Overview of the recent advances in porcine epidemic diarrhea vaccines.

Vaccination is the most effective means of preventing and controlling porcine epidemic diarrhea (PED). Conventional vaccines developed from porcine epidemic diarrhea virus (PEDV) GI-a subtypes (CV777 and SM98) have played a vital role in preventing classical PED. However, with the emergence of PEDV mutants in 2010, conventional PEDV GI-a subtype-targeting vaccines no longer provide adequate protection against PEDV GII mutants, thereby making novel-type PED vaccine development an urgent concern to be addressed. Novel vaccines, including nucleic acid vaccines, genetically engineered subunit vaccines, and live vector vaccines, are associated with several advantages, such as high safety and stability, clear targeting, high yield, low cost, and convenient usage. These vaccines can be combined with corresponding ELISA kits to differentiate infected from vaccinated animals, which is beneficial for disease confirmation. This review provides a detailed overview of the recent advancements in PED vaccines, emphasizing on the research and application evaluation of novel PED vaccines. It also considers the future directions and challenges in advancing these vaccines to widespread use in clinics.

Effect of feeding fermented distiller's grains diets on immune status and metabolomics of spleen and mesenteric lymph nodes in finishing cattle.

To explore the effect of feeding fermented distiller's grains (FDG) diets on spleen and mesenteric lymph nodes (MLN) immune status and metabolomics in finishing cattle, eighteen Guanling crossbred cattle (18 months old, 250.0 ± 25 kg) were randomly divided into 3 groups: a basal diet (Control) group, an FDG-15% group, and an FDG-30% group (containing 0%, 15% and 30% FDG to partially replace the concentrates, respectively). After 75 days, the spleens and MLN were collected for detection of relative spleen weight, immune parameters, and metabolomic analysis. Compared with the Control group, FDG-30% group significantly increased (P<0.05) the relative spleen weight. In addition, the level of IL-17A in the spleen of the FDG-30% group was significantly higher than that of the FDG-15% group. Metabolomic analysis showed that differential metabolites (VIP＞1, P＜0.05) of spleen and MLN in FDG-15% and FDG-30% groups are mostly lipids and lipid molecules. KEGG analysis illustrated that choline metabolism in cancer, glycerophospholipid metabolism, biosynthesis of unsaturated fatty acids and insulin resistance were metabolic pathways in spleen shared by FDG-15% group vs.Control group and FDG-30% group vs.Control group, and choline metabolism in cancer was a metabolic pathway in MLN shared by FDG-15% group vs.Control group and FDG-30% group vs.Control group. These results suggest that feeding FDG may promote spleen development by regulating choline metabolism in cancer, glycerophospholipid metabolism, biosynthesis of unsaturated fatty acids and insulin resistance. Additionally, it may affect MLN development by regulating choline metabolism in cancer. SIGNIFICANCE: Fermented distiller's grains (FDG) is a high quality alternative to feed because it is rich in beneficial microorganisms and nutrients. The spleen and mesenteric lymph nodes (MLN) are important peripheral immune organs in animals, whose status reflects the health of the animal. However, there are few reports on the effect of feeding FDG diets on spleen and MLN immune status and metabolomics in domestic animals. In this study, we found that feeding FDG may promote spleen development by regulating choline metabolism in cancer, glycerophospholipid metabolism, biosynthesis of unsaturated fatty acids and insulin resistance metabolic pathways, and may affect MLN development by regulating choline metabolism in cancer. This study extends our understanding of the metabolomics of the spleen and MLN in FDG and helps to further understand of the immunomodulatory effects of the FDG diet.

Probiotic-Fermented Distillers Grain Alters the Rumen Microbiome, Metabolome, and Enzyme Activity, Enhancing the Immune Status of Finishing Cattle.

