Agaricus subrufescens fermented rye affects the development of intestinal microbiota, local intestinal and innate immunity in suckling-to-nursery pigs.

BACKGROUND: Agaricus subrufescens is considered as one of the most important culinary-medicinal mushrooms around the world. It has been widely suggested to be used for the development of functional food ingredients to promote human health ascribed to the various properties (e.g., anti-inflammatory, antioxidant, and immunomodulatory activities). In this context, the interest in A. subrufescens based feed ingredients as alternatives for antibiotics has also been fuelled during an era of reduced/banned antibiotics use. This study aimed to investigate the effects of a fermented feed additive -rye overgrown with mycelium (ROM) of A. subrufescens-on pig intestinal microbiota, mucosal gene expression and local and systemic immunity during early life. Piglets received ROM or a tap water placebo (Ctrl) perorally every other day from day 2 after birth until 2 weeks post-weaning. Eight animals per treatment were euthanized and dissected on days 27, 44 and 70. RESULTS: The results showed ROM piglets had a lower inter-individual variation of faecal microbiota composition before weaning and a lower relative abundance of proteobacterial genera in jejunum (Undibacterium and Solobacterium) and caecum (Intestinibacter and Succinivibrionaceae_UCG_001) on day 70, as compared to Ctrl piglets. ROM supplementation also influenced gut mucosal gene expression in both ileum and caecum on day 44. In ileum, ROM pigs showed increased expression of TJP1/ZO1 but decreased expression of CLDN3, CLDN5 and MUC2 than Ctrl pigs. Genes involved in TLR signalling (e.g., TICAM2, IRAK4 and LY96) were more expressed but MYD88 and TOLLIP were less expressed in ROM pigs than Ctrl animals. NOS2 and HIF1A involved in redox signalling were either decreased or increased in ROM pigs, respectively. In caecum, differentially expressed genes between two groups were mainly shown as increased expression (e.g., MUC2, PDGFRB, TOLLIP, TNFAIP3 and MYD88) in ROM pigs. Moreover, ROM animals showed higher NK cell activation in blood and enhanced IL-10 production in ex vivo stimulated MLN cells before weaning. CONCLUSIONS: Collectively, these results suggest that ROM supplementation in early life modulates gut microbiota and (local) immune system development. Consequently, ROM supplementation may contribute to improving health of pigs during the weaning transition period and reducing antibiotics use.

Exploring the effect of a microencapsulated citrus essential oil on in vitro fermentation kinetics of pig gut microbiota.

Essential oils (EOs) have emerged as a potential alternative to antibiotics in pig breeding due to their antimicrobial properties. Citrus EOs, a common by-product of the orange juice industry, can be an interesting alternative from a financial perspective due to their huge offer in the global market. Thus, the effect of a citrus EO, and specifically different formulations of Brazilian Orange Terpenes (BOT), on pig gut microbiota was evaluated by means of an in vitro fermentation model simulating different sections of the pig gut (stomach, ileum, and colon). Treatments consisted in: BOT in its unprotected form (BOT, 1.85 and 3.70 mg/mL), microencapsulated BOT (MBOT, 3.50 and 7.00 mg/mL), colistin (2 µg/mL), and a control. BOT and MBOT altered in a similar way the total bacterial 16S rRNA gene copies in the stomach only from 18 h of incubation onwards, and no metabolite production in terms of short-chain fatty acids (SCFAs) was detected. In ileal and colonic fermentations, BOT and MBOT affected ileal and colonic microbiota in terms of total bacterial 16S rRNA gene copies, reduced phylogenetic diversity, and altered composition (p < 0.05) as evidenced by the significant reduction of certain bacterial taxa. However, more pronounced effects were found for MBOT, indicating its higher antimicrobial effects compared to the unprotected BOT, and suggesting that the antibacterial efficiency of the unprotected BOT was probably enhanced by microencapsulation. Furthermore, MBOT stimulated lactate production in ileal fermentations and greatly stimulated overall SCFA production in colonic fermentations. This indicates that besides the shifts in ileal and colonic microbiota by the delivered EO (BOT), the wall material of microcapsules (chitosan/modified starch) might have worked as an additional carbon source with prebiotic functioning, stimulating growth and metabolic activity (SCFAs) of colonic bacteria.

Effects of E scherichia coli Nissle 1917 on the Porcine Gut Microbiota, Intestinal Epithelium and Immune System in Early Life.

