Postbiotic-based recombinant receptor activator of NF-κB ligand enhanced oral vaccine efficiency in chicken.

Functional M cells are differentiated by receptor activator of NF-κB ligand (RANKL) and capture of luminal antigens to initiate immune responses. We aimed to use postbiotic-based recombinant chicken RANKL (cRANKL) to promote M cell differentiation and test the efficacy of oral vaccines. Chicks were divided into three groups that were administered phosphate-buffered saline (PBS), cell extracts of wild-type Lactococcus lactis subsp. lactis IL1403 (WT_CE), or cell extracts of recombinant L. lactis expressing cRANKL (cRANKL_CE). The expression of the M cell marker was measured, and the gut microbiome was profiled. The efficiency of the infectious bursal disease (IBD) vaccine was tested after 12 consecutive days of administering cRANKL_CE. The chickens that were administered cRANKL_CE (p = 0.038) had significantly higher Annexin A5 (ANXA5) mRNA expression levels than those in the PBS group (PBS vs. WT_CE, p = 0.657). In the gut microbiome analysis, no significant changes were observed. However, the relative abundance of Escherichia-Shigella was negatively correlated (r = - 0.43, p = 0.019) with ANXA5 mRNA expression in Peyer's patches. cRANKL_CE/IBD (p = 0.018) had significantly higher IBD-specific faecal IgA levels than PBS/IBD (PBS/IBD vs. WT_CE/IBD, p = 0.217). Postbiotic-based recombinant cRANKL effectively improved the expression of M cell markers and the efficiency of oral vaccines. No significant changes were observed in the gut microbiome after administration of postbiotic-based recombinant cRANKL. This strategy can be used for the development of feed additives and adjuvants. KEY POINTS: • Postbiotic-based recombinant cRANKL enhanced the expression of ANXA5 in chicken. • The relative abundance of Escherichia-Shigella was negatively correlated with ANXA5 expression. • Postbiotic-based recombinant cRANKL effectively improved the efficiency of oral vaccine.

Omega-3-Rich Fish-Oil-Influenced Mouse Gut Microbiome Shaped by Intermittent Consumption of Beef.

Beef consumption can provide various amino acids, lipids, vitamins, and minerals; however, excessive intake causes metabolic disorders and increases the probability of obesity, atherosclerosis, and colorectal cancer. The intake of omega-3 fatty acids can ameliorate metabolic disorders by lowering blood glucose and triglyceride levels. In the present study, we investigated the effect of omega-3-rich fish oil on body performance and the gut microbiome in a beef-rich diet. Four-week-old C57BL/6 mice were distributed into four groups (chow diet [Chow], chow with beef diet [Beef], chow with omega-3 diet [Cw3], and chow with beef and omega-3 diet [Bw3]). We observed that body weight was unaltered between groups, and serum triglyceride levels were reduced in the omega-3 supplemented groups. The beta diversity indices, unweighted UniFrac distance (P = 0.001), and Jaccard distance (P = 0.001) showed statistically significant differences, and the principal coordinates analysis plot showed a clear separation between groups. In addition, the taxonomic comparison revealed that beef consumption increased numerous potentially pathogenic bacteria, including Escherichia-Shigella, Mucispirillum, Helicobacter, and Desulfovibrio, which were decreased following omega-3 supplementation. Metabolic comparison based on 16S rRNA revealed that energy and glucose metabolism were higher in omega-3 supplemented groups. Our findings suggest that the omega-3 supplementation under intermittent beef consumption contributes to changes in the gut microbiome and microbial metabolic pathways.

Oral Administration of Mice with Cell Extracts of Recombinant Lactococcus lactis IL1403 Expressing Mouse Receptor Activator of NF-kB Ligand (RANKL).