A total of 30 Simmental crossbred cattle (6.50 months old, 265.0 ± 22.48 kg) were randomly divided into three groups, with 10 heads per group, and fed for 45 days. The diet treatments consisted of the Control group without PFDG supplementation, the PFDG-15% group with 15% PFDG substituting for 15% concentrate, and PFDG-30% group with 30% PFDG substituting for 30% concentrate. The results showed that compared with the Control group, the average daily gain (ADG) of the cattle in the PFDG-30% group decreased significantly (0.890 vs. 0.768 kg/d, p = 0.005). The serum malondialdehyde content of cattle in the PFDG-15% and PFDG-30% groups decreased significantly (p = 0.047) compared to that of the Control group. However, the serum superoxide dismutase activity of cattle in the PFDG-30% group was significantly higher than that of the Control group (p = 0.047). Meanwhile, both the PFDG-15% and PFDG-30% groups (1758.47 vs. 2061.30 µg/mL) showed higher serum levels of immunoglobulin G, while the interleukin-10 concentration was lower in the PFDG-30% group (p = 0.027). In addition, the PFDG-15% and PFDG-30% groups shifted the rumen microbiota by improving the abundances of F082 (related to propionic acid production) and fiber-degrading bacteria (Lachnospiraceae_UGG-009 and Prevotellaceae_UCG-001) and reducing the abundance of the disease-associated bacteria Selenomonas. A Kyoto encyclopedia of genes and genomes (KEGG) analysis illustrated that three key metabolic pathways, including phenylalanine metabolism, pyrimidine metabolism, and tryptophan metabolism, were enriched in the PFDG-15% group, but eight key metabolic pathways, including arachidonic acid metabolism, were enriched in the PFDG-30% group. Importantly, both the PFDG-15% and PFDG-30% groups increased (p < 0.01) the activities of cellulase, lipase, and protease in the rumen. Finally, the different bacterial abundance in the rumen was associated with changes in the ADG, serum antioxidant capacity, immune status, rumen enzyme activity, and metabolites. These results suggest that PFDG alters rumen microbiome abundance, metabolome, and enzyme activity for enhancing serum antioxidant capacity and the immune status, but when the supplemental level reaches 30%, it has a negative effect on ADG and the anti-inflammatory factors in finishing cattle.

The effect of feeding fermented distillers' grains diet on the intestinal metabolic profile of Guanling crossbred cattle.

Fermented distiller's grains (FDG)-based diets are nutritious and can improve the growth and intestinal immunity in livestock. However, there is limited research examining the effect of feeding FDG-based diets on changes in intestinal metabolites and related pathways in livestock. In this study, nine Guanling crossbred cattle (Guizhou Guanling Yellow cattle × Simmental cattle) were selected and randomly divided into a basal diet (BD) group and two experimental groups fed with FDG replacing 15% and 30% of the daily ration concentrates (FDG-Case A and FDG-Case B), respectively, with three cattle in each group. Fresh jejunum (J) and cecum (C) tissues were collected for metabolomic analysis. Differential metabolites and metabolic pathways were explored by means of univariate and multivariate statistical analysis. Compared with the J-BD group, 30 and 100 differential metabolites (VIP > 1, p < 0.05) were obtained in the J-FDG-Case A group and J-FDG-Case B group, respectively, and the J-FDG-Case B vs. J-FDG-Case A comparison revealed 63 significantly differential metabolites, which were mainly divided into superclasses including lipids and lipid-like molecules, organoheterocyclic compounds, and organic acids and derivatives. Compared with the C-BD, 3 and 26 differential metabolites (VIP > 1, p < 0.05) were found in the C-FDG-Case A group and C-FDG-Case B group, respectively, and the C-FDG-Case B vs. C-FDG-Case A comparison revealed 21 significantly different metabolites, which were also mainly divided into superclasses including lipids and lipid-like molecules, organoheterocyclic compounds, and organic acids and derivatives. A total of 40 metabolic pathways were identified, with a significance threshold set at p < 0.05. Among them, 2, 14, and 18 metabolic pathways were significantly enriched in the J-FDG-Case A vs. J-BD, J-FDG-Case B vs. J-BD, and J-FDG-Case B vs. J-FDG-Case A comparisons, respectively. Meanwhile, 1, 2, and 3 metabolic pathways were obtained in the C-FDG-Case A vs. C-BD, C-FDG-Case B vs. C-BD, and C-FDG-Case B vs. C-FDG-Case A comparisons, respectively. Furthermore, four significant metabolic pathways, namely insulin resistance, biosynthesis of unsaturated fatty acids, linoleic acid metabolism, and primary bile acid biosynthesis, were significantly enriched in Guanling crossbred cattle fed FDG diets. These results suggest that feeding FDG diets may promote the growth and intestinal immunity of Guanling crossbred cattle by regulating metabolic patterns of lipid compounds and related metabolic pathways. This study sheds light on the potential metabolic regulatory mechanisms of FDG diets and offers some references for their use in livestock feed.