Early in life and particularly around weaning, piglets are susceptible to infections because of abrupt social, environmental, and dietary changes. Dietary interventions with probiotic bacteria have gained popularity because of the increased awareness of the direct link between diet and health. In this study, piglets received the probiotic strain Escherichia coli Nissle 1917 (EcN) or a control treatment perorally from day 2 after birth until 2 weeks post-weaning. To investigate spatio-temporal effects of EcN on the gut microbiota composition, intestinal epithelial gene expression and immune system, feces, digesta, blood, scraping material and mesenteric lymph node tissue were collected at different time points. In addition, oral vaccinations against Salmonella enterica serovar Typhimurium were administered on days 21 and 45 of the study to assess the immunocompetence. EcN-treated pigs showed a reduced diversity of taxa within the phylum Proteobacteria and a lower relative abundance of taxa within the genus Treponema during the pre-weaning period. Moreover, EcN induced T cell proliferation and Natural Killer cell activation in blood and enhanced IL-10 production in ex vivo stimulated mesenteric lymph node cells, the latter pointing toward a more regulatory or anti-inflammatory state of the local gut-associated immune system. These outcomes were primarily observed pre-weaning. No significant differences were observed between the treatment groups with regards to body weight, epithelial gene expression, and immune response upon vaccination. Differences observed during the post-weaning period between the treatment groups were modest. Overall, this study demonstrates that the pre-weaning period offers a 'window of opportunity' to modulate the porcine gut microbiota and immune system through dietary interventions such as EcN supplementation.

Environmentally enriched housing conditions affect pig welfare, immune system and gut microbiota in early life.

BACKGROUND: Conventional pig housing and management conditions are associated with gastrointestinal pathophysiology and disease susceptibility in early life. Developing new strategies to reduce both therapeutic and prophylactic antibiotic use is urgent for the sustainable swine production globally. To this end, housing methodology providing effective environmental enrichment could be a promising alternative approach to reduce antibiotic usage, as it has been proven to positively influence pig welfare and immune status and reduce susceptibility to infections. It is, however, poorly understood how this enriched housing affects systemic and local pulmonary immune status and gut microbiota colonization during early life. In the present study, we compared the effects of two housing conditions, i.e., conventional housing: (CH) versus enriched housing (EH), on immune status and gut microbiota from birth until 61 days of age. RESULTS: The expected benefits of enrichment on pig welfare were confirmed as EH pigs showed more positive behaviour, less aggression behaviour during the weaning transition and better human animal relation during the post weaning phase. Regarding the pigs' immune status, EH pigs had higher values of haemoglobin and mean corpuscular volume in haematological profiles and higher percentages of T cells and cytotoxic T cells in peripheral blood. Furthermore, EH pigs showed higher ex vivo secretion of IL1ß and TNF-α after lipopolysaccharide stimulation of whole blood than CH pigs. The structure of the developing faecal microbiota of CH and EH pigs significantly differed as early as day 12 with an increase in the relative abundance of several bacterial groups known to be involved in the production of short chain fatty acids, such as Prevotella_2, Christensenellaceae_R_7_group and Ruminococcus gauvreauii group. Furthermore, the main difference between both housing conditions post weaning was that on day 61, CH pigs had significantly larger inter-individual variation of ileal and colonic microbiota than EH pigs. In addition to housing, other intrinsic factors (e.g., sex) were associated with gut microbiota development and immune competence. CONCLUSIONS: In addition to the known welfare benefits for pigs, environmentally enriched housing also positively drives important aspects of the development of the immune system and the establishment of gut microbiota in early life. Consequently, EH may contribute to increasing productivity of pigs and reducing antibiotic use.

Chryseobacterium chengduensis sp. nov. isolated from the air of captive giant panda enclosures in Chengdu, China.

A Gram-negative, aerobic, non-motile, rod-shaped bacterial strain, designated 25-1(T), was isolated from the air inside giant panda enclosures at the Chengdu Research Base of Giant Panda Breeding, China. Strain 25-1(T) grew optimally at pH 7.0-8.0, at 28-30 °C and in the presence of NaCl concentrations from 0.0% to 0.5 %. 16S rRNA gene sequence analysis indicated that strain 25-1(T) belongs to the genus Chryseobacterium within the family Flavobacteriaceae and is related most closely to C. carnis G81(T) (96.4% similarity), C. lathyri RBA2-6(T) (95.8% similarity), and C. zeae JM1085(T) (95.8% similarity). Its genomic DNA G+C molar composition was 36.2%. The major cellular fatty acids were iso-C15:0 (44.0%), iso-C17:0 3OH (19.8%) and C16:1 ω7c/16:1 ω6c (12.7%). The only isoprenoid quinone was menaquinone 6 (MK-6). The major polar lipids were phosphatidylethanolamine, two unidentified amino lipids and two unidentified lipids. The DNA-DNA relatedness between strain 25-1(T) and C. lathyri RBA2-6(T) was 38%. Phenotypic, genotypic, and phylogenetic characteristics indicated that strain 25-1(T) is a novel member of the genus Chryseobacterium, for which the name C. chengduensis sp. nov. is proposed. The type strain is 25-1(T) (CCTCC AB2015133(T)=DSM 100396(T)).

Lysobacter chengduensis sp. nov. Isolated from the Air of Captive Ailuropoda melanoleuca Enclosures in Chengdu, China.