Receptor activator of NF-kB ligand (RANKL) is known to play a major role in bone metabolism and the immune system, and its recombinant form has been expressed in bacterial systems for research since the last two decades. However, most of these recombinant forms are used after purification or directly using living cells. Here, there were cell extracts of recombinant Lactococcus lactis expressing mouse RANKL (mRANKL) used to evaluate its biological activity in mice. Mice were divided into three groups that were fed phosphate-buffered saline (PBS), wild-type L. lactis IL1403 (WT_CE), and recombinant L. lactis expressing mRANKL (mRANKL_CE). The small intestinal transcriptome and fecal microbiome were then profiled. The biological activity of mRANKL_CE was confirmed by studying RANK-RANKL signaling in vitro and in vivo. For small intestinal transcriptome, differentially expressed genes (DEGs) were identified in the mRANKL_CE group, and no DEGs were found in the WT_CE group. In the PBS vs. mRANKL_CE gene enrichment analysis, upregulated genes were enriched for heat shock protein binding, regulation of bone resorption, and calcium ion binding. In the gut microbiome analysis, there were no critical changes among the three groups. However, Lactobacillus and Sphingomonas were more abundant in the mRANKL_CE group than in the other two groups. Our results indicate that cell extracts of mRANKL_CE can play an effective role without a significant impact on the intestine. This strategy may be useful for the development of protein drugs.

Oral Immunization of Mice with Cell Extracts from Recombinant Lactococcus lactis Expressing SARS-CoV-2 Spike Protein.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread all over the world and became a pandemic that named coronavirus disease-2019 (COVID-19). At present, several intramuscular vaccines have been successfully developed and mass vaccination has progressed in many countries. The aim of the study is to develop and examine an oral vaccine against COVID-19 with recombinant Lactococcus lactis IL1403, a strain of lactic acid bacteria, expressing SARS-CoV-2 spike (S) protein receptor-binding domain (RBD) S1 subunit as an immunizing antigen. PBS or cell extracts from recombinant L. lactis were orally administered into mice (control VS treatment), and formation of antigen-specific antibodies and changes in the gut microbiome were analyzed. Intracellular antigen was detected, but its secretion was not successful. After immunization, antigen-specific serum IgG and fecal IgA levels were 1.5-fold (P = 0.002) and 1.4-fold (P = 0.016) higher in the immunized mice (treatment) than control, respectively. Gut microbiome profiles were clearly separated between the two groups when analyzed for beta diversity with overall similarity. At the genus level, while Coprococcus (P = 0.036) and unclassified genus of Ruminococcaceae (P = 0.037) in treatment were more abundant than control, rc4-4 (P = 0.013) and Stenotrophomonas (P = 0.021) were less abundant. Our results indicate that cell extract containing SARS-CoV-2 antigen can induce mice to produce antigen-specific antibodies without overall changes in the gut microbiome. This strategy may be useful for the development of other oral viral vaccines.

Draft genome of the Korean smelt Hypomesus nipponensis and its transcriptomic responses to heat stress in the liver and muscle.

Pond smelt (Hypomesus nipponensis) is a cold-freshwater fish species and a winter economic aquaculture resource in South Korea. Because of its high susceptibility to abnormal water temperature from global warming, a large number of smelt die in hot summers. Here, we present the first draft genome of H. nipponensis and transcriptomic changes in molecular mechanisms or intracellular responses under heat stress. We combined Illumina and PacBio sequencing technologies to generate the draft genome of H. nipponensis. Based on the reference genome, we conducted transcriptome analysis of liver and muscle tissues under normal (NT, 5°C) vs. warm (HT, 23°C) conditions to identify heat stress-induced genes and gene categories. We observed a total of 1987 contigs with N50 of 0.46 Mbp, with the largest contig (3.03 Mbp) in the assembled genome. A total of 20,644 protein-coding genes were predicted, and 19,224 genes were functionally annotated: 15,955 genes for Gene Ontology terms and 11,560 genes for KEGG Orthology. We conducted the lost and gained genes analysis compared with three species that: human, zebrafish, and salmon. In the lost genes analysis, we detected that smelt lost 4461 (22.16%), 2825 (10.62%), and 1499 (3.09%) genes compare with above three species, respectively. In the gained genes analysis, we observed that smelt gained 1133 (5.49%), 1670 (8.09%), and 229 (1.11%) genes compared with the above species, respectively. From transcriptome analysis, a total of 297 and 331 differentially expressed genes (DEGs) with a false discovery rate <0.05 were identified in the liver and muscle tissues, respectively. Gene enrichment analysis of DEGs indicates that upregulated genes were significantly enriched for lipid biosynthetic process (GO:0008610, P < 0.001) and regulation of apoptotic process (GO:0042981, P < 0.01), and genes were downregulated by immune responses such as myeloid cell differentiation (GO:0030099, P < 0.001) in the liver under heat stress. In muscle tissue, upregulated genes were enriched for hypoxia (GO:0001666, P < 0.05), transcription regulator activity (GO:0140110, P < 0.001), and calcium-release channel activity (GO:0015278, P < 0.01), and genes were downregulated for a nicotinamide nucleotide biosynthetic process (GO:0019359, P < 0.01). The results of KEGG pathway analysis were similar to that of gene enrichment analysis. The draft genome and transcriptomic of H. nipponensis will be a useful genetic resource for functional and evolutionary studies. Our findings will improve understanding of molecular mechanisms and heat responses and be useful for predicting survival of the smelt and its closely related species under global warming.