Partially Alternative Feeding with Fermented Distillers' Grains Modulates Gastrointestinal Flora and Metabolic Profile in Guanling Cattle.

Fermented distillers' grains (FDG) are commonly used to enhance the health and metabolic processes of livestock and poultry by regulating the composition and activity of the intestinal microbiota. Nevertheless, there is a scarcity of research on the effects of the FDG diet on the gastrointestinal microbiota and its metabolites in cattle. This study examines the impact of FDG dietary supplements on the gastrointestinal flora and metabolic profile of Guanling cattle. Eighteen cattle were randomly assigned to three treatment groups with six replicates per group. The treatments included a basal diet (BD), a 15% concentrate replaced by FDG (15% FDG) in the basal diet, and a 30% concentrate replaced by FDG (30% FDG) in the basal diet. Each group was fed for a duration of 60 days. At the conclusion of the experimental period, three cattle were randomly chosen from each group for slaughter and the microbial community structure and metabolic mapping of their abomasal and cecal contents were analyzed, utilizing 16S rDNA sequencing and LC-MS technology, respectively. At the phylum level, there was a significant increase in Bacteroidetes in both the abomasum and cecum for the 30%FDG group (p < 0.05). Additionally, there was a significant reduction in potential pathogenic bacteria such as Spirochetes and Proteobacteria for both the 15%FDG and 30%FDG groups (p < 0.05). At the genus level, there was a significant increase (p < 0.05) in Ruminococcaceae_UCG-010, Prevotellaceae_UCG-001, and Ruminococcaceae_UCG-005 fiber degradation bacteria. Non-target metabolomics analysis indicated that the FDG diet significantly impacted primary bile acid biosynthesis, bile secretion, choline metabolism in cancer, and other metabolic pathways (p < 0.05). There is a noteworthy correlation between the diverse bacterial genera and metabolites found in the abomasal and cecal contents of Guanling cattle, as demonstrated by correlation analysis. In conclusion, our findings suggest that partially substituting FDG for conventional feed leads to beneficial effects on both the structure of the gastrointestinal microbial community and the metabolism of its contents in Guanling cattle. These findings offer a scientific point of reference for the further use of FDG as a cattle feed resource.

Impact of feeding dried distillers' grains with solubles diet on microbiome and metabolome of ruminal and cecal contents in Guanling yellow cattle.

