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 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.

Insights into the Gut and Skin Microbiome of Freshwater Fish, Smelt (Hypomesus nipponensis).

Freshwater smelt (Hypomesus nipponensis) is a planktivorous fish found in the river of South Korea, Japan, China, and Russia. Because of its specific characteristics living in the cold temperature, this species is economically valuable in the various countries that held winter festival. The body size of the smelt is too small, so people consumed raw smelt during the winter festival sometimes. However, the microbial studies of smelt are nonexistent. Here, we characterized and compared the bacterial communities in the gut and skin of freshwater smelts. We amplified, sequenced, and analyzed the V4 regions of bacterial 16S rRNA genes from freshwater smelts. The microbial diversity in the skin (375 OTUs) was much greater than that in the gut (250 OTUs). At the phylum level, Proteobacteria (gut: 51.5%; skin: 52.9%), Firmicutes (gut: 30.6%; skin: 25.4%), Bacteroidetes (gut: 7.7%; skin: 14.7%), and Actinobacteria (gut: 5.2%; skin: 3.8%) were predominant in both organs. At the genus level, Sphingomonas (gut: 24.9%; skin: 4.4%, P < 0.01) was more abundant in the gut, whereas Acinetobacter (gut: 0.8%; skin: 11.8%, P = 0.02) and Pseudomonas (gut: 0.3%; skin: 2.1%, P = 0.01) were more abundant in the skin. Both beneficial (Lactobacillus) and harmful (Staphylococcus and Streptococcus) bacteria were detected in both organs, even under freshwater conditions. These results revealed that smelts have their own unique microbial communities in the gut and skin. Our findings broaden the understanding of planktivorous freshwater fish microbiomes and provide new insights into fish microbiomes for ensuring food safety.

Differences in microbiome and virome between cattle and horses in the same farm.

OBJECTIVE: The ecosystem of an animal farm is composed of various elements, such as animals, farmers, plants, feed, soil, and microorganisms. A domesticated animal's health is largely connected with the reservoir of bacteria and viruses in animal farms. Although a few studies have focused on exploring the gut microbiome of animals, communities of microbiota and viruses in feedlots have not been thoroughly investigated. METHODS: Here, we collected feces and dust samples (4 groups: cattle feces, C_F; horse feces, H_F; cattle dust, C_D; and horse dust, H_D) from cattle and horse farms sharing the same housing and investigated their microbiome/virome communities by Illumina sequencing. RESULTS: Dust groups (C_D and H_D) showed higher microbial diversity than feces groups (C_F and H_F) regardless of animal species. From the microbial community analysis, all the samples from the four groups have major phyla such as Proteobacteria (min 37.1% to max 42.8%), Firmicutes (19.1% to 24.9%), Bacteroidetes (10.6% to 22.1%), and Actinobacteria (6.1% to 20.5%). The abundance of Streptococcus, which commonly recognized as equine pathogens, was significantly higher in the horse group (H_D and H_F). Over 99% among the classified virome reads were classified as Caudovirales, a group of tailed bacteriophages, in all four groups. Foot-and-mouth disease virus and equine adenovirus, which cause deadly diseases in cattle and horse, respectively, were not detected. CONCLUSION: Our results will provide baseline information to understand different gut and environmental microbial ecology between two livestock species.

Evaluating the association between body weight and the intestinal microbiota of weaned piglets via 16S rRNA sequencing.