A novel bacterial strain, designated as CF21(T), was isolated from the air of Ailuropoda melanoleuca enclosures in China. Cells were gram-negative, aerobic, non-motile, and rod shaped. Strain CF21(T) grew at 10-40 °C (optimum 28-30 °C) and pH 6.0-9.0 (optimum pH 7.0-8.0) and in the presence of NaCl concentrations ranging from 0.0% (w/v) to 2.0 % (optimum 0.0-1.0%). 16SrRNA gene sequence analysis indicated that strain CF21(T) belonged to genus Lysobacter within class Gammaproteobacteria and was most closely related to Luteimonas dalianensi OB44-3(T) (95.8% similarity), Lysobacter ruishenii CTN-1(T) (95.1%), Lysobacter spongiicola KMM329(T) (94.8 %), and Lysobacter daejeonensis GH1-9T (94.6%). The genomic G+C DNA content was 68.72 mol%. Major cellular fatty acids of CF21(T) were iso-C16:0 (30.22%), iso-C15:0 (25.70%), and the sum of 10-methyl C16 : 0 and/or iso-C17 : 1ω9c (21.94%). The prominent isoprenoid quinone was ubiquinone 8 (Q-8). Primary polar lipids included diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, and an unknown phospholipid. DNA sequence relatedness between strain CF21(T) and L. ruishenii CTN-1(T) was 56%, which was clearly below the 70% threshold for prokaryotic species delineation. These analyses indicated that CF21(T) is a novel member of genus Lysobacter, for which the name Lysobacter chengduensis sp. nov. is proposed. The type strain is CF21(T) (=CGMCC1.15145(T) = DSM 100306(T)).

Sensitive detection of Porcine circovirus-2 by droplet digital polymerase chain reaction.

Sensitive detection of Porcine circovirus-2 (PCV-2) is very important for surveillance of postweaning multisystemic wasting syndrome. Droplet digital polymerase chain reaction (ddPCR) is novel PCR method that can achieve high precision. Our study aimed to develop a sensitive assay utilizing ddPCR to detect PCV-2. Specificity of the assay was confirmed by the failure of amplification of DNA of other relevant viruses. The detection limit for ddPCR was 25 copies/µL, a 4-fold greater sensitivity than TaqMan real-time PCR. Both methods showed a high degree of linearity (R(2) = ~1), although TaqMan real-time PCR showed less sensitivity than ddPCR for clinical detection. Our findings indicate that ddPCR might represent a promising platform for detecting PCV-2 viral loads.

Promoter methylation, mRNA expression of goat tumor­associated genes and mRNA expression of DNA methyltransferase in enzootic nasal tumors.

The aim of the present study was to investigate the promoter methylation status and mRNA expression of goat tumor­associated genes, in addition to the mRNA expression of DNA methyltransferase genes in enzootic nasal tumors (ENT). Methylation­specific polymerase chain reaction and SYBR Green reverse transcription­quantitative polymerase chain reaction were used to detect the methylation status and the mRNA expression levels of DNA methyltransferases (DNMTs), O6­methylguanine­DNA methyltransferase (MGMT), the tumor suppressor genes P73, P53, GADD45G, CHFR and THBS1, the transcription factor CEBPA, the proto­oncogenes KRAS, NRAS and C­myc and EGFR in 24 nasal tumor tissue samples and 20 normal nasal epithelia tissue samples. The associations between promoter methylation and DNMT, and promoter methylation and mRNA expression of the genes were analyzed. The results indicated that the expression levels of DNMT1 increased by 56% compared with those in normal nasal epithelial tissues, while MGMT, DNMT3a and DNMT3b had similar expression levels in the two tissue types. The expression levels of P53 decreased by 36.8% and those of THBS1 by 43%, while C­myc increased by 2.9­fold and CEBPA by 2­fold compared with that in normal nasal epithelial tissues. GADD45G, P73, CHFR and NRAS were observed to have similar expression levels in the two tissue types. However, no expression was observed for EGFR and KRAS. CHFR, GADD45G and THBS1 were identified to be methylated in tumor suppressor genes. The methylation expression rate of the CHFR gene was ~60% in the two tissue types and for THBS1 it was 100% in the nasal tumor tissues as opposed to 20% in the normal nasal epithelial tissues. The exhaustive methylation expression rate of GADD45G was 62.5% and the partial methylation expression rate was 37.5% in nasal tumor tissue, while no methylation was observed in normal nasal epithelial tissues. C­myc was the only gene identified to be methylated amongst proto­oncogenes. The methylation expression rate of C­myc was 87.5% in nasal tumor tissues and 15% in normal nasal epithelial tissues. The methylation expression rate of CEBPA was 100% in nasal tumor tissues and 40% in normal nasal epithelial tissues. The methylation expression rate of the EGFR gene was ~80% in the two tissues. In summary, the present study identified abnormal methylation of the C­myc, CEBPA, GADD45G and THBS1 genes in nasal tumor tissues. The expression levels of DNMT1, C­myc and CEBPA were upregulated and the expression of P53 and THBSI were downregulated in nasal tumor tissues, with a significant difference between the two groups (P<0.05). Therefore, it is suggested that these six genes may be used as diagnostic marker candidates for ENT. The results may serve as a foundation for screening of tumor­specific markers for early diagnosis of ENT and further investigate the epigenetic mechanisms of enzootic nasal tumor virus (ENTV)­induced nasal epithelium cell carcinoma.