Dried distillers' grains with solubles (DDGS) are rich in nutrients, and partially alternative feeding of DDGS effectively reduces cost of feed and improves animals' growth. We used 16S rDNA gene sequencing and LC/MS-based metabolomics to explore the effect of feeding cattle with a basal diet (BD) and a Jiang-flavor DDGS diet (replaces 25% concentrate of the diet) on microbiome and metabolome of ruminal and cecal contents in Guanling yellow cattle. The results showed that the ruminal and cecal contents shared the same dominance of Bacteroidetes, Firmicutes and Proteobacteria in two groups. The ruminal dominant genera were Prevotella_1, Rikenellaceae_RC9_gut_group, and Ruminococcaceae_UCG-010; and the cecal dominant genera were Ruminococcaceae_UCG-005, Ruminococcaceae_UCG-010, and Rikenellaceae_RC9_gut_group. Linear discriminant analysis effect size analysis (LDA > 2, P < 0.05) revealed the significantly differential bacteria enriched in the DDGS group, including Ruminococcaceae_UCG_012, Prevotellaceae_UCG_004 and Anaerococcus in the ruminal contents, which was associated with degradation of plant polysaccharides. Besides, Anaerosporobacter, Anaerovibrio, and Caproiciproducens in the cecal contents were involved in fatty acid metabolism. Compared with the BD group, 20 significantly different metabolites obtained in the ruminal contents of DDGS group were down-regulated (P < 0.05), and based on them, 4 significantly different metabolic pathways (P < 0.05) were enriched including "Linoleic acid metabolism," "Biosynthesis of unsaturated fatty acids," "Taste transduction," and "Carbohydrate digestion and absorption." There were 65 significantly different metabolites (47 were upregulated, 18 were downregulated) in the cecal contents of DDGS group when compared with the BD group, and 4 significantly different metabolic pathways (P < 0.05) were enriched including "Longevity regulating pathway," "Bile secretion," "Choline metabolism in cancer," and "HIF-1 signaling pathway." Spearman analysis revealed close negative relationships between the top 20 significantly differential metabolites and Anaerococcus in the ruminal contents. Bacteria with high relevance to cecal differential metabolites were Erysipelotrichaceae_UCG-003, Dielma, and Solobacterium that affect specific metabolic pathways in cattle. Collectively, our results suggest that feeding cattle with a DDGS diet improves the microbial structure and the metabolic patterns of lipids and carbohydrates, thus contributing to the utilization efficiency of nutrients and physical health to some extent. Our findings will provide scientific reference for the utilization of DDGS as feed in cattle industry.

Plasma Metabolomic Profiling after Feeding Dried Distiller's Grains with Solubles in Different Cattle Breeds.

Dried distiller's grains with solubles (DDGS) are rich in nutrients and can enhance animals' growth and immunity. However, there are few reports on the effects of a diet of DDGS on plasma metabolism and the related action pathways in domestic animals. In this study, groups of Guanling yellow cattle (GY) and Guanling crossbred cattle (GC) having a basal diet served as the control groups (GY-CG and GC-CG), and DDGS replacing 25% of the diet of GY and GC served as the replacement groups (GY-RG and GC-RG), with three cattle in each group. Plasma samples were prepared for metabolomic analysis. Based on multivariate statistical and univariate analyses, differential metabolites and metabolic pathways were explored. Twenty-nine significantly different metabolites (p < 0.05) were screened in GY-RG compared with those in GY-CG and were found to be enriched in the metabolic pathways, including choline metabolism in cancer, linolenic acid metabolism, and amino acid metabolism. Nine metabolites showed significant differences (p < 0.05) between GC-RG and GC-CG and were mainly distributed in the metabolic pathways of choline metabolism in cancer, glycerophospholipid metabolism, prostate cancer metabolism, and gonadotropin-releasing hormone (GnRH) secretion. These results suggest that a DDGS diet may promote healthy growth and development of experimental cattle by modulating these metabolic pathways. Our findings not only shed light on the nutritional effects of the DDGS diet and its underlying mechanisms related to metabolism but also provide scientific reference for the feed utilization of DDGS.

Effect of feeding a dried distillers' grains with solubles diet on the metabolism of the intestinal wall in Guanling crossbred cattle: a preliminary assessment.