Due to the ban on the use of antimicrobial growth promoters in livestock feeds, understanding the relationship between intestinal microbiota and the physiology of the host has become very important for improving livestock performance. In this study, we investigated the relationship between intestinal microbiota and body weights of weaned piglets. Lighter (n = 9) and heavier (n = 9) 9-week-old weaned piglets were selected from approximately one hundred individuals based on their body weights. Their fecal microbial communities were analyzed by sequencing the V4 region of the 16S rRNA gene. The microbial richness estimators of the heavier piglets were significantly higher than those of the lighter piglets. At the phylum level, the microbiota of the heavier group had significantly higher levels of Firmicutes and a higher Firmicutes-to-Bacteroidetes ratio than that of the lighter group. At the genus level, the levels of several genera, such as Anaerococcus and Lactococcus, were significantly different in the two groups. In particular, the lighter group had significantly higher levels of opportunistic pathogenic bacteria, such as Anaerotruncus and Bacteroides, compared with those of the heavier group. Moreover, the levels of bacteria expressing the components of several metabolic pathways were significantly different in the two groups. The microbiota of the heavier group had a significantly higher involvement in three KEGG pathways concerned with xenobiotic degradation than that of the lighter group. These results may provide insights into host-microbe interactions occurring in the piglet intestine and will be useful in establishing a strategy for improving growth performance in the swine industry.

Development of a Rapid Identification Method for the Differentiation of Enterococcus Species Using a Species-Specific Multiplex PCR Based on Comparative Genomics.

Enterococci are lactic acid bacteria that are commonly found in food and in animal gut. Since 16 S ribosomal RNA (rRNA) sequences, genetic markers for bacterial identification, are similar among several Enterococcus species, it is very difficult to determine the correct species based on only 16 S rRNA sequences. Therefore, we developed a rapid method for the identification of different Enterococcus species using comparative genomics. We compared 38 genomes of 13 Enterococcus species retrieved from the National Center of Biotechnology Information database and identified 25,623 orthologs. Among the orthologs, four genes were specific to four Enterococcus species (Enterococcus faecalis, Enterococcus faecium, Enterococcus hirae, and Enterococcus durans). We designed species-specific primer sets targeting the genes and developed a multiplex PCR using primer sets that could distinguish the four Enterococcus species among the nine strains of Enterococcus species that were available locally. The multiplex PCR method also distinguished the four species isolated from various environments, such as feces of chicken and cow, meat of chicken, cow, and pigs, and fermented soybeans (Cheonggukjang and Doenjang). These results indicated that our novel multiplex PCR using species-specific primers could identify the four Enterococcus species in a rapid and easy way. This method will be useful to distinguish Enterococcus species in food, feed, and clinical settings.

Multiview Monitoring of Individual Cattle Behavior Based on Action Recognition in Closed Barns Using Deep Learning.

Cattle behavior recognition is essential for monitoring their health and welfare. Existing techniques for behavior recognition in closed barns typically rely on direct observation to detect changes using wearable devices or surveillance cameras. While promising progress has been made in this field, monitoring individual cattle, especially those with similar visual characteristics, remains challenging due to numerous factors such as occlusion, scale variations, and pose changes. Accurate and consistent individual identification over time is therefore essential to overcome these challenges. To address this issue, this paper introduces an approach for multiview monitoring of individual cattle behavior based on action recognition using video data. The proposed system takes an image sequence as input and utilizes a detector to identify hierarchical actions categorized as part and individual actions. These regions of interest are then inputted into a tracking and identification mechanism, enabling the system to continuously track each individual in the scene and assign them a unique identification number. By implementing this approach, cattle behavior is continuously monitored, and statistical analysis is conducted to assess changes in behavior in the time domain. The effectiveness of the proposed framework is demonstrated through quantitative and qualitative experimental results obtained from our Hanwoo cattle video database. Overall, this study tackles the challenges encountered in real farm indoor scenarios, capturing spatiotemporal information and enabling automatic recognition of cattle behavior for precision livestock farming.

Improving Known-Unknown Cattle's Face Recognition for Smart Livestock Farm Management.