Effects of Lactobacillus salivarius isolated from feces of fast-growing pigs on intestinal microbiota and morphology of suckling piglets.

The study determined the effects of Lactobacillus salivarius (LS) administered early in the life of suckling piglets on their growth performance, gut morphology, and gut microbiota. Thirty litters of 3-day-old crossbreed piglets were randomly assigned to one of the three treatments, and treatments were commenced on day 3 after birth. During the whole period of the experiment, the piglets were kept with their mothers and left to suckle ad libitum while being supplemented with a milk formula with or without the bacterial probiotic supplemented. The control group (CON) was not treated with probiotics, the HLS group was treated with LS144 (HLS) screened from feces of fast-growing pigs with high body mass index (BMI) while the NLS group was supplemented with LS160 (NLS) screened from feces obtained from pigs of normal BMI. At the weaning time, a higher abundance of Actinobacteria, Lentisphaerae, and Elusimicrobia phyla were observed in NLS piglets, whereas the abundance of Fibrobacteres phylum was significantly reduced in NLS and HLS piglets compared with the CON. A greater abundance of Lactobacillus was detected in the HLS treatment compared with the CON. The abundance of Bacteroides and Fibrobacter was higher in the CON piglets compared with the HLS and NLS piglets. Compared with the CON group, the oral administration of LS significantly increased the number of Lactobacillus and villus height in the duodenum, jejunum, and ileum. Moreover, the villus height of the duodenum was significantly improved in the HLS treatment compared with the NLS treatment. Based on the findings in the neonatal piglet model, we suggest that oral supplementation of LS, particularly LS isolated from high BMI pigs, could be beneficial by improving the intestinal villus height.

Crocin supplementation during oocyte maturation enhances antioxidant defence and subsequent cleavage rate.

The purpose of the present study was to assess the effect of crocin supplementation during oocyte maturation on the antioxidant defence and anti-apoptotic ability and subsequent developmental competence of porcine oocytes. Oocytes were cultured in media containing 0, 300, 400 or 500 µg/ml of crocin. Upon maturation, the maturation rates, reactive oxygen species (ROS) and glutathione (GSH) levels, mRNA expression of genes (SOD, CAT, GPx, Bcl-2, BAX and Caspase3), expression of cleaved caspase3 and subsequent embryo cleavage rates were measured. Results indicated that the maturation rate of the 400 µg/ml group was 86.80% (p < 0.01). The ROS concentration of the 500 µg/ml group was the lowest (p < 0.01). The GSH concentration of the 400 µg/ml group was the highest (p < 0.01). The SOD, CAT and GPx mRNA expression levels were the highest in the 300, 400 and 500 µg/ml groups, respectively, with the expression levels of all genes being significantly higher than that of the control group (p < 0.01). The Bcl-2/BAX mRNA expression ratio in 400 and 500 µg/ml groups significantly higher than other groups and significantly decreased caspase3 expression level (p < 0.01). The expression level of cleaved caspase3 in the 500 µg/ml treatment group was the lowest, significantly lower than that of the control group (p < 0.01). The cleavage rate of the 400 µg/ml group was 62.50% (p < 0.01). These experimental results show that the supplementation of in vitro culture medium with 400 µg/ml of crocin significantly enhanced the antioxidant defence and anti-apoptotic ability and subsequent cleavage rate of porcine embryo.