Dried distillers' grains with solubles (DDGS)-based diets are nutritious and can improve the inflammations and intestinal immunity in livestock. However, there is limited research examining the effect of feeding DDGS-based diets on changes in intestinal metabolites and related pathways in livestock. In this study, six Guanling crossbred cattle (Guizhou Guanling Yellow cattle × Simmental cattle) were selected and divided into a basal diet (BD) group and an experimental group fed with DDGS replacing 25% of the daily ration concentrates (DDGS) (n=3), respectively. Fresh jejunum (J), ileum (I) and cecum (C) tissues were collected for metabolomic analysis. Differential metabolites and metabolic pathways were explored by means of univariate and multivariate statistical analysis. In comparison to the J-BD group, 123 differential metabolites (VIP > 1, p < 0.05) were identified in the J-DDGS group, which (top 20) were mainly divided into superclasses, including lipids and lipid-like molecules, organic acids and derivatives, and organoheterocyclic compounds. Compared with the I-BD group, 47 differential metabolites were obtained in the I-DDGS group, which were mainly divided into superclasses, including lipids and lipid-like molecules and organic acids and derivatives. The C-DDGS vs. C-BD comparison revealed 88 differential metabolites, which were mainly divided into superclasses, including lipids and lipid-like molecules, organic oxygen compounds, and nucleosides. A total of 34 significant metabolic pathways were found (p < 0.05, -log(p) > 1.3). Among them, 3 significant pathways were significantly enriched in the J-DDGS group, 11 significant pathways were significantly enriched in the I-DDGS group, and 20 significant pathways were significantly enriched in the C-DDGS group. Importantly, primary bile acid biosynthesis, linoleic acid metabolism, and arachidonic acid metabolism correlated with intestinal inflammation and immunity by regulating gut microbiota, prostaglandin synthesis, and cell signaling. The data suggest that DDGS-fed cattle unregulated three metabolic pathways mentioned above and that a DDGS-based diet was able to maintain a balance of these three metabolic pathways, thus resulting in improvement of intestinal inflammation and enhanced immunity in cattle. In conclusion, the DDGS diet has the potential to improve intestinal inflammation and enhance the immunity of Guanling crossbred cattle by regulating the metabolic patterns of lipids and lipid-like molecules, organic acids and derivatives, and related metabolic pathways. These results allude to potential metabolic regulatory mechanisms of DDGS diets and also provide a theoretical basis for the application of DDGS in livestock feed.

Leptospira interrogans infection leads to IL-1ß and IL-18 secretion from a human macrophage cell line through reactive oxygen species and cathepsin B mediated-NLRP3 inflammasome activation.

Leptospirosis is a worldwide zoonosis caused by spirochetes from the genus Leptospira. Although there is a large diversity of clinical signs and symptoms, a severe inflammatory response is common to all leptospirosis patients. The mechanism of IL-1ß secretion during Leptospira infection has been previously studied in mouse macrophages. However, the outcome of Leptospira infection is very different in human and murine macrophages, and the mechanisms responsible for IL-1ß secretion in human macrophages had not been investigated. This study therefore examines the effects of Leptospira interrogans infection on inflammasome activation and proinflammatory cytokine expression in human macrophages. Increased mRNA and protein expression of NLRP3 was observed by real time RT-PCR and flow cytometry at 1 h after co-cultivation. Enzyme-linked immunosorbent assay (ELISA) determination showed that IL-1ß and IL-18 are released in the culture supernatants at 1 h after cultivation. The inhibition assay showed that glybenclamide (a K+ efflux inhibitor that blocks NLRP3 inflammasome activation) and N-benzyloxycarbony-Val-Ala-Asp (O-methyl)-fluoromethylketone (Z-VAD-FMK; a caspase-1 inhibitor) and NLRP3 depletion with siRNAs reduced the levels of IL-1ß and IL-18 release. Moreover, the levels of IL-1ß and IL-18 production decreased in CA-074 (a cathepsin B inhibitor) and NAC (an anti-oxidant) pretreated human macrophages, compared to untreated controls. This study suggests that L. interrogans infection leads to reactive oxygen species (ROS)- and cathepsin B-dependent NLRP3 inflammasome activation, which subsequently mediates caspase-1 activation and IL-1ß and IL-18 release.

The role of reactive oxygen intermediates in the intracellular fate of Leptospira interrogans in the macrophages of different hosts.

BACKGROUND: Pathogenic species of Leptospira cause leptospirosis, a global zoonotic disease. Our previous work showed that leptospires survive and replicate in human macrophages but are killed in murine macrophages. However, the mechanism responsible for the different intracellular fates of leptospires within the macrophages of different hosts remains unclear. RESULTS: The present study demonstrates that infection with Leptospira interrogans caused significant up-regulation of reactive oxygen species (ROS) and superoxide in J774A.1 cells but did so to a lesser extent in THP-1 cells. The up-regulation of ROS and superoxide was significantly inhibited by the NADPH oxidase inhibitor apocynin. The damaged leptospires and remnants of leptospires within membrane-bound vacuoles were significantly inhibited by apocynin in J774A.1 cells but were less inhibited in THP-1 cells. In addition, apocynin significantly prevented damage to leptospires and the co-localization of L. interrogans with lysosomes in J774A.1 cells but did so to a lesser extent in THP-1 cells. Furthermore, the relative fluorescence intensity levels of intracellular leptospires and the viability of the intracellular leptospires increased in apocynin pretreated J774A.1 and THP-1 cells after 2 h of infection. CONCLUSIONS: The present study, based on our previous findings, further demonstrated that ROS contributed substantially to the bactericidal ability of mouse macrophages to kill intracellular leptospires. However, ROS did not contribute as much in human macrophages, which partially explains the different intracellular fates of L. interrogans in human and mouse macrophages.