Accurate identification of individual cattle is of paramount importance in precision livestock farming, enabling the monitoring of cattle behavior, disease prevention, and enhanced animal welfare. Unlike human faces, the faces of most Hanwoo cattle, a native breed of Korea, exhibit significant similarities and have the same body color, posing a substantial challenge in accurately distinguishing between individual cattle. In this study, we sought to extend the closed-set scope (only including identifying known individuals) to a more-adaptable open-set recognition scenario (identifying both known and unknown individuals) termed Cattle's Face Open-Set Recognition (CFOSR). By integrating open-set techniques to enhance the closed-set accuracy, the proposed method simultaneously addresses the open-set scenario. In CFOSR, the objective is to develop a trained model capable of accurately identifying known individuals, while effectively handling unknown or novel individuals, even in cases where the model has been trained solely on known individuals. To address this challenge, we propose a novel approach that integrates Adversarial Reciprocal Points Learning (ARPL), a state-of-the-art open-set recognition method, with the effectiveness of Additive Margin Softmax loss (AM-Softmax). ARPL was leveraged to mitigate the overlap between spaces of known and unknown or unregistered cattle. At the same time, AM-Softmax was chosen over the conventional Cross-Entropy loss (CE) to classify known individuals. The empirical results obtained from a real-world dataset demonstrated the effectiveness of the ARPL and AM-Softmax techniques in achieving both intra-class compactness and inter-class separability. Notably, the results of the open-set recognition and closed-set recognition validated the superior performance of our proposed method compared to existing algorithms. To be more precise, our method achieved an AUROC of 91.84 and an OSCR of 87.85 in the context of open-set recognition on a complex dataset. Simultaneously, it demonstrated an accuracy of 94.46 for closed-set recognition. We believe that our study provides a novel vision to improve the classification accuracy of the closed set. Simultaneously, it holds the potential to significantly contribute to herd monitoring and inventory management, especially in scenarios involving the presence of unknown or novel cattle.

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.

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.

Ecological Effects of Benzyl Chloride on Different Korean Aquatic Indigenous Species Using an Artificial Stream Mesocosm Simulating a Chemical Spill.

In this study, an artificial stream mesocosm consisting of a head tank, faster-flowing riffle section, gravel section, pool section, lower-run section, and tail tank was installed to simulate a chemical spill in a river. The responses of freshwater periphyton algae, crustacea (Moina macrocopa), freshwater worm (Limnodrilus hoffmeisteri), benthic midge (Glyptotendipes tokunagai), and fish (Zacco platypus and Aphyocypris chinensis) were observed after exposure to benzyl chloride (classified as an accident preparedness substance, APS) at concentrations of 1, 2, and 4 µL/L for 22.5 h. Higher concentrations increased the inhibition (photosynthetic efficiency decrease) of periphyton algae and the mortality of M. macrocopa, whereas the reproduction of the female cladoceran decreased in the 4 µL/L treatment. Mortality of fish did not occur or was lower (≤20%) at all concentrations; however, toxic symptoms were observed for some time after chemical exposure termination and later, symptoms receded. G. tokunagai mortality increased at all concentrations except the control after seven days, and no significant toxic effects were observed in L. hoffmeisteri. The hazardous concentration of benzyl chloride was calculated as 94 µg/L. This study showed the different sensitivities of each species to benzyl chloride. The findings can assist in environmental risk assessment of APSs after chemical spills to protect Korean aquatic species.

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.

Changes in the Microbial Community of the Mottled Skate (Beringraja pulchra) During Alkaline Fermentation.

