Difference analysis of intestinal microbiota and metabolites in piglets of different breeds exposed to porcine epidemic diarrhea virus infection.

The gut microbial composition of the Luchuan (LC) piglet, one of China's native breeds, has rarely been studied, especially when compared to other breeds. This study developed a porcine epidemic diarrhea virus (PEDV) infection model in LC and Largewhite (LW) piglets, and analyzed the patterns and differences of intestinal microbial communities and metabolites in piglets of these two breeds after infection. The diarrhea score, survival time, and distribution of viral antigens in the intestine of piglets infected with PEDV differed among breeds, with the jejunal immunohistochemistry score of LW piglets being significantly higher than that of LC piglets (P < 0.001). The results of 16S rRNA sequencing showed differences in microbial diversity and community composition in the intestine of piglets with different breeds between PEDV infection piglets and the healthy controls. There were differences in the species and number of dominant phyla and dominant genera in the same intestinal segment. The relative abundance of Shigella in the jejunum of LC piglets after PEDV infection was significantly lower than that of LW piglets (P < 0.05). The key microorganisms differed in the microbiota were Streptococcus alactolyticus, Roseburia faecis, Lactobacillus iners, Streptococcus equi, and Lactobacillus mucosae (P < 0.05). The non-targeted metabolite analysis revealed that intestinal metabolites showed great differences among the different breeds related to infection. Spearman correlation analysis was conducted to examine any links between the microbiota and metabolites. The metabolites in the intestine of different breeds related to infection were mainly involved in arginine biosynthesis, synaptic vesicle cycle, nicotinic acid and nicotinamide metabolism and mTOR signaling pathway, with significantly positive or negative correlations (P < 0.05) between the various microorganisms. This study provides a theoretical foundation for investigating the application of core microorganisms in the gut of piglets of different breeds in the digestive tracts of those infected with PEDV, and helps to tackle the antimicrobial resistance problem further.

Genetic variation analysis of the sigma B protein gene of novel duck reovirus in southeastern China from 2011 to 2020.

The novel duck reovirus (NDRV) disease first appeared in China in 2011. Infected ducks may be of various ages and breeds. Sigma B protein, which is the key component of NDRV's outer capsid, may trigger group-specific neutralizing antibodies linked to NDRV infection, pathogenicity, and immune defense. The sigma B protein gene of fourteen NDRV field strains was amplified by RT-PCR and cloned into the pMD-18 T vector for sequencing to examine the genetic variation in sigma B proteins of NDRVs in southeastern China between 2011 and 2020. The sigma B protein gene of the fourteen NDRV southeastern strains included in this analysis had 96.3 %-99.8 % nucleotide, and 96.2 %-99.7 % deduced amino acid sequence homology. Phylogenetic analysis revealed that the fourteen southeastern strains belonged to a well-supported lineage that included NDRV and Muscovy duck reovirus (MDRV) strains. However, Avian reovirus (ARV) formed a distinct genetic lineage in the gene tree. The sigma B protein gene sequences of NDRV strains found in southeastern China are substantially conserved, according to these findings. There is no significant geographical difference between NDRV southeastern strains and DRV strains in other regions of China. Our findings will add to the molecular epidemiological picture of NDRV strain spread in southeastern China between 2011 and 2020, laying the groundwork for potential in-depth research on vaccine collection and comprehensive prevention.

Small RNA expression patterns in seminal plasma exosomes isolated from semen containing spermatozoa with cytoplasmic droplets versus regular exosomes in boar semen.

Multiple physiological pathways are controlled by exosomes. Exosomes may be found in seminal plasma where they carry functional molecules to the sperm, such as microRNAs (miRNAs). Sperm cytoplasmic droplets (CDs) are remnants of cytoplasm, and their migration is a morphological characteristic of epididymal maturation. However, miRNA expression patterns in seminal plasma exosomes found in semen containing spermatozoa with CDs versus regular exosomes in boar semen have not been examined. In this study, seminal plasma exosomes were isolated from semen containing spermatozoa with CDs and miRNA expression profiles were analyzed. A total of 348 known and 206 new miRNAs were identified. Sixteen miRNAs were significantly differentially expressed. Of these, 13 miRNAs (ssc-miR-101, ssc-miR-148a-5p, ssc-miR-184, ssc-miR-202-3p, ssc-miR-221-5p, ssc-miR-2483, ssc-miR-29a-3p, ssc-miR-29c, ssc-miR-31, ssc-miR-362, ssc-miR-500-5p, ssc-miR-542-3p, and ssc-miR-769-5p) were significantly upregulated, whereas three miRNAs (ssc-miR-1249, ssc-miR-155-5p, and ssc-miR-296-5p) were significantly downregulated. GO and KEGG pathway analyses showed that these targeted genes were enriched for functions such as metabolic process, reproductive process, proteasome, ubiquitin mediated proteolysis, and oxidative phosphorylation. Therefore, seminal plasma exosomes are predicted to play a key role in the regulation of sperm CDs.

[Study on elastic limit and relevant enclasp force of non-precious metal casting clasp].

OBJECTIVE: To investigate the elastic limit and relevant enclasp force of the non-precious metal casting clasp. METHODS: Casting clasp samples of five cobalt-chromium alloys and one 18 - 8 nickel-chromium alloy were made from prefabricated clasp wax by invesing, casting, sandblasting, and ultrasonic cleaning. The process of casting clasp samples deflected by loading and returned by unloading was tested and electric signals were collected by an omnipotent material machine. The analog electric signal was converted to digital signal by an analog to digital converter and stored in a computer. The elastic limit and the relevant enclasp force were analyzed using a relative software. RESULTS: The elastic limit and the relevant enclasp force of the casting clasp made from the 18 - 8 nickel-chromium alloy were smallest and those of the clasps made from the cobalt-chromium alloys in various brands were different. The range of the elastic limit of the cobalt-chromium alloy casting clasp with the length of 5.0 mm in undercut was 0.28 mm-0.33 mm and the relevant enclasp force was 14.42 g-19.28 g. CONCLUSIONS: In clinic, we should select the suitable undercut deepness wherein the cobalt-chromium alloy casting clasps, according to different brands of the casting alloy, undercut length, undercut slope, and the clasp thickness.