[Development and Application of Reverse Transcription Loop-mediated Isothermal Amplification Method for Detection of Porcine Rotavirus of Swine].

We wished to establish a method for rapid and sensitive detection of reverse transcription loop-mediated isothermal amplification(RT-LAMP)for the rapid and sensitive detection of porcine rotavirus (PoRV). According to the published PoRV VP7 sequences in GenBank,6specific primers were designed. According to the concentrations of foward and reverse primers, Bst DNA polymerase, Mg(2+), and dNTP, reaction conditions were optimized. Results revealed the concentration ratio of foward and reverse primers to be 200 nmol/L:2, 400 nmol (1:12), Bst DNA polymerase concentration to be 0.64U/µL,Mg2+concentration to be 2.5mmol/L, and dNTP concentration to be 1.0mmol/L in 1hat 60℃.The amplification effect achieved a "ladder" effect, with amplified bands being shown only for PoRV. RT-LAMP was specific and did not elicit a cross reaction with porcine epidemic diarrhea virus, transmissible gastroenteritis virus of pigs, or classical swine fever virus. The sensitivity of RT-LAMP was 1.0×10(2) copies/µL. After the reaction, inspection by the naked eye revealed positive amplification products to appears as cloudy-white precipitates, and addition of SYBR Green I showed a color change. These data demonstrate that RT-LAMP is suitable for the rapid and sensitive detection of PoRV.

[Screening and identification of receptor reacting with nucleocapsid protein of duck enteritis virus].

To clarify the pathogenesis of Duck enteritis virus (DEV), the cDNA library of duck's liver infected by DEV and a bait plasmid containing DEV nucleocapsid protein (NP) gene were constructed, then the receptor was screened from the cDNA library plasmid by the yeast two-hybrid system and verified by GST pull-down test. The results showed that the capacity of the primary cDNA library was 1 x 106 CFU with insertion size from 0.5 to 1 kb, and the bait plasmid of pGBKT7-NP showed no self-activation. The receptor reacting with DEV NP in duck liver was initially confirmed as the protein kinase C inhibitor (PKCI). These results provide new clues for further investigation on pathogenesis of DEV.

[Field investigation of sparganosis in frogs in Huaxi area].

Frogs were caught from 4 towns in Huaxi of Guiyang and dissected. The collected spargana were used to infect young dogs for species identification. Results showed that the wild frogs were identified as Rana nigromaculata, and the infection rate was 16% (131/818) with an average intensity infection of 3.44 per frog, The tapeworm obtained from an infected dog was specified as Spirometra mansoni.

[Development and application of TaqMan-MGB real-time quantitative PCR assay for detection of goat pox virus].

The complete gene sequences of eight capripoxvirus strains in GenBank were aligned and analyzed with DNAStar software. We selected a size of 64 bp gene fragment that was located in gp064 region of goat pox virus (GPV) genome, and designed a pair of primers and a TaqMan-MGB probe against the gene fragment with Primer Express 2.0 software. Then, the fluorescence quantitative PCR (FQ-PCR) assay was developed and the standard curve of different dilution series was described. We extracted the DNA samples from clinical skin pox, scab and GPV infected materials of artificial challenge animals. The FQ-PCR assay has been performed for all kinds of DNA samples. The results showed that the FQ-PCR assay was sensitive, specific, stable and could be used for clinical diagnosis. This method provided an important tool for rapid diagnosis of goat pox clinically, and for study GPV pathogenesis in the course of disease occurrence, development and convalescence.