Beringraja pulchra, Cham-hong-eo in Korean, is a mottled skate which is belonging to the cartilaginous fish. Although this species is economically valuable in South Korea as an alkalinefermented food, there are few microbial studies on such fermentation. Here, we analyzed microbial changes and pH before, during, and after fermentation and examined the effect of inoculation by a skin microbiota mixture on the skate fermentation (control vs. treatment). To analyze microbial community, the V4 regions of bacterial 16S rRNA genes from the skates were amplified, sequenced and analyzed. During the skate fermentation, pH and total number of marine bacteria increased in both groups, while microbial diversity decreased after fermentation. Pseudomonas, which was predominant in the initial skate, declined by fermentation (Day 0: 11.39 ± 5.52%; Day 20: 0.61 ± 0.9%), while the abundance of Pseudoalteromonas increased dramatically (Day 0: 1.42 ± 0.41%; Day 20: 64.92 ± 24.15%). From our co-occurrence analysis, the Pseudoalteromonas was positively correlated with Aerococcaceae (r = 0.638) and Moraxella (r = 0.474), which also increased with fermentation, and negatively correlated with Pseudomonas (r = -0.847) during fermentation. There are no critically significant differences between control and treatment. These results revealed that the alkaline fermentation of skates dramatically changed the microbiota, but the initial inoculation by a skin microbiota mixture didn't show critical changes in the final microbial community. Our results extended understanding of microbial interactions and provided the new insights of microbial changes during alkaline fermentation.

Changes in Gut Microbial Community of Pig Feces in Response to Different Dietary Animal Protein Media.

Beef, pork, chicken and milk are considered representative protein sources in the human diet. Since the digestion of protein is important, the role of intestinal microflora is also important. Despite this, the pure effects of meat and milk intake on the microbiome are yet to be fully elucidated. To evaluate the effect of beef, pork, chicken and milk on intestinal microflora, we observed changes in the microbiome in response to different types of dietary animal proteins in vitro. Feces were collected from five 6-week-old pigs. The suspensions were pooled and inoculated into four different media containing beef, pork, chicken, or skim milk powder in distilled water. Changes in microbial communities were analyzed using 16S rRNA sequencing. The feces alone had the highest microbial alpha diversity. Among the treatment groups, beef showed the highest microbial diversity, followed by pork, chicken, and milk. The three dominant phyla were Proteobacteria, Firmicutes, and Bacteroidetes in all the groups. The most abundant genera in beef, pork, and chicken were Rummeliibacillus, Clostridium, and Phascolarctobacterium, whereas milk was enriched with Streptococcus, Lactobacillus, and Enterococcus. Aerobic bacteria decreased while anaerobic and facultative anaerobic bacteria increased in protein-rich nutrients. Functional gene groups were found to be over-represented in protein-rich nutrients. Our results provide baseline information for understanding the roles of dietary animal proteins in reshaping the gut microbiome. Furthermore, growth-promotion by specific species/genus may be used as a cultivation tool for uncultured gut microorganisms.

Pan-Genomics of Lactobacillus plantarum Revealed Group-Specific Genomic Profiles without Habitat Association.

Lactobacillus plantarum is a lactic acid bacterium that promotes animal intestinal health as a probiotic and is found in a wide variety of habitats. Here, we investigated the genomic features of different clusters of L. plantarum strains via pan-genomic analysis. We compared the genomes of 108 L. plantarum strains that were available from the NCBI GenBank database. These genomes were 2.9-3.7 Mbp in size and 44-45% in G+C content. A total of 8,847 orthologs were collected, and 1,709 genes were identified to be shared as core genes by all the strains analyzed. On the basis of SNPs from the core genes, 108 strains were clustered into five major groups (G1-G5) that are different from previous reports and are not clearly associated with habitats. Analysis of group-specific enriched or depleted genes revealed that G1 and G2 were rich in genes for carbohydrate utilization (L-arabinose, L-rhamnose, and fructooligosaccharides) and that G3, G4, and G5 possessed more genes for the restriction-modification system and MazEF toxin-antitoxin. These results indicate that there are critical differences in gene content and survival strategies among genetically clustered L. plantarum strains, regardless of habitats.

Effect of Ground Chopi (Zanthoxylum piperitum) on Physicochemical Traits and Microbial Community of Chicken Summer Sausage during Manufacture.

Changes in microbial community and physicochemical traits of chicken summer sausage made from spent layer thigh added with different level (0%, 0.1%, 0.3%, and 0.5% w/w) of ground chopi (Zanthoxylum piperitum) during manufacture were analyzed. The microbial community was profiled and analyzed by sequencing 16S rRNA gene using Illumina MiSeq. Samples were taken from raw sausage batter, after 15 h of fermentation, 8 h of cooking including cooling down, and 7 d of drying. The final pH of the sausage was reduced by the addition of ground chopi. However, no clear effect on water activity was observed. Ground chopi inhibited the development of red curing color after fermentation as it exhibited antimicrobial effect. However, the effect on species richness and microbial composition after cooking was unclear. Ground chopi delayed lipid oxidation during manufacture and the effect was dependent on the addition level. Fermentation reduced the species richness with a dominancy of lactic acid bacteria. The profile of microbiota in the raw batter was different from other stages, while the closest relationship was observed after cooking and drying. Proteobacteria was predominant, followed by Firmicutes and Bacteroidetes in raw samples. Firmicutes became dominating after fermentation and so forth, whereas other predominant phylum decreased. At genus level, unclassified Lactobacillales was the most abundant group found after fermentation and so forth. Therefore, the overall microbial composition aspects were mainly controlled during fermentation by the abundance of lactic acid bacteria, while bacterial counts and lipid oxidation were controlled by cooking and the addition of ground chopi.

Tracing of the fecal microbiota of commercial pigs at five growth stages from birth to shipment.

The intestinal microbiota affect various physiological traits of host animals such as brain development, obesity, age, and the immune system. In the swine industry, understanding the relationship between intestinal microbiota and growth stage is essential because growth stage is directly related to the feeding system of pigs, thus we studied the intestinal microbiota of 32 healthy pigs across five sows at 10, 21, 63, 93, and 147 d of ages. The intestinal microbiota were altered with growth of pigs and were separated into three distinct clusters. The relative abundance of several phyla and genera were significantly different between growth stages. We observed co-occurrence pattern of the intestinal microbiota at each growth stage. In addition, we predicted the functions of the intestinal microbiota and confirmed that several KEGG pathways were significantly different between growth stages. We also explored the relationship between the intestinal microbiota and innate factors such as the maternal effect and gender. When pigs were young, innate factors affected on construction of intestinal microbiota, however this tendency was disappeared with growth. Our findings broaden the understanding of microbial ecology, and the results will be used as a reference for investigating host-microbe interactions in the swine industry.

Prohibition of antibiotic growth promoters has affected the genomic profiles of Lactobacillus salivarius inhabiting the swine intestine.

After the introduction of a ban on the use of antibiotic growth promoters (AGPs) for livestock, the feeding environment, including the composition of animal intestinal microbiota, has changed rapidly. We hypothesized that the microbial genomes have also been affected by this legal prohibition, and investigated an important member of the swine gut microbiota, Lactobacillus salivarius, with a pan-genomic approach. Here, we isolated 21 L. salivarius strains composed of 6 strains isolated before the AGP prohibition (SBPs) and 15 strains isolated after the AGP prohibition (SAPs) at an interval of a decade, and the draft genomes were generated de novo. Several genomic differences between SBPs and SAPs were identified, although the number and function of antibiotic resistance genes were not different. SBPs showed larger genome size and a higher number of orthologs, as well as lower genetic diversity, than SAPs. SBPs had genes associated with the utilization of L-rhamnose and D-tagatose for energy production. Because these sugars are also used in exopolysaccharide (EPS) synthesis, we tried to identify differences in biofilm formation-associated genes. The genes for the production of EPSs and extracellular proteins were different in terms of amino acid sequences. Indeed, SAPs formed dense biofilm and survived better than SBPs in the swine intestinal environment. These results suggest that SAPs have evolved and adapted to protect themselves from new selection pressure of the swine intestinal microenvironment by forming dense biofilms, adopting a distinct antibiotic resistance strategy. This finding is particularly important to understand the evolutionary changes in host-microbe interaction and provide detailed insight for the development of effective probiotics for